PRAKTIKUM KIMIA PROSES

KINETIKA REAKSI ENZIMATIS

1. Laporannya dirapiin
lagi ya
2. Jurnalnya dimasukin
disini ya
3. Lembar pengesahan
ditaruh habis daftar
pustaka ya

Disusun oleh

1. Haqiqi Hadziq Fikri (D500180060)

2. Dewi Kristiana (D500180062)

3. Nanda Mila Afida (D500180065)

Kelompok : 8B

Tanggal : 10 Mei 2020

LABORATORIUM TEKNIK KIMIA

PROGRAM STUDI TEKNIK KIMIA
UNIVERSITAS MUHAMMADIYAH SURAKARTA
2020

DAFTAR ISI
BAB 1 TUJUAN PERCOBAAN 1

BAB 2 PENDAHULUAN
1
A. Laktosa 1 B. Sukrosa 1 C. Enzim
2
D. Kinetika Enzim 3
E. Model kinetik Lineweaver-Buck 3
BAB 3 METODOLOGI 7
A. Alat 7 B. Bahan 8
C. Gambar Alat
9

BAB 4 Cara Kerja 11

Diagram Alir 13

BAB 5 HASIL
18
A. Hasil Percobaan 18 B. Pembahasan
19
C. Kesimpulan 22
DAFTAR PUSTAKA 23
BAB 6 LAMPIRAN 26
A. Data Hasil Percobaan 26
B. Perhitungan 27
C. Galat 30

I. TUJUAN PERCOBAAN
Menentukan konstanta Lineweaver-Bruk reaksi hidrolisis laktosa
dengan menggunakan enzim invertase dan yeast.

II. PENDAHULUAN
A. Laktosa
 Laktosa (saccharum lactis) atau disebut juga gula susu merupakan
disakarida yang berasal dari susu sapi (Bos taurus). Kadar laktosa
dalam susu sapi sekitar 5%. Jika dihidrolisis, laktosa akan
menghasilkan glukosa dan galaktosa (Sukardiman,dkk ,2020).
Laktosa merupakan sumber energi yang masuk hampir setengah
keseluruhan kalori yang terdapat pada susu (35-45%). Selain itu
laktosa juga diperlukkan untuk absorbs kalsium. Hasil hidrolisis
laktosa yang berupa galaktosa adalah senyawa yang penting untuk
pembentukansebrosida. Galaktosa juga dapat dibentuk oleh tubuh dari
sukrosa di hati (Sinuhaji,2006).

B. Glukosa
Glukosa adalah sumber energi utama bagi tubuh. Hormon yang
mempengaruhi kadar glukosa adalah insulin dan glukagon yang
berasal dari pankreas.Insulin dibutuhkan untuk permeabilitas membran
sel terhadap glukosa dan untuktransportasi glukosa ke dalam sel
(Joyce, 2013).
Glukosa merupakan salah satukarbohidrat yang sangat penting dan
dibutuhkan sebagai sumber energi danmerupakan bahan bakar utama
bagi otak dan sel darah merah. Glukosa dapatdiperoleh dari makanan
yang mengandung karbohidrat (Marks, 1996).

C. Enzim
Enzim adalah protein yang khusus disisntesis oleh sel hidup untuk
mengkatalis reaksi yang berlangsung didalamnya. Oleh karena itu,
banyak sekali bio- atalisator yang dibentuk oleh jumlah maupun
jenisnya yang tidak dapat terhitung banyaknya (Martoharsono, 2006).

Kebanyakan enzim yang terdapat di dalam alat-alat atau

organorgan organisme hidup berupa larutan koloidal dalam cairan
tubuh, seperti air ludah, darah, cairan lambung dan cairan pankreas.
Enzim terdapat di bagian dalam sel. Hal ini terikat erat dengan
protoplasma.
Enzim juga ada di dalam mitokondria dan ribosom.
(Sumarjo,2009)

1
 2

Semua enzim murni yang telah diamati sampai saat ini adalah
protein, dan aktivitas katalitiknya bergantung kepada strukturnya
sebagai protein (Lehninger,1982).

Karena enzim merupakan protein, maka kemungkinan kenaikan

suhu dapat menyebabkan terjadinya proses denaturasi, apabila hal
tersebut terjadi, maka bagian sisi aktif enzim akan terganggu dan
menyebabkan konsentrasi enzim menjadi berkurang sehingga
kecepatan reaksinya pun akan menurun (Ariandi, 2016).

Beberapa enzim hanya terdiri dari protein, tapi kebanyakan enzim

mengandung non protein, seperti karobohidrat, lipid, logam, fosfat atau
beberapa bahan organik (Deman, 1997).
Enzim yang lengkap disebut haloenzim, bagian protein disebut
apoenzim dan bagian non protein disebut kofaktor. Senyawa yang
diubah dalam reaksi yang dikatalis enzim disebut substrat. Dalam
reaksi enzim, substrat bergabung dengan haloenzim dan dilepas dalam
bentuk dimodifasi (Deman, 1997).
Atas dasar jenis reaksinya enzim dibagi menjadi enam golongan,
yaitu(Martoharsono, 2006):
1. Oksida-reduktase
Golongan enzim oksida-reduksi dibagi menjadi 5 sub
golongan mengkatalis substrat yang bergugus fungsional >CHOJ;
>C=O; >C=CH; >CH-NH2 dan >CH-NH-, enzim ini berperan
dalam reaksi oksida reduksi.
2. Transferase
Pada golongan enzin transferase ini dijumpai 8 sub
golongan. Beberapa diantaranya adalah enzim yang memindahkan
gugus, berkarbon satu, aldehid atau ketonik, asilfosfat, dan gugus
yang mengandung substart. Enzimini berperan dalam, reaksi
pemindahan gugus.
3. Hydrolase
Enzim yang kerjanya secara menghidrolisis substrat masih
dibagi menjadi enzim yang menghidrolisis senyawa yang
berikatan, misalnya ester, glikosidik, peptide, okatan C-N dan
anhidrida.
4. Liase
Golongan enzim ini mempunyai 3 sub-golongan yaitu yang
mengkatalis reaksi adisis terhadap ikatan >C=C<; C=O dan C=N-

.
 3

5. Lisomerase
Enzim golongan ini mengkatalis semua reaksi isomerase
termasuk reaksi rasemasi.

6. Ligase
Enzim ligase merupakan sekelompok enzim pembentuk
ikatan dengan bantuan energi yang bersal dari pemecahan ATP.
Golongan enzim ini disebut juga sebagai sentase.

Enzim berfungsi sebagai katalisator yaitu mepercepat reaksi

dengan menurunkan energi aktivasi dan sifatnya yang khas. Terdapat
enzim yang mampu mengkatalis satu substrat saja, danada pula yang
bersifat streo spesifik, karena enzim mengkatalisis reaksi-reaksi dalam
sistem biologis, maka enzim disebut biokatalisator (Murray & Rodwell,
2009).

D. Kinetika Enzim
Kinetika enzim adalah aktivitas enzim yang didasarkan oleh
konsentrasi substrat. Pada konsentrasi substrat yang amat rendah,
kecepatan reaksipun amat rendah, tetapi kecepatan ini akan meningkat
dengan meningkatnya konsentrasi substrat. Jika kita menguji pengaruh
konsentrasi substrat yang terus meningkat setiap saat kita mengukur
kecepatn awal reaksi yang dikatalisis ini, kita akan menemukan bahwa
kecepatan ini meningkat dengan nilai yang semakin kecil. Pada
akhirnya akan tercapai titik batas, dan setelah titik ini dilampaui,
kecepatan reaksi hanya akan meningkat sedemikian kecil dengan
bertambahnya konsentrasi substrat. Pada batas ini yang disebut
kecepatan maksimum ( Vmaks ) , enzim menjadi jenuh karena
substratnya dan tidak dapat berfungsi lebih cepat(Lehninger, 1982).

Reaksi Enzima :

Analisa kuantitatif kinetika reaksi enzim dapat dilakukan dengan

dua azas pendekatan : azas keseimbangan menurut Michaelis- Menten,
dan azas teori keadaan tunak (steady state theory) menurut Briggs-
haldane. (Wirahadikusumah, 1981)
 4

Fungsi khusus enzim antara lain yaitu (Martoharsono, 2006):

1. Merendahkan energi aktivasi
Suatu zat A akan berubah menjadi zat B, jika sekurang –
kurangnya sebagai zat A mendapatkan cukup energi sehingga
dapat berada dalam keadaan aktif atau dalam keadaan transisi,
yaitu kemudian bisa berubah menjadi zat B. energi ini disebut
energi aktivasi.
2. Kecepatan reaksi
Ada tiga keadaan kadar substrat berpengaruh atas kecepatan
dan laju reaksi yaitu:
1. Reaksi tingkat nol
2. Reaksi tingkat satu
3. Reaksi tingkat dua

E. Model Kinetik Lineweaver-Burk

Persamaan Michaelis-Menten dapat ditransformasi secara aljabar
menjadi bentuk lain yang lebih bermanfaat didalam pemetaan data
percobaan. Suatu transformasi umum diturunkan secara sederhana
dengan membuat kebalikan dari kedua sisi persamaan
MichaelisMenten sebagai berikut (Lehninger , 1982).

1 Kᴍ 1 1

= + 𝑣𝑜
𝑉𝑚𝑎𝑘𝑠[𝑆]
𝑉𝑚𝑎𝑘𝑠

persamaan tersebut adalah transformasi persamaan

MichaelisMenten yang disebut persamaan Lineweaver-Burk. Bagi
enzim-enzim yang mengikuti hubungan Michaelis-Menten secara
benar, pemetaan
1 terhadap akan menhasilkan garis lurus yang memiliki sudut
𝑣𝑜
𝐾ᴍ 1
. Perpotongan garis terhadap sumbu y atau sumbu sebesar
𝑉𝑚𝑎𝑘𝑠 𝑣𝑜
1 dan perpotongan garis terhadap sumbu x atau sumbu
adalah
𝑉𝑚𝑎𝑘𝑠

sebesar - 1 (Lehninger , 1982).

𝐾ᴍ

Lineweaver-Burk merupakan persamaan tentang hubungan antara

 5

1/[S] vs 1/VO+ kurve yang didapat adalah garis lurus dengan slope =
Km/Vmaks , sedangkan intersep= 1/Vmaks. Persamaan tersebut dapat
dinyatakan sebagai berikut : ( Hargono , 2019).

Keunggulan dari Lineweaver-Burk ini yaitu menampilkan visual

dengan cepat dari invers bentuk konsentrasi substrat dan kecepatan
reaksi. Dan untuk memahami konsep penghambatan enzim. Berikut
merupakan Plot bentuk Reguler dari Lineweaver-Burk (Khyade,2019):

Gambar 1. Bentuk Reguler Lineweaver-Burk Plot (Plot dua timbal balik

).
III. ALAT DAN BAHAN
A. Alat
Berikut ini merupakan alat yang digunakan dalam
melakukan percobaan kinetika reaksi enzim.
Table 1. alat-alat yang digunakan dalam percoban kinetika reaksi
enzimatis.

Ukuran
No Nama alat Jumlah
(ml)
1 Botol timbang - 1
2 Buret 50 1
3 Corong kaca - 1
4 Erlenmeyer 50 3
5 Gelas beker 250 3
6 Gelas plastic - 4
7 Gelas ukur 25,1 1,1
8 Hot plate - 1
9 Kaca arloji - 1
10 Karet hisap - 1
11 Klem - 1
12 Labu ukur 100,25 3,1
13 Pengaduk kaca - 1
14 Pipet tetes - 1
15 Pipet ukur 10 2
16 Pipet volume 10 1
17 Statif - 1
18 Thermometer - 1
19 Waterbath - 1

B. Bahan
Berikut merupakan bahan yang digunakan dalam percobaan
kinetika reaksi enzimatis.
 7

Table 2. Bahan yang digunakan dalam percobaan kinetika reaksi

enzimatis.
Massa Volume ρ Kadar
No Nama bahan
(g) (ml) (g/mol) (%)
1 Aquades - Secukupnya - -
2 Buffer asetat - 40 - -
3 Fehling A - 75 - -
4 Fehling B - 75 - -
5 Fermipan 10 - - -
Glukosa 12 - - -
6
Indikator MB - 30 tetes - -
7
Laktosa 19 - - -
8
9 NaOH 1 - - 99

C. Gambar Alat
 Berikut ini merupakan gambar alat yang digunakan dalam percobaan
kinetika reaksi enzimatis.
1. Alat titrasi

2
3

Keterangan :

1. Buret
2. Erlenmeyer
3. Hot plate
4. Klem
5. Kran Buret
6. Statif

Gambar 2. Rangkaian alat titrasi

2. Alat penentuan kecepatan reaksi (waterbath)

 9

1
4 5 6

Keterangan :
1. Larutan
2. Pengukur suhu
3. Tempat inkubasi
4. Tombol Pengatur Suhu
5. Tombol Power
6. Waterbath

Gambar 3. Rangkaian alat penentuan kecepatan reaksi IV. Cara

Kerja
Berikut merupakan cara kerja yang dilakukan dalam percobaan
kinetika reaksi enzimatis.
1. Pembuatan Laktosa
Larutan laktosa sebanyak 19 g diambil melalui kaca arloji dan
ditimbang melalui neraca analitik. Setelah itu, dimasukkan ke gelas
beker disertai penambahan aquades secukupnya.Lalu, dimasukkan ke
labu ukur 250 mL disertai penambahan aquades hingga tanda batas
dan dikocok hingga homogen.
2. Pembuatan Yeast (Fermipan)
Yeast (Fermipan) sebanyak 10 g ditimbang melalui kaca arloji
lalu, dilarutkan ke gelas beker disertai penambahan aquades
secukupnya. Kemudian, dimasukkan ke labu ukur 100 mL disertai
penambahan aquades hingga tanda batas dan dikocok hingga
homogen.
3. Pembuatan glukosa
Sukrosa sebanyak 12 g ditimbang melalui kaca arloji, lalu
dimasukkan ke gelas beker disertai penambahan aquades
secukupnya. Kemudian, dimasukkan ke labu ukur 100 mL disertai
penambahan aquades hingga tanda batas dan dikocok hingga
homogen.
4. Pembuatan NaOH
Padatan NaOH sebanyak 1 g ditimbang melalui botol timbang
lalu dimasukkan ke gelas beker disertai penambahan aquades
secukupnya. Lalu, dimasukkan ke labu ukur 100 mL disertai
penambahan aquades hingga tanda batas dan dikocok hingga
homogen.
5. Penentuan Kecepatan Reaksi
4 gelas plastic disiapkan, kemudian masing-masing gelas diisi
dengan larutan buffer asetat 10 ml ditambahkan larutan laktosa
40,30,20,10 ml. Selanjutnya ditambahkan aquades 10,20,30,40 ml.
Kemudian dimasukkan kedalam water batch dengan suhu 38 derajat
C selama 74 menit. Pada selang waktu 10 menit dimasukkan yeast
ke dalam masing-masing gelas sebanyak 10 ml. Setelah menit
ke68,70,72,74 dimasukkan larutan NaOH sebanyak 1 ml untuk
menghentikan reaksi, lalu gelas plastik diambil dari water batch.
6. Analisis Glukosa
Setelah proses inkubasi selesai, sampel diambil sebanyak 10 ml
kemudian diencerkan dengan aquades pada labu ukur 100 ml.
Sampel yang sudah diencerkan dimasukkan ke dalam buret.
Selanjutnya Fehling A dan Fehling B diambil masing-masing 5 ml
dimasukkan ke dalam Erlenmeyer dan dipanaskan sampai mendidih.
10
Setelah mendidih ditetesi dengan 2 tetes Methyl Blue (MB),
kemudian dititrasi dalam keadaan panas sampai terjadi perubahan
warna dari biru menjadi merah bata. Kemudian catat volume dan
ulangi titrasi sebanyak 3 kali pada masing-masing sampel.
7. Peneraan Fehling
Glukosa sebanyak 12 gram dilarutkan dengan aquades ke
dalam labu ukur 100 ml. Kemudian larutan sukrosa dimasukkan ke
dalam buret. Fehlinng A dan Fehling B masing-masing 5 ml
dimasukkan ke Erlenmeyer dan dipanaskan sampai kering
mendidih. Setelah kering, ditetesi 2 tetes methyl blue dan dititrasi
dalam keadaan panas sampai terjadu perubahan warna dari biru
menjadi merah bata. Titrasi diulangi sebanyak 3 kali.
11
 12

Diagram Alir

1. Pembuatan larutan laktosa

Kaca Arloji Gelas Beker Aquades

19 gram laktosa
secukupnya

Labu ukur Aquades

250ml hingga tanda
batas

Kocok hingga

homogen
Gambar 4. Diagram alir pembuatan larutan laktosa

2. Pembuatan larutan glukosa

12 gram glukosa Kaca Arloji

Gelas Aquades
Beker
secukupnya

Aquades Labu ukur

hingga tanda batas 100ml

Kocok hingga
homogen

Gambar 5. Diagram alir pembuatan larutan glukosa.

 13

3. Pembuatan larutan yeast

Kaca Aquades
10 gram fermipan Gelas Beker
Arloji secukupnya

Labu ukur Aquades

100ml hingga tanda
batas

Kocok hingga
homogen

Gambar 6. Diagram alir pembuatan larutan yeast

4. Pembuatan larutan NaOH

Botol
Gelas Beker
Timbang

Labu ukur
Aquades
100ml
hingga tanda
batas

Aquades
1 gram NaOH secukupnya
Kocok hingga
homogen

Gambar 7. Diagram alir pembuatan larutan NaOH

 14

5. Penentuan Kecepatan Reaksi

Buffer Asetat 10ml 10ml 10ml 10ml

Laktosa 10ml 20ml 30ml 40ml
Aquades 40ml 30ml 20ml 10ml

Cup Cup Cup Cup

plastik plastik plastik plastik

Water bath 38°C

Inkubasi selama 74 menit

Ditambah 10 ml yeast
selang waktu 10 menit

Pertambahan NaOH 1ml

dimenit ke 68 dan tunggu 2
menit

Gambar 8. Diagram alir penentuan kecepatan reaksi

 15

6. Analisis Kadar Glukosa

Larutan sampel
Fehling A 5 ml + masingmasing diambil 10

Fehling B 5 ml ml

Labu ukur 100 ml

Erlenmeyer

Buret
Hot plate

Setelah mendidih + 2 tetes

indikator Methyl Blue

Titrasi

Sampai terjadi perubahan warna dari biru tua

menjadi merah bata
 16

Catat volume

Ulangi 3 Kali

Gambar 9. Diagram alir analisis kadar glukosa

7. Peneraan Fehling

Fehling A 5ml +
Larutan Glukosa
Fehling B 5 ml
 17

Labu ukur 100 ml

Erlenmeyer

Buret
Hot plate

Dipanaskan sampai mendidih

Ditambahkan 2 tetes
indikator MetilBiru (MB)

Dititrasi

Sampai terjadi perubahan warna dari biru

menjadi merah bata

Catat volumenya

Ulangi 3

Gambar 10. Diagram alir peneraan Fehling

V. HASIL DAN PEMBAHASAAN
A. Hasil Percobaan
Pada percobaan kinetika reaksi enzimatis yang telah dilakukan
diperoleh hasil data sebagai berikut. Tabel 3. Data penentuan
kecepatan rekasi.
waktu
No laktosa (ml) buffer asetat (ml) aquades (ml) inkubasi
(menit)
1 10 10 40 74
2 20 10 30 74
3 30 10 20 74
4 40 10 10 74

Tabel 4. Data analisis kadar glukosa.

volume (ml) volume rata-rata
No sampel (ml)
1 2 3
1 1 84,5 84,2 84,5 84,4
2 2 64,5 64,3 64,7 64,5
3 3 50 50,4 50,3 50,2333
4 4 21,5 21,4 21,5 21,4667

Tabel 5. Data peneraan fehling.

volume (ml) volume
No Larutan
1 2 3 ratarata (ml)
1 Fehling A 5 5 5 5
2 Fehling B 5 5 5 5
3 Glukosa 1,5 1,5 1,4 1,4667

17
B. Pembahasan
 19

Pada percobaan ini, kami menggunakan enzim invertase dari

yeastsebagai katalis. Enzim invertase merupakan enzim yang
bekerja khusus, dalam rekasi hidrolisis laktosa menjadi sukrosa dan
galaktosa.
Dalam praktikum kinetika reaksi enzimatis ini, kami melakukan
percobaan dengan 3 tahapan yaitu penentuan kecepatan reaksi,
analisis kadar sukrosa, dan peneraan fehling. Tahapan pertama,
penentuan kecepatan reaksi dilakukan dengan memberi larutan
buffer asetat, aquades dan larutan laktosa dengan variasi volume ke
dalam gelas plastic. Kemudian dimasukkan ke dalam inkubator
dengan suhu 38˚ C, hal ini karena enzim membutuhkan suhu yang
optimum untuk bekerja. Apabila menggunakan suhu yang terlalu
tinggi mengakibatkan enzim mengalami kerusakan, dan sebaliknya
jika menggunakan suhu yang terlalu rendah maka enzim alan
mengalami denaturasi. Pemberian larutan buffer asetat bertujuan
untuk menjaga pH enzim agar tetap optimum (5-9) sehingga reaksi
berjalan maksimal.
Inkubasi larutan sampel dilakukan selama 74 menit dengan
penambahan yeast selama selang 10 menit pertama. Yeast
ditambahkan karena yeast merupakan enzim invertase yang
mempercepat reaksi hidrolisis laktosa menjadi sukrosa dan
galaktosa. Reaksi yang terjadi sebagai berikut :

Enzim invertase

C12H22O11+H2O
(C6H12O6+C6H12O6…………(laktosa)) (air) 9) (Sukrosa))
(Galaktosa)

10 menit terkahir inkubasi dihentikan yang sebelumitu

ditambahkan 1 ml NaOH 1% kedalam masing-masing gelas plastik
selang waktu 2 menit, yang berfungsi sebagai inhibitor, yaitu
penghambat reaksi untuk control reaksi sel.

Selanjutnya, analisis kadarglukosa sampel yang dikeluarkan

dari inkubator diambil 10 ml dan diencerkan dalam 100 ml aquades,
kemudian dititrasi dengan fehling A dan fehling B yang sebelumnya
dididihkan terlebih dahulu serta ditambah 2 tetes methyl blue. Titrasi
dilakukan hingga larutan mencapai titik akhir titrasi yang ditandai
dengan perubahan warna dari biru menjadi merah bata dengan
endapan. Endapan merah bata timbul karena fehling bereaksi dengan
glukkosa. Fehling A merupakan larutan CuSO4 dalam air, ion Cu 2+
 20

direduksi menjadi ion Cu 2+ dalam suasana basa akan terdapat

endapan berwarna merah bata. Reaksi yang terjadi yaitu :

2Cu 2+ +2OH Cu2O+H2O…………………………(10)

Pada hasil percobaan penambahanyeast pada sampel

dalam waktu yang berbeda yaitu pada 10 menit pertama, kedua,
ketiga, dan keempat tidak mempengaruhi kecepatan reaksi. Namun
jumlah sukrosa yang ditambahkan dalam sampel masing-masing
yaitu 10,20,30,40 ml yang mempengaruhi kecepatan reaksi. Karena
semakin tinggi konsentrasi sukrosa yang ditambahkan maka akan
meningkatkan kadar substrat, dan peningkatan kadar substrat juga
akan menyebabkan kecepatan reaksi meningkat. Hal ini juga akan
menyebabkan kecepatan reaksi meningkat.

Hak ini dapat dilihat pada grafik hubungan 1/s dan 1/v sebagai
berikut:

Gambar 9. Grafik hubungan antara 1/s dengan 1/v

Grafik diatas menunjukkan hubungan antara konsentrasi

substrat 1/S dengan 1/V. pada dasar teori percobaan kinetika reaksi
enzimatis bahwa semakin tinggi konsentrsi substrat maka kecepatan
reaksinya juga semakin tinggi dengan ditandai kenaikan grafik
hubungan 1/s dan 1/v. sedangkan pada grafik yang diperoleh dari
percobaan menunjukkan sebaliknya Hal ini dikarenakan pada saat
titrasi kurang teliti terhadap perubahan warna yang terjadi. Pada
grafik diatas diperoleh persamaan y = -9,4601x + 726,11 dengan
nilai R2= 0,7245, sehingga nilai Vm =1,80261^-5 dan Km =
 21

1,247192429. Hal ini menunjukkan bahwa kecepatan reaksi yang

dikatalis enzim bergantung pada konsentrasi substrat. Nilai Vm
tergantung pada konsentrasi enzim.

Perbedaan hasil yang diperoleh dipengaruhi oleh

faktorfaktor, seperti konsentrasi substrat dan variasi waktu yang
digunakan kadang lebih atau kurang dari waktu yang ditentukan.

Tahap ketiga, peneraan fehling, larutan fehling A dan

larutan fehling B dititrasi dalam keadaaan mendidih dan diberi 2
tetes indicator methyl blue dengan larutan sukrosa standar hingga
tercapai titik akhir titrasi dengan terjadinya perubahan warna dari
biru menjadi merah bata dengan endapan. Hasil dari peneraan
fehling ini digunakan untuk menghitung konversi glukosa.
VI. KESIMPULAN
Dari percobaan kinetika reaksi enzimatis yang telah dilakukan dapat diambil
kesimpulan sebagai berikut :
1. Faktor – faktor yang mempengaruhi kerja enzim adalah :
a. Konsentrasi enzim
b. Suhu
c. Konsentrasi substrat
d. Pengaruh pH
2. Dari grafik Lineweaver-Bruk diperoleh hasil :
a. y = -9,4601x + 726,11
b. R2= 0,7245.
c. Vm = 0.00137
d. Km =-0.01296
 21
DAFTAR PUSTAKA

Ariandi. 2016. Pengenalan Enzim Amilase (Alpha-Amylase) dan Reaksi

Enzimatisnya Menghidrolisis Amilosa Pati Menjadi Glukosa. ” Jurnal
dinamika”. 7 (1) : 74 – 32.

Deman, M., J. 1997. Kimia Makanan. Bandung : ITB.

Hargono. 2019. Kinetika Hidrolisis Pati Singkong Manis (Manhot esculenta) Pada Suhu
Rendah. “Jurnal Teknik Kimia”. 4(1) : 11-15.

Joyce, L. 2013. Pedoman Pemeriksaan Laboratorium & Diagnostik Edisi 6. Jakarta

: EGC.

Khyade, V., B., Seema, K., D., dan Manali, R., S. 2019. The Indian Square for
Enzyme Kinetics Plot); It’s Inverse Form and Other Additional Form of
Plots (Equations). “International Journal of Scientific Research in
Chemistry (IJSRCH)”. 4(1) : 39 – 56.

Lehninger. 1982. Dasar-Dasar Biokimia Jilid 1. Bogor: Erlangga.

Marks, B. D; A. D. Marks dan C. M. Smith. 1996. Biokimia Kedokteran Dasar :

Sebuah Pendekatan Klinis. Alih Bahasa Brahm U. Pandit. Jakarta :
EGC Penerbit Buku Kedokteran.

Martoharsono, S. 2006. Biokimia I. Yogyakarta: UGM Press.

Murray, R., K., G., D., K., dan Rodwell, V., W. 2009. Biokimia harper (27 ed.). Jakarta:
Buku Kedokteran EGC.

Sanuhaji, A., B. 2006. Intoleransi Laktosa. ”Majalah Kedokteran Nusantara” . 39

(4) : 9 – 424.

Sumarjo, D. 2009. Pengantar Kimia Buku Panduan Kuliah Mahasiswa Kedokteran.

Jakarta : Buku Kedokteran EGC.
Wirahadikusumah, Muhamad. 1977. Biokimia Protein, enzim & asam nukleat.
Bandung : Penerbit ITB.
 22

Surakarta,12 Mei 2020

Asisten Pembibing Praktikan,

1. Haqiqi Hadziq Fikri (D500180060)

2. Dewi Kristiana (D500180062)

3. Nanda Mila Afida (D500180065)
(Arofa Fauziyatad Daroeni)

Mengetahui,

Dosen Pembimbing

(Dr. Akida Mulyaningtyas, S.T., M.Sc.)

VII. LAMPIRAN
A. Data Hasil Percobaan
Berikut merupakan data hasil percobaan kinetika reaksi enzimatis.

Tabel 3. Data penentuan kecepatan rekasi

No laktosa Buffer Aquades (ml) Waktu inkubasi
(ml) asetat (ml) (menit)
1. 10 10 40 74
2. 20 10 30 74
3. 30 10 20 74
4. 40 10 10 74

Tabel 4. Data analisis kadar glukosa

No Sampel Volume(ml) Volume ratarata
I II III (ml)
1. I 84,5 84,2 84,5 84,4
2. II 64,5 64,3 64,7 64,5
3. III 50 50,4 50,3 50,2333
4. IV 21,5 21,4 21,5 21,4667

Tabel 5. Data peneraan fehling.

No Larutan Volume (ml) Volume rata-rata
I II III (ml)
1. Fehling A 5 5 5 5
2. Fehling B 5 5 5 5
3. Glukosa 1,5 1,5 1,4 1,4667

23
B. Perhitungan
 24

1. Menentukan kadar substrat

Diketahui : Massa laktosa = 19 gram
Volume pengencer = 250 ml
Ditanya : Kadar substrat …….? Dijawab
:
𝑀𝑎𝑠𝑠𝑎 𝑙𝑎𝑘𝑡𝑜𝑠𝑎
Konsentrasi laktosa =
𝑣𝑜𝑙𝑢𝑚𝑒 𝑝𝑒𝑛𝑔𝑒𝑛𝑐𝑒𝑟

=19 𝑔𝑟𝑎𝑚
250 𝑚𝑙

= 0.076 gr/ml
Volume larutan = V laktosa + V buffer asetat + V aquades
= 10 +10 +40
= 60 ml
𝑘𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑠𝑖 𝑙𝑎𝑘𝑡𝑜𝑠𝑎 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑎𝑞𝑢𝑎𝑑𝑒𝑠
Kadar substrat =
𝑣𝑜𝑙𝑢𝑚𝑒 𝑙𝑎𝑟𝑢𝑡𝑎𝑛
𝑔𝑟
0.88 𝑚𝑙 𝑥 40 𝑚𝑙
=
60 𝑚𝑙

= 0.05066
Dengan rumus yang sama maka didapatkan nilai kadar substrat pada
setiap sampel yaitu,
Table 6. Kadar substrat pada setiap sampel
Volume Volume
Aquades Larutan Konsentrasi Kadar
No. Sampel
(ml) (ml) substrat substrat

1 I 40 60 0.088 0.01267
2 II 30 60 0.088 0.02533
3 III 20 60 0.088 0.038
4 IV 10 60 0.088 0.05067

2. Menentukan kecepatan reaksi

Diketahui : Massa glukosa : 12 gr
Volume pengenceran : 100 ml

𝑚𝑎𝑠𝑠𝑎 𝑠𝑢𝑘𝑟𝑜𝑠𝑎
• kadar sukrosa standar =
𝑣𝑜𝑙𝑢𝑚𝑒 𝑝𝑒𝑛𝑔𝑒𝑛𝑐𝑒𝑟
12 𝑔𝑟𝑎𝑚
= = 0.12 gram/mol
100 𝑚𝑙
 • konversi sukrosa
(x) diketahui : V1 (volume pengenceran)
 26

V2 (volume titrasi)
C (kadar glukosa)
Vo ( volume peneraan fehling)
V3 (volume substrat)
S (kadar substrat)

Ditanya : konversi glukosa (x)?

Jawab :
𝑉1 (
).
𝑐.𝑉𝑜
Konversi glukosa (x) = 𝑉2
𝑉3.𝑆

=
(10).(0.012666)

=1,64629

• kecepatan reaksi (V)

𝐾𝑜𝑛𝑣𝑒𝑟𝑠𝑖 𝑔𝑙𝑢𝑘𝑜𝑠𝑎 (𝑥)
V =
𝑤𝑎𝑘𝑡𝑢 𝑖𝑛𝑘𝑢𝑏𝑎𝑠𝑖

=
=0.022247

Dengan menggunakan rumus yang sama maka didapatkan nilai dari sampe yang
berbeda yaitu,

Tabel 7. Data hasil percobaan konversi glukosa (x) untuk setiap sampel
C
V1 V2 V3
No (g/ml S (g/ml) Vo (ml) X
(ml) (ml) (ml)
)
0.02
1 100 2 0.12 60 0.01267 0.022247 1.64629
0.00
2 100 5 0.12 60 0.02533 0.022247 0.41157
0.00
3 100 2 0.12 60 0.038 0.022247 0.18292
0.00
4 100 1 0.12 60 0.005067 0.022247 0.10289
 27

Table 8. data hasil percobaan kecepatan reaksi (V) pada setiap sampel
No Sampel S (g/ml) X V V/S 1/S 1/V
1 I 0.0126 1.6462 0.0222 0.0794 78.9473 44.9493
2 II 0.0253 0.4115 0.0055 0.1184 39.4736 179.7975
3 III 0.038 0.1829 0.0024 0.4633 26.3157 404.5445
4 IV 0.0506 0.1028 0.0013 0.7539 19.7368 719.1903

3. Menentukan Km dan Vm Diketahui : V = 𝑉𝑚 (𝑠) → 1 = 𝐾𝑚 .

1
+ 1

𝐾𝑚 +(𝑠) 𝑉 𝑉 𝑆 𝑉𝑚

y = ax + b Ditanya
:Vm dan Km?
Dijawab :

Dari grafik diperoleh y = -9,4601x + 726.11

𝐾𝑚 1 1
= -9,4601 Vm→ = = 0.00137
𝑉𝑚 𝑉𝑚 726,11

Km=-9,4601 x Vm

=-9,4601 x 0.00137

=-0.01296
 28

C. Galat
a. Penetuan kadar glukosa
1. Sampel I
• Rata-rata = = 84.4

• Galat acak

=0.00166

• Galat sistematis
ΣS= ± 𝑥 1
= 0.5

• Galat campuran
Σ = √((Σ𝑅)2 + (Σ𝑆)2

= 0.12500

2. Sampel II

• Rata-rata =
=64.5

• Galat acak

ΣR =
= 197.2266

• Galat sistematis
ΣS = ± 𝑥 1
= 0.5

• Galat campuran
Σ =√(Σ𝑅)2 + (Σ𝑆)2

= 019449.30
 29

3. Sampel III

• Rata-rata =
= 50.23

• Galat acak
ΣR =

= 659.4016

• Galat sistematis
ΣS = ± 𝑥 1
= 0.5

• Galat campuran

Σ = √(Σ𝑅)2 + (Σ𝑆)2

= 217405. 40

4. Sampel IV

• Rata-rata =
= 21.46

• Galat acak
ΣR =

= 0

• Galat sistematis
ΣS = ± 𝑥1
= 0.5

• Galat campuran
Σ = √(Σ𝑅)2 + (Σ𝑆)2
= √(02 + (0.5)2 = 0.125
30
 LAPORAN SEMENTARA

KINETIKA REAKSI ENZIMATIS

I. Judul : Kinetika Reaksi Enzimatis hari, tanggal

: Sabtu, 9 Mei 2020 kelompok/shift : 8B/Sabtu Pagi
Anggota : 1. Haqiqi Hadziq Fikri (D500180060)
2. Dewi Kristiana (D500180062)
3. Nanda Mila Afida (D500180065)
Asisten : Arofa Fauziyatad Daroeni

II. Data Percobaan

1. Penetapan Kecepatan Reaksi
Tabel 1. Data Penetapan Kecepatan Reaksi
No laktosa (ml) Buffer asetat (ml) Aquades (ml) Waktu inkubasi
(menit)
1. 10 10 40 74
2. 20 10 30 74
3. 30 10 20 74
4. 40 10 10 74

2. Penentuan Kadar Glukosa

Tabel 2. Data Penentuan Kadar Glukosa
No Sampel Volume(ml) Volume rata-rata (ml)
I II III
1. I 84,5 84,2 84,5 84,4
2. II 64,5 64,3 64,7 64,5
3. III 50 50,4 50,3 50,2333
4. IV 21,5 21,4 21,5 21,4666

3. Percobaan Peneraan Fehling

Tabel 3. Data Percobaan Peneraan Fehling
No Larutan Volume (ml) Volume rata-rata
I II III (ml)
1. Fehling A 5 5 5 5
2. Fehling B 5 5 5 5
3. Glukosa 1,5 1,5 1,4 1,4666

Surakarta,12Mei 2020

Asisten Pembimbing Praktikan,

1. Haqiqi Hadziq Fikri (D500180060)
 2. Dewi Kristiana (D500180062)
3. Nanda Mila Afida (D500180065)
(Arofa Fauziyatad Daroeni)

Mengetahui,

Laboran

(Hartini, S.T)
 Laboratorium Teknik Kimia
Program Studi Teknik Kimia Fakultas Teknik
Pengisian BKB3 merupakan syarat yang harus dipenuhi sebelum bekerja di
Laboratorium sebagai asesmen terhadap berbagai resiko pekerjaan yang melibatkan
Universitas Muhammadiyah Surakarta
bahan yang berbahaya dan beracun (B3). B3 dapat berupa bahan utama, produk, dan
produk antara maupun produk samping dari proses. Borang ini harus diisi secara
lengkap, disetujui oleh pembimbing atau orang yang bertanggung jawab terhadap
pekerjaan yangdilakukan.
2.1: Bahan berbahaya yang digunakan ataudihasilkan
Bahan yang berbahaya Frase Resiko (Frase (Ambang Batas
(Hazardous Materials) (Risk Phrases) keselamatan) keselamatan)
Safety Phrases Workplace
exposure limit

Judul Proyek/Penelitian/ kegiatan Kinetika reaksi enzimatis (WEL)

(Title of activity)
1.1 deskripsi singkat tentang proyek/penelitian/kegiatan
Pembimbing/peneliti
Mahasiswa dapat menjelaskanutama/ Akidareaksi
kondisi oprasi pada Mulyaningtyas, S.T.,formaldehid,
sinsesa urea M.sc dapat menganalisis kad
penanggung jawab (Suvisor/principal
formaldehid dan kadar resin dalam percobaan sintesa urea formaldehid. Dan dapat menentukan pH pada tah
investigator)
reaksi dan hasil percobaan sintesa urea formaldehid.

Program studi/Study program Teknik kimia

Tanggal pengisian/Date of 12 Mei 2020

assessment

Lokasipenelitian/eksperimen
(Nama gedung danruang)/location of
work

Bagian 1 Proyek/penelitian/kegiatan

Bagian 2 Potensi bahaya

 Bahan kimia NaoH, yeast ,roti, sukrosa H314,H402 P260,P264,P273,P2
80
Carcinogens, H314
mutagens or
reproductive toxins
(penyebab kanker,
mutasi gen, dan
beracun terhadap
sistem reproduksi)

Dusts or fumes P280,P273

(Debu kimia atau
uap/asap)

Asphyxiants/gang guan P304,P340

pernafasan

Bahan lain yang NaoH H402 P305,P260,P264

berbahaya bagi
kesehatan

Bagian 3 Resiko terhadap kesehatan

3.1: Penyakit atau kondisi yang disebabkan oleh bahan yang berbahayatersebut
Menyebabkan luka bakar kulit parah dan kerusakan mata

3.2: Kemungkinan Rute masuk ke tubuh manusiamelalui

Pernafasan Mulut/makan suntikan absorpsi/penyerapan lainnya Pilih semua yang

sesuai

3.3: Skala penggunaan bahan berbahaya tersebut

skala kecil skala sedang skalabesar prakteklapangan hewan Pilih semua yang
sesuai
Tanaman
Maintenance Cleaning Other
The substance will only be used in the laboratory during experiments.

3.4: Frekuensi penggunaan bahan berbahayatersebut

Harian Mingguan Bulanan lainnya Pilih salah satu

On the average daily for a span of one month.

3.5: Jumlah maksimum atau konsentrasi yangdipakai

Bisadiabaikan rendah sedang tinggi Pilih salah satu

3.6: Dampak dari bahanberbahaya

Bagian 4 Alat kendali untuk mengurangi resiko
Bisadiabaikan rendah sedang tinggi Pilih salah satu

4.7: Manajemen Limbah danpembuangan

3.7: Siapa saja yang terdampak oleh
cairan bahaya danresiko
padatan Gas Cair campuran
anorganik Organik
Staf Mahasiswa pengunjung masyarakatumum anak muda (<18th) *Ibubaru lainnya
lainnya

3.8: Penilaian resiko terhadap kesehatan (sebelum penggunaan mediakendali)

4.8: Memonitorpenyebaran
Tingkat resiko Bisadiabaikan Rendah sedang/rendah sedang Pilih salah satu

tinggi
4.9: Pengawasan terhadapkesehatan
3.9: Penilaian resiko terhadap lingkungan (sebelum penggunaan mediakendali)
Level of risk Bisa diabaikan rendah Sedang/rendah Sedang Pilih salah satu
4.10: Instruksi, Training, dan Supervisi
tinggi
Instruksi khusus diperlukan untuk melakukan pekerjaan dengan selamat (Jika ya, isi detilnya di ya
bawah)

4.1: TempatPenyimpanan
Training khusus diperlukan untuk melakukan pekerjaan dengan selamat (Jika ya, isi detilnya di ya
Laboratorium
bawah) Ruangan areaterkendali penyimpanan Pilih semua yang
sesuai
box Lemariasam ruangan yangberventilasi Akses yangterkontrol
lainnya
A: Pekerjaan tidak dapat/tidak boleh dilakukan tanpa pengawasan langsung dari ya
pembimbing/supervisor (Jika ya, isi detilnya di bawah)
4.2: Pengendalianlainnya
B: Pekerjaan tidak dapat/tidak boleh dilakukan tanpa persejuan/izin dari ya
Cuci kulit yang (Jika
pembimbing/supervisor terpapar
ya, secara menyeluruh
isi detilnya di bawah)

4.3: Penyimpanan bahan yang berbahayatersebut

C: Pekerjaan dapat/ boleh dilakukan tanpa pengawasan langsung dari pembimbing/supervisor ya
(Jika ya,Dilaboratorium dalam ruangan
isi detilnya di bawah)
4.4: Transportasi of bahan yang berbahayatersebut
Pembimbing
Menggunakan alat

4.5: Peralatan Pelindung Diri(PPD)

JasLab Keseluruhan Chemicalsuit baju sekalipakai Apron Pilih semua yang

sesuai
Kaus tangan kacamata kacamata/Goggles pelindung muka Kaus tangan
Alatpelindungkepala sepatukeselamatan lainnya

4.6: Peralatan pelindung pernafasan(PPP)

Topeng sekalipakai topeng penyaring(filter) Pelindung setengah muka Pilih semua yang
pelindung seluruh muka Alat bantu pernafasan alat pernafasan sesuai

Other
 5.1: Prosedur dalam keadaan GawatDarurat

5.2: Tumpahan sedikit

Prosedur yang Dibersihkan dengan tissue

harus dilakukan

Tindakan lainnya Lakukan evakuasi dan amankan area yang berbahaya ya

Melaporkan ke penanggung jawab (eg principal investigator / school safety ya

officer etc)

5.3: Tumpahan yang banyak

Prosedur yang Dibersihkan dengan tissue
harus dilakukan

Tindakan lainnya Lakukan evakuasi terhadap lokasi ya

Telepon petugas sekuriti dan pemadam kebakaran ya

Melaporkan ke penanggung jawab (eg principal investigator / school safety ya

officer etc)

5.4: Pemadamkebakaran
Carbon dioksida Air Powder Foam Blanket Automatic fire suppression
lainnya

5.5: Pertolongan pertama

Dibersihkan dengan air yang terkena, jangan memberi apapun kemulut

5.6: Daftar kontak pada kondisidarurat

Nama Kedudukan/posisi dalm proyek Telephone

Bagian 5 Prosedur dalam keadaan Gawat Darurat

Bagian 6Persetujuan
6.1: Pengisiborang
Nama Tanda tangan Tanggal
Trisnawati 5 april 2020
6.2: Penanggung jawab/penelitiutama
Nama Tanda tangan Tanggal

Matriks EstimasiResiko
Tingkat keparahan Kemungkinan keterjadian
Tinggi Sedang Rendah terabaikan

Parah Tinggi Tinggi Sedang Effectively zero

Sedang Tinggi Sedang Sedang /Rendah Effectively zero
Rendah Sedang / Rendah Rendah Rendah Effectively zero
Terabaikan Effectively zero Effectively zero Effectively zero Effectively zero
 International Journal of Scientific Research in Chemistry (IJSRCH)
© 2019 IJSRCH | Volume 4 | Issue 1 | ISSN : 2456-8457

The Indian Square for Enzyme Kinetics Through the Regular

Lineweaver-Burk Plot (Double Reciprocal Plot); It’s Inverse Form and
Other Additional Form of Plots (Equations)
Vitthalrao Bhimasha Khyade, Seema Karna Dongare, Manali Rameshrao Shinde
Science Association, Shardabai Pawar Mahila Mahavidyalaya, Shardanagar Tal. Baramati Dist. Pune,
Maharashtra, India
ABSTRACT

Each enzyme deserves a specific Michaelis-Menten constant (Km), which is determined through the double
reciprocal plot, also recognized as Lineweaver-Burk plot. This constant of Michaelis-Menten (Km) is
concentration of substrate [S] and it avails the velocity (v) of reaction to proceed up to half of it’s maximal or
Vmax. The regular Lineweaver-Burk plot and it’s inverse form are designated as y 1 and y2 lines respectively.
Present attempt is considering additional plots or the lines keeping the concept in regular Lineweaver-Burk
plot constant. These plots include: y 3 and y4. In slope and intercept form the lines y 3 and y4 expressed as: y3=
[(Km÷Vmax)(X)] + [(km+1)÷(Vmax)] and y4= -[(Vmax÷Km)(X)] + [(Vmax -1)÷(Km)]. In addition; y 5 = X + 0 and
y6 = - X + 1 are the two reference lines are also considered in this attempt. The line y 3 intersect the reference
line y5 at the point “A”, the x- co-ordinate and y- co-ordinate of which are equal to each other and correspond
to: [(Km+1)÷(Vmax+Km)]. The line y1 intersect the reference line y6 at the point “B”, the x- co-ordinate and y-
co-ordinate of which respectively correspond to: [(Vmax – 1) ÷ (Vmax + Km)] and [(Km + 1) ÷ (Vmax + Km)].
The point at which the reference line y 5 attains [(Vmax – 1) ÷ (Vmax + Km)] is labeled as the point “C”. The X –
co-ordinate and Y – co-ordinate of the point “C” corresponds to: [(Vmax – 1) ÷ (Vmax + Km)] [(Km + 1) ÷
(Vmax + Km)] respectively. The point of intersection of the line y 2 and the reference line y6 is labeled as the
point “D”. The X – co-ordinate and Y – co-ordinate of the point “D” corresponds to: [(Km + 1) ÷ (Vmax + Km)]
and [(Vmax – 1) ÷ (Vmax + Km)] respectively. The length of segment AB=BC=CD=DA and it correspond to:
[(Vmax - Km - 2) ÷ (Vmax + Km)]. The point “A”; “B”; “C” and “D” constitute the vertices of square ABCD. The
resulting mathematical square, herewith labeled as: “Indian Square For Enzyme Kinetics”. Each of the four
vertices (corners) have known coordinates in terms of Vmax and Km, the key indices in enzyme kinetics.
Keywords : Indian Square, Enzyme Kinetics, Mathematical Approach

I. INTRODUCTION The diagonals of a square bisect each other and meet

In geometry, a square is a regular quadrilateral, which
means that it has four equal sides and four equal
angles (90-degree angles, or (100-gradian angles or
right angles) ( Weisstein, Eric). It can also be defined
as a rectangle in which two adjacent sides have equal
length. A square with vertices ABCD would be
at 90°. The diagonals of a square bisect its angles.
Opposite sides of a square are both parallel and equal
in length. All four angles of a square are equal. Each is
360°/4 = 90°, so every angle of a square is a right angle.
All four sides of a square are equal. The diagonals of a
square are equal (Zalman Usiskin and Jennifer Griffin,
2008).

denoted ◻ {\displaystyle \square } as “Square: ABCD”.

IJSRCH19412 | Received : 10 Jan 2019 | Accepted : 25 Jan 2019 | January-February-2019 [ 4 (1) : 39-56]
39
 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56
The velocity (v) of biochemical reaction catalyzed by
the enzyme vary according to the status of factors
like: concentration of the substrate [S]; hydrogen ion
concentration; temperature; concentration of the
respective enzyme; activators and inhibitors. There is
no linear response of velocity (v) of biocatalyzed
reaction to the concentration of the substrate [s]. This
may be due to saturable nature of enzyme catalyzed
biochemical reactions. If the initial velocity (v) or rate
of the enzyme catalyzed biochemical reaction is
expressed in terms of substrate-concentration of [S], it
appears to increase. That is to say, initial velocity (v)
of the enzyme catalyzed biochemical reaction get
increase according to the increase in the
concentration of substrate [S]. This tendency of
increase in initial velocity (v) of the enzyme catalyzed
biochemical reaction according to the increase in the
concentration of substrate [S] is observed up to certain
level of the concentration of substrate [S]. At this
substrate concentration [S], the enzyme exhibit
saturation and exert the initial velocity (v) of the
biocatalyzed reaction to achieve maximum velocity
(Vmax). Hans Lineweaver and Dean Burk (1934)
suggested the double reciprocal plot for presenting the
information in the form of readings or the data on the
concentration of substrate [S] and rate or velocity (v)
of the biocatalyzed reaction. In enzyme kinetics,
double reciprocal plot suggested by Hans Lineweaver
and Dean Burk is well esteemed graphical
presentation of the data on concentration of the
substrate [S] and velocity (v) of the biocatalyzed
reaction recognized as, the “Lineweaver–Burk plot”.
This plot deserve wide applicability. The most
significant application of Lineweaver–Burk plot lies in
the determination of concentration of substrate [S]
which is responsible for achievement of the half the
maximum rate or the velocity (Vmax ÷ 2) of the
biochemical reaction catalyzed by the enzymes. The
“Km” or Michaelis constant is the concentration of
substrate [S] responsible for yield of the reaction rate,
which is corresponding to exactly half the rate or
velocity of maximal (Vmax ÷ 2) for enzyme involved
biochemical reaction. For practical purposes, this
International Journal of Scientific Research in Chemistry (www.ijsrch.com) | Volume 4 | Issue 1
“Km” or Constant of Michaelis is the reading
pertaining [S] that allows velocity to achieve half with
reference to maximum rate or velocity (Vmax). The
affinity of enzymes for their substrate vary. Generally,
the enzyme with a higher Km value has little bit
lower affinity for its substrate. According to Keith J.
Laidler (1997), enzymes with lower affinity for their
substrate, requires a greater volume of substrate or
substrate concentration for the purpose to achieve
maximum rate or velocity of enzyme involved
biochemical reactions.
The wide range of applicability is the distinguishing
feature of Lineweaver–Burk plot. In the past, there
was no computer facilities as today. In such a critical
situation, the parameters of enzyme kinetics, Km and
Vmax served a lot through this Lineweaver-Burk plot
for fortified concept of enzyme kinetics. In this
Lineweaver-Burk plot, reading the inverse of
maximum velocity of biocatalyzed reaction (1÷
Vmax) take the position of y-intercept (Fig.1). The
negative value of inverse of Km (1÷Km) take the
position of xintercept. The quick visual impression of
the inverse form of substrate concentration and rate
or velocity of reaction is one more advantage of
Lineweaver-Burk plot. And this feature help for
understanding the concept of enzyme inhibition.
Accordingly, mathematical equation suggested by
Lineweaver and
𝟏 𝐊𝐦 𝟏
Dean Burk (1934) can be written as: 𝐕 =𝐕𝐦𝐚𝐱 𝐗 𝐒 +
𝟏
𝐕𝐦𝐚𝐱
Fig. 1: Regular form of Linrweaver-Burk Plot (Double
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 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56
Km 1 1
Reciprocal Plot) y1= = Vmax X S + Vmax
This Lineweaver–Burk plot deserve wide applicability.
It is useful for the determination of K m , the most
significant factor in enzyme kinetics. The intercept on
y – axis of “Lineweaver–Burk-Plot” is the reciprocal of
Vmax or (1/ Vmax ). And intercept on X – axis of
“Lineweaver–Burk-Plot” is the reciprocal of - K m or
(−1/Km ). Reciprocals of both, [S] and (v) are utilized
in the Lineweaver-Burk plot. That is to say,
1 1 this plot is
pertaining V and S . Therefore,
“Lineweaver–Burk-Plot” is also termed as a double
reciprocal graph. This attempt through the
“Lineweaver-Burk-Plot”, is giving quick, concept or
idea of the biochemical reaction. It also allow to
understand the mechanism of activation of enzyme
and inhibition of enzyme. Researchers including
authors of present attempt designating the double
reciprocal plot as a Nobel Plot. Most of researchers
entertaining the enzyme kinetics through this double
reciprocal plot are non-mathematical academicians.
Present attempt is trying it’s best to minimize the
errors in understanding the concepts in enzyme
kinetics through modification in the
“LineweaverBurk-Plot”.
Each and every method is with positive and negative
points of advantages. According to Hayakawa, et al
(2006), there is distortion of error structure through
this double reciprocal plot of “Lineweaver-BurkPlot”.
It is therefore, method of graphical presentation of
“Lineweaver–Burk-Plot” (double-reciprocal-plot)
appears to attempt to minimize errors. This may yield
easier method of calculation of constants or
parameters of enzyme kinetics. On this line of
improvement of method of calculation of constants or
parameters of enzyme kinetics, much more work is
already exist. According to Hayakawa, et al (2006),
methods of improvement in the calculation of
constants or parameters of enzyme kinetics are under
the title, “non-linear regression or alternative linear
International Journal of Scientific Research in Chemistry (www.ijsrch.com) | Volume 4 | Issue 1
forms of equations”. And they include: the plot of
“Hans-Woolf”; the plot of “Eadie-Hofste”; such and
the others (Greco and Hakala, 1979).
Dick (2011) explained type of inhibition of enzyme
activity or stoping the working of enzymes. Of course,
this discussion is based exclusively on “Lineweaver–
Burk-Plot” (reciprocals ob both the axes) is able to
group or classify the inhibitors of actions of enzymes.
Accordingly, the inhibitors of enzyme can basically be
grouped into the types like: The “Competitive
Inhibitors”; “Non-competitive inhibitors” and
“uncompetitive inhibitors”. The inhibitors of enzyme
of “Competitive” class deserve one and the same point
of intersection on the Y-axis. It clearly means,
inhibitors of enzyme of “Competitive” class are not
affecting on maximal rate or velocity of reaction
(competitive inhibitors provide protection the
maximum velocity Vmax. They keep this maximum
velocity Vmax non-affected). But, slopes of equations
are not same. Slopes are different slopes. The
inhibitors of enzyme of “Non-competitive” class
deserve one and the same point of intersection on the
X-axis. It clearly means, inhibitors of enzyme of
“Non-competitive” class are not affecting on the Km,
the [S] for half the maximal rate or velocity of
reaction (Km is remains unaffected by noncompetitive
inhibitors. The inverse of Km doesn't change). The
non-competitive inhibition produces plots with the
same x-intercept (−1/Km ) as uninhibited enzyme (Km
is unaffected) but different slopes and y-intercepts.
Uncompetitive inhibition causes different intercepts
on both the y- and x-axes (Berg, et al, 2002). John E.
Dowd and Douglas Briggs (1965) reviewed the
literature on “Estimates of Michaelis – Menten kinetic
constants through the use of different linear
transformation” and listed some problems with
Lineweaver–Burk plot (double reciprocal plot).
Accordingly, Lineweaver–Burk plot (double
reciprocal graph) is appearing in most of the new as
well older books of biochemistry. It seems in having
prone to error. There may be mistake in
understanding the expected for researchers. The
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readings of inverse of “v” are on Y – axis. The readings Fig. 2: Regular form of Linrweaver-Burk Plot (Double
of inverse of “[S]” are on X – axis. The lower values of Reciprocal Plot) along with it’s Inverse form y2
both the readings ( inverse of “v” and inverse of “[S]” ) Vmax 1 1

are occupying higher (signifiacant) position in graph. = Km X v − Km

And… and… higher values of both the readings

( inverse of “v” and inverse of “[S]” ) are occupying (C). If the 1÷S and 1÷v for given enzyme catalyzed
lower (non-significant) position in graph. Both the biochemical reaction are inverses of each other, then
conditions may be interpreted wrongly. the domain of 1÷S is equal to the range of 1÷v and the
range of 1÷v is equal to the domain of 1÷S.
MATHEMATICAL PROPERTIES OF EQUATION
FOR INVERSE FORM OF ENZYME KINETICS: (D).The 1÷S and 1÷v for given enzyme catalyzed
biochemical reaction deserve symmetry, then there is
(A). The mathematical equation for regular a similar type symmetry in between the real form of
Lineweaver Burk plot is explaining binary relation equation and it’s inverse form of equation.
1
between the inverse of substrate concentration and S
(E).The co-ordinates of the point of intersection of
1
1 Km 1 1 1
and V of enzyme involved processes. It’s inverse form
both the equations V = Vmax X S + Vmax and S
of equation is making this mathematical association of Vmax 1 1 1
= Xv Km
1 1 − correspond to: ( ,
1
S and V more fortified. It means, characters of Km Vmax−Km

inverse form of mathematical equation for kinetics of

Vmax−Km ).
enzyme matches to the characters of converse
associations.
(F). The 1÷S and 1÷v for given enzyme catalyzed
biochemical reaction may deserve “one-to-one”.
Even if a function 1÷S is not one-to-one relation
with 1÷v ; it is attainable for the purpose of
definition of inverse form of equation through
limiting the areas or the domain.

v (G). The inverse form of equation in enzyme

(B). Theoretically, mathematical equation for regular
Lineweaver Burk plot for a given enzyme is with
unique inverse function. That is to say, for each
concentration of substrate, there is a unique value for
velocity of enzyme catalyzed biochemical reaction.
kinetics is a function that reverses real form of
mathematical equation for regular Lineweaver
Burk plot. If the equation for (1÷v), applied to
input (1÷ S), yields a consequence or result of
(1÷v). After appertaining its inverse equation (1÷S) to
(1÷v) gives the outcome 1÷S, and contrariwise.
1 1
(H). If ( S , v ) is a point on the graph of the original
1
equation of Lineweaver–Burk plot, then the point ( S

This proceed ahead since the inverse form of equation ,

1
must be the converse association.
) must be a point on the graph of the inverse
function of enzyme kinetics. The Lineweaver–Burk

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plot and it’s inverse function are mirror images of = 𝐊𝐦 𝐗 𝐯 − 𝐊𝐦 ) are the mathematical inverse functions
each other with respect to the line y=x. for each other (Seema K. Dongare, et al, 2018). Let us
APPLICATIONS OF MATHEMATICAL INVERSE
have a attempt to intersect the line y1 and y2 for
FUNCTION (EQUATION) FOR ENZYME KINETICS:
obtaining the x- and y- co-ordinates for the point of
Applications related to enzyme catalyzed processes intersection.
deserve ubiquitous nature. This ubiquitous nature is
both in natural alive condition and in laboratory y1 = y 2
experimental conditions. Detailed study of kinetics of 𝐊𝐦 𝟏 𝐕𝐦𝐚𝐱 𝟏
enzyme catalyzed reactions remains controversial. Step – 1: 𝐕𝐦𝐚𝐱 𝐗 + 𝐕𝐦𝐚𝐱 = 𝐊𝐦 𝐗 − 𝐊𝐦
Michaelis–Menten equation expect reaching a
𝟏 𝟏 𝐕𝐦𝐚𝐱 𝐊𝐦
nonchanging position of response for further side
limit of experimentation. At this condition of Step – 2: 𝐕𝐦𝐚𝐱 + 𝐊𝐦 = [ 𝐊𝐦 − 𝐕𝐦𝐚𝐱 ] X
experimentation, enzyme concentration far beyond 𝐕𝐦𝐚𝐱 + 𝐊𝐦 (𝐕𝐦𝐚𝐱−𝐊𝐦)(𝐕𝐦𝐚𝐱+𝐊𝐦)
molar concentration of sites that liable for [
Step – 3: 𝐕𝐦𝐚𝐱.𝐊𝐦 = 𝐊𝐦.𝐕𝐦𝐚𝐱
accessibility. It needs large amount of substrate.
]X
Substantial study at laboratory level is going to prove
(𝐕𝐦𝐚𝐱 + 𝐊𝐦) 𝐊𝐦 𝐕𝐦𝐚𝐱
the concept in expectation of Michaelis–Menten
equation. This situation may be the limiting factor Step – 4: 𝐕𝐦𝐚𝐱.𝐊𝐦 (𝐕𝐦𝐚𝐱−𝐊𝐦)(𝐕𝐦𝐚𝐱+𝐊𝐦) =X
applicability of the concept in expectation of
𝟏
Michaelis–Menten equation. In such situation, it
Step – 5: (𝐕𝐦𝐚𝐱−𝐊𝐦) =X
become exclusively impossible for practical
accessibility of the concept in expectation of
Michaelis–Menten equation. 𝟏
The regular Michaelis–Menten equation (𝐕
At the point of non-accessibility of the concept in 𝐊𝐦 𝟏 𝟏
expectation of Michaelis–Menten equation, “inverse = 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 ) explains the influence of
function or equation for enzyme kinetics” deserve concentration of substrate [S] on velocity of enzyme
applicability. Here it is essential to mention that, catalyzed biochemical reaction. It’s inverse function
“inverse function or equation for enzyme kinetics” is may explain the influence of velocity of enzyme
giving contrivance for analysis of kinetics that are catalyzed biochemical reaction (v) on concentration
involving enzymes. It may establish contrivance for of substrate [S]. That is to say, inverse function of the
bridging the concept of kinetics of enzyme related 𝟏
regular Michaelis–Menten equation and ( 𝐒
reactions reaching the steady or “Non-changing
𝐕𝐦𝐚𝐱 𝟏 𝟏
Position” It reveals compactness of attack of enzyme
= 𝐊𝐦 𝐗 𝐯 − 𝐊𝐦 ) is going to explain the role of product
with it’s active site corresponding to the site of
of enzyme catalyzed biochemical reaction in
substrate. More over, the “inverse function or
controlling the rate of reaction. The regular
equation for enzyme kinetics” explain “Species 𝟏 𝐊𝐦 𝟏 𝟏
Specific Nature of Enzymes”. Michaelis–Menten equation ( 𝐕 = 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 ) is
demonstrating the Km (Michaelis constant), the
Intersection of the line y1 and line y2 (Fig. 2):
substrate concentration [S] at which the velocity (v)
𝟏 𝐊𝐦 𝟏 𝟏 𝟏 of the enzyme catalyzed biochemical reaction attain
The lines y1 ( 𝐕 = 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 ) and y2 ( 𝐒 half of it’s maximum (Vmax ÷ 2). And … and … the
𝐕𝐦𝐚𝐱 𝟏 𝟏

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inverse function is demonstrating the [1÷ (Vmax. –
Km)], (Baramati Constant), point on both, the regular
𝟏 𝐊𝐦
Michaelis–Menten equation ( 𝐕 = 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 )
𝟏 𝐊𝐦
it’s inverse function ( 𝐕 = 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 ). At this point
[1÷ (Vmax. – Km)], (Baramati Constant), both the
equations are equal with each other. This point [1÷
(Vmax. – Km)], (Baramati Constant) is going to serve
the saturation of enzyme molecules and the substrate
molecules in enzyme catalyzed biochemical reaction.
Through the reverse the mathematical steps and to get
inverse of substrate concentration (1÷S) back from
some output value, say inverse of respective velocity
(1÷v), it is necessary to carry out the steps exactly in
opposite sequence. It means that, it is prime need to
subtract the inverse of maximum velocity (1÷Vmax)
from inverse of respective velocity
(1÷v) and then multiply the result by
) and it’s inverse function ( = 𝐗 + ).
𝐕𝐦𝐚𝐱 𝐕 𝐕𝐦𝐚𝐱 𝐒 𝐕𝐦𝐚𝐱
𝟏 𝟏
At this point [1÷ (Vmax. – Km)], (Baramati
Constant), both the equations are equal with each
𝟏 𝟏 and
other. This point [1÷ (Vmax. – Km)], (Baramati
Constant) is going to serve the saturation of enzyme
molecules and the substrate molecules in enzyme
catalyzed biochemical reaction. The attempt on the
inverse function for enzyme kinetics of present
attempt may open a new chapter to classify the
enzymes on the basis of mathematical approach
(Seema K. Dongare, et al, 2018).
ONE MORE ATTEMPT ON THE ESTABLISHMENT
OF THE EQUATIONS ( y3; y4 ; y5 and y6 ) FOR THE
ENZYME KINETICS:
In this attempt, four new plots or the lines with their
respective equations are going to be considered.
𝐕𝐦𝐚𝐱 These new lines include: y3; y4 ; y5 and y6.
𝐊𝐦 . This is

𝟏 (I). Attempt for the line y3 (Fig.3): Let us

going to yield the equation correspond to: 𝐒
𝐕𝐦𝐚𝐱 𝟏 𝟏
= 𝐊𝐦 𝐗 𝐯 − 𝐊𝐦 (Fig.2). The 1÷S and 1÷v for given
enzyme catalyzed biochemical reaction deserve
symmetry. That is to say, the real form of
“Lineweaver-Burk-Plot” and it’s inverse form derived
in the present attempt, are exhibiting the symmetry.
first consider the line y3. The slope and intercept
on y- axis of this y3 line are considered as:
− 𝐾𝑚 𝐾𝑚+1
𝑉𝑚𝑎𝑥 and 𝑉𝑚𝑎𝑥 respectively. Therefore, the
mathematical equation in “Slope-intercept” form
(in the form of typical y= m x +c) of this line y3 can
be written as: − 𝐾𝑚 𝐾𝑚+1

The co-ordinates of the point of intersection of both y3 = 𝑉𝑚𝑎𝑥 x + 𝑉𝑚𝑎𝑥

(Real form of Lineweaver Burk plot and it’ Inverse If we replace the “x” by (1÷ S) and substitute the Km
𝟏 𝐊𝐦 𝟏 𝟏
= [S (Vmax –v) ÷ v ]; the mathematical equation for
𝟏 form) the equations 𝐕 = 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 and
𝐒 the line y3 is going to transform into:
𝐕𝐦𝐚𝐱 𝟏 𝟏 𝟏
= 𝐊𝐦 𝐗 𝐯 − 𝐊𝐦 correspond to: ( 𝐕𝐦𝐚𝐱−𝐊𝐦 , − 𝑆(𝑉𝑚𝑎𝑥−𝑣) 𝑆(𝑉𝑚𝑎𝑥−𝑣)+𝑣
𝟏
𝐕𝐦𝐚𝐱−𝐊𝐦 ). The inverse function of the regular = 𝑣 𝑉𝑚𝑎𝑥 𝑠 + 𝑣 𝑉𝑚𝑎𝑥
𝟏 𝐕𝐦𝐚𝐱 𝟏
𝟏 Michaelis–Menten equation and ( 𝐒 = 𝐊𝐦 𝐗 𝐯 − 𝐊𝐦 ) is Simplification will yields into
going to serve to understand the concept on role of
product in enzyme involved reaction. The [1÷ (Vmax. − (𝑉𝑚𝑎𝑥−𝑣) 𝑆(𝑉𝑚𝑎𝑥−𝑣)+𝑣

– Km)], (Baramati Constant), point on both, the

𝟏 𝐊𝐦 = 𝑣 𝑉𝑚𝑎𝑥 + 𝑣 𝑉𝑚𝑎𝑥
𝟏 regular Michaelis–Menten equation ( 𝐕 = 𝐕𝐦𝐚𝐱
𝐗 𝐒+ 𝑆(𝑉𝑚𝑎𝑥−𝑣)+𝑣−(𝑉𝑚𝑎𝑥− 𝑣)
𝟏 𝟏 𝐊𝐦 𝟏 𝟏
= 𝑣 𝑉𝑚𝑎𝑥

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(𝑉𝑚𝑎𝑥−𝑣)(𝑠−1)+𝑣
It means, that as X increases, the Y tends to decrease
= 𝑣 𝑉𝑚𝑎𝑥 and here in present attempt, it attains to it’s
𝐾𝑚+1 minimum, that is (
It definitely means for plotting the y3; it is necessary 𝐾𝑚 ). Negative correlation

to consider X = (1÷ S) and represents a significant relationship between the

variables x and y, which, depending on what they are
(𝑉𝑚𝑎𝑥−𝑣)(𝑠−1)+𝑣
modeling, can be understood as input and output, or
Y= 𝑣 𝑉𝑚𝑎𝑥
cause and effect.

Through replacing the values of Vmax; V and S, it is

The ranges for X and Y for the line y3 are zero to one
possible to calculate the respective values of Y. This is
and (1÷ Vmax) to and 𝑉𝑚𝑎𝑥
𝐾𝑚+1
going to serve the purpose of plotting this new line
. Thus, the line y3 is going to help to imagine the
(y3) along with y1 and y2.
maximum and minimum values, both for X and Y.
Intersection of the line y3 and line y1 (Fig. 3 ):
𝐊𝐦 𝟏 𝟏
The line y1 ( 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 ) and the line y2
𝐕𝐦𝐚𝐱 𝟏 − 𝐊𝐦 𝐊𝐦+𝟏
( 𝐊𝐦 𝐗 − 𝐊𝐦 ) and y3 ( 𝐕𝐦𝐚𝐱 𝐗 + 𝐕𝐦𝐚𝐱 ) are shown in the
figure 3. Let us have a attempt to intersect the line y 2
and y3 for obtaining the x- and y- co-ordinates for the
point of intersection.

Fig. 3: Regular form of Linrweaver-Burk Plot

Y 1 = y3
(Double Reciprocal Plot) (y1) along with it’s
𝐊𝐦 𝟏 𝟏 − 𝐊𝐦 𝐊𝐦+𝟏
Inverse form (y2) and (y3).
Step – 1: 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 = 𝐕𝐦𝐚𝐱 𝐗 + 𝐕𝐦𝐚𝐱
Limitations and Significance of the Line (y3): 𝟏 𝐊𝐦+𝟏 − 𝐊𝐦 −𝐊𝐦
Line y3 is a line with a negative slope. This line is −
Step – 2: 𝐕𝐦𝐚𝐱 𝐕𝐦𝐚𝐱 =[ 𝐕𝐦𝐚𝐱 + 𝐕𝐦𝐚𝐱 ]X
trending downward from left to right. In other words,
𝟏−𝐊𝐦−𝟏 (−𝟐 𝐊𝐦)
the line's rise to run ratio is a negative value. Slope of Step – 3: 𝐕𝐦𝐚𝐱 = [ 𝐕𝐦𝐚𝐱 ] X
a line explains the pattern of change occurring in a
system. It also indicate the direction of change, −𝐊𝐦 (−𝟐 𝐊𝐦)
Step – 4: 𝐕𝐦𝐚𝐱 = [ 𝐕𝐦𝐚𝐱 ] X
whether positive or negative. The lines y1 and y2 (Fig.
1 and 2) are with a positive slope. Both of them are −𝐊𝐦 𝐕𝐦𝐚𝐱
going up from left to right. While, the line y 3 (Fig. 3) Step – 5: 𝐕𝐦𝐚𝐱 − 𝟐 𝐊𝐦 =X
is with a negative slope. It is going down from left to 𝟏
right. Step – 6: 𝟐 = X

The line y3 is representing a negative correlation Intersection of the line y3 and line y2 (Fig. 3 ):
between two variables [X = (1÷ S) and Y =
𝐕𝐦𝐚𝐱 𝟏 − 𝐊𝐦 𝐊𝐦+𝟏

(𝑉𝑚𝑎𝑥−𝑣)(𝑠−1)+𝑣 𝑣
The lines y2 ( 𝐊𝐦 𝐗 − 𝐊𝐦 ) and y3 ( 𝐕𝐦𝐚𝐱 𝐗 + 𝐕𝐦𝐚𝐱 ) are
𝑉𝑚𝑎𝑥 ]. shown in the figure 3. Let us have a attempt to

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 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56

intersect the line y2 and y3 for obtaining the x- and y-

co-ordinates for the point of intersection. If we replace the “x” by (1÷ v) and substitute the Km =
[S(Vmax –v) ÷ v]; the mathematical equation for the
Y 2 = y3 line y4 is going to transform into:
𝐕𝐦𝐚𝐱 𝟏 − 𝐊𝐦 𝐊𝐦+𝟏
− 𝑣 𝑉𝑚𝑎𝑥 𝑣 (𝑉𝑚𝑎𝑥−1)
Step – 1: 𝐊𝐦 𝐗 − 𝐊𝐦 = 𝐕𝐦𝐚𝐱 𝐗 + 𝐕𝐦𝐚𝐱
= 𝑆(𝑉𝑚𝑎𝑥−𝑣) 𝑣 + 𝑆(𝑉𝑚𝑎𝑥−𝑣)
𝐊𝐦+𝟏 𝟏 𝐕𝐦𝐚𝐱 𝐊𝐦
Step – 2: 𝐕𝐦𝐚𝐱 + 𝐊𝐦 = [ 𝐊𝐦 + 𝐕𝐦𝐚𝐱 ] X
Simplification will yields into
𝐊𝐦(𝐊𝐦+𝟏)+ 𝐕𝐦𝐚𝐱 (𝐕𝐦𝐚𝐱.𝐕𝐦𝐚𝐱+𝐊𝐦.𝐊𝐦)
− 𝑉𝑚𝑎𝑥 𝑣 (𝑉𝑚𝑎𝑥−1)
[
Step – 3: 𝐕𝐦𝐚𝐱.𝐊𝐦 = 𝐊𝐦.𝐕𝐦𝐚𝐱
= 𝑆(𝑉𝑚𝑎𝑥−𝑣) + 𝑆(𝑉𝑚𝑎𝑥−𝑣)
]X
𝐊𝐦(𝐊𝐦+𝟏)+ 𝐕𝐦𝐚𝐱 𝐕𝐦𝐚𝐱.𝐊𝐦 𝑣 (𝑉𝑚𝑎𝑥−1)− 𝑉𝑚𝑎𝑥

= 𝑆(𝑉𝑚𝑎𝑥−𝑣)
Step – 4: 𝐕𝐦𝐚𝐱.𝐊𝐦 (𝐕𝐦𝐚𝐱.𝐕𝐦𝐚𝐱+𝐊𝐦.𝐊𝐦) =X
𝐊𝐦(𝐊𝐦+𝟏)+ 𝐕𝐦𝐚𝐱
𝑣 𝑉𝑚𝑎𝑥−𝑣 − 𝑉𝑚𝑎𝑥
Step – 5: (𝐕𝐦𝐚𝐱.𝐕𝐦𝐚𝐱+𝐊𝐦.𝐊𝐦) =X
= 𝑆(𝑉𝑚𝑎𝑥−𝑣)

𝑉𝑚𝑎𝑥 (𝑣−1)−𝑣

(II). Attempt for the line y4 (Fig. 4): = 𝑆(𝑉𝑚𝑎𝑥−𝑣)

It definitely means for plotting the y4; it is necessary

to consider X = (1÷ v) and

𝑉𝑚𝑎𝑥 (𝑣−1)−𝑣

Y = 𝑆(𝑉𝑚𝑎𝑥−𝑣)

Through replacing the values of Vmax; V and S, it is

possible to calculate the respective values of Y. This is
going to serve the purpose of plotting this new line
Fig. 4: Regular form of Linrweaver-Burk Plot (Double
(y4) along with y1; y2 and y3.
Reciprocal Plot) (y1) along with it’s Inverse form (y2);
(y3) and (y4).
Limitations and Significance of the Line (y4):
Line y4 is a line with a negative slope. This line is
Let us consider the line y4. The slope and intercept on
tending downward from left to right. In other words,
− 𝑉𝑚𝑎𝑥 y-
axis of this y4 line are considered as: 𝐾𝑚 and
the line's rise to run ratio is a negative value. Slope of
𝑉𝑚𝑎𝑥−1 a line explains the pattern of change occurring in a
respectively. Therefore, the mathematical
𝐾𝑚 system. It also indicate the direction of change,
equation in “Slope-intercept” form (in the form of whether positive or negative. The lines y1 and y2 (Fig.
typical y= m x +c) of this line y4 can be written as: 1 and 2) are with a positive slope. Both of them are
− 𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥−1 going up from left to right. While, the line y3 and y4
Y4 = 𝐾𝑚 x+ 𝐾𝑚

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(Fig. 4) are with a negative slope. Both the lines y 3 𝐕𝐦𝐚𝐱 𝟏 − 𝐕𝐦𝐚𝐱
The line y2 ( 𝐊𝐦 𝐗 − 𝐊𝐦 ) and y4 ( 𝐊𝐦 x+
and y4 going down from left to right.
𝐕𝐦𝐚𝐱−𝟏
) are shown in the figure 4. Let us have a
𝐊𝐦
The line y4 is representing a negative correlation attempt to intersect the line y 2 and y4 for obtaining
between two variables [X = (1÷ v) and the x- and y- co-ordinates for the point of
𝑉𝑚𝑎𝑥 (𝑣−1)−𝑣
intersection. y4 = y2
Y= 𝑆(𝑉𝑚𝑎𝑥−𝑣) ].
− 𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥−1 𝑽𝒎𝒂𝒙 𝟏 Step – 1:
𝐾𝑚 x+ 𝐾𝑚 = 𝑲𝒎 𝑿 − 𝑲𝒎
It means, that as X increases, the Y tends to decrease 𝑉𝑚𝑎𝑥−1 𝟏 𝑽𝒎𝒂𝒙 𝑽𝒎𝒂𝒙
and here in present attempt, it attains to it’s minimum, Step – 2: 𝐾𝑚 + 𝑲𝒎 = [ 𝑲𝒎 + 𝑲𝒎 ] X
𝑉𝑚𝑎𝑥−1
𝑉𝑚𝑎𝑥−1+1 𝟐𝑽𝒎𝒂𝒙 Step –
that is 𝑉𝑚𝑎𝑥 .
3: 𝐾𝑚 = [ 𝑲𝒎 ] X
Negative correlation represents a significant 𝑉𝑚𝑎𝑥 𝟐𝑽𝒎𝒂𝒙 Step –
relationship between the variables x and y, which, 4: 𝐾𝑚 = [ 𝑲𝒎 ] X
𝑉𝑚𝑎𝑥 𝐾𝑚
depending on what they are modeling, can be
understood as input and output, or cause and effect. Step – 5: 𝐾𝑚 2 𝑉𝑚𝑎𝑥

The range of values for X and Y for the line y 4 are = X Step – 6: =X
𝑉𝑚𝑎𝑥−1 𝑉𝑚𝑎𝑥−1
zero to 𝑉𝑚𝑎𝑥 and from 𝐾𝑚 to zero. Intersection of the line y4 with line y3 (Fig. 4 ):
𝐊𝐦 𝟏 𝟏 − 𝐕𝐦𝐚𝐱
Thus, the line y4 is going to help to imagine the The line y1 ( 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 ) and y4 ( 𝐊𝐦 x+
maximum and minimum values, both for X and Y. 𝐕𝐦𝐚𝐱−𝟏

More over, the line y4 is inverse form of the line y3. ) are shown in the figure 4. Let us have a
𝐊𝐦

Intersection of the line y4 with line y1 (Fig. 4 ): attempt to intersect the line y 1 and y4 for obtaining
the x- and y- co-ordinates for the point of
𝐊𝐦 𝟏 𝟏 − 𝐕𝐦𝐚𝐱
intersection.
The line y1 ( 𝐕𝐦𝐚𝐱 𝐗 𝐒 + 𝐕𝐦𝐚𝐱 ) and y4 ( 𝐊𝐦 x+
𝐕𝐦𝐚𝐱−𝟏 y4 = y 3
) are shown in the figure 4. Let us have a
𝐊𝐦 − 𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥−1 − 𝐾𝑚 𝐾𝑚+1 Step – 1:
attempt to intersect the line y 1 and y4 for obtaining 𝐾𝑚 x+ 𝐾𝑚 = 𝑉𝑚𝑎𝑥 x + 𝑉𝑚𝑎𝑥
the x- and y- co-ordinates for the point of − 𝑉𝑚𝑎𝑥.𝑉𝑚𝑎𝑥+𝐾𝑚.𝐾𝑚 𝐾𝑚+1 𝑉𝑚𝑎𝑥−1

intersection. y4 = y1 Step – 2: [ 𝐾𝑚.𝑉𝑚𝑎𝑥 ] X = 𝑉𝑚𝑎𝑥 - 𝐾𝑚

− (𝑉𝑚𝑎𝑥.𝑉𝑚𝑎𝑥− 𝐾𝑚.𝐾𝑚 )
− 𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥−1 𝑲𝒎 𝟏
Step – 3: [ 𝐾𝑚.𝑉𝑚𝑎𝑥 ]X=
Step – 1: 𝐾𝑚 x + 𝐾𝑚 = 𝑽𝒎𝒂𝒙 𝑋 + 𝑽𝒎𝒂𝒙
𝐾𝑚 (𝐾𝑚+1)− 𝑉𝑚𝑎𝑥 (𝑉𝑚𝑎𝑥−1)
− 𝑉𝑚𝑎𝑥 𝑲𝒎 𝟏 𝑉𝑚𝑎𝑥−1
𝑉𝑚𝑎𝑥.𝐾𝑚
Step – 2: [ 𝐾𝑚 - 𝑽𝒎𝒂𝒙 ]X = 𝑽𝒎𝒂𝒙 - 𝐾𝑚
− (𝑉𝑚𝑎𝑥.𝑉𝑚𝑎𝑥− 𝐾𝑚.𝐾𝑚 )
− 𝑉𝑚𝑎𝑥.𝑉𝑚𝑎𝑥 − 𝐾𝑚.𝐾𝑚 𝑲𝒎−𝑽𝒎𝒂𝒙(𝑽𝒎𝒂𝒙−𝟏)
Step – 4: [ 𝐾𝑚.𝑉𝑚𝑎𝑥 ]X=
Step – 3: [ 𝐾𝑚.𝑉𝑚𝑎𝑥 ]X = 𝑽𝒎𝒂𝒙.𝑲𝒎 −[𝑉𝑚𝑎𝑥 (𝑉𝑚𝑎𝑥−1)− 𝐾𝑚(𝐾𝑚+1)]
− (𝑉𝑚𝑎𝑥.𝑉𝑚𝑎𝑥+𝐾𝑚.𝐾𝑚)
𝑉𝑚𝑎𝑥.𝐾𝑚
Step – 4: [ 𝐾𝑚.𝑉𝑚𝑎𝑥 ]X = [𝑉𝑚𝑎𝑥 (𝑉𝑚𝑎𝑥−1)− 𝐾𝑚(𝐾𝑚+1)]
−[𝑽𝒎𝒂𝒙(𝑽𝒎𝒂𝒙−𝟏)−𝑲𝒎])
Step – 5: X = 𝑉𝑚𝑎𝑥.𝑉𝑚𝑎𝑥−𝐾𝑚.𝐾𝑚
𝑽𝒎𝒂𝒙.𝑲𝒎
[𝑽𝒎𝒂𝒙(𝑽𝒎𝒂𝒙−𝟏)−𝑲𝒎])

Step – 5: X = (𝑽𝒎𝒂𝒙.𝑽𝒎𝒂𝒙+𝑲𝒎.𝑲𝒎) (III). Attempt for the lines y5 (Fig. 5 ):

Intersection of the line y4 with line y2 (Fig. 4 ):

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Let us first consider the line y5. The slope and Y 5 = y3

intercept on y- axis of this y5 line are considered as: − 𝐾𝑚 𝐾𝑚+1

one and zero respectively. Therefore, the Step -1: X + 0 = 𝑉𝑚𝑎𝑥 x + 𝑉𝑚𝑎𝑥
1 𝐾𝑚 𝐾𝑚+1
mathematical equation in “Slope-intercept” form (in
Step -2: [1 + 𝑉𝑚𝑎𝑥] X = 𝑉𝑚𝑎𝑥
the form of typical y= m x +c) of this line y5 can be
𝑉𝑚𝑎𝑥+ 𝐾𝑚 𝐾𝑚+1 Step
written as:
-3: [ 𝑉𝑚𝑎𝑥 ]X = 𝑉𝑚𝑎𝑥
𝐾𝑚+1 𝑉𝑚𝑎𝑥
Y5 = x + 0 Step -4: X = 𝑉𝑚𝑎𝑥 x 𝑉𝑚𝑎𝑥 + 𝐾𝑚
This y5 is the only line in this present attempt 𝐾𝑚+1

allowing to replace the X by any suitable parameter in Step -5: X = 𝑉𝑚𝑎𝑥+ 𝐾𝑚

the enzyme kinetics.
This y5 is the only line in this present attempt Let us label this point of the intersection of the line y5
allowing to replace the X by any suitable parameter with the line y3 as: “A”. Both, the X – co-ordinate and
in the enzyme kinetics. The value for X is directly Y – co-ordinate of the point correspond to : X =
𝐾𝑚+1
proportional to the value of Y (With proportional
constant is equal to one). The minimum value for X 𝑉𝑚𝑎𝑥+ 𝐾𝑚
for the line y5 is zero. There is no upper limit for this
line y5. Now let us have a look on the intersection of the line
y5 with the y4.
Y 5 = y4
− 𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥− 1 Step -1:
X+0= 𝐾𝑚 x+ 𝐾𝑚

1 𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥− 1 Step

-2: [1 + 𝐾𝑚 ] X = 𝐾𝑚
𝑉𝑚𝑎𝑥+ 𝐾𝑚 𝑉𝑚𝑎𝑥− 1
Step -3: [ 𝐾𝑚 ]X= 𝐾𝑚
𝑉𝑚𝑎𝑥− 1 𝐾𝑚

Step -4: X = 𝐾𝑚 x 𝑉𝑚𝑎𝑥 + 𝐾𝑚

𝑉𝑚𝑎𝑥− 1
Fig. 5: Regular form of Linrweaver-Burk Plot
Step -5: X = 𝑉𝑚𝑎𝑥+ 𝐾𝑚
(Double Reciprocal Plot) (y1) along with it’s
Inverse form (y2); (y3); (y4) and (y5).
Let us label this point of the intersection of the line y5
with the line y4 as: “C”.
Intersection of the line y5 with other lines in the
Both, the X – co-ordinate; Y – co-ordinate of this
attempt (Fig. 5 ):
point (“C”) are one and same and correspond to: X =
𝑉𝑚𝑎𝑥− 1
The figure – 5 in the present is well explaining the
intersection of the line y5 with the other lines. The 𝑉𝑚𝑎𝑥+ 𝐾𝑚

point of intersection of line y5 with the line y1 and y2
is one and the same. X – and Y – co-ordinates of the
point of intersection of line y5 with the line y1 and y2
are same and correspond to: [(1÷ (Vmax – Km)]. Now
let us have a look on the intersection of the line y 5
with the y3.
(IV). Attempt for the lines y6 (Fig. 6):
Let us first consider the line y6. The slope and
intercept on y- axis of this y6 line are considered as:
one and one respectively. Therefore, the
mathematical equation in “Slope-intercept” form (in

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the form of typical y= m x +c) of this line y 6 can be Step -1: 𝑉𝑚𝑎𝑥 x + 𝑉𝑚𝑎𝑥 = - X + 1
written as: 𝐾𝑚 1
1
Y6 = - x + 1 Step -2: [ 𝑉𝑚𝑎𝑥 + ] X = 1 - 𝑉𝑚𝑎𝑥
This y6 is the line in this present attempt allowing to 𝐾𝑚+𝑉𝑚𝑎𝑥 𝑉𝑚𝑎𝑥−1 Step
replace the X by suitable parameter in the enzyme -3: [ 𝑉𝑚𝑎𝑥 ] X = 𝑉𝑚𝑎𝑥
kinetics. 𝑉𝑚𝑎𝑥−1 𝑉𝑚𝑎𝑥

The value for X is directly proportional to the value Step -4: X = 𝑉𝑚𝑎𝑥 x 𝑉𝑚𝑎𝑥+𝐾𝑚
𝑉𝑚𝑎𝑥−1
of Y (With proportional constant is equal to one).
Step -5: X = 𝑉𝑚𝑎𝑥+𝐾𝑚
The minimum value and maximum value for both, X
and Y for the line y6 correspond to zero and one
Let us label this point of the intersection of the line y6
respectively.
with the line y1 as: “B”. Both, the X – co-ordinate and
The line y5 and line y6; both are the inverse form of
Y – co-ordinate of the this point “B” correspond to : X
each other.
𝑉𝑚𝑎𝑥− 1

= 𝑉𝑚𝑎𝑥+ 𝐾𝑚
Now let us have a look on the intersection of the line
y6 with the y2.
Y 2 = y6
𝑉𝑚𝑎𝑥 1
Step -1: 𝐾𝑚 x - 𝐾𝑚 = - X + 1
𝑉𝑚𝑎𝑥 1
1
Step -2: [ 𝐾𝑚 + ] X = 1 + 𝐾𝑚
𝐾𝑚+𝑉𝑚𝑎𝑥 𝐾𝑚+ 1 Step
-3: [ 𝐾𝑚 ] X = 𝐾𝑚
𝐾𝑚+ 1 𝐾𝑚
Step -4: X = 𝐾𝑚 x 𝑉𝑚𝑎𝑥+𝐾𝑚
Fig. 6: Regular form of Linrweaver-Burk Plot (Double
𝐾𝑚+ 1
Reciprocal Plot) (y1) along with it’s Inverse form (y2); Step -5: X = 𝑉𝑚𝑎𝑥+𝐾𝑚
(y3); (y4); (y5) and (y6).
Intersection of the line y6 with other lines in the Let us label this point of the intersection of the line y6
attempt (Fig. 6 ): with the line y1 as: “D”. The X – co-ordinate of the
The figure – 6 in the present is well explaining the 𝐾𝑚+ 1 this
intersection of the line y6 with the other lines. The point “D” correspond to : X = 𝑉𝑚𝑎𝑥+ 𝐾𝑚
point of intersection of line y6 with the line y3 and y4 The Y – co-ordinate of the this point “D” correspond
is one and the same. X – co-ordinate of the point of to :
intersection of line y6 with the line y3 and y4 1 𝐾𝑚+ 1 𝑉𝑚𝑎𝑥+𝐾𝑚− 𝐾𝑚−1 𝑉𝑚𝑎𝑥−1

correspond to: [1 - (1÷ (Vmax – Km)] = [(Vmax – Km Y = 1 − 𝑉𝑚𝑎𝑥+ 𝐾𝑚

-1) ÷ (Vmax – Km)]. Y – co-ordinate of the point of = 𝑉𝑚𝑎𝑥+ 𝐾𝑚 = 𝑉𝑚𝑎𝑥+ 𝐾𝑚
intersection of line y6 with the line y3 and y4
correspond to: [ (1÷ (Vmax – Km)]. (V). Attempt for the Indian Square For Enzyme
Kinetics (Fig. 7 and 8):
Now let us have a look on the intersection of the line
y6 with the y1. The present attempt tried it’s best to derive and to set
Y 1 = y6 some new equations keeping the meaning and
𝐾𝑚 1

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concept expected by the well esteemed standard

Lineweaver-Burk plot, the double reciprocal plot for
the enzyme kinetics.

Let us refer (have a look) on the fig.7. This fig. 7 is

nothing but the result of the present attempt on
derivation of some new equations keeping the
meaning and concept expected by the well esteemed
standard Lineweaver-Burk plot, the double reciprocal
plot for the enzyme kinetics constant. The equations
in this attempt include: y 3; y4; y5 and y6. The line y1 Fig. 8 : Position of Indian Square For Enzyme Kinetics
itself is the original Lineweaver-Burk Plot (Double in Linrweaver-Burk Plot (Double Reciprocal Plot).
reciprocal plot). The line y2 is the inverse form of line
y1 ; the original Lineweaver-Burk Plot (Double The X- co-ordinates and Y- co-ordinates of the four
reciprocal plot). The point of intersection of the line points (A,B,C,D) of the square obtained by the
y5 and y6 is here, in the present attempt is labeled as, intersection of various lines in the present attempt are
“O”. listed below:

The X – co-ordinate of the point “A” correspond to:

Km+1

Vmax+ Km

The Y – co-ordinate of the point “A” correspond to:

Km+1

Vmax+ Km

The X – co-ordinate of the point “B” correspond to:

𝑉𝑚𝑎𝑥− 1

Fig. 7 : Indian Square For Enzyme Kinetics Through 𝑉𝑚𝑎𝑥+ 𝐾𝑚

The Linrweaver-Burk Plot (Double Reciprocal Plot) The Y – co-ordinate of the point “B” correspond to:
𝐾𝑚+1
(y1) and The Other Lines In the Present Attempt (y2;
y3; y4; y5 and y6). 𝑉𝑚𝑎𝑥+ 𝐾𝑚
The X – co-ordinate of the point “C” correspond to:
The attempt on the intersection of the line y 5 and y3 is 𝑉𝑚𝑎𝑥− 1

yielding the point “A” as appearing in fig. 8. The

𝑉𝑚𝑎𝑥+ 𝐾𝑚
attempt on the intersection of the line y 6 and y1 is
yielding the point “B” as appearing in fig. 8. The The Y – co-ordinate of the point “C” correspond to:
attempt on the intersection of the line y 5 and y4 is Vmax− 1

yielding the point “C” as appearing in fig. 8. The

Vmax+ Km
attempt on the intersection of the line y 6 and y6 is
The X – co-ordinate of the point “D” correspond to:
yielding the point “D” as appearing in fig. 8. 𝐾𝑚+ 1

𝑉𝑚𝑎𝑥+𝐾𝑚

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 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56

The Y – co-ordinate of the point “D” correspond to:
𝑉𝑚𝑎𝑥−1
𝑉𝑚𝑎𝑥+ 𝐾𝑚
The distance between the point “A” and point “B”
(OR the length of segment AB) can be obtained by
subtraction of the X- co-ordinate of the point “A”
from the X – co-ordinate of the point “B”. Therefore,
the length of segment (AB) correspond to:
The distance between the point “D” and point “A” (OR
the length of segment DA) can be obtained by
subtraction of the Y- co-ordinate of the point “A”
from the Y – co-ordinate of the point “D”.
Therefore, the length of segment (DA) correspond to:
𝑉𝑚𝑎𝑥−1 𝐾𝑚+1
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚 - 𝑉𝑚𝑎𝑥+ 𝐾𝑚
𝑉𝑚𝑎𝑥− 1−𝐾𝑚−1

𝑉𝑚𝑎𝑥− 1 𝐾𝑚+ 1 = 𝑉𝑚𝑎𝑥+ 𝐾𝑚

𝑉𝑚𝑎𝑥− 𝐾𝑚−2
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚 - 𝑉𝑚𝑎𝑥+ 𝐾𝑚
𝑉𝑚𝑎𝑥− 1− 𝐾𝑚−1
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚
From the above steps of attempts of obtaining the
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚
𝑉𝑚𝑎𝑥− 𝐾𝑚−2 distance between the points of intersection of various
lines or length of segment (AB; BC; CD and DA); all
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚
the four sides of rectangle ABCD are equal and
correspond to:
The distance between the point “B” and point “C”
𝑉𝑚𝑎𝑥− 𝐾𝑚−2
(OR the length of segment BC) can be obtained by
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚
subtraction of the Y- co-ordinate of the point “B”
from the Y – co-ordinate of the point “C”. Therefore,
Therefore, rectangle ABCD is the square.
the length of segment (BC) correspond to:
This square (ABCD) resulted from the intersection of
𝑉𝑚𝑎𝑥− 1 𝐾𝑚+1 the line y3 and line y5 ; line y1 and line y6 ; line y4 and
y5 ; line y2 and line y6 ; here, through this present is
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚 - 𝑉𝑚𝑎𝑥+ 𝐾𝑚
𝑉𝑚𝑎𝑥− 1−𝐾𝑚−1 designated as “Indian Square For Enzyme Kinetics”.
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚
𝑉𝑚𝑎𝑥− 𝐾𝑚−2 (VI). Properties and Significance of Indian Square For
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚 Enzyme Kinetics:
1. The regular form of Lineweaver-Burk plot (the
The distance between the point “C” and point “D” (OR line y1 [equation: (1÷v) = (Km÷Vmax) (1÷S) +
the length of segment CD) can be obtained by (1÷Vmax)] intersect the line y6 (equation: - X + 1)
subtraction of the X- co-ordinate of the point “D” at the point “B” of the proposed “Indian Square
from the X – co-ordinate of the p2oint “C”. For Enzyme Kinetics”. The X – co-ordinate and Y
– co-ordinate of this point “B” correspond to:
Therefore, the length of segment (CD) correspond to: [(Vmax – 1) ÷ (Vmax + Km)] and [(Km + 1) ÷
= (Vmax + Km)] respectively.
𝑉𝑚𝑎𝑥− 1 𝐾𝑚+ 1
2. Subtraction of Y- co-ordinate of the point “B”
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚 - 𝑉𝑚𝑎𝑥+𝐾𝑚 from the X – co-ordinate of the point “B” is going
𝑉𝑚𝑎𝑥− 1−𝐾𝑚−1
to yield the length of each side of the proposed
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚 “Indian Square For Enzyme Kinetics”.
𝑉𝑚𝑎𝑥− 𝐾𝑚−2
∴ [(Vmax – 1) ÷ (Vmax + Km)] - [(Km + 1) ÷
= 𝑉𝑚𝑎𝑥+ 𝐾𝑚 (Vmax + Km)] = [(Vmax – Km – 2) ÷ (Vmax +
Km)].

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 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56
The length of each side of the proposed “Indian
Square For Enzyme Kinetics” correspond to:
[(Vmax – Km – 2) ÷ (Vmax + Km)].
3. The length of segment AB; the segment BC; the
segment CD and the segment DA of the proposed
“Indian Square For Enzyme Kinetics” are equal
and correspond to: [(Vmax – Km – 2) ÷ (Vmax +
Km)].
4. The point “A” of the proposed “Indian Square For
Enzyme Kinetics” is the point of intersection of
the line y3 and the line y5. The X – co-ordinate
and Y – co-ordinate of the point “A” of the
proposed “Indian Square For Enzyme Kinetics”
correspond to: [(Km +1) ÷ (Vmax + Km)] and [(Km
+1) ÷ (Vmax + Km)] respectively. (Both, the X –
co-ordinate and Y – co-ordinate of the point “A”
of the proposed “Indian Square For Enzyme
Kinetics” are equal to each other and correspond
to: [(Km +1) ÷ (Vmax + Km)] ).
5. The point “C” of the proposed “Indian Square For
Enzyme Kinetics” is the point of intersection of
the line y4 and the line y5. The X – co-ordinate
and Y – co-ordinate of the point “C” of the
proposed “Indian Square For Enzyme Kinetics”
correspond to: [(Vmax - 1) ÷ (Vmax + Km)] and
[(Vmax - 1) ÷ (Vmax + Km)] respectively. (Both,
the X – co-ordinate and Y – co-ordinate of the
point “C” of the proposed “Indian Square For
Enzyme Kinetics” are equal to each other and
correspond to: [(Vmax - 1) ÷ (Vmax + Km)] ).
6. The point “D” of the proposed “Indian Square For
Enzyme Kinetics” is the point of intersection of
the line y2 and the line y6. The X – co-ordinate
and Y – co-ordinate of the point “D” of the
proposed “Indian Square For Enzyme Kinetics”
correspond to: [(Km + 1) ÷ (Vmax + Km)] and
[(Vmax - 1) ÷ (Vmax + Km)] respectively.
7. The length of segment AB=BC=CD=DA and it
correspond to: [(Vmax - Km - 2) ÷ (Vmax + Km)].
The point “A”; “B”; “C” and “D” constitute the
vertices of square ABCD.
International Journal of Scientific Research in Chemistry (www.ijsrch.com) | Volume 4 | Issue 1
8. The resulting mathematical square, herewith
labeled as: “Indian Square For Enzyme Kinetics”.
Each of the four vertices (corners) have known
coordinates in terms of Vmax and Km, the key
indices in enzyme kinetics.
9. The value of [(Km + 1) ÷ (Vmax + Km)] is greater
than [(1÷Vmax)] and less than [(1÷2)]. This range
can be written as: [(1÷Vmax)] < [(Km + 1) ÷ (Vmax
+ Km)] < [(1÷2)].
10. The value of [(Vmax - 1) ÷ (Vmax + Km)] is
greater than [(1÷2)] and less than [(Vmax -1) ÷
Vmax)]. This range can be written as: [(1÷2)] <
[(Vmax - 1) ÷ (Vmax + Km)] < [(Vmax -1)
÷Vmax)].
II. CONCLUSION
Each enzyme deserve a specific Michaelis-Menten
constant (Km), which is determined through the
double reciprocal plot, also recognized as
LineweaverBurk plot. This constant of Michaelis-
Menten (Km) is concentration of substrate [S] and it
avails the velocity (v) of reaction to proceed up to half
of it’s maximal or Vmax. The regular Lineweaver-
Burk plot and it’s inverse form are designated as y 1
and y2 lines respectively. Present attempt is
considering additional plots or the lines keeping the
concept in regular Lineweaver-Burk plot constant.
These plots include: y3 and y4. In slope and intercept
form the lines y3 and y4 expressed as: y3= -
[(Km÷Vmax)(X)] +
[(km+1)÷(Vmax)] and y4= -[(Vmax÷Km)(X)] + [(Vmax
-1)÷(Km)]. In addition; y5 = X + 0 and y 6 = - X + 1 are
the two reference lines are also considered in this
attempt. The line y3 intersect the reference line y5 at
the point “A”, the x- co-ordinate and y- co-ordinate of
which are equal to each other and correspond to:
[(Km+1)÷(Vmax+Km)]. The line y1 intersect the
reference line y6 at the point “B”, the x- co-ordinate
and y- co-ordinate of which respectively correspond
to: [(Vmax – 1) ÷ (Vmax + Km)] and [(Km + 1) ÷
(Vmax + Km)]. The point at which the reference line
y5 attains [(Vmax – 1) ÷ (Vmax + Km)] is labeled as the
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 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56
point “C”. The X – co-ordinate and Y – coordinate of
the point “C” correspond to: [(Vmax – 1) ÷ (Vmax +
Km)] [(Km + 1) ÷ (Vmax + Km)] respectively. The
point of intersection of the line y2 and the reference
line y6 is labeled as the point “D”. The X – co-ordinate
and Y – co-ordinate of the point “D” correspond to:
[(Km + 1) ÷ (Vmax + Km)] and [(Vmax – 1) ÷ (Vmax +
Km)] respectively. The length of segment
AB=BC=CD=DA and it correspond to:
[(Vmax - Km - 2) ÷ (Vmax + Km)]. The point “A”;
“B”; “C” and “D” constitute the vertices of square
ABCD. The value of [(Km + 1) ÷ (Vmax + Km)] is
greater than [(1÷Vmax)] and less than [(1÷2)]. This
range can be written as: [(1÷Vmax)] < [(Km + 1) ÷
(Vmax + Km)] < [(1÷2)]. The value of [(Vmax - 1) ÷
(Vmax + Km)] is greater than [(1÷2)] and less than
[(Vmax -1) ÷ Vmax)]. This range can be written as:
[(1÷2)] < [(Vmax - 1) ÷ (Vmax + Km)] < [(Vmax -1)
÷Vmax)]. The resulting mathematical square,
herewith labeled as: “Indian Square For Enzyme
Kinetics”. Each of the four vertices (corners) have
known coordinates in terms of Vmax and Km, the
key indices in enzyme kinetics.
III. ACKNOWLEDGEMENTS
The academic support received from the
administrative staff of Agricultural Development
Trust Baramati Shardanagar, (Malegaon Colony, Post
Box No. – 35, Tal. Baramati; Dist. Pune – 413115
Maharashtra, India) during the tenure of 19 October,
2018 to 31 December, 2018 deserve appreciations and
exert a grand salutary influence.
IV. REFERENCES
[1]. W., Weisstein, Eric. "Square".
mathworld.
http://mathworld.wolfram.com/Square.html
Weisstein, Eric W. "Square." From MathWorld--
A Wolfram Web Resource.
http://mathworld.wolfram.com/Square.html
International Journal of Scientific Research in Chemistry (www.ijsrch.com) | Volume 4 | Issue 1
[2]. Zalman Usiskin and Jennifer Griffin, "The
Classification of Quadrilaterals. A Study of
Definition", Information Age Publishing, 2008,
p. 59, ISBN 1-59311-695-0.
[3]. "Problem Set 1.3". jwilson.coe.uga.edu. Retrieved
2017-12-12.
[4]. Josefsson, Martin, "Properties of equidiagonal
quadrilaterals" Forum Geometricorum, 14
(2014), 129-144.
[5]. "Maths is Fun - Can't Find
It (404)". www.mathsisfun.com.
Retrieved 2017-12-12.
[6]. Chakerian, G.D. "A Distorted View of Geometry."
Ch. 7 in Mathematical Plums (R. Honsberger,
editor). Washington, DC:
Mathematical Association of America, 1979: 147.
[7]. 1999, Martin Lundsgaard Hansen, thats IT (c).
"Vagn Lundsgaard Hansen". www2.mat.dtu.dk.
Retrieved 2017-12-12.
[8]. "Geometry classes, Problem 331. Square, Point on
the Inscribed Circle, Tangency Points. Math
teacher Master Degree. College, SAT Prep.
Elearning, Online math tutor, LMS".
gogeometry.com. Retrieved 2017-12-12.
[9]. Park, Poo-Sung. "Regular polytope distances",
Forum Geometricorum 16, 2016, 227-232.
http://forumgeom.fau.edu/FG2016volume16/FG
2
01627.pdf
[10]. John H. Conway, Heidi Burgiel, Chaim
Goodman-Strauss, (2008) The Symmetries of
Things, ISBN 978-1-56881-220-5 (Chapter 20,
Generalized Schaefli symbols, Types of
symmetry of a polygon pp. 275-278).
[11]. Wells, Christopher J. "Quadrilaterals".
www.technologyuk.net. Retrieved 2017-12-12.
Clark, W. E. and Suen, S. "An Inequality Related
to Vizing's Conjecture." Electronic J.
Combinatorics 7, No. 1, N4, 1-3, 2000.
http://www.combinatorics.org/Volume_7/Abstra
cts/v7i1n4.html.
53
 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56
[12]. Coxeter, H. S. M. and Greitzer, S. L. Geometry
Revisited. Washington, DC: Math. Assoc. Amer.,
p. 84, 1967.
[13]. Detemple, D. and Harold, S. "A Round-Up of
Square Problems." Math. Mag. 69, 15-27, 1996.
[14]. Dixon, R. Mathographics. New York: Dover, p.
16, 1991.
[15]. Eppstein, D. "Rectilinear Geometry."
http://www.ics.uci.edu/~eppstein/junkyard/rect.
html.
[16]. Fischer, G. (Ed.). Plate 1 in Mathematische
Modelle aus den Sammlungen von Universitäten
und Museen, Bildband. Braunschweig,
Germany: Vieweg, p. 2, 1986.
[17]. Fukagawa, H. and Pedoe, D. "One or Two
Circles and Squares," "Three Circles and
Squares," and "Many Circles and Squares
(Casey's Theorem)." §3.1-3.3 in Japanese Temple
Geometry Problems. Winnipeg, Manitoba,
Canada: Charles Babbage Research Foundation,
pp. 37-42 and 117-125, 1989.
[18]. Gardner, M. The Sixth Book of Mathematical
Games from Scientific American. Chicago, IL:
University of Chicago Press, pp. 165 and 167,
1984.
[19]. Guy, R. K. "Rational Distances from the Corners
of a Square." §D19 in Unsolved Problems in
Number Theory, 2nd ed. New York:
SpringerVerlag, pp. 181-185, 1994.
[20]. Harris, J. W. and Stocker, H. "Square." §3.6.6 in
Handbook of Mathematics and Computational
Science. New York: Springer-Verlag, pp. 84-85,
1998.
[21]. Kern, W. F. and Bland, J. R. Solid Mensuration
with Proofs, 2nd ed. New York: Wiley, p. 2,
1948.
[22]. Yaglom, I. M. Geometric Transformations I. New
York: Random House, pp. 96-97, 1962.
[23]. Lineweaver, H; Burk, D. (1934). "The
determination of enzyme dissociation
constants". Journal of the American Chemical
International Journal of Scientific Research in Chemistry (www.ijsrch.com) | Volume 4 | Issue 1
Society. 56 (3): 658–666.
doi:10.1021/ja01318a036.
[24]. Hayakawa, K.; Guo, L.; Terentyeva, E.A.; Li,
X.K.; Kimura, H.; Hirano, M.; Yoshikawa, K.;
Nagamine, T.; et al. (2006). "Determination of
specific activities and kinetic constants of
biotinidase and lipoamidase in LEW rat and
Lactobacillus casei (Shirota)". Journal of
Chromatography B. 844 (2): 240–50.
doi:10.1016/j.jchromb.2006.07.006. PMID
16876490.
[25]. Greco, W.R.; Hakala, M.T. (1979). "Evaluation of
methods for estimating the dissociation constant
of tight binding enzyme inhibitors" (PDF).
Journal of Biological Chemistry. 254 (23): 12104–
12109. PMID 500698.
[26]. Dowd, John E.; Riggs, Douglas S. (1965). "A
comparison of estimates of Michaelis–Menten
kinetic constants from various linear
transformations" (pdf). Journal of Biological
Chemistry. 240 (2): 863–869.
[27]. Keith J. Laidler (1997). New Beer in an Old
Bottle: Eduard Buchner and the Growth of
Biochemical Knowledge, pp. 127–133, ed. A.
Cornish-Bowden, Universitat de València,
Valencia, Spain, 1997 http://bip.cnrs-
mrs.fr/bip10/newbeer/laidler.pdf
[28]. Dick RM (2011). "Chapter 2. Pharmacodynamics:
The Study of Drug Action". In Ouellette R, Joyce
JA. Pharmacology for Nurse Anesthesiology.
Jones & Bartlett Learning. ISBN 978-0-7637-
8607-6
[29]. Berg, Jeremy M.; Tymoczko, John L.; Stryer,
Lubert (2002). "The Michaelis-Menten Model
Accounts for the Kinetic Properties of Many
Enzymes". Biochemistry. 5th edition. W H
Freeman.
[30]. John E. Dowd and Douglas Briggs (1965).
Estimates of Michaelis – Menten kinetic
constants from various linear transformation.
The Journal of Biological Chemistry Vol. 240 No.
2 February, 1965.
54
 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56
http://www.jbc.org/content/240/2/863.full.pdf
[31]. Hall, Arthur Graham; Frink, Fred Goodrich
(January 1909). "Chapter II. The Acute Angle
[14] Inverse trigonometric functions". Written at
Ann Arbor, Michigan, USA. Trigonometry. Part
I: Plane Trigonometry. New York, USA: Henry
Holt and Company / Norwood Press / J. S.
Cushing Co. - Berwick & Smith Co., Norwood,
Massachusetts, USA. p. 15. Retrieved 2017-08-
12.
https://en.wikipedia.org/wiki/Inverse_function
[32]. Scheinerman, Edward R. (2013). Mathematics:
A Discrete Introduction. Brooks/Cole. p. 173.
ISBN 978-0840049421.
[33]. Keisler, Howard Jerome. "Differentiation" (PDF).
Retrieved 2015-01-24. §2.4
[34]. Scheinerman, Edward R. (2013). Mathematics:
A Discrete Introduction. Brooks/Cole. p. 173.
ISBN 978-0840049421.
[35]. Korn, Grandino Arthur; Korn, Theresa M.
(2000) [1961]. "21.2.-4. Inverse Trigonometric
Functions". Mathematical handbook for
scientists and engineers: Definitions, theorems,
and formulars for reference and review (3 ed.).
Mineola, New York, USA: Dover Publications,
Inc. p. 811;. ISBN 978-0-486-41147-7.
[36]. Oldham, Keith B.; Myland, Jan C.; Spanier,
Jerome (2009) [1987]. An Atlas of Functions:
with Equator, the Atlas Function Calculator (2
ed.). Springer Science+Business Media, LLC.
doi:10.1007/978-0-387-48807-3. ISBN 978-0387-
48806-6. LCCN 2008937525.
[37]. Briggs, William; Cochran, Lyle (2011).
Calculus / Early Transcendentals Single Variable.
AddisonWesley. ISBN 978-0-321-66414-3.
[38]. Devlin, Keith J. (2004). Sets, Functions, and
Logic / An Introduction to Abstract Mathematics
(3rd ed.). Chapman & Hall / CRC Mathematics.
ISBN 978-1-58488-449-1.
[39]. Fletcher, Peter; Patty, C. Wayne (1988).
Foundations of Higher Mathematics. PWS-Kent.
ISBN 0-87150-164-3.
International Journal of Scientific Research in Chemistry (www.ijsrch.com) | Volume 4 | Issue 1
[40]. Lay, Steven R. (2006). Analysis / With an
Introduction to Proof (4 ed.). Pearson / Prentice
Hall. ISBN 978-0-13-148101-5.
[41]. Smith, Douglas; Eggen, Maurice; St. Andre,
Richard (2006). A Transition to Advanced
Mathematics (6 ed.). Thompson Brooks/Cole.
ISBN 978-0-534-39900-9.
[42]. Thomas, Jr., George Brinton (1972). Calculus and
Analytic Geometry Part 1: Functions of One
Variable and Analytic Geometry (Alternate ed.).
Addison-Wesley.
[43]. Wolf, Robert S. (1998). Proof, Logic, and
Conjecture / The Mathematician's Toolbox. W.
H. Freeman and Co. ISBN 978-0-7167-3050-7.
[44]. Stryer L, Berg JM, Tymoczko JL (2002).
Biochemistry (5th ed.). San Francisco:
W.H.Freeman. ISBN 0-7167-4955-6.
[45]. Schomburg I, Chang A, Placzek S, Söhngen C,
Rother M, Lang M, Munaretto C, Ulas S, Stelzer
M, Grote A, Scheer M, Schomburg D (January
2013). "BRENDA in 2013: integrated reactions,
kinetic data, enzyme function data, improved
disease classification: new options and contents
in BRENDA". Nucleic Acids Research. 41
(Database issue): D764–72.
doi:10.1093/nar/gks1049. PMC 3531171. PMID
23203881
[46]. Radzicka A, Wolfenden R (January 1995). "A
proficient enzyme". Science. 267 (5194): 90–931.
Bibcode:1995Sci...267...90R.
doi:10.1126/science.7809611. PMID 7809611.
[47]. Callahan BP, Miller BG (December 2007). "OMP
decarboxylase—An enigma persists". Bioorganic
Chemistry. 35 (6): 465–9.
doi:10.1016/j.bioorg.2007.07.004. PMID
17889251.
[48]. Michaelis L, Menten M (1913). "Die Kinetik der
Invertinwirkung" [The Kinetics of Invertase
Action]. Biochem. Z. (in German). 49: 333–369.;
Michaelis L, Menten ML, Johnson KA, Goody RS
(2011). "The original Michaelis constant:
translation of the 1913 Michaelis-Menten paper".
55
 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56

Biochemistry. 50 (39): 8264– 9. No. 1, 2017, pp. 1-22.ISSN 2454-390X

doi:10.1021/bi201284u. PMC 3381512. PMID
21888353.
[49]. Briggs GE, Haldane JB (1925). "A Note on the
Kinetics of Enzyme Action". The Biochemical
Journal. 19 (2): 339–339. doi:10.1042/bj0190338.
PMC 1259181. PMID 16743508.
[50]. Ellis RJ (October 2001). "Macromolecular
crowding: obvious but underappreciated".
Trends in Biochemical Sciences. 26 (10): 597–
604. doi:10.1016/S0968- 0004(01)01938-7. PMID
11590012.
[51]. Kopelman R (September 1988). "Fractal reaction
kinetics". Science. 241 (4873): 1620 [52].
Bibcode:1988Sci...241.1620K.
doi:10.1126/science.241.4873.1620.
17820893.
[53]. Segel, L.A.; Slemrod, M. (1989). "The
quasisteady-state assumption: A case study in
perturbation". ThermochimActa31 (3): 446–477.
doi:10.1137/1031091.
[54]. Leskovac, V. (2003). Comprehensive enzyme
kinetics. New York: Kluwer Academic/Plenum
Pub. ISBN 978-0-306-46712-7.
[55]. Greco, W.R.; Hakala, M.T. (1979). "Evaluation
of methods for estimating the dissociation
constant of tight binding enzyme inhibitors,".J
BiolChem254 (23): 12104–12109. PMID 500698.
www.iaiest.com
[58]. Seema Karna Dongare, Manali Rameshrao
Shinde, Vitthalrao Bhimasha Khyade (2018).
Mathematical Inverse Function (Equation) For
Enzyme Kinetics. Research Journal of Recent
Sciences. Res. J. Recent Sci. Vol. 7(12), 9-15,
December
(2018).
PMID
www.isca.in, www.isca.me
[59]. Seema Karna Dongare, Manali Rameshrao
Shinde, Vitthalrao Bhimasha Khyade (2018).
Mathematical Inverse Function (Equation) For
Enzyme Kinetics. International Journal of
Scientific Research in Chemistry (IJSRCH) |
Online ISSN: 2456-8457 © 2018 IJSRCH |
Volume 3 | Issue 4: 35– 42.
http://ijsrch.com/archive.php?v=3&i=6&pyear=2
018

[56]. Hayakawa, K.; Guo, L.; Terentyeva, E.A.; Li,

X.K.; Kimura, H.; Hirano, M.; Yoshikawa,K.;
Nagamine, T. et al. (2006). "Determination of Cite this article as :
specific activities and kinetic constants of
biotinidase and lipoamidase in LEW rat and Vitthalrao Bhimasha Khyade, Seema Karna Dongare,
Lactobacillus casei (Shirota)". J Chromatogr B M
AnalytTechnol Biomed Life Sci844 (2): 240– an
50.doi:10.1016/j.jchromb.2006.07.006. PMID ali
16876490. R
[57]. Vitthalrao B. Khyade; Vivekanand V. Khyade a
and Sunanda V. Khyade (2017). The Baramati m
Quotient for the accuracy in calculation of Km es
value for Enzyme Kinetics. International hr
Academic Journal of Innovative Research Vol. 4, ao

International Journal of Scientific Research in Chemistry (www.ijsrch.com) | Volume 4 | Issue 1 56

 Vitthalrao Bhimasha Khyade et al. Int J Sci Res Chemi. January-February-2019; 4 (1) : 39-56

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International Journal of Scientific Research in Chemistry (www.ijsrch.com) | Volume 4 | Issue 1 59

Jurnal Dinamika, April 2016, halaman 74-82 Vol. 07. No. 1
ISSN 2087 - 7889

PENGENALAN ENZIM AMILASE (ALPHA-AMYLASE) DAN REAKSI

ENZIMATISNYA MENGHIDROLISIS AMILOSA PATI MENJADI GLUKOSA

Ariandi

Program Studi Biologi, Fakultas Sains

Universitas Cokroaminoto Palopo
Email: ariandi.manda@gmail.com

ABSTRAK

Amilase (Alpha-amylase) adalah enzim yang mengkatalisis hidrolisis dari alpha-1,

4glikosidik amilosa pati menghasilkan glukosa. Jumlah glukosa yang dihasilkan selama
reaksi enzimatis diukur dengan menggunakan pereaksi dinitrosalycilic acid (DNS) pada
panjang gelombang 550 nm. Semakin tinggi nilai absorbansi yang dihasilkan, semakin
banyak pula gula pereduksi (glukosa) yang terkandung dalam sampel.Larutan DNS
yang awalnya berwarna kuning akan bereaksi dengan gula reduksi sehingga
menimbulkan warna jingga kemerahan. Kurva standar glukosa nilai persamaan y=
0,0034x + 0,1818, R²=0,9875yang berarti data tersebut termasuk teliti.Berdasarkan data
absorbansi glukosa tereduksi yang dihasilkan dari hidrolisis pati oleh enzim
alphaamilaseterlihat bahwa semakin lama waktu kinerja enzim amilase, semakin
menurun nilai absorbansinya yang berarti kadar glukosanya semakin menurun
(fluktuatif), kemungkinan kenaikan suhu menyebabkan terjadinya proses denaturasi,
bagian sisi aktif enzim akan terganggu dan menyebabkan konsentrasi enzim menjadi
berkurang sehingga kecepatan reaksinya pun akan menurun. Pengujian pati sisa;
persamaan untuk kurva standar pati, y= 8.6x+0.021, nilai R2=0,9908, semua larutan
sampel berwana kuning dan nilai absorbansinya sangat rendah (hampir mendekati nol),
hal ini berarti kemungkinan hampir semua pati yang terkandung dalam larutan telah
terhidrolisis oleh enzim alpha amilase menjadi glukosa. Apabila terdapat amilosa pati
dalam larutan akan berpengaruh dalam pembentukan intentitas warna warna biru-hitam,
hal ini disebabkan oleh adanya molekul iodium (Ion-ion triiodida) yang terikat dalam
kumparan helix amilosa pati.

Kata Kunci: Alpha-amylase, glukosa, amilosa pati

dari enzim amilase adalah untuk memecah
PENDAHULUAN pati dalam makanan sehingga mereka dapat

Amilase diklasifikasikan sebagai digunakan oleh tubuh. Amilase juga

saccharidase (enzim yang memotong disintesis dalam buah tanaman selama

polisakarida). Amilase merupakan enzim pematangan, menyebabkan buah menjadi

pencernaan, terutama dilakukan oleh lebih manis.

pankreas dan kelenjar ludah. Fungsi utama

 Ariandi (2016)
Enzim amilase banyak digunakan dalam dekstrin, oligosakarida dan monosakarida.
industri. Hal ini digunakan dalamindustri Alphaamilase adalah endo-amilase.
pembuatan dan fermentasi bir untuk Exoamylases menghidrolisis alpha
konversi pati menjadi gula terfermentasi. 1,4glikosidik linkage hanya dari
Pada industri tekstil, amilase digunakan nonpereduksi ujung rantai polisakarida luar.
untuk merancang tekstil, kemudian pada Exoamylases termasuk beta-amilase dan
industri deterjen, amilase tercampur dengan glucoamylases (gamma-amilase, amyloglu-
enzim protease dan lipase sebagai pencuci cosidases) (Aiyer, 2005).
noda pakaian dan dalam industri makanan Mekanisme kerja enzim α-amilase terdiri
digunakan untuk pembuatan sirup manis, dari dua tahap, yaitu : tahap pertama
untuk meningkatkan konten diastase degadasi amilosa menjadi maltosa dan
tepung, untuk modifikasi makanan bayi, maltotriosa yang terjadi secara acak.
dan menghilangkan pati dalam produksi Degadasi ini terjadi sangat cepat dan diikuti
jelly. dengan menurunnya viskositas dengan
Amilase adalah enzim yang cepat. Tahap kedua terjadi pembentukan
mengkatalisis hidrolisis dari alpha- glukosa dan maltosa sebagai hasil akhir dan
1,4glikosidik polisakarida untuk tidak acak. Keduanya merupakan kerja
menghasilkan dekstrin, oligosakarida, enzim α-amilase pada molekul amilosa.
maltosa, dan D-glukosa. Amilase bisa Pada molekul amilopektin kerja α-amilase
berasal dari hewan, jamur, dan sumber akan menghasilkan glukosa, maltosa dan
tanaman. Pancreatin dan pancrelipase satu seri α-limit dekstrin, serta
mengandung amilase yang berasal dari oligosakarida yang terdiri dari empat atau
pankreas hewan, pankreas biasanya babi. lebih glukosa yang mengandung ikatan α-
Amilase juga berasal dari malt barley dan 1,6-glikosidik (Winarno, 2010).Tujuan dari
jamur Aspergillus oryzae (Wang, 2009). penelitian ini adalah untuk mengukur kadar
Ada beberapa tipe amilase yang berbeda glukosa yang terbentuk dari reaksi

Enzim ini diklasifikasikan sesuai dengan enzimatis alfa alpha amilase dan mengukur
cara memotong ikatan glysosidic. Alpha- kadar pati sisanya.
amilase menghidrolisis alpha 1,4- TINJAUAN PUSTAKA
glikosidik, secara acak menghasilkan
Ada beberapa tipe amilase yang berbeda
Enzim ini diklasifikasikan sesuai dengan

75
 Pengenalan Enzim Amilase (Alpha-Amylase) dan Reaksi Enzimatisnya
Menghidrolisis Amilosa Pati Menjadi Glukosa

cara memotong ikatan glikosidik. Alpha- nonpereduksi ujung rantai polisakarida luar.
amilase menghidrolisis alpha 1,4- Exoamylases termasuk beta-amilase dan
glikosidik, secara acak menghasilkan glucoamylases (gamma-amilase, amyloglu-
dekstrin, oligosakarida dan monosakarida. cosidases) (Aiyer, 2005).
Alphaamilase adalah endo-amilase.

Exoamylases menghidrolisis alpha

1,4glikosidik linkage hanya dari
METODE
Enzim α-amilase memiliki gugus
karboksil dan nitrogen pada sisi aktifnya. Proses pengujian hidrolisis pati: Mengisi
Substrat membentuk komplek adsorpsi setiap tabung reaksi dengan larutan pati
dengan enzim dimana posisi ikatan 0,05%, kemudian menambahkan 0,5 ml
glukosidik dalam posisi saling berhadapan larutan enzim alpha-amilase yang telah
dengan gugus karboksil dan kelompok terencerkan 1000 kali. Selanjutnya
imidazol. Karboksil anion menyerang memasukkan sampel ke dalam penangas air
bagian nukleofil C (1) dari substrat yang pada suhu 90oC dan mengambil tabung
bertujuan untuk menetralkan rantai ion pada waktu 0, 10, 20, 30, 40, 50, 60 menit.
amidazol. Pada reaksi deglukosilasi, Pengamatan gula yang terbentuk:
kelompok imidazol menjadi dasar untuk mengambil sampel pada setiap tabung
memisahkan komponen air pada posisi C sebanyak 1 ml dan menambahkan 3 ml
larutan DNS,
kemudian
memanaskannya
selama 5 menit pada
air mendidih lalu
mendinginkannya.
Mengukur absorbansi
Gambar 1. Struktur kompleks pati dan Iodine. sampel dengan
(Ophardt, Charles E. 2003. Elmhurst College; Virtual Chembook).
spektrofotometer
(1) (Naz, 2002). Aktivitas enzim α-amilase
pada panjang gelombang 550 nm.
dapat diukur berdasarkan penurunan kadar
Membuat kurva standar glukosa pada
pati yang larut atau jumlah gula pereduksi
konsentrasi 100, 150, 200, 250, dan 300
yang terbentuk (Judoamidjojo et al. 1992)
ppm.

76
 Ariandi (2016)
Pengamatan pati sisa: mengambil enzimatis dengan enzim alpha amilase
sampel pada setiap tabung sebanyak 1 ml untuk mendegradasi ikatan tersebut. Enzim
dan menambahkan 0,1 ml larutan Iod alpha amilase dapat memecah pati secara
konsentrasi 0,2% kemudian mengocoknya acak dari tengah atau bagian dalam molekul
sampai homogen dan menambahkan 3 ml pati.
aqudes. Mengukur absorbansi sampel
dengan spektrofotometer pada panjang Pengamatan Glukosa yang terbentuk
gelombang 660 nm. Membuat kurva
standar pati pada konsentrasi 0,015; 0,020; Pengamatan glukosa yang terbentuk dari
0,250; dan 0,030%. reaksi enzimatis alpha amilase dengan cara
mengambil 1 ml cairan supernatan. Enzim
HASIL DAN PEMBAHASAN alpha amilase akan bekerja dengan cara
bereaksi dengan molekul substrat (pati),
Molekul amilosa sebagian besar terdiri sehingga akan menghasilkan senyawa
dari rantai tunggal dengan 500 sampai glukosa. Enzim amilase menghidrolisis
20.000 ikatan α-1,4-D-glukosa. ikatan glikosidik β-1,4, sehingga amilosa
Amilosa dapat membentuk “extended terurai menjadi glukosa (Lynd, 2002).
shape” cenderung berakhir menjadi Setelah itu ditambahkan 3 ml DNS dan
kumpuran heliks. Heliks tunggal amilosa menginkubasinya pada suhu 100oC selama
memiliki ikatan hidrogen antara atom ±5 menit. Jumlah glukosa yang dihasilkan
oksigen nomor 2 dan atom oksigen nomor selama reaksi enzimatis diukur dengan
6 pada permukaan luar helix dengan menggunakan pereaksi asam dinitro salisilat
mengarah ke dalam cincin oksigen atau dinitrosalycilic acid (DNS) pada
(Wang, 2009). panjang gelombang 550 nm. Semakin tinggi
Ikatan alpha-1,4-D-glukosa dalam nilai absorbansi yang dihasilkan,
amilosa pati akan kita lakukan reaksi
semakin banyak pula gula pereduksi sampel. (glukosa) yang
terkandung dalam

77
 Pengenalan Enzim Amilase (Alpha-Amylase) dan Reaksi Enzimatisnya
Menghidrolisis Amilosa Pati Menjadi Glukosa

1.4
y = 0,0034x + 0,1818
1.2
R² = 0,9875
1

Absorbansi
0.8
0.6
0.4
0.2
0
0 50 100 150 200 250 300 350
Konsentrasi (ppm)

Grafik 1. Kurva standar glukosa

Tabel 1. Konsentrasi Glukosa yang terbentuk dari hidrolisis pati
Waktu Sampel 1 Sampel 2
(Menit)
Absorbansi Konsentrasi Absorbansi Konsentrasi
0 0.786 177,706 0.803 182,706
10 0.547 107,412 0.668 143,000
20 0.524 100,647 0.638 134,176
30 0.510 96,529 0.692 150,059
40 0.463 82,706 0.638 134,176
50 0.479 87,412 0.643 135,647
60 0.433 73,882 0.648 137,118

200.000
180.000
160.000
Konsentrasi glukosa

140.000
120.000
100.000
80.000
60.000
Sampel 1
40.000
Sampel 2
20.000
0.000
0 10 20 30 40 50 60 70
Waktu (menit)

Grafik 2. Kurva konsentrasi glukosa yang terbentuk dari hidrolisis pati

Berdasarkan kurva standar glukosa = 0,0034x + 0,1818, R²=0,9875 yang diatas terlihat
bahwa nilai persamaan y berarti data tersebut termasuk teliti.

78
 Ariandi (2016)
Walaupun nilai R belum mencapai 0,99, 3,5dinitrosalicylic acid (DNS). DNS
kemungkinan hal ini disebabkan oleh faktor merupakan senyawa aromatis yang akan
teknis (keakuratan/ketelitian alat) dan bereaksi dengan gula reduksi maupun
ketidak-telitian praktikan dalam melakukan komponen pereduksi lainnya untuk
pengukuran, seperti dalam proses membentuk 3-amino-5-nitrosalicylic acid,
pemipetan larutan dengan menggunakan suatu senyawa yang mampu menyerap
pipet mikro yang tidak teliti. dengan kuat radiasi gelombang
Berdasarkan data absorbansi glukosa elektromagnetik pada 540 nm. Semakin
yang terbentuk yang dihasilkan dari banyak komponen pereduksi yang terdapat
hidrolisis pati oleh enzim alpha-amilase, dalam sampel, maka akan semakin banyak
data terlihat bahwa semakin lama waktu pula molekul 3-amino-5-nitrosalicylic acid
pemanasan kinerja enzim amilase, semakin yang terbentuk dan mengakibatkan serapan
menurun nilai absorbansinya, yang berarti semakin tinggi (Sazciet.al. 1986)
kadar glukosanya semakin menurun Reaksi dengan DNS yang terjadi
(fluktuatif). Berdasarkan teori seharusnya merupakan reaksi redoks pada gugus
semakin lama enzim bekerja pada suhu aldehid gula dan teroksidasi menjadi gugus
tinggi yang optimal (enzim termofilik), karboksil. Sementara itu DNS sebagai
maka reaksi enzim berlangsung lebih cepat. oksidator akan tereduksi membentuk 3-
o
Setiap peningkatan suhu 1 C dapat amino dan 5nitrosalicylic acid. Reaksi ini
meningkatkan rata-rata reaksi lebih 10% berjalan dalam suasana basa. Bila terdapat
sampai mencapai suhu optimal, setelah itu gula reduksi pada sampel, maka larutan
enzim menjadi tidak aktif (Illanes, 2008 DNS yang awalnya berwarna kuning akan
dalam Heryanto, 2012). Selain itu, karena bereaksi dengan gula reduksi sehingga
enzim merupakan protein, maka menimbulkan warna jingga kemerahan
kemungkinan kenaikan suhu dapat (Sastrohamidjojo, 2005)
menyebabkan terjadinya proses denaturasi,
apabila hal tersebut terjadi, maka bagian Pengamatan Pati Sisa
sisi aktif enzim akan terganggu dan
menyebabkan konsentrasi enzim menjadi Amilosa patiberbentuk helix tunggal
berkurang sehingga kecepatan reaksinya memiliki bentuk mirip dengan siklodekstrin
pun akan menurun dengan memiliki permukaan bagian dalam
Metode penentuan komposisi gula yang relatif hidrofobik yang dapat berikatan
reduksi dalam sampel menggunakan molekul dengan air, yang relatif mudah
pereaksi asam dinitro salisilat atau

79
 Pengenalan Enzim Amilase (Alpha-Amylase) dan Reaksi Enzimatisnya
Menghidrolisis Amilosa Pati Menjadi Glukosa

hilang akan digantikan oleh lipid hidrofobik Pengujian pati dengan melakukan
atau molekul aromatik penambahan Iodium-reagen KI pada larutan
(Aiyer, 2005). pati. Jika amilosa pati terdapat dalam
Karakteristik ini dapat mengikatkan larutan, maka akan menghasilkan warna
rantai amilosa dengan molekul Iodium biru-hitam. Jika amilosa pati tidak hadir,
(misalnya, polyiodides, rantai I3- dan I5dan maka warna akan tetap oranye atau kuning.
membentuk struktur seperti I93-dan I153-). Untuk Amilopektin pati, selulosa, ataupun
Setiap lingkaran helix amilosa disakarida seperti sukrosa yang terdapat
dapatmengikat sekitar dua atom iodium dan dalam larutan tidak akan memberikan efek
warna biru dihasilkan karena adanya warna.
interaksi donor-akseptor antara air dan
polyiodides yang kekurangan elektron
(Ophardt, 2003).
Berdasarkan data yang didapatkan,
nilai kurva standar pati R2=0,9908, nilai
tersebut termasuk teliti, dengan nilai
persamaan y=8.6x + 0.021.Berdasarkan data,
semua larutan sampel berwana kuning dan
nilai absorbansinya sangat rendah mendekati
nol, hal ini berarti hampir semua pati yang
terkandung dalam larutan telah terhidrolisis
oleh enzim alpha amilase menjadi glukosa.
Hal ini kemungkinan

0.3
0.25
Absorbansi

0.2
0.15 y = 8.6x + 0.021
R² = 0.9908
0.1
0.05
0 80
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035
Konsentrasi (%)

Grafik 3. Kurva standar pati

Ariandi
(2016)
diseb el
abkan langs
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81
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 Pengen
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82