Biokimia Untuk Kimia

DAFTAR ISI
2020
Halaman
Daftar Isi 2
Kata Pengantar 3
I. DIKTAT
Tata Tertib Laboratorium Biokimia
Pembuatan larutan buffer
II. Uji lipid
4
6
14
PRAKTIKUM
III. Uji asam amino dan protein
IV. Penentuan kadar protein dengan metoda Lowry
V. Percobaan oksidasi biologi
23
35
43
VI. Percobaan enzim
VII. Urine
VIII. Percobaan empedu
BIOKIMIA 50
60
69
IX. Selulase UNTUK MAHASISWA JURUSAN KIMIA 78
FMIPA UNIVERSITAS RIAU
DISUSUN OLEH:
TIM BIOKIMIA JURUSAN KIMIA

FMIPA UNIVERSITAS RIAU
**
DOSEN PENANGGUNG JAWAB
KELAS: A: Dra.
SILVERA DEVI SY, M.Si & Dra. ANDI DAHLIATY, MS
B: Dra. ANDI DAHLIATY, MS & YUANA NURULITA,
Ph.D C: Dra. SILVERA DEVI SY, M.Si &
Dra. ANDI DAHLIATY, MS
Biokimia i
10/2/2020
KATA PENGANTAR
Diktat praktikum ini disusun sebagai pedoman dalam melakukan praktikum Biokimia
di Jurusan Kimia, FMIPA, Universitas Riau. Materi yang terdapat pada praktikum ini
mencakup dasar-dasar terpenting dalam Biokimia yang dapat dikembangkan lebih lanjut pada
penelitian seperti penentuan kadar protein berbagai materi biologik dan teknik penelitian
enzim.
Pada diktat praktikum ini terdapat tujuan pelaksanaan praktikum, teori singkat untuk
memperdalam pemahaman mahasiswa/i mengenai materi yang dibahas serta dilengkapi
dengan prosedur kerja. Diktat ini juga berisi lembaran khusus untuk menulis Tinjauan
Pustaka, Hasil Pengamatan, serta Hasil dan Pembahasan tiap Bab yang dipraktekkan di
Laboratorium. Diharapkan diktat praktikum ini dapat membantu mahasiswa/idalam
mempersiapkan dan melaksanakan praktikum dengan lebih baik, terarah, dan terencana.
Kami mengucapkan terimakasih pada semua pihak yang telah memberikan kontribusi
dalam pembuatan diktat praktikum ini. Ucapan terima kasih juga kami sampaikan kepada
Pranata Laboratorium Pendidikan Biokimia dan para asisten yang turut membantu kelancaran
penyeleng garaan praktikum Biokimia di Jurusan Kimia, FMIPA, Universitas Riau.
Pekanbaru, Februari 2020

Tim Penyusun Diktat Praktikum Biokimia Dasar
Dra. Andi Dahliaty, MS
Dra. Silvera Devi Sy, M.Si
Yuana Nurulita, Ph.D
Mengetahui:
Ketua Jurusan Kimia Kepala Laboratorium Biokimia
FMIPA Universitas Riau
Dr. Muhdarina, M.Si Dra. Silvera Devi Sy, M.Si

NIP. 19610814 198702 2 001 NIP. 19601220 198702 2 005
ii
DAFTAR ISI
KATA PENGANTAR..............................................................................................................ii
DAFTAR ISI............................................................................................................................iii
TATA TERTIB LABORATORIUM BIOKIMIA................................................................iv
I. PEMBUATAN LARUTAN BUFFER..............................................................................1

1.1 Tujuan.............................................................................................................................1
1.2 Pendahuluan....................................................................................................................1
1.3. Prosedur Kerja.................................................................................................................4
II. IDENTIFIKASI LIPID.................................................................................................9
2.1. Tujuan.............................................................................................................................9
2.2. Pendahuluan....................................................................................................................9
2.3. Prosedur Kerja..............................................................................................................12
III. IDENTIFIKASI ASAM AMINO DAN PROTEIN.......................................................18
3.1. Tujuan...........................................................................................................................18
3.2. Pendahuluan..................................................................................................................18
3.3. Prosedur Kerja...............................................................................................................21
IV. PENENTUAN KADAR PROTEIN SECARA LOWREY............................................30
4.1. Tujuan...........................................................................................................................30
4.2. Pendahuluan..................................................................................................................30
4.3. Prosedur Kerja...............................................................................................................33
V. PERCOBAAN OKSIDASI BIOLOGI.......................................................................38
5.1. Tujuan...........................................................................................................................38
5.2. Pendahuluan..................................................................................................................38
5.3. Prosedur Kerja...............................................................................................................41
VI. PERCOBAAN ENZIM...................................................................................................45
6.1. Tujuan...........................................................................................................................45
6.2. Pendahuluan..................................................................................................................45
6.3. Prosedur kerja................................................................................................................45
VII. URIN................................................................................................................................55
7.1. Tujuan............................................................................................................................55
7.2. Pendahuluan...................................................................................................................55
7.3. Prosedur Kerja...............................................................................................................55
VIII. PERCOBAAN EMPEDU.............................................................................................64
8.1. Tujuan............................................................................................................................64
8.2. Pendahuluan...................................................................................................................64
8.3. Prosedur Kerja...............................................................................................................64
IX. SELULASE.......................................................................................................................73
9.1. Tujuan...........................................................................................................................73
9.2. Pendahuluan...................................................................................................................73
9.3. Alat dan Bahan...............................................................................................................73
iii
TATA TERTIB LABORATORIUM BIOKIMIA
KEWAJIBAN MAHASISWA PESERTA PRAKTIKUM

I. Waktu
1. Praktikan harus datang 15 menit sebelum praktikum dimulai.
2. Praktikan tidak diperkenankan masuk laboratorium sebelum izin dari asisten.
3. Praktikan harus mempelajari penenuntun sebelumnya dan bersiap untuk diuji tentang
objek yang akan dilakukan.
II. Alat dan Reagensia

1. Praktikan harus menyediakan sendiri baju praktikum yang dipakai sewaktu praktikum
2. Semua alat yang akan dipakai harus dipinjam dengan bon dan harus dikembalikan
dalam keadaan bersih serta lengkap/utuh
3. Botol-botol reagen harus terletak pada tempat yang ditentukan dan tidak boleh dibawa
ke meja masing-masing
4. Botol reagen yang kosong / habis segera beritahu asisten
5. Sebelum praktikum dimulai, kelompok praktikan harus memeriksa kesesuaian jumlah
alat dengan daftar alat yang telah diperiksa Pranata Laboratorium Pendidikan (PLP).
Bila ada ketidaksesuaian harus segera dilaporkan. Pelaporan kekurangan alat setelah
praktikum berlangsung dianggap sebagai alat hilang yang harus diganti kelompok
praktikum yang bersangkutan.
6. Jika ada alat yang hilang atau pecah selama praktikum, maka alat tersebut harus
diganti secepatnya, atau paling lambat sebelum akhir semester (semester yang sedang
berlangsung) oleh kelompok mahasiswa yang bersangkutan. Kelompok mahasiswa
yang lalai mengganti alat akan dinyatakan gagal dan nilai tidak dikeluarkan. Setiap
penggantian alat harus ditunjukkan kepada asisten yang lalu menandatanganinya, dan
melaporkan pengembalian alat tersebut kepada Kepala Laboratorium Biokimia
III. Kebersihan Laboratorium

1. Tidak diperkenankan membuang sampah ditempat cucian, sampah harus dibuang
ditempat yang telah disediakan
2. Jika ada zat yang tumpah agar segera disiram/dibersihkan dengan pencuci yang sesuai
iv
3. Jika ada terjadi kecelakaan segera beritahu asisten
IV. Laporan dan Lain-lain

1. Praktikan harus membuat laporan pengamatan setelah selesai praktikum hari itu dan
menyerahkannya kepada asisten untuk diperiksa dan ditandatangani.
2. Menyerahkan laporan lengkap pada waktu praktikum berikutnya
3. Praktikan yang tidak masuk 2 kali berturut-turut atau 3 kali tidak masuk dianggap
mengundurkan diri
4. Tidak masuk karena sakit harus dengan surat keterangan dokter dan izin harus
diketahui Penasehat Akademis
v
I. PEMBUATAN LARUTAN BUFFER
I.1 Tujuan
1. Mengetahui, memahami dan dapat membuat larutan buffer
2. Mengetahui pengaruh pengenceran terhadap pH larutan
3. Mengetahui penambahan sedikit asam dan sedikit basa terhadap pH larutan
4. Membandingkan penentuan pH larutan buffer secara teoritis dan penggunaan
dengan pH meter.
I.2 Pendahuluan
Larutan buffer (penyangga) merupakan larutan yang terdiri dari campuran asam
lemah dan garamnya seperti Na2HPO4 – NaH2PO4 atau basa lemah dengan garamnya,
misalnya NH3 - NH4Cl. Larutan buffer dapat juga dapat didefenisikan sebagai campuran
asam lemah dengan basa konjugasinya atau basa lemah dengan asam konjugasinya.
Larutan buffer dapat mempertahankan pH suatu system karena dengan penambahan
sedikit asam dan basa atau pengenceran tidak akan mempengaruhi pH system. Larutan
buffer ini banyak digunakan dalam penelitian Biokimia terutama untuk penelitian yang
berhubungan dengan protein, enzim, dan asam nukleat (DNA, RNA).
Larutan buffer yang sering digunakan dalam penelitian Biokimia:
Buffer pH range
Citric Acid – Na2HPO4 2.6–7.6
Citric Acid – Sodium Citrate 3.0–6.2
Sodium Acetate – Acetic Acid 3.7–5.6
Na2HPO4 – NaH2PO4 5.8–8.0
Sodium Carbonate – Sodium Bicarbonate 9.2–10.8
6
TUGAS JURNAL
*Deskripsikan definisi dan karakteristik buffer, serta cara perhitungan dan
pembuatan larutan Na2HPO4 0.5 M dan NaH2PO4 0.5 M (Volume masing-masing
larutan dalam masing-masing 1 liter) menggunakan rumus .
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
7
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
8
1.3. Prosedur Kerja
1.3.1. Alat
1. Gelas ukur 100 mL
2. Batang pengaduk
3. Gelas kimia 250 mL
4. Botol semprot
5. Gelas ukur 10 mL dan 25 mL
6. pH meter
1.3.2. Bahan:
1. Larutan Na2HPO4
2. Larutan NaH2PO4
3. Larutan HCl
4. Larutan NaOH
5. Akuades
Tabel berikut adalah panduan pembuatan buffer pada beberapa nilai pH dengan cara
menambahkan sejumLah volume dari masing-masing larutan stok Na 2HPO4 dan
NaH2PO4.
o
Buffer Sodium Phosphat 20 C
Volume dari 0,5 M Volume dari 0,5 M

pH
Na2HPO4 ( mL ) NaH2PO4 ( mL )
5,8 7,9 92,1
6,0 12,0 88,0
6,2 17,8 82,2
6,4 25,5 74,5
6,6 35,2 64,8
6,8 46,3 53,7
7,0 57,7 42,3
7,2 68,4 31,6
7,4 77,4 22,6
7,6 84,5 15,5
7,8 89,6 10,4
8,0 93,2 6,8
Cara Kerja
Untuk membuat larutan buffer phosphat 0,05 M pada pH 6,0 sebanyak 100 mL
1. Larutan Na2HPO4 1,2 mL dimasukkan ke dalam gelas kimia
9
2. Kemudian tambahkan larutan NaH2PO4 8,8 mL
3. Setelah itu tambahkan akuades hingga ±80 mL
4. Ukur pH hingga didapatkan pH 6,0 dengan penambahan larutan HCl atau NaOH
5. Setelah didapatkan pH 6.0, encerkan hingga 100 mL
6. Hasil ini kemudian dibagi menjadi 3 bagian
a. Bagian I: tambahkan sedikit asam dan ukur pHnya
b. Bagian II: tambahkan sedikit basa dan ukur pHnya
c. Bagian III: tambahkan sedikit akuades dan ukur pHnya
TUGAS
1. Bagaimana cara membuat larutan buffer asetat 0,05 M pH 4
Ka = 1,8.10-5 sebanyak 100 mL
2. Cara membuat buffer phosphat pH 7 dengan konsentrasi 0,5 mmol dalam 100
mL
pKa=7,2
3. Jelaskan dengan kenapa larutan buffer dengan penambahan sedikit asam atau
basa pH tidak mengalami perubahan?
10
HASIL PENGAMATAN
No Zat Kimia pH
1 Larutan Na2HPO4 + Larutan NaH2PO4 + akuades
2 Larutan Na2HPO4 + Larutan NaH2PO4 + akuades + larutan
HCl* atau NaOH*
3 Larutan buffer dibagi 3
a. Bagian I + sedikit asam
b. Bagian II + sedikit basa
c. Bagian III + sedikit akuades
*coret yang tidak digunakan
Pekanbaru, ...................................
2020
Asisten,
11
HASIL DAN PEMBAHASAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
12
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
13
II. IDENTIFIKASI LIPID
2.1. Tujuan
1. Uji kelarutan lipid
2. Uji akrolein untuk mengidentifikasi adanya lipid dalam sampel
3. Penentuan berat jenis minyak
4. Penentuan kadar air dalam lemak
5. Penentuan asam lemak bebas
2.2. Pendahuluan
Lipid merupakan salah satu biomolekul kompleks utama yang ditemukan
dalam sel dan jaringan makhluk hidup. Sifat dari lipid antara lain adalah tidak larut
dalam air, akan tetapi larut dalam pelarut non polar/organik seperti eter, kloroform,
heksan, asetonitril, 1,4 dioksan, isopropanol. Lipid terbentuk dari reaksi asam lemak
dengan gliserol yang dikenal dengan triasilgliserida (triasilgliserol). Asam lemak
dalam lipid dapat berbentuk asam lemak jenuh dan asam lemak tidak jenuh.
Keberadaan gliserol dalam lipid dapat diidentifikasi dengan menggunakan senyawa
KHSO4. Identifikasi gliserol bergantung pada dehidrasi dan oksidasi gliserol menjadi
akrolein
H2C OH HC O
HC OH + KHSO4 C H + KHSO4. 2 H2O

H2C OH C H
H
Jika minyak dipanaskan hingga temperatur tinggi, dapat terdegradasi menjadi gliserol
dan membentuk akrolein. Itu sebabnya minyak panas seringkali berbau tajam.
14
TUGAS
JURNAL
*Reaksi pembentukan senyawa lipid, definisi asam lemak, reaksi oksidasi asam
lemak,
pentingnya penentuan berat jenis pada lipid.
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
15
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
16
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
2.3. Prosedur Kerja
2.3.1. Alat
1. Tabung reaksi
2. Batang pengaduk
3. Penjepit kayu
4. Botol semprot
5. Lampu spiritus
6. Gelas ukur 10 mL atau 25 mL
2.3.2. Bahan Uji Kelarutan

1. Minyak kelapa (minyak goreng)
2. Alkohol tehnis
3. Chloroform
Cara kerja:
1. Sediakan 3 buah tabung reaksi dan masing-masing tabung diisi dengan :
Tabung 1 : 3 mL air
Tabung 2 : 3 mL alkohol tehnis
Tabung 3 : 3 mL kloroform
2. Selanjutnya masukkan kedalam setiap tabung 2 tetes minyak goreng
3. Kocok campuran di dalam ke 3 tabung reaksi ini dengan kuat
4. Perhatikan apakah terjadi pemisahan atau tidak dari ketiga cairan didalam
tabung-tabung reaksi tersebut, amati dan catat.
2.3.3. Bahan Uji Gliserol dalam Minyak:

1. KHSO4
2. Gliserol
17
3. Minyak kelapa ( minyak goreng )
Cara Kerja:
1. Sediakan 2 tabung reaksi yang bersih dan kering. Dalam tabung reaksi A
masukkan 10 tetes minyak kelapa, dan kedalam tabung reaksi B masukkan
10 tetes gliserol
2. Kedalam masing-masing tabung reaksi masukkan 0,25 gr KHSO4
3. Panaskan masing-masing tabung reaksi tersebut dengan api dari lampu
spiritus
4. Perhatikan bau yang tercium!
2.3.4. Bahan Untuk Penentuan Berat Jenis Minyak

1. Beberapa jenis Minyak
Cara kerja
1. Timbang piknometer kosong hingganya berat konstan (catat)
2. Masukkan sampel minyak ke dalam piknometer.
3. Kemudian timbang kembali piknometer yang berisi sampel (catat)
4. Lakukan cara kerja yang sama untuk sampel minyak yang lainnya .
5. Tentukan berat jenis sampel minyak.
2.3.5. Bahan Untuk Penentuan ALB ( Asam Lemak Bebas )

1. Minyak
2. Alkohol
3. Benzen
4. NaOH 0,1 N
Cara Kerja
1. Timbang 5 gr sampel Minyak , masukkan kedalam erlenmeyer.

2. Kemudian tambahkan 25 mL campuran alkohol benzol ( 1 : 1 ) dan dikocok
3. Tanbahkan beberapa tetes indikator fenolftalein, kocok kembali
18
4. Selanjutnya larutan ini di titrasi dengan NaOH 0,1 N sampai titik akhir titrasi.
ml NaOH pentiter x N NaOH x 256 x 100

% ALB =
Berat contoh x 1000
19
HASIL PENGAMATAN
Uji Lipid
1. Tes Kelarutan
No Zat Kimia Pengamatan
1 5 mL akuades + 4 tetes minyak goreng aduk
2 5 mL alkohol + 4 tetes minyak goreng aduk
3 5 mLkloroform + 4 tetes minyak goreng aduk
2. Identifikasi gliserol dalam lipid

1 10 tetes minyak kelapa + 0,5 gr KHSO4
2 10 tetes gliserol + 0,5 gr KHSO4
3.Penentuan Berat Jenis Minyak

Minyak 1
Piknometer kosong ditimbang ........................................
Piknometer + sampel ditimbang ......................................
BJ = ρ m = massa jenis v = volume
m
ρ=
v BJ = ...................................................
Minyak 2
m
ρ=
v BJ = ...................................................
Minyak 3
20
m
ρ=
v BJ = ...................................................
5. Penentuan ALB ( Asam Lemak Bebas )

% ALB = .................................................................
Pekanbaru, ...........................................2
020
Asisten,
21
PEMBAHASAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
22
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
23
III. IDENTIFIKASI ASAM AMINO DAN PROTEIN
3.1. Tujuan
1. Mengetahui unsur-unsur yang terdapat di dalam asam amino dan protein
2. Mengidentifikasi gugus α amino pada asam amino dan protein
3. Mengidentifikasi adanya ikatan peptida
4. Mengetahui adanya senyawa monohidroksi benzen pada asam amino
3.2. Pendahuluan
Protein mengandung unsur C, H, O, N, dan S. Protein adalah senyawa polimer
yang disusun dari monomer-monomer asam amino yang dihubungkan oleh ikatan
peptida. Asam amino adalah senyawa asam karboksilat yang atom Cα mengandung
gugus amina.
Larutan protein apabila ditambah dengan ninhidrin dan kemudian dipanaskan,
maka akan terbentuk senyawa kompleks yang berwarna. Ninhidrin juga memberikan
reaksi warna dengan proteosa, pepton, dan peptida yang memiliki gugus asli -amino:
Larutan protein apabila ditambah dengan ninhidrin, kemudian dipanaskan akan

terbentuk senyawa kompleks yang berwarna. Ninhidrin juga memberikan reaksi warna
dengan proteosa, pepton, dan peptida yang memiliki gugus asli -amino.
Keberadaan ikatan peptida di dalam protein dapat di identifikasi dengan
menggunakan reagen biuret. Hal ini disebabkan karena apabila protein direaksikan
dengan reagen Biuret (mengandung senyawa CuSO4 dan NaOH ) dalam suasana basa
akan menghasilkan larutan berwana biru violet.
Selain uji biuret dapat juga dilakukan uji Milon dan reagen yang digunakan
dalam uji Millon adalah larutan mercuri dan ion mercuro dalam asam nitrat dan
asam nitrit. Uji sampel yang memberikan hasil positif dengan reagen Millon adalah
sampel yang memiliki derivat mono hidroksibenzen misalnya tirosin dan fenol.
- +
Reaksi ini kurang baik dengan adanya ion Cl dan NH4 . Oleh karena itu uji ini tidak
dapat dipakai untuk analisa protein di dalam urine.
Protein dapat diendapkan dengan berbagai cara, antara lain dengan
menggunakan asam komplek TCA, pelarut organik dan kristal dari garam-garam
anorganik. Efektivitas pengendapan protein tergantung pada jenis zat kimianya.
24
TUGAS JURNAL
*Struktur asam amino, struktur umum protein, pembagian asam amino, konsep
pengendapan protein dengan menggunakan beberapa zat kimia.
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
25
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
26
3.3. Prosedur Kerja
3.3.1. Alat:
1. Tabung reaksi
2. Batang pengaduk
3. Penjepit kayu
4. Botol semprot
5. Lampu spiritus
6. Gelas ukur 10 mL atau 25 mL
3.3.2. Bahan Identifikasi unsur-unsur yang terdapat dalam protein

1. Larutan protein (putih telur)
2. NaOH kristal
3. NaOH 10 %
4. HgCl2 1 %
5. Lakmus merah
Cara Kerja:
1. Larutan protein ±3 tetes dimasukkan ke dalam tabung reaksi bersih dan
kering, kemudian dipanaskan, dan cium bau yang dihasilkan dari proses
pemanasan!.
2. Larutan protein ±4 tetes dimasukkan ke dalam tabung reaksi dan ditambahkan
1 keping NaOH padat. Panaskan hati-hati. Cium adanya bau ammonia atau
perubahan warna lakmus merah yang diletakkan dimulut tabung..
3. a. Larutan protein ±4 tetes dimasukkan ke dalam tabung reaksi
b. Kemudian ditambahkan larutan NaOH 10 % sebanyak 2 mL.
c. Campuran segera dipanaskan/dididihkan
d. Selanjutnya ditambahkan 4 tetes HgCl2 1%, maka akan terjadi larutan
berwarna hitam.
27
Pertanyaan sebagai tugas yang harus dijawab di dalam laporan praktikum saudara :
1. Kegosongan menunjukkan adanya unsur apa? dan terbentuknya bintik-bintik
air di atas tabung menunjukkan adanya unsur apa lagi? Bau ammonia dan
perubahan warna lakmus merah menjadi biru menunjukkan adanya unsur apa?
2. Endapan apa yang terbentuk bila putih telur yang telah dihidrolisa dengan NaOH
10 % direaksikan dengan HgCl2 1 % dan menyebabkan larutan menjadi hitam ?
3.3.3. Bahan Uji Ninhidrin

1. Larutan Ninhidrin 0,1 % dalam alkohol tehnis
2. Larutan protein ( putih telur : air = 1 : 5 )
Cara Kerja :
1. Larutan protein ±3 mL dimasukkan ke dalam tabung reaksi.
2. Kemudian tambahkan 0,5 mL larutan ninhidrin 0,1%
3. Selanjutnya dipanaskan hingga mendidih. Amati dan catat warna yang
terjadi!
1. Warna apa yang terbentuk ?
2. Gugus apa yang memberikan uji positif ?
3.3.4. Bahan Uji Biuret

1. Larutan NaOH 10%
3. Urea yang dipanaskan
4. Larutan CuSO4 0,1%
5. Urea
6. Saliva ( 1 : 100 )
28
Cara kerja
1. Larutan protein ± 3 mL dimasukkan ke dalam tabung reaksi
2. Kemudian tambahkan 1 mL NaOH 10% dan aduk
3. Selanjutnya tambahkan 1 tetes CuSO4 0,1% dan kocok baik-baik.
4. Apabila tidak dihasilkan warna biru ungu, tambahkan lagi CuSO4 sampai
terbentuk warna (maksimal 10 tetes)
5. Percobaan diulang kembali dengan mengganti larutan protein dengan larutan
urea
Pertanyaan sebagai tugas yang harus dijawab di dalam laporan praktikum :

1. Mangapa harus dihindarkan kelebihan pemakaian CuSO4 ?
2. Kenapa urea juga positif dengan uji biuret?
3.3.5. Bahan Uji Millon

1. Reagen Millon: gunakan reagen Millon yang telah siap pakai dari pabrik atau
buat reagen Millon dengan melarutkan 10 gr mercuri dalam 20 mL asam
nitrat pekat. Apabila telah melarut semua dan uap coklat tidak kelihatan lagi,
encerkan dengan 60 mL air, dan kemudian disimpan.
Cara kerja :
1. Larutan protein ± 3 mL dimasukkan ke dalam tabung reaksi
2. Kemudian tambahkan 4 atau 5 tetes reagen Millon
3. Selanjutnya campuran ini dipanaskan. (Jika reagen yang digunakan terlalu
banyak, maka warna akan hilang pada pemanasan).
4. Warna merah dari larutan menandakan reaksi uji Millon positif
a. Apa yang terjadi jika garam mercuri ditambahkan ke dalam
protein?
b. Larutan protein yang bagaimana yang akan memberikan uji negatif?
29
Mengapa?
3.3.6. Bahan Pengendapan Protein oleh Asam Kompleks

1. Larutan TCA ( Tricloroasetat ) 7% , tempatkan dalam botol bertutup gabus.
Cara kerja :
1. Larutan protein ±3 mL dimasukkan ke dalam tabung reaksi
2. Kemudian tambahkan 1 s/d 4 tetes larutan TCA 7% (hati-hati jangan sampai
kena tangan, kalau TCA terkena tangan segera basuh dengan air. TCA adalah
asam yang sangat korosif dan keras).
3. Jika masih belum terbentuk endapan, tambahkan lagi TCA tetes demi tetes
sehingga terbentuk endapan.
3.3.7. Bahan Pengendapan dengan Pelarut Organik

2. Alkohol teknis
Cara kerja:
1. Larutan protein ±2 mL dimasukkan ke dalam sebuah tabung reaksi
2. Kemudian tambahkan 10 mL alkohol tehnis dan aduk baik-baik.
3. Endapan yang dihasilkan,di ambil sedikit dan periksa kelarutannya dalam air
Pertanyaan
1. Apa tujuan percobaan tahap 3? Jelaskan!
3.3.8. Bahan Pengendapan dengan garam (Salting Out)

1. Larutan protein ( putih telur ; air = 1 : 5 )
2. Ammonium Sulfat
3. Reagen Biuret
4. Kertas saring
30
Cara kerja :
1. Larutan protein ±3 mL dimasukkan ke dalam tabung reaksi
2. Kemudian ditambahkan Kristal ammonium sulfat sedikit demi sedikit
sampai terbentu endapan
3. Endapan ini kemudian disaring. (endapan dan filtrate)
4. Endapan dimasukkan ke dalam tabung reaksi kemudian ditambahkan air
sampai larut. Larutan ini kemudian diuji dengan uji biuret
5. Filtrat diuji dengan uji biuret
Pertanyaan:
Apa yang dapat anda simpulkan dari percobaan 4 dan 5
31
HASIL PENGAMATAN
1. Analisa unsur-unsur yang terdapat dalam senyawa asam amino dan protein

1 3 tetes protein
2 4 tetes protein + 1 keping NaOH
3 4 tetes protein + 2 mL NaOH 10 % + 4 tetes HgCl2 1 %
2. Test Biuret

1 2 mL albumin 2 % + 2 mL NaOH + 3 - 5 tetes CuSO4 0,5%
2 2 mL urea 2 % + 2 mL NaOH + 3 - 5 tetes CuSO4 0,5%
3 2 mL urea yang dipanaskan + 2 mL NaOH + 3-5 tetes CuSO4 0,5%
4 2 mL saliva 2 % + 2 mL NaOH + 3 - 5 tetes CuSO4 0,5%
5 2 mL pati + mL NaOH + 3 - 5 tetes CuSO4 0,5%
3. Test Ninhidrin
1 2 mL albumin 2 % + 5 tetes ninhidrin waterbath 10’
2 2 mL (NH4)SO4 Kuat + 5 tetes ninhidrin waterbath 10’
4 . Uji Millon
5 tetes Millon + 3 mL protein
5. Pengendapan protein oleh asam kompleks
3 mL larutan protein + 4 tetes TCA 7 % sampai terbentuk endapan
6. Pengendapan dengan pelarut organik
2 mL protein + 10 mL alkohol aduk
32
Bila ada endapan uji kelarutan dalam air
7. Salting Out
1 5 mL putih telur + (NH4)2SO4 jenuh saring
Endapan + akuades
2 Filtrat + (NH4)2SO4 kristal saring
Endapan + akuades
1 mL filtrat + 1 mL NaOH + 3 tetes CuSO4 0,5%
Pekanbaru, ....................................
.2020
Asisten,
33
HASIL DAN PEMBAHASAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
34
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
35
IV. PENENTUAN KADAR PROTEIN SECARA LOWREY
(FOLIN CIOCALTEAU TEST)
4.1. Tujuan
1. Dapat menentukan kadar protein dari sampel saliva
2. Memahami prinsip dasar penentuan kadarprotein dari sampel berdasarkan
metode lowry
4.2. Pendahuluan
Metoda Lowrey merupakan pengembangan dari metoda Biuret dengan
mengkombinasikan pereaksi Biuret dan Folin Ciocalteau. Penentuan kadar protein
dengan cara Biuret berdasarkan atas pengukuran serapan cahaya oleh ikatan
kompleks yang berwarna biru ungu. Ini terjadi apabila protein bereaksi dengan
tembaga dalam lingkungan alkali. Warna yang timbuldisebabkan oleh warna hasil
reaksi Biuret, disamping warna yang disebabkan oleh asam amino tirosina dan
tripthopan yang ada dalam protein.
Dalam metoda Lowrey terjadi dua tahapan reaksi. Reaksi tahap pertama di
metoda Biuret Cu+2-protein dalam suasana alkalis akan tereduksi menjadi Cu+. Pada tahap
kedua, ion Cu+akan mereduksi reagen Folin Ciocaltea, kompleks phosphomolibdat-
phosphotungstat menghasilkan heteropoly-molybdenum blue. Warna biru ini dapat
dideteksi dengan alat spektrofotometer dengan panjang gelombang 500 s/d 750 nm.
Penentuan kadar protein secara Lowrey lebih peka daripada dengan cara Biuret, karena
hanya dapat mengukur protein dengan konsentrasi di atas 1 – 10 mg/mL. Seperti juga
+
cara Biuret, cara Lowrey dapat diganggu oleh adanya ion NH4 di dalam larutan.
36
TUGAS
JURNAL
*Prinsip kerja dari spektrofotometer dan hal-hal yang harus diperhatikan dalam
analisis kuantitatif dengan mengunakan spektrofotometer
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
37
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
38
4.3. Prosedur Kerja
4.3.1. Alat :
1. Pipet ukur 1 mL
2. Tabung reaksi; Batang pengaduk
3. Kertas label
4. Botol semprot
5. Lembaran-lembaran kertas saring panjang
6. Buret lengkap dengan klem
7. Gelas kimia 250 mL dan 1000 mL
8. Ball pipetor untuk mengisap reagen Folin Ciocalteau yang beracun
9. Spektrofotometer sinar tampak
4.3.2. Bahan :
1. Reagen A : buat 200 mL 2% Na 2CO3 dalam 0,1 N NaOH. Reagen ini bisa
disimpan lama
2. Reagen B : 0,5 % CuSO4.5 H2O dalam 1 % Natrium Kalium Tartarat atau
dinatrium Tartarat atau dikalium Tartarat. Reagen ini bisa disimpan lama
3. Reagen Folin Ciocalteau : Periksa normalitas dari reagen Folin Ciocalteau yang
telah jadi dari pabrik dengan dititrasi dengan NaOH 1 N menggunakan indikator
fenolftalein, atau dengan membacanya bila telah tertera pada label pabrik. Bila
normalitas asam reagen tersebut lebih dari 1 N, encerkan reagen tersebut dengan
air hingga normalitasnya menjadi 1 N
4. Larutan standar protein: buatlah 100 mL larutan serum albumin standar dengan
konsentrasi 1000 g/mL dengan cara sebagai berikut : timbang 100 mg albumin
standar dan larutkan dengan 100 mL akuades secara kuantitatif. Catat
konsentrasinya dan tempelkan pada botol tempat menyimpan larutan standar
tersebut
5. Larutan sampel : 1 mL saliva yang diencerkan yang diencerkan dengan akuades
menjadi 100 mL.
39
6. Reagen C (dibuat pada saat praktikum akan dimulai, harus fresh ) : campur 50 mL
reagen A dengan 1 mL reagen B. Buang setelah 1 hari (reagen C tidak bisa
disimpan pada waktu lebih lama dari 1 hari)
7. Akuades
Cara Kerja
1. Sediakan 7 tabung reaksi yang kering

2. Dengan menggunakan pipet ukur 1 mL, kedalam tabung-tabung
reaksi tersebut masukkan larutan-larutan sebagai berikut :
Tabung 1 : 0,3 mL larutan protein standar + 0,7 mLakuades, hingga c menjadi 0,3
x c larutan standar semula
Tabung 2 : 0,4 mL larutan protein standar + 0,6 mLakuades, hingga c menjadi 0,4
Tabung 3 : 0,6 mL larutan protein standar + 0,4 mL akuades, hingga c menjadi 0,6
Tabung 4 : 0,8 mL larutan protein standar + 0,2 mL akuades, hingga c menjadi 0,8
Tabung 5 : 1,0 mL larutan protein standar, sehingga c tetap sama dengan larutan
standar
Tabung 6: Blanko 1 mL akuades
Tabung 7: Sampel saliva (1:100)
3. Jangan lupa mencatat secara tepat harga c larutan protein
standar yang tercantum pada botolnya
4. Kedalam ketujuh tabung reaksi tersebut masing-masing
dimasukkan 5 mL reagen C dengan menggunakan buret yang telah disediakan,
kocok dan biarkan pada suhu kamar selama paling sedikit 10 menit.
5. Setelah paling sedikit 10 menit, tambahkan 0,5 mL reagen Folin
Ciocalteau kedalam masing-masing tabung reaksi dengan menggunakan pipet ukur
1 mL yang telah disediakan disamping botol reagen. Hisap dengan bantuan ball
pipettor yang disediakan untuk penggunaan bersama. Kocok dengan batang
pengaduk. Setiap kali mau memindahkan batang pengaduk dari satu batang ke
tabung lainnya, maka batang pengaduk itu harus terlebih dahulu dibilas dengan
akuades dan keringkan dengan lap
40
6. Diamkan ketujuh tabung reaksi tersebut selama 30 menit pada
suhu kamar
7. Setelah 30 menit, ukur serapan dari larutan-larutan tersebut
pada panjang gelombang 500 nm.
HASIL PENGAMATAN
Tugas laporan
1. Buatlah kurva standar dan tentukan kadar protein larutan sampel yang diberikan.
2. Apakah kebaikan dan keburukan metode Lowry ini ?
3. Protein apakah yang terdapat dalam saliva?
Pengamatan
No Tabung Absorban
1 Tabung 1
2 Tabung 2
3 Tabung 3
4 Tabung 4
5 Tabung 5
6 Blanko
7 Sampel Saliva
Pekanbaru, .......................................202
0
Asisten,
41
PEMBAHASAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
42
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
43
V. PERCOBAAN OKSIDASI BIOLOGI
5.1. Tujuan
1. Menentukan pengaruh pemanasan terhadap kesegaran susu.
2. Memahami perbedaan reaksi yang terjadi pada susu segar dan susu yang
telah dipanaskan
5.2. Pendahuluan
Susu segar disamping mengandung senyawa-senyawa (nutriasi) yang sangat
bermanfaat bagi kehidupan dan juga mengandung mikroba, untuk menghilangkan
bakteri ini biasanya digunakan pasteurisasi. Enzim dehidrogenase yang terdapat dalam
susu segar akan mengkatalis pelepasan H dari formaldehyde dan kemudian ion H+ ini
akan bereaksi dengan metilen blue membentuk leuko metilen blue, reaksi ini
berlangsung secara anaerob. Apabila di dalam susu ada mikroba, maka oksigen terlarut
akan digunakan oleh mikroba menyebabkan system (susu) kekurangan oksigen. Susu
o
yang dipanaskan sampai 70 C (pasteurmilk) menyebabkan enzim dehydrogenase tidak
aktif
44
TUGAS JURNAL
*Mekanisme kerja enzim dehydrogenase, Reaksi perubahan warna yang terjadi
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
45
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
46
V.3. Prosedur Kerja
5.3.1. Alat:
1. Tabung reaksi
2. Pipet takar
3. Pipet tetes
4. Waterbath
5.3.2. Bahan :
1. Susu segar
2. Susu yang telah dipasteurisasi
3. Metilen Blue 0,02%
4. Formaldehid 0,4%
Cara Kerja :
1. Sediakan 2 tabung reaksi(A dan B)
2. Pada tabung reaksi A ditambahkan 5 mL susu segar dan tabung reaksi B
ditambahkan 5 mL susu yang telah dipasteurisasi.
3. Kemudian ke dalam masing-masing tabung reaksi ditambahkan 3 tetes metilen
blue dan 1 mL formaldehyde, kemudian dikocok perlahan.
o
4. Kedua tabung reaksi ini diletakkan ke dalam waterbath 65 C selama 30 menit.
5. Amati yang terjadi
47
HASIL PENGAMATAN

1 5 mL susu segar + 3 tetes metilen blue + 1 mL formaldehid,
masukkan dalam waterbath 65oC 30 menit
2 5 mL susu yang telah dipasteurisasi + 3 tetes metilen blue +
1 mL formaldehid, masukkan dalam waterbath 65oC
30 menit
Pekanbaru, ....................................
.2020
Asisten,
48
PEMBAHASAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
49
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
50
VI. PERCOBAAN ENZIM
( Pengaruh Suhu dan pH terhadap aktivitas enzim )
6.1. Tujuan
1. Membuktikan bahwa kecepatan reaksi enzimatik sampai suhu tertentu sebanding
dengan kenaikan suhu. Reaksi enzimatik mempunyai suhu optimum.
2. Membuktikan bahwa keasaman ( pH ) mempengaruhi kecepatan reaksi
enzimatik
6.2. Pendahuluan
Secara konvensional penamaan enzim sesuai dengan nama substratnya dan enzim
ini merupakan senyawa biokatalisator yang dapat mempercepat terjadinya suatu reaksi
kimia. Enzim pada umumnya adalah suatu protein sehingga enzim juga sangat sensitif
terhadap suhu dan pH. Masing-masing enzim bertindak sebagai katalisator secara
maksimal pada suhu dan pH optimum dan apabila enzim berada pada suhu dan pH
ekstrim protein akan terdenaturasi dan proses ini akan mempengaruhi aktivitas enzim
tersebut.
6.3. Prosedur kerja

6.3.1. Alat:
1. Tabung reaksi dan Gelas piala
2. Pipet tetes dan Pipet takar
3. Stopwatch
4. Waterbath
5. Termometer
6. Rak tabung reaksi
7. Spektrofotometer
6.3.2. Bahan :
1. Liur sebagai sumber amilase
2. Larutan pati 0,4 mg/mL
3. Larutan iodium dalam pati
51
4. Larutan pati 0,4 mg/mL dalam berbagai pH ( 3, 5, 7, 9 Dan 11 )
5. Larutan Iodium
6.3.3. Cara kerja Pengaruh Suhu Terhadap Aktivitas Enzim

1. Encerkan air liur 100X dengan air suling
2. Siapkan 6 pasang tabung reaksi yang bersih
LARUTAN TABUNG B TABUNG U
Larutan Pati 1 1
Inkubasi masing-masing pasangan tabung tersebut pada setiap suhu
yang digunakan (1,2,3,4,5,6 ) selama 5 menit
Liur diencerkan 100X - 200l
Aquades 200l -
Campur baik-baik. Diamkan selama 1 menit.
Hentikan reaksi enzim dengan merendamkan tabung reaksi ke dalam air
mendidih ( 100oC ) selama 5 menit, kemudian dinginkan
Larutan iodium 1 1
Aquades 8mL 8mL
mL
mL
1. Pasangan I ditempatkan dalam bejana berisi es ( 0o C )
2. Pasangan II ditempatkan dalam bejana berisi air yang suhunya
dipertahankan tetap pada suhu 25o C
3. Pasangan III ditempatkan di rak tabung pada suhu ruang
4. Pasangan IV ditempatkan dalam penangas air yang suhunya
5. Pasangan V ditempatkan dalam penangas air yang suhunya
6. Pasangan VI ditempatkan dalam penangas air mendidih ( 100o C )
Segera baca serapan ( A ) pada panjang gelombang 680 nm. Hitung selisih serapan
(  A ) antara tabung B ( A pada t = 0 menit ) dengan tabunng U dari setiap suhu.
52
Buatlah tabel berikut ini :
SUHU AB AU  A/menit ( v )
0o C
25oC
Suhu Ruang
37o C
60o C
100o C
3. Buatlah kurva yang menggambarkan hubungan kecepatan reaksi enzimatik

(v=A/menit(v)
6.3.4. Cara kerja Pengaruh pH Terhadap Aktivitas Enzim

1. Encerkan liur 100X dengan air suling
2. Siapkan 6 pasang tabung reaksi yang bersih. Tiap pasang tabung diberi tanda B
( Blanko ) dan U ( Uji )
3. Pipet ke dalam tiap-tiap tabung :
LARUTAN TABUNG B TABUNG U
Larutan pati dalam berbagai pH 1 mL 1 mL
o
Inbkubasi pada suhu 37 C paling sedikit 5 menit
Liur diencerkan 100X - 200 l
Aquades 200l -
Campur baik-baik. Diamkan selama 1 menit.
Hentikan reaksi enzim dengan merendamkan tabung reaksi ke dalam air
mendidih ( 100oC ) selama 5 menit, kemudian dinginkan
Larutan Iodium 1 mL 1 mL
Akuades 8 mL 8 mL
4. Segera baca serapan ( A ) pada panjang gelombang 680 nm. Hitung A antara
tabung B ( A pada t = 0 menit ) dengan tabung U.
5. Buatlah tabel berikut ini :
PH AB AU Δ A/menit ( v )
3
5
53
7
9
11
6. Buat kurva yang menggambarkan hubungan antara kecepatan reaksi enzimatik

( Δ A/menit ( v ) )
54
TUGAS JURNAL
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
55
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
56
HASIL PENGAMATAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
57
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
Pekanbaru, ......................................202
0
Asi
sten,
58
PEMBAHASAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
59
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
60
.......................................................................................................................................................
.......................................................................................................................................................
VII. URIN
7.1. Tujuan
1. Mahasiswa memahami cara analisa karakter urin seperti berat jenis dan glukosa urin
2. Mahasiswa memahami sifat urin serta hubungannya dengan fungsi ginjal dan
metabolism tubuh
7.2. Pendahuluan
Urin adalah cairan yang berisi zat-zat kimia terlarut dari sisa metabolisme
(seperti urea), garam terlarut, materi organik antara lain glukosa ( diabetes ) yang
diekskresikan oleh ginjal, oleh karena itu berat jenis dari urin tidak sama dengan air.
Materi yang terkandung di dalam urin dapat diketahui melalui urinalisis.
Struktur molekul urea:
Gula pereduksi seperti glukosa akan mereduksi ion kupri dalam suasana basa,
dengan penambahan arsenomolibdat akan menghasilkan warna biru (molybdenum
blue). Kekuatan intensitas (absorbansi) warna biru tersebut sesuai dengan konsentrasi
glukosa yang teroksidasi. Absorbsi larutan ini diukur pada panjang gelombang 660 nm
menggunakan alat spektrofotometer. Konsentrasi glukosa dapat ditentukan dengan
menggunakan hukum Lambert-Beer dengan bantuan beberapa seri konsentrasi larutan
standar glukosa.
7.3. Prosedur Kerja
7.3.1. Alat
61
1. Urinometer 2. Spektrofotometer 3. Gelas kimia 100 mL.
4. Gelas piala 250 mL 5. Gelas kimia 500 mL 6. Buret lengkap
7. Gelas ukur 50 mL 8. Tabung reaksi 9. Gelas kimia 250 mL
10. Pipet ukur 1 mL 11. Kelereng 12. Lampu spiritus
13. Batang pengaduk 14. Waterbath 15. Ball Pipettor
7.3.2. Bahan Pengukuran Berat Jenis
a) Urin
Cara kerja:
1. Masukkan urin ke dalam gelas ukur sampai 50 mL
2. Masukkan urinometer ke dalam gelas ukur yang telah berisi urin
dengan cara memutarnya, dengan tujuan supaya jangan lengket pada dinding
gelas ukur
3. Setelah urinometer diam (tidak berputar lagi) pada permukaan urine, bacalah
skala yang ditunjukkan oleh urinometer pada permukaan urine
7.3.2. Bahan Pengukuran Konsentrasi Glukosa dalam Urin

a) Larutan standar glukosa 100 µg/mL. Larutan ini dibuat secara kuantitatif
menggunakan labu takar.
b) Reagen warna Arsenomolibdat : 12,5 gr ammoniummolibdat dalam 200 mL
aquadest, ditambah 10,5 mL H2SO4 pa. Kemudian masukkan ke dalam
labu ukur 250 mL. Larutkan 1,5 gr disodium hydrogen arsenat dalam 12,5
mL aquadest. Tuang larutan disodioum arsenat ke dalam larutan molibdat.
Encerkan campuran ini hingga larutan menjadi 250 mL. Simpan larutan ini
dalam botol gelap dan inkubasi pada suhu 37oC selama 24 sampai 48 jam atau
dalam suhu kamar selama 72 jam 3.
c) Reagen A : 1,2 gr KNaTaratarat, 2,4 gr Na2CO3 anhydrous, 1,6 gr
NaHCO3, 14,4 gr Na2SO4 anhydrous dilarutkan di dalam 80 mLair dengan
menggunakan pemanasan.
d) Reagen B : Larutkan 2 gr CuSO4. 5H2O dan 18 gr Na2SO4 anhydrous ke
dalam 100 mL air, aduk hingga larut semuanya dan teteskan 1 s/d 2 tetes
H2SO4 pa
e) Reagen Nelson Somogyi: Reagen ini harus dibuat pada saat mau praktikum,
karena pemakaiannya harus dalam keadaan segar dan reagen ini tidak bisa
disimpan untuk jangka waktu yang lebih lama dari satu hari. Cara membuat
62
reagen nelson Somogyi adalah dengan mencampurkan 4 volume reagen A
dengan 1 volume reagen B. Untuk satu kali praktikum buat 50 mL larutan
Nelson Somogyi yaitu dengan mencampurkan 40 mL reagen A dengan 10
mL reagen B.
f) Larutan sampel urin
g) Air dingin/air es dan es batu
Cara kerja :
1. Sediakan 7 tabung reaksi yang kering
2. Dengan menggunakan pipet ukur 1 mL, ke dalam tabung-tabung reaksi tersebut

masukkan larutan-larutan sebagai berikut :
Tabung 1 : 0,2 mL larutan glukosa standar + 0,8 mL aquadest, sehingga c
menjadi 0,2 x c larutan standar semula
Tabung Blanko: 1 mL aquadest
Tabung Sampel: 1 mL larutan urin
3. Jangan lupa mencatat secara tepat harga c larutan standar glukosa yang
tercantum pada botolnya
4. Ke dalam ketujuh tabung reaksi tersebut masing-masing masukkan 1 mL reagen
NelsonSomogyi. tutup ke tujuh tabung reaksi tersebut dengan kelereng
5. Letakkan ke tujuh tabung reaksi tersebut dalam penangas air mendidih selama 20
menit. Setelah itu dinginkan ke tujuh tabung reaksi tersebut secara cepat di
dalam air es/air dingin sampai suhu zat di dalam tabung-tabung tersebut sama
dengan suhu kamar.
63
6. Setelah ke tujuh tabung reaksi tersebut dingin, tambahkan 1 mL reagen
Arsenomolibdat ke dalam masing-masing tabung reaksi dengan dengan
menggunakan pipet ukur 5 mL yang disediakan (untuk dipakai bersama dengan
kelompok lain). Larutan ini beracun, sebaiknya diisap dengan bantuan ball
pipettor, yang juga disediakan untuk dipakai bersama kelompok lain.
7. Selanjutnya ke dalam ketujuh tabung reaksi tersebut masing-masing masukkan
7 mL akuades dengan bantuan buret yang telah disediakan.
8. Aduk larutan dalam ke tujuh tabung reaksi tersebut dengan batang pengaduk.
Setiap kali akan memindahkan batang pengaduk dari satu tabung ke tabung lain,
maka batang pengaduk tersebut terlebih dahulu dibilas dengan akuades dan
keringkan dengan lap.
9. Ukur absorbansi larutan-larutan tersebut pada panjang gelombang 660 nm
64
TUGAS JURNAL
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
65
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
.........................................................................................................................................................
66
HASIL PENGAMATAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
Pekanbaru, .....................................202
0
Asisten,
67
PEMBAHASAN
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
68
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
.......................................................................................................................................................
69
VIII. PERCOBAAN EMPEDU
8.1. Tujuan
1. Mahasiswa memahami fungsi, komposisi, dan sifat getah empedu
2. Mahasiswa mampu mengidentifikasi keberadaan pigmen empedu dalam suatu
sampel
8.2. Pendahuluan
Kandung empedu menyimpan cairan empedu yang diproduksi oleh hati
diantara waktu-waktu makan. Selama pencernaan, kandung empedu akan berkontraksi
dan mengalirkan getah empedu secara cepat kedalam duodenum melalui
duktuskoledokus. Komposisi getah empedu hati berbeda dari getah empedu kandung
empedu yang lebih pekat. Dari percobaan dan penelusuran literature untuk bab ini
diharapkan mahasiswa dapat mengetahui komposisi dan sifat getah empedu meliputi
emulsifikasi, netralisasi asam, dan ekskresinya. Sifat fisika dari empedu adalah berupa
bau, warna dan pH.
8.3. Prosedur Kerja

1. Identifikasi pH
Bahan dan reagen: empedu sapi /ayam
Alat-alat : Tabung reaksi dan Indikator Universal
Cara kerja:
1. Masukkan cairan empedu ke dalam tabung reaksi
2. Celupkan Indikator Universal ke dalam cairan tersebut
3. Angkat dan baca dengan mencocokkan dengan standar warna yang ada pada
pH universal
2. Identifikasi BJ Empedu
Berat jenis adalah perbandingan relatif antara massa jenis sebuah zat dengan
massa jenis air murni (1 g/cm 3 atau 1000 kg/m3). Berat jenis (Bj) dan Massa jenis (Mj)
70
adalah sama karena berat jenis adalah perbandingan antara massa jenis suatu benda
dengan massa jenis air, sedangkan massa jenis air diketahui = 1 maka berat jenis suatu
benda dibagi dengan 1 maka Bj = Mj. Beberapa benda padat jika dimasukkan ke dalam
air akan mengalami peristiwa: 1) tenggelam apabila Bj zat padat > Bj air. 2) Terapung
apabila Bj zat padat < Bj air. 3) melayang Bj zat padat = Bj air .
Bahan dan reagen : Empedu

Alat-alat : 1. Gelas ukur
2.Termometer
3. Urinometer
Carakerja:
1. Masukkan empedu ke dalam gelas ukur 10 ml
2. Masukkan Urinometer ke dalam larutan empedu tersebut dengan memutarnya
3. Hitung BJ dengan jalan membaca skala yang ada pada urinometer
3. Identifikasi sifat Emulsi

Emulsi merupakan jenis koloid campuran antara partikel-partikel suatu zat cair
(fase terdispersi) dengan zat cair lainnya (fase pendispersi) Emulsi tediri dari fase
terdispersi, fase pendispersi, dan emulgator. Fungsi pembentukan emulsi antara lain:
meningkatkan kelarutan, dan meningkatkan kestabilan.
Bahan dan reagen : 1. Minyak kelapa; 2. Cairan empedu; 3. Aquadest

Alat-alat : Tabung reaksi, Gelas ukur, Pipet tetes
Cara kerja:
1. Sediakan 2 buah tabung reaksi, beri label E untuk eksperimen dan C untuk
kontrol
2. Masukkan 1 ml aquadest ke dalam masing-masing tabung
3. Tambahkan 1ml cairan empedu ke dalam Tabung E
4. Tambahkan 1ml minyak kelapa ke dalam Tabung C
5. Aduk kedua tabung secara bersamaan selama1 menit, kemudian biarkan
6. Perhatikan warna emulsinya yang lebih stabil
71
4. PETENKOFER UNTUK ASAM EMPEDU
Prinsip Dasar: Asam empedu dengan furfural akan membentuk cincin berwarna pada
bidang batas cairan.
Bahan dan reagen : 1. LarutanGlukosa5%
2. H2SO4 pa.
3. Larutan empedu encer (1 :10)
Alat-alat : 1. Tabung reaksi.
2. Gelas piala.
3. Gelas ukur.
4. Pipet tetes
Carakerja:
1. Sediakan satu buah tabung reaksi, masukkan 2,5 mL empedu encer
2. Tambahkan 2,5 mL larutan glukosa 5 %
3. Miringkan tabung reaksi tersebut sambil tambahkan 2,5 mL H2SO4 pa pada
dindingnya dengan hati-hati sehingga terbentuk 2 lapisan
4. Dinginkan dalam gelas piala yang berisi air dingin
5. Perhatikan warna yang ada pada kedua batas cairan
5. .PIGMEN-PIGMEN EMPEDU
Prinsip Dasar:
Sebagian besar pigmen-pigmen empedu berasal dari hasil penghancuran
eritrosit, pigmen utama dan terbanyak adalah bilirubin dan biliferdin. Yang pertama
berwarna merah/kuning coklat dan yang kedua berwarna hijau. Hasil oksidasi dari
pigmen-pigmen berikutnya akan membentuk bermacam-macam warna.
A. Uji Gmelin
Bahan dan reagen : 1. Asam Nitrat pekat .
2. Larutan empedu encer
Alat-alat : 1. Tabung reaksi
72
2. Pipet takar
Carakerja:
1. Sediakan satu buah tabung reaksi, masukkan 3 ml asam nitrat pekat
2. Tambahkan 3ml empedu encer memakai pipet. Harus dengan hati-hati sehingga
keduanya tidak bercampur
3. Perhatikan warna-warna yang terlihat pada perbatasan kedua cairan tersebut
4. Uji kepekaan percobaan ini dengan menggunakan empedu yang lebih encer
(1:50)
5. Catat warna-warnanya
B. Uji smith
Bahan dan reagen : 1. Empedu encer.
2. Iodium 0,5% dalam alkohol
Alat-alat : 1. Tabung reaksi
2. Pipet tetes.
3. Gelas piala.
4. Pipet takar
Carakerja:
1. Sediakan satu buah tabung reaksi, masukkan sedikit empedu
2. Masukkan beberapa tetes larutan iodium sehingga membentuk lapisan diatas
cairan tersebut
3. Perhatikan cincin berwarna hijau tua atau biru kehijau-hijauan dibawah lapisan
iodium.
73
TINJAUANPUSTAKA
*Tambahkan dalam bagian ini bagaimana peranan empedu dalam proses pencernaan
makanan; dan prinsip reaksi pengujian
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
..........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
74
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
75
HASILPENGAMATAN
PercobaanEmpedu
Bau Empedu: ................................
Warna Empedu: ................................
pH Empedu: ...............................
BJ empedu
Masukkan urinometer kedalam empedu, baca skala urinometer: .........................
Emulsi empedu
Zat kimia Pengamatan
Tabung E =1 ml aq + 1ml cairan empedu,aduk selama 1 mnt
Tabung C= 1ml aq + 1ml minyak kelapa, aduk selama 1mnt
Petenkofer untuk asam empedu

2,5 ml empedu + 2,5 ml glukosa 5% kemudian miringkan sambil + H2SO4
...........
UjiGmelin
3 ml asam nitrat + 3 ml empedu ........................................
3 ml asam nitrat + 3 ml empedu (1: 50 ) .............................
UjiSmith
Empedu +beberapa tetes lar. iodium ........................................
Pekanbaru, ......................................202
0
76
Asisten,
77
PEMBAHASAN
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
78
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
79
IX. SELULASE
IX.1. Tujuan
1. Memahami prinsip aktivitas enzim dan gula reduksi
2. Mengetahui cara menghitung aktivitas enzim
9.2. Pendahuluan
Selulase adalah satu dari beberapa enzim hidrolase yang dapat mengkatalisa
penguraian selulosa menjadi molekul-molekul kecil (glukosa, sellobiosa ). Aktivitas
enzim selulase adalah kemampuan 1 ml enzim untuk menghidrolisis selulosa
menghasilkan µmol glukosa atau gula pereduksi. Konsentrasi gula pereduksi yang
dihasilkan ini dapat ditentukan dengan metoda Nelson Somogyi.
Karena keterbatasan peralatan, maka pada percobaan ini 2 kelompok digabung
menjadisatu.
9.3. Alat dan Bahan

Peralatan:
A. Untuk setiap gabungan 2 kelompok:
1. Tabung reaksi 2. Pipet ukur1 ml
3. Batang pengaduk 4. Botol semprot
5. Lembaran-lembaran kertas saring panjang 6. Gelas kimia 500 ml
0
7. Waterbath termostat temperatur 40 C 8. Kelereng
9. Rak tabung reaksi steinless steel

10. Spektrofotometer sinar tampak
B. Untuk dipakai bersama kelompok lain:

1. Buret
2. Ballpipettor untuk mengisap reagen Arsenomolibdat yang beracun
80
Bahan dan reagen yang disiapkan asisten:
1. Reagen warna Arsenomolibdat (lihat percobaan VII)
2. Reagen A (lihat percobaan VII)
3. ReagenB (lihat percobaan VII)
4. Reagen Nelson Somogyi: Reagen ini harus dibuat pada saat mau praktikum,
karena pemakaiannya harus dalam keadaan segar, dan reagen ini tidak bisa
disimpan untuk jangka waktu yang lebih lama dari satu hari. Cara membuat
reagen Nelson Somogyi adalah dengan mencampurkan 4 volume reagen A
dengan 1 volume reagen B.Untuk satu kali praktikum buat 50 ml larutan
Nelson Somogyi yaitu dengan mencampurkan
40 ml reagen A dengan 10 ml reagenB.
5. Bufer Asetat 0,05 M pH5,5 ( dibuat dengan bantuan pH meter)
6. Larutan substrat: larutkan 0,5 g Carboxymethylcellulosa (CMC) dalam 100 ml
buffer asetat pH 5,5.
7. Larutan EDTA: Larutkan 0,0672 g garam dinatrium EDTA (BM=336) dalam
100 ml buffer asetat pH 5,5.
8. Larutan CuSO4: Larutkan 250g CuSO4.5H2O (BM=249,6) dalam 100 ml
buffer asetat pH 5,5.
9. Es batu untuk menyimpan larutan enzim
10. Larutan standar glukosa100 μg/ml dalam larutan buffer asetat pH 5,5
11. 20 ml larutan enzim berkonsentrasi 0,0004 mg/ml. Taruh larutan enzim ini
dalam tabung reaksi yang dicelupkan dalam gelas kimia yang berisi es. Larutan
enzim dibuat pada saat praktikum akan dimulai.
81
Cara Kerja :
3. Siapkan tabung reaksi (kering dan bersih), masing-masing tabung
reaksi diberi label, dan ke dalam masing-masing tabung reaksi dimasukkan
bahan seperti tertera pada table berikut:
No LABEL ISI TABUNG REAKSI

1 Blanko 2 ml Bufer Asetat pH 5,5 (0,05M)
2 Kontrol Biarkan kosong
3 Uji 1 1 ml Substrat
4. Masukkan ke 5 tabung reaksi tersebut ke dalam waterbath termostat

bertemperatur 400 C, biarkan selama 5 menit.
3. Setelah 5 menit berada dalam warterbath, tanpa mengeluarkan tabung-tabung
reaksi tersebut, tambahkan 1 ml enzim selulase (dengan bantuan pipet ukur 1 ml)
ke dalam masing-masing tabung reaksi uji dan kontrol.
4. Kemudian di aduk dengan dengan batang pengaduk yang telah disediakan secara
berlahan. Satu batang pengaduk untuk satu tabung reaksi. Hati-hati mengaduknya,
jangan sampai larutan berbusa, karena akan merusak enzim..
5. Biarkan tabung-tabung tersebut selama 30 menit dalam waterbath, sambil sekali-
kali diaduk pelan
6. Setelah 30 menit, semua tabung reaksi dikeluarkan dari waterbath dan ke dalam
tabung uji, kontrol, dan blanko ditambahkan masing-masing 1 ml reagen Nelson-
Somogyi (untuk menghentikan kerja enzim). Dan khiusus untuk tabung kontrol di
tambahkan 1 ml substrat
7. Selanjutnya semua mulut tabung reaksi di tutup dengan kelereng dan semua
tabung reaksi ini dimasukkan ke dalam beaker glass (penangas air) yang berisi air
panas biarkan + 20 menit
8. Selanjutnya semua tabung reaksi di dinginkan hingga suhu ruang. Kemudian
ditambahkan masing-masing 1ml larutan arsenomolibdat aduk dan biarkan selama
5 menit
9. Selanjutnya pada masing-masing tabung reaksi ditambahkan 6 ml akuades
(menggunakan buret) dan biarkan kembali selama 30 menit. Jika terdapat endapan
82
maka harus disentrifugasi ( 9500 rpm selama 10 menit)
10. Selanjutnya setelah penambahan aquades campuran ini di vortex dan dibiarkan
kembali selama 20 sampai dengan 30 menit
11. Sebagai larutan standar digunakan larutan glukosa dengan berbagai konsentrasi
12. Absorbansi dari masing- masing larutan diukur dengan alat spektrofotometer
UV-Visibel Genesis 10S pada panjang gelombang 540 nm.
Data Absorbansi yang diperoleh digunakan untuk menentukan konsentrasi gula

pereduksi hasil hidrolisis substrat (CMC) oleh enzim sellulase. Aktivitas enzim
ekuivalen dengan gula pereduksi yang dihasilkan permililiter (µmol gula
pereduksi/ml).
Tugas laporan :
1. Hitung aktivitas enzim pada temperatur 400 C, pH 4,5 seabagai berikut :
Aktivitas enzim = µmol gula pereduksi uji - µmol gula pereduksi kontrol
Volume enzim x waktu inkubasi (menit)
2. Apa gunanya pra-inkubasi 5 menit dalam waterbath bertemperatur 40 0 C dari

tabung-tabung kontrol, Uji 1, Uji 2 dan Uji 3?
3. Kenapa pada tabung kontrol penambahan substrat dilakukan setelah reaksi
enzim dihentikan dengan penabahan reagen Nelson Somogyi ?
TINJAUAN PUSTAKA
*pada bagian ini deksripsikan tentang klasifikasi enzim berdasarkan reaksi yang
83
dikatalisis, factor yang mempengaruhi kerja enzim, dan gula reduksi
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
..........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
84
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
HASIL PENGAMATAN
85
No Tabung Absorban
1 Tabung 1
2 Tabung 2
3 Tabung 3
4 Tabung Blanlo
5 Tabung Kontrol
Pekanbaru, ............................................2020
Asisten,
86
Diktat Praktikum Biokimia 2018
PEMBAHASAN
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
........................................................................................................................................... ...........
................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
.......................................................................................................................................... ............
...............................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
........................................................................................................................................... ...........
................................................................................................................................
Diktat Praktikum Biokimia 2018
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
........................................................................................................................................... ...........
................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
...........................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................
............................................................................................................................................

Biokimia Untuk Kimia

Diunggah oleh

Informasi Dokumen

Judul Asli

Hak Cipta

Format Tersedia

Bagikan dokumen Ini

Bagikan atau Tanam Dokumen

Opsi Berbagi

Apakah menurut Anda dokumen ini bermanfaat?

Apakah konten ini tidak pantas?

Hak Cipta:

Format Tersedia

Biokimia Untuk Kimia

Diunggah oleh

Hak Cipta:

Format Tersedia

DAFTAR ISI

TIM BIOKIMIA JURUSAN KIMIA

Pekanbaru, Februari 2020

Dr. Muhdarina, M.Si Dra. Silvera Devi Sy, M.Si

I. PEMBUATAN LARUTAN BUFFER..............................................................................1

KEWAJIBAN MAHASISWA PESERTA PRAKTIKUM

II. Alat dan Reagensia

III. Kebersihan Laboratorium

IV. Laporan dan Lain-lain

Larutan buffer dapat mempertahankan pH suatu system karena dengan penambahan

berhubungan dengan protein, enzim, dan asam nukleat (DNA, RNA).

Larutan buffer yang sering digunakan dalam penelitian Biokimia:

Volume dari 0,5 M Volume dari 0,5 M

*coret yang tidak digunakan

HC OH + KHSO4 C H + KHSO4. 2 H2O

2.3.2. Bahan Uji Kelarutan

2.3.3. Bahan Uji Gliserol dalam Minyak:

2.3.4. Bahan Untuk Penentuan Berat Jenis Minyak

2.3.5. Bahan Untuk Penentuan ALB ( Asam Lemak Bebas )

1. Timbang 5 gr sampel Minyak , masukkan kedalam erlenmeyer.

ml NaOH pentiter x N NaOH x 256 x 100

2. Identifikasi gliserol dalam lipid

3.Penentuan Berat Jenis Minyak

5. Penentuan ALB ( Asam Lemak Bebas )

Larutan protein apabila ditambah dengan ninhidrin, kemudian dipanaskan akan

3.3.2. Bahan Identifikasi unsur-unsur yang terdapat dalam protein

3.3.3. Bahan Uji Ninhidrin

3.3.4. Bahan Uji Biuret

Pertanyaan sebagai tugas yang harus dijawab di dalam laporan praktikum :

3.3.5. Bahan Uji Millon

3.3.6. Bahan Pengendapan Protein oleh Asam Kompleks

3.3.7. Bahan Pengendapan dengan Pelarut Organik

3.3.8. Bahan Pengendapan dengan garam (Salting Out)

No Zat Kimia Pengamatan

No Zat Kimia Pengamatan

5. Pengendapan protein oleh asam kompleks

3 mL larutan protein + 4 tetes TCA 7 % sampai terbentuk endapan

6. Pengendapan dengan pelarut organik

2 mL protein + 10 mL alkohol aduk

1. Sediakan 7 tabung reaksi yang kering

No Zat Kimia Pengamatan

6.3. Prosedur kerja

6.3.3. Cara kerja Pengaruh Suhu Terhadap Aktivitas Enzim

3. Buatlah kurva yang menggambarkan hubungan kecepatan reaksi enzimatik

6.3.4. Cara kerja Pengaruh pH Terhadap Aktivitas Enzim

6. Buat kurva yang menggambarkan hubungan antara kecepatan reaksi enzimatik

7.3. Prosedur Kerja

7.3.2. Bahan Pengukuran Konsentrasi Glukosa dalam Urin

1. Sediakan 7 tabung reaksi yang kering

2. Dengan menggunakan pipet ukur 1 mL, ke dalam tabung-tabung reaksi tersebut

Tabung Sampel: 1 mL larutan urin

8.3. Prosedur Kerja

Bahan dan reagen : Empedu

3. Identifikasi sifat Emulsi

Bahan dan reagen : 1. Minyak kelapa; 2. Cairan empedu; 3. Aquadest

Tabung E =1 ml aq + 1ml cairan empedu,aduk selama 1 mnt

Tabung C= 1ml aq + 1ml minyak kelapa, aduk selama 1mnt

Petenkofer untuk asam empedu

9.3. Alat dan Bahan

9. Rak tabung reaksi steinless steel

B. Untuk dipakai bersama kelompok lain:

No LABEL ISI TABUNG REAKSI

4. Masukkan ke 5 tabung reaksi tersebut ke dalam waterbath termostat