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)

Bobot x skor

1 Format 5

2 Abstract 10

3. Introduction 20

4. Materials and Methods 10

5. Result and discussion 30

6. Conclusion 10

7. Acknowledgment 5

8. References 5

9. Clear 5

Total score 100

Singaraja, 13 April 2015

Penilai,

Dr. I Nyoman Tika, M.Si

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 three-dimensional 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 + Sk1

k2

ESk3

k4

E + P

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).

Figure 1. Relationship between substrate concentration and Vo plot directly

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

plot of Lineweaver Burk

Figure 1 filled the equation of Michaelis-

Menten as follows.

[S]K

[S]VoV

M

max

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]

3k

2k

[P]4

k

3k

2k

[S]1

k

[E]

[ES]

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

=

1

32

k

kk . The equation becomes:

[S]

MK

[ES]

[E]

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],

[S]

MK

1[ES]

t[E]

[ES]

[ES]t[E]

[ES]

[E]

1[S]

MK

[ES]

t[E]

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.

1[S]

MK

V

Vso

[ES]

t[E]

V

V maxmax

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):

M

max

K

[S]VV

Because KM is 1

32

k

kk so that Ks =

1

32

k

kk

So KM will always be equal to or greater than Ks. Judging from the equation:

maxmax

M

M

max

V

1

[S]

1

V

K

V

1so

[S]K

[S]VV

Above equation is identical to the straight line equation y = ax + b

Where y =maxmax

M

V

1band

V

Ka;

[S]

1x;

V

1

oV

1is

maxV

1, the crossing point on the axis

[S]

1

isMK

1 . Equal to the slope of the line

maks

M

V

K

Lineweaver-Burk plot is also very useful for

Vmax

Vo

Vmax

2

0

0

KM 2KM 3KM 4KM 5KM

[S]

KM/Vmax

-1/KM

1/V

1/Vmax

01/[S]

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

No Tubes Casein

(mL)

Phosphate

Buffer (mL)

Trypsin

(mL)

I t = 0 minute 0.1 5.9 1

t = 20 minutes 0.1 5.9 1

II t = 0 minute 0.5 5.5 1

t = 20 minutes 0.5 5.5 1

III t = 0 minute 1.0 5.0 1

t = 20 minutes 1.0 5.0 1

IV t = 0 minute 3.0 3.0 1 t = 20 minutes 3.0 3.0 1

V t = 0 minute 5.0 1.0 1

t = 20 minutes 5.0 1.0 1

RESULT AND DISCUSSION

Result

After measuring the absorbance of each solutions at = 650 nm by using

spectrophotometer is obtained data as follow:

Table 2. The Absorbance of each solutions at = 650 nm

Test

Tubes

Absorbance

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

was measured by using spectronic 20+. Absorbance measurement results for each

colored solutions are presented in following

table.

Table 3. The result of absorbance measurement in each solution at = 650 nm

Test

Tubes

Absorbance Absorbance

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

1/Vo in each test tube is represent in following table:

Table 4. The value of 1/Vo in each test tube

Test Tube A 1/A = 1/Vo

(mole/minutes)

I 0.210 4.762

II 0.364 2.747

III 0.444 2.252 IV 0.500 2.000

V 0.564 1.773

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

2.0008 grams in 100 mL of distilled water, so

the concentration of casein as substrate as

follow:

[Casein] = mL100

gram2.0008

= 0.02 gr/mL = 20 mg/mL After dilution process until reach the volume of

7 mL, so the concentration of casein as

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:

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.

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.

maxmax

M

V

1

[S]

1

V

K

oV

1

So, based on the linear equation the value of

maxV

1is the value at x = 0, so

y = 0.8248x + 1.926

y = 0.8248 (0) + 1.926

y = 1.926

maxV

1= 1.926

Vmax = 0.5191.926

1 mole/minutes

While the intersection of [S]

1 axis

is -MK

1, this

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

Therefore -MK

1= -2.335 and KM =

2.335

1

=0.428 mg/mL

y = 0.8248x + 1.926R = 0.9814

0

1

2

3

4

5

6

0 1 2 3 4

1/V

o(m

ole

/min

ute

s)

1/[S] (mg/mL)

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

Series1

Linear (Series1)

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