Diktat Praktikum Biokimia 2022

2022
2022
1
DAFTAR ISI
Halaman
Daftar Isi 2
Kata Pengantar 3
Tata Tertib Laboratorium Biokimia 4
I. Pembuatan larutan buffer 6
II. Uji lipid 14
III. Uji asam amino dan protein 23
IV. Penentuan kadar protein dengan metoda Lowry 35
V. Percobaan oksidasi biologi 43
VI. Percobaan enzim 50
VII. Urine 60
VIII. Percobaan empedu 69
IX. Selulase 78
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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 biologi 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/i dalam mempersiapkan dan
melaksanakan praktikum dengan lebih baik, terarah, dan terencana.
Kami mengucapkan terima kasih 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
penyelenggaraan praktikum Biokimia di Jurusan Kimia, FMIPA, Universitas Riau.
Pekanbaru, Januari 2022

Tim Penyusun Diktat Praktikum Biokimia Dasar
Dra. Silvera Devi Sy, M.Si
Dra. Andi Dahliaty, MS
Yuana Nurulita, PhD
Yuli Haryani, MSc, Apt
Riryn Novianty, M.Si
Noviza Delfira
3
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 laboran. 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
3. Jika ada terjadi kecelakaan segera beritahu asisten
4
IV. Laporan dan Lain-lain
1. Praktikan harus membuat laporan sementara setelah selesai praktikum hari itu dan
menyerahkannya kepada asisten
2. Menyerahkan laporan lengkap pada waktu praktikum berikutnya, selambat-lambatnya
seminggu setelah praktikum
3. Setelah selesai praktikum buku laporan sementara harus diparaf oleh asisten
4. Praktikan yang tidak masuk 2 kali berturut-turut atau 3 kali tidak masuk dianggap
mengundurkan diri
5. Tidak masuk karena sakit harus dengan surat keterangan dokter dan izin harus diketahui
Penasehat Akademis
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I. PEMBUATAN LARUTAN BUFFER
1.1 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.
1.2 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
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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 .
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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 Na2HPO4 dan NaH2PO4.
Buffer Sodium Phosphat 20oC
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
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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
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?
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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, ................................... 2021

Asisten,
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HASIL DAN PEMBAHASAN
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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.
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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. Kloropform
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
3. Minyak kelapa ( minyak goreng )
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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
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
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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 = r m = massa jenis v = volume
m
r= BJ = ...................................................
v
Minyak 2
BJ = r m = massa jenis v = volume
m
r= BJ = ...................................................
v
Minyak 3
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20
BJ = r
m = massa jenis v = volume
m
r= BJ = ...................................................
v
5. Penentuan ALB ( Asam Lemak Bebas )

% ALB = .................................................................
Pekanbaru, ........................................... 2021

Asisten,
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PEMBAHASAN
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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 a-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 a-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.
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TUGAS JURNAL
*Struktur asam amino, struktur umum protein, pembagian asam amino, konsep
pengendapan protein dengan menggunakan beberapa zat kimia.
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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.
26
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 )
27
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? Mengapa?
28
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
Cara kerja :
1. Larutan protein ±3 mL dimasukkan ke dalam tabung reaksi
2. Kemudian ditambahkan Kristal ammonium sulfat sedikit demi sedikit sampai
29
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
30
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 mLsaliva 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
31
6. Pengendapan dengan pelarut organik
2 mL protein + 10 mL alkohol aduk
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, ..................................... 2021

Asisten,
32
HASIL DAN PEMBAHASAN
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34
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.
35
TUGAS JURNAL
*Prinsip kerja dari spektrofotometer dan hal-hal yang harus diperhatikan dalam analisis
kuantitatif dengan mengunakan spektrofotometer
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37
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 % Na2CO3 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
1N
4. Larutan standar protein: buatlah 100 mL larutan serum albumin standar dengan
konsentrasi 1000 µg/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.
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
38
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 x c
Tabung 3 : 0,6 mL larutan protein standar + 0,4 mL akuades, hingga c menjadi 0,6 x c
Tabung 4 : 0,8 mL larutan protein standar + 0,2 mL akuades, hingga c menjadi 0,8 x c
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
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.
39
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,....................................... 2021
Asisten,
40
PEMBAHASAN
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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 yang dipanaskan sampai 70oC (pasteurmilk) menyebabkan enzim
dehydrogenase tidak aktif
43
TUGAS JURNAL
*Mekanisme kerja enzim dehydrogenase, Reaksi perubahan warna yang terjadi
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5.3. 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.
4. Kedua tabung reaksi ini diletakkan ke dalam waterbath 650C selama 30 menit.
5. Amati yang terjadi
46
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, ..................................... 2021

Asisten,
47
PEMBAHASAN
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49
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
4. Larutan pati 0,4 mg/mL dalam berbagai pH ( 3, 5, 7, 9 Dan 11 )
5. Larutan Iodium
50
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 mL 1 mL
Inkubasi masing-masing pasangan tabung tersebut pada setiap suhu yang
digunakan (1,2,3,4,5,6 ) selama 5 menit
Liur diencerkan 100X - 200µl
Aquades 200µl -
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
Aquades 8 mL 8 mL
1. Pasangan I ditempatkan dalam bejana berisi es ( 00 C )

2. Pasangan II ditempatkan dalam bejana berisi air yang suhunya dipertahankan tetap pada
suhu 250 C
3. Pasangan III ditempatkan di rak tabung pada suhu ruang
4. Pasangan IV ditempatkan dalam penangas air yang suhunya dipertahankan tetap pada
suhu 370 C
5. Pasangan V ditempatkan dalam penangas air yang suhunya dipertahankan tetap pada
suhu 600 C
6. Pasangan VI ditempatkan dalam penangas air mendidih ( 1000 C )
Segera baca serapan ( A ) pada panjang gelombang 680 nm. Hitung selisih serapan
( D A ) antara tabung B ( A pada t = 0 menit ) dengan tabunng U dari setiap suhu.
51
Buatlah tabel berikut ini :
SUHU AB AU D A/menit ( v )
00 C
250 C
Suhu Ruang
370 C
600 C
1000 C
3. Buatlah kurva yang menggambarkan hubungan kecepatan reaksi enzimatik

(v=DA/menit(v)
52
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
Inbkubasi pada suhu 37oC paling sedikit 5 menit
Liur diencerkan 100X - 200 µl
Aquades 200µl -
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
7
9
11
6. Buat kurva yang menggambarkan hubungan antara kecepatan reaksi enzimatik (Δ

A/menit ( v ) )
53
TUGAS JURNAL
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55
HASIL PENGAMATAN
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Pekanbaru, ...................................... 2021
Asisten,
57
PEMBAHASAN
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59
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. Pendahulan
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
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
60
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 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.
61
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.
62
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
63
TUGAS JURNAL
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HASIL PENGAMATAN
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PEMBAHASAN
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68
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/cm3 atau 1000 kg/m3). Berat jenis (Bj) dan Massa jenis (Mj)
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
69
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
70
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
2. Pipet takar
71
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.
72
TINJAUANPUSTAKA
*Tambahkan dalam bagian ini bagaimana peranan empedu dalam proses pencernaan
makanan; dan prinsip reaksi pengujian
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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, ...................................... 2021

Asisten,
75
PEMBAHASAN
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77
IX. SELULASE
9.1. 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
7. Waterbath termostat temperatur 400C 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
78
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.
79
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
80
9. Selanjutnya pada masing-masing tabung reaksi ditambahkan 6 ml akuades
(menggunakan buret) dan biarkan kembali selama 30 menit. Jika terdapat endapan
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 400 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 ?
81
TINJAUAN PUSTAKA
*pada bagian ini deksripsikan tentang klasifikasi enzim berdasarkan reaksi yang
dikatalisis, factor yang mempengaruhi kerja enzim, dan gula reduksi
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83
HASIL PENGAMATAN
No Tabung Absorban
1 Tabung 1
2 Tabung 2
3 Tabung 3
4 Tabung Blanlo
5 Tabung Kontrol
Pekanbaru, ............................................ 2021

Asisten,
84
PEMBAHASAN
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Diktat Praktikum Biokimia 2022

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Pekanbaru, Januari 2022

KEWAJIBAN MAHASISWA PESERTA PRAKTIKUM

3. Jika ada terjadi kecelakaan segera beritahu asisten

asam nukleat (DNA, RNA).

Larutan buffer yang sering digunakan dalam penelitian Biokimia:

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

5,8 7,9 92,1

*coret yang tidak digunakan

Pekanbaru, ................................... 2021

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 )

Pekanbaru, ........................................... 2021

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

3.3.7. 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

2 mL protein + 10 mL alkohol aduk

Bila ada endapan uji kelarutan dalam air

Pekanbaru, ..................................... 2021

1. Sediakan 7 tabung reaksi yang kering

No Zat Kimia Pengamatan

Pekanbaru, ..................................... 2021

6.3. PROSEDUR KERJA

1. Pasangan I ditempatkan dalam bejana berisi es ( 00 C )

3. Buatlah kurva yang menggambarkan hubungan kecepatan reaksi enzimatik

Campur baik-baik. Diamkan selama 1 menit.

6. Buat kurva yang menggambarkan hubungan antara kecepatan reaksi enzimatik (Δ

7.3. Prosedur Kerja

1. Urinometer 2. Spektrofotometer 3. Gelas kimia 100 mL.

7.3.2. Bahan Pengukuran Konsentrasi Glukosa dalam Urin

b) Reagen warna Arsenomolibdat : 12,5 gr ammoniummolibdat dalam 200 mL

c) Reagen A : 1,2 gr KNaTaratarat, 2,4 gr Na2CO3 anhydrous, 1,6 gr NaHCO3, 14,4

d) Reagen B : Larutkan 2 gr CuSO4. 5H2O dan 18 gr Na2SO4 anhydrous ke dalam 100

1. Sediakan 7 tabung reaksi yang kering

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

Pekanbaru, ...................................... 2021

9.3. Alat dan Bahan

7. Waterbath termostat temperatur 400C 8. Kelereng

B. Untuk dipakai bersama kelompok lain:

No LABEL ISI TABUNG REAKSI