BIOCHEMISTRY EXPERIMENT

ARTICLE
THE DETERMINATION OF ENZYME REACTION KINETIC
(Vmax and KM) OF TRYPSIN IN CATELIZE CASEIN REACTION

By
Ni Kadek Wahyuni Antari
1213031002
A

CHEMISTRY EDUCATION DEPARTMENT

FACULTY OF MATHEMATICS AND NATURAL SCIENCES
UNIVERSITAS PENDIDIKAN GANESHA
2015

Rubrik Penilaian Artikel Praktikum

Biokimia S1

Nama mahasiswa

: Ni Kadek Wahyuni Antari

NIM

: 1213031002

Tanggal

: 13 April 2015

Mata Acara praktikum

: Kinetika Reaksi Enzim

No.

Aspek yang dinilai

Bobot

Skor

(%)

(0-100)

Format

Abstract

10

3.

Introduction

20

4.

Materials and Methods

10

5.

Result and discussion

30

6.

Conclusion

10

7.

Acknowledgment

8.

References

9.

Clear

Total score

100

Singaraja, 13 April 2015

Penilai,

Dr. I Nyoman Tika, M.Si

Bobot x skor

THE DETERMINATION OF ENZYME REACTION KINETIC (Vmax and KM) OF

TRYPSIN IN CATELIZE CASEIN REACTION
Ni Kadek Wahyuni Antari
Chemistry Education Department, FMIPA, UNDIKSHA
Udayana Street Singaraja, Bali
Email: wahyuni.antari@yahoo.co.id
Abstract
Enzyme is a protein that has a specific three-dimensional structure that is able to catalyze biological reactions
(biocatalytic activation). In the kinetics of enzyme reaction, the value of Vmax and KM are determined by using the
graphical relationship between the rates of enzymatic reaction with substrate concentration. The objective of this
experiment was to determine the Kinetics of trypsin enzyme in catalyze casein reactions by calculating the value of
Km and Vmax. for incubation time 20 minutes and 0 minute using Lineweaver-Burk plot. The method used in this
experiment was qualitative and quantitative analysis. Then, the instrument used is spectronic 20+. The result of
this experiment revealed that the value of Km for the enzymatic reaction with the enzyme trypsin and casein
substrate with t = 0 min and t = 20 min was 0.428 mg/mL and Vmax rates of 0.519 mole/minutes respectively.
Keywords: Enzyme, enzyme activities, concentration and reaction rate

INTRODUCTION
Enzyme is biocatalyst that able
increase reaction rate in biology system and
enzymes do not change during reaction. An
enzyme is a protein that has a particular threedimensional structure that is able to catalyze
biological reactions (biocatalytic activity).
Enzymes can increase the reaction rate due to
the presence of the enzyme reaction that occurs
will have a lower activation energy than the
usual reaction. Enzymes assist in providing a
reaction to the reaction pathway has a lower
activation energy for the transition of substrate
into products as compared to the process
without a catalyst, so that the enzyme is often
referred to as a catalyst (Tika, 2010).
Trypsin is one kind of enzyme, which
is a protein that speeds up a certain biochemical
reaction. It has different names, such as
proteinase, proteolytic, and tripsina enzymes.
Trypsin is found in the small intestine. It can
also be made from fungus, plants, and bacteria.
But it is usually made for commercial purposes
from the pancreas of livestock. Trypsin is given
to people who lack enzymes needed for
digestion. It is also given in combination with
bromelain and routine for treatment of
osteoarthritis. Some people apply trypsin
directly to wounds and ulcers to remove dead
tissue and improve healing (WebMD, 2014).
Enzymes have specific shapes and
structures that determine their functions. The
enzymes active site is very selective, allowing
only certain substances to bind. If the shape of

an enzyme is changed in any way, or the protein

denatured, then the binding site also changes,
thus disrupting enzymatic functions (Murray,
1995). Measurement of enzyme levels can be
done in two ways, namely: (1) as compared
with pure enzyme, (2) measuring the reaction
rate its catalyzed. At the lab is done by
determining the levels of the enzyme reaction
rate catalyzed (Tika, 2010).
Enzyme activity is defined as the
amount of enzyme that will convert a certain
amount of S to P in a specified period of time
under conditions of constant temperature and
pH (Wirahadikusuma, 2001). Circumstances
where the maximum initial reaction rate (Vmax)
was achieved on saturated substrate conditions.
This can be explained by the postulate of
reaction as follows.

E+S

k1
k2

ES

k3

E+P

k4

Where E, S, and P are each enzyme, substrate,

and reaction products respectively.
The effect of substrate concentration
when substrate concentration is increased two
times, then the initial reaction velocity (Vo) has
doubled. Michaelis and Menten stated that the
enzymatic reaction at various concentrations of
substrates having two phases: 1) when [S] is
low, not all of the active enzyme bound to the
enzyme, 2) on [S] is high, the active side was
entirely bound to the substrate. At this time the

enzyme has been working at full capacity

(Poedjadi,1994).
Vmax
Vo
Vmax

[ES]

[ES]

2KM

KM

3KM 4KM 5KM

[S]

Figure 1. Relationship between substrate

concentration and Vo plot directly

1/V

KM/Vmax
-1/KM
1/Vmax

1/[S]

Figure 2. Relationship between 1/Vo and 1/[S]

plot of Lineweaver Burk
Figure 1 filled the equation of MichaelisMenten as follows.
V [S]
Vo max
K M [S]
Where: Vo is reaction rate enzyme and
substrate [S] and KM is constant of Michaelis
Menten (mol/L) and Vmax is maximum rate of
enzyme
KM is a measure of the ES complex
stability, the speed of decomposition of the
complex ES with ES Kompeks formation rates.
The specificity of a substrate that is catalyzed
by first looking at the formation of complex
enzyme substrate (ES), which then decomposes
into enzyme and products (E and P). In this case
k1, k2, k3,k4 is the reaction rate constant. Initially
is achieved disintegration of the state of the
enzyme rate equal to the speed of formation of
ES.
k1[S][E] + k4 [E][P] = k2 [ES] + k3 [ES]

k 2 k3

k 4 [P]

k2 k3
. The equation becomes:
k1

[E]

k1 [S]

k 2 k3
In these conditions the concentration of P
(product) is very little that is ignored. Constants
k1, k2, k3 and k4 is written as a constant KM. KM
[E]

KM

[S]
If the total amount or concentration of
enzyme [E]t is considered as the sum of the free
enzyme, [E] and joined the substrate [ES] is the
concentration [E] = [E]t - [ES],
K
[E] [E]t [ES] [E]t

1 M
[ES]
[ES]
[ES]
[S]
[E]t K M

1
[ES] [S]
So the maximum rate (Vmax) when the
enzymes are all in the form of complexes with
substrates. While Vo is proportional to [ES]. It
can be written as follows.
Vmax [E]t
Vmax K M

so

1
V
[ES]
V
[S]
In Figure 1 it can see that KM is
expressed as moles per liter. And KM is very
large then the equation is written as follows
(Redhana, 2003):

Vmax [S]
KM

Because KM is

k2 k3
k k3
so that Ks = 2
k1
k1

So KM will always be equal to or greater than

Ks. Judging from the equation:
V

Vmax [S]
K M [S]

so

KM 1
1

V Vmax [S] Vmax
1

Above equation is identical to the straight line

equation y = ax + b
K
1
1
1
Where y = ; x
; a M and b
V
[S]
Vmax
Vmax

1
1
1
is
, the crossing point on the axis
[S]
Vo Vmax
K
1
is
. Equal to the slope of the line M
Vmaks
KM
Lineweaver-Burk plot is also very useful for

determining the type inhibition enzymatic

reactions that occur in (Tika, 2010).
MATERIALS AND METHOD
This experiment was conducted at
Organic Chemistry Laboratory of Chemistry
Education Department, Faculty of Mathematics
and Natural Sciences, Ganesha Education
University on 30th March 2015.
Materials
Materials used in this experiment were
TCA solution 20% (30 mL), casein solution 2%
(20 mL), phosphate buffer 0.1 M (pH 8.0) (42
mL), trypsin solution (10 mL), NaOH solution
0.5 M (40 mL), follin-ciocalteu reagnet (10
mL), and distilled water.
Equipment
Equipment used in this experiment
were test tubes (10 units), test tube rack (1 unit),
volumetric pipette 5 mL (2 units), volumetric
pipette 10 mL (2 units), graduated cylinder 5
mL (2 units), graduated cylinder 10 mL (2
units), stirrer rod (2 units), drop pipette (3
units), beaker glass 500 mL (2 units), centrifuge
(1 unit), spectrophotometer (1 unit), volumetric
flask 100 mL (2 units), and stopwatch.
Procedures
This experiment was carried out
accordance with the procedures adopted from
Penuntun
Praktikum
Biokima
book
developed by I Wayan Redhana and Siti
Maryam (2003: 26-28). There are several
procedures was conducted in this experiment,
as follow:
Making TCA Solution 20%
As much as 20 mL of pure TCA was taken and
diluted until rich the scale of 100 mL.
Making Casein Solution 2%
Casein powder was weighed as much as 2.0008
grams and mixed with water until reach the
scale of 100 mL.
Makin Trypsin solution
Trypsin solution was made by dissolved
enzymefort as much as 0.5001 grams into 50
mL of water.
Making Folin-Ciocalteu Reagent
As much as 1 mL of pure folin-ciocalteu was
taken and diluted until reach the scale volume
of 10 mL.
Making Phosphate Buffer 0.1 M (pH 8.0)

As much as 94 mL of K2HPO4 solution 1 M was

mixed with 6 mL of KH2PO4 solution 1 M and
the pH was checked by pH meter.
Making NaOH solution 0.5 M
As much as 0.2002 grams of solid NaOH was
weighed and dissolved with water until reach
the scale volume of 100 mL.
Incubation t = 20 minutes
Five test tubes were prepared and give label 1,
2, 3, 4, 5. A total of 5 mL of casein solution 2
% were added into all test tubes and incubated
in water bath for 5 minutes at 35oC. Then it was
added a solution of phosphate buffer and
trypsin solution while stirring slowly. After
that, it was incubated in water bath for 20
minute at 35oC calculated from the enzyme is
added. The reaction was stopped by addition of
3 mL 20% TCA solution is accompanied with
strong stirring. Furthermore, it was settling for
30 minutes in ice water so that the deposition of
protein and trypsin could perfectly happen.
After that, the solution was centrifuged for 10
minutes and then filtered. The filtrate obtained
then done according to Anson's method, in
which as many as 2 mL of filtrate was taken and
then added 4 mL of NaOH solution 0.5 M and
1 mL of folin-cioceltau reagent. It was allowed
to stand for 10 minutes. Finally, the absorbance
of each solution in test tube was measured by
using a spectrophotometer.
Incubation t = 0 minute
Buffer phosphate solution was entered into test
tube and then it was added by trypsin solution.
After that, it was added by 3 mL 20% TCA
solution and incubate in water bath for 30
minutes at 35oC. Last one was added 1% casein
solution as much as 5 mL and stirred strong. It
was incubated for 30 minutes in ice water
calculated from the casein solution is added.
After that, the solution was centrifuged for 10
minutes and then filtered. The filtrate obtained
then done according to Anson's method, in
which as many as 2 mL of filtrate was taken and
then added 4 mL of NaOH solution 0.5 M and
1 mL of folin-cioceltau reagent. It was allowed
to stand for 10 minutes. Finally, the absorbance
of each solution in test tube was measured by
using a spectrophotometer.
The composition of solution in each test tube
was followed the following table.

Table 1. Addition of solution to each tube

Casein
No
Tubes
(mL)
t = 0 minute
0.1
I
t = 20 minutes
0.1
t = 0 minute
0.5
II
t = 20 minutes
0.5
t = 0 minute
1.0
III
t = 20 minutes
1.0
t = 0 minute
3.0
IV
t = 20 minutes
3.0
t = 0 minute
5.0
V
t = 20 minutes
5.0

Phosphate
Buffer (mL)
5.9
5.9
5.5
5.5
5.0
5.0
3.0
3.0
1.0
1.0

Trypsin
(mL)
1
1
1
1
1
1
1
1
1
1

RESULT AND DISCUSSION

Result
After measuring the absorbance of each
spectrophotometer is obtained data as follow:
solutions at = 650 nm by using
Table 2. The Absorbance of each solutions at = 650 nm
Absorbance
Test
Tubes
t = 0 minute
t = 20 minutes
I
0.050
0.260
II
0.056
0.420
III
0.066
0.510
IV
0.090
0.590
V
0.116
0.680
Discussion
In this experiment was analyzed the
influence of substrate concentration on enzyme
activity. In this analysis done two variations,
namely the time spent at 0 minutes and 20
minutes, which given the time variation of this
enzyme shows a length of binding to the
substrate until finally precipitated by TCA. This
enzyme reaction kinetics are determined by the
value of Vmax and Km. Vmax and Km is the value
determined in a way to graph the relationship
between the reaction rate against substrate
concentration.
In this experiment which was used as
the substrate namely casein and an enzyme
trypsin. Trypsin is a proteolytic enzyme, this
enzyme catalyzes the hydrolysis of the bond
peptide, where in the peptide bonds were
broken in the substrate casein is located on the
carboxyl side of lysine or arginine residues are
charged.
Before starting the experiment, it was
prepared ten test tubes filled with a solution of
casein, trypsin and acetate buffer with different
concentrations then given two different
treatments. Five of the ten test tubes given
incubation time 0 minutes and five others were

treated test tube incubation for 20 minutes. For

incubation 20 minutes, firstly carried out the
initial incubation for 5 min at 35oC. The
purpose of this incubation process is to create
condition the substrate at the optimal
temperature (35oC). After it was added with
phosphate buffer solution (pH = 8.0) and
trypsin solution. The addition of trypsin
solution aims to catalyze the hydrolysis of
peptide bonds that exist in casein. The next
solution was incubated for 20 minutes, in which
the incubation of trypsin allows the enzyme
bound to the substrate. After the incubation
process is completed, then the solution was
added a solution of 20% TCA and was
accompanied by stirring. The purpose of the
addition of TCA solution is to stop the enzyme
activity that can be analyzed the influence of
substrate concentration on the rate of enzymatic
reactions. In addition, all of test tubes were
incubate in ice water to accelerate the reaction
stop. The ability of TCA to stop the enzyme
activity is due to the acidic solution of TCA so
as to lower the pH of the solution so as to
denaturate either protein or casein on trypsin.

Regarding the solution to the long

incubation time 0 min, there was little
difference in work procedures. Where in the
fifth test tube were added first phosphate buffer
and trypsin solutions, then continue added
solution of TCA. Having experienced the
process of incubation and then added to the
casein solution with different volume.
After TCA solution stop the enzyme
activity, the solution was centrifuged for 10
minutes and then filtered. The filtrate obtained
was then done by the method of Anson where
as many as 2 mL of filtrate was taken and then
added with 4 mL of 0.5 M NaOH solution and

Folin cioceltau reagent. These reagents can be

reduced by the phenolic group on the amino
acid tyrosine in the filtrate are produce
tungstate and molybdate that has blue color.
The blue color produce in each test tube from
test tube 1 until 5 after the filtrate was done by
Ansons method is darker, in which the first test
tube contained light blue solution, second test
tube contained turkish solution, third test tube
contained bluish solution, forth test tube
contained dark blue solution, and the last test
tube contained blackish blue solution as shown
as following figure.

(a)
(b)
Figure 3. The color of solution after trating by Ansons method for (a) t = 0 minutes (b) t = 20 minutes.
The color from test tube 1 until 5 is more darker (bluish until blackish blue solution)
Then the absorbance of each colored solution
colored solutions are presented in following
was measured by using spectronic 20+.
table.
Absorbance measurement results for each
Table 3. The result of absorbance measurement in each solution at = 650 nm
Absorbance
Test
Absorbance
Tubes
t = 0 minute
t = 20 minutes
I
0.050
0.260
0.210
II
0.056
0.420
0.364
III
0.066
0.510
0.444
IV
0.090
0.590
0.500
V
0.116
0.680
0.564
Based on the result above, the value of 1/Vo can
be calculated by using the value of absorbance
because 1/A = 1/Vo. The result calculation of
Table 4. The value of 1/Vo in each test tube
Test Tube

I
II
III
IV
V

0.210
0.364
0.444
0.500
0.564

Then, to make the graph of enzymatic reaction

kinetic is need to calculate the concentration of
substrate or in this case is the concentration of
casein solution in each test tube after diluting
process. The mass of casein is added is equal to

1/Vo in each test tube is represent in following

table:

1/A = 1/Vo
(mole/minutes)
4.762
2.747
2.252
2.000
1.773
2.0008 grams in 100 mL of distilled water, so
the concentration of casein as substrate as
follow:

[Casein] =

substrate in each test tube can be known by

using the equation V1 x M1 = V2 x M2. The result
of calculation toward the concentration of
casein in each test tube represent as following
table:

2.0008gram

100 mL
= 0.02 gr/mL = 20 mg/mL
After dilution process until reach the volume of
7 mL, so the concentration of casein as
Table 5. The concentration of casein solution as substrate in each test tube after diluting process
Test Tube
[S]
1/[S] (mg/mL)
I
0.286
3.496
II
1.429
0.700
III
2.857
0.350
IV
8.571
0.117
V
14.286
0.070

After the value of 1/Vo and 1/[S] is obtained, it

can be made the curve of relationship between

1/Vo and 1/[S] as the following figure.

The Curve of Relationship between 1/Vo and 1/[S]

1/Vo (mole/minutes)

y = 0.8248x + 1.926
R = 0.9814

5
4
3

Series1

Linear (Series1)

1
0
0

1/[S] (mg/mL)
Figure 4. The graph of relationship between 1/Vo and 1/[S]
The resulting graph between 1/Vo and 1/[S] is
produce a straight line. The linear equation of
the graph is y = 0.8248x + 1.926 with R =
0.9814.
Then, by combining this linear
equation with equation from Lineweaver-Burk,
so the value of 1/Vmaks, 1/[S], and -1/Km could
be obtained. The equation of Lineweaver-Burk
is as follow.

KM 1
1

Vo Vmax [S] Vmax
1

So, based on the linear equation the value of

1
is the value at x = 0, so
Vmax
y = 0.8248x + 1.926
y = 0.8248 (0) + 1.926
y = 1.926

1
Vmax

= 1.926

Vmax =

1
1.926

0.519 mole/minutes

While the intersection of

, this
[S]
KM
value is equal with value when y = 0, so
y = 0.8248x + 1.926
0 = 0.8248x + 1.926
-1.926 = 0.8248x
x = -2.335
1
1
Therefore = -2.335 and KM =
KM
2.335
=0.428 mg/mL

axis is -

CONCLUSION
Based on the discussion above, it can
be conclude that: (1) Kinetics of enzyme
reactions can be studied and determined by
determining the value of Km and Vmax. (2) The
Value of Km for the enzymatic reaction with the
enzyme trypsin and casein substrate with t = 0
min and t = 20 min was 0.428 mg/mL and
0.519 mole/minutes.
ACKNOWLEDGEMENT
In writing this article, the author has of
a lot of support, guidance and encouragement
from many quarters. For this reason, the author
respectfully thanks for Dr. I Nyoman Tika,
M.Si as lecturer on the practicum, Ms. Dewi as
lecturer assistant, Mr. Dewa Subamia as the
laboratory assistant and also all member of
RKBI12 of chemistry class for always as good
partners.

REFERENCES
Murray, Robert K, dkk. 1995. Biokimia Harper.
Terjemahan. Andri Hartono. Jakarta:
Buku Kedokteran EGC.
Poedjadi, Anna dan Titin Supriyanti. 1994.
Dasar-Dasar Biokimia. Jakarta:
Universitas Indonesia
Redhana, I Wayan dan Siti Maryam. 2003.
Penuntun Praktikum Biokimia.
Singaraja : IKIP Negeri Singaraja
Tika, I Nyoman. 2010. Penuntun Praktikum
Biokimia. Singaraja: Universitas
Pendidikan Ganesha
WebMD. (2014). WebMD. Retrieved April 22,
2014, from http://www.webmd.com/
Wirahadikusuma, Muhamad. 2001. Biokimia
:Protein, Enzim, dan Asam Nukleat.
Bandung : Penerbit ITB