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: Justyn Jaworski

: 2012007558

: (8 )

: 2014 11 25
: Biosensors and Enzyme Kinetics
: In this laboratory, students will identify the Michaelis-Menton constant (K M) for N-methyl
tryptophan oxidase and determine its Vmax.
:
(a) Enzyme Catalyst

- Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes.1
While catalyst is a substance that speeds up a chemical reaction, but is not consumed by the reaction.2
- In biology, the active site is a small port in an enzyme where substrate molecules bind and undergo
a chemical reaction. This chemical reaction occurs when a substrate collides with and slots into the
active site of an enzyme.3 In our experiment, the substrate is L-abrine and the enzyme is N-methyl
tryptophan oxidase (MTOX).
(b) Enzyme Kinetics: The Enzyme Substrate Complex
- A theory to explain the catalytic action of enzymes was proposed by the Swedish chemist Savante
Arrhenius in 1888. He proposed that the substrate and enzyme formed some intermediate substance,
which is known as the enzyme substrate complex. Only a specific substrate(s) can bond in that site
and this makes the enzyme specific to that substrate.
- The reaction can be represented as:

Figure 1: Enzyme-substrate complex


(c) Detection for L-abrine
- In order to detect for the presence of L-abrine, an enzyme, N-methyl tryptophan oxidase (MTOX) is
used to monitor it. The reaction is shown as below:
L-abrine + O2 Tryptophan + Formaldehyde + H2O2
- From the equation above, we can observe that one of the products is H 2O2. H2O2 can be easily
detected by using horseradish peroxidase (HRP) to oxidize TMB into a blue colored product. As a
result, the hydrogen peroxide is reduced to water and the oxidized form of TMB. Therefore, detection
of L-abrine can be determined by observing the oxidation of TMB.
(d) Beer-Lambert Law and Lineweaver-Burk Plot
- The BeerLambert law, also known as Beer's law relates the attenuation of light to the properties of
the material through which the light is traveling.4 The equation is shown as below:
A= l c
A: absorbance (no units)
: molar absorptivity with units of L mol-1 cm-1

1 http://en.wikipedia.org/wiki/Enzyme_catalysis
2 http://www.chemicool.com/definition/catalyst.html
3 http://en.wikipedia.org/wiki/Active_site
4 http://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law

l : path length of the sample, cm


c: concentration of the compound in solution, mol L-1
- The Lineweaver-Burke plot has the form y = mx + b, and is the formula for a straight line.5
1 = KM
V
Vma x

1
[S]

1
Vmax

V: reaction velocity (the reaction rate)


[S]: concentration of substrate
KM: dissociation constant for ES
Vmax: maximum rate of product formed/time
:
- L-abrine
- Distilled Water
- 2x Tris Buffer (ph 6)
- H2SO4 (will be used by the instructor)
- TMB (3,3',5,5'-Tetramethylbenzidine)
- N-methyltryptophan oxidase (5.8 uM stock)
- Horesradish Peroxidase
- Apparatus and glasswares
* Disposable Microcentrifuge Tubes
* Disposable plastic pipette tip
* Pipettor
:
1. Varying the amount of L-abrine (constant N-methyl tryptophan oxidase concentration)
2. Prepare 4 different concentrations of L-abrine (ie., 32.8uM, 65.6uM, 164uM, 328uM) from the
3.28 mM stock. Prepare 4 samples for each concentration (that is a total of 16 tubes)
4. Add 30uL of Tris Buffer to 30uL of the L-abrine solution.
5. Add 0.2 uL of N-methyl tryptophan oxidase and mix.
6. After 1, 10, 20, and 30 minutes add 60uL of TMB substrate and 0.5uL HRP and stop the reaction
after 5 minute by adding 120 uL of H2SO4.
7. Measure the absorbance at 450 nm for each sample.
:
The result of color changes are shown as below:
Reaction

Color change

L-abrine + MTOX

Colorless

L-abrine + MTOX + TMB

Light blue

5 PPT of Biosensors and Enzyme Kinetics

L-abrine + MTOX + TMB + H2SO4

Yellow

From the result above, we can observe that the color of reactant starts to change from
colorless to light blue due the oxidation of TMB. After that, H 2SO4 is added to prevent fading color of
the reactant.
Absorbance measured at 450nm in 1cm path length of oxidized
TMB (240uL)
[S], Molar concentration of L-Abrine in
60uL reaction catalyzed by MTOX
enzyme (units are in moles/L)

5 minute

10 minutes

20 minutes

30 minutes

0.000164

0.416

0.538

0.819

1.062

0.000082

0.314

0.414

0.549

0.695

0.0000328

0.192

0.243

0.304

0.429

0.0000164

0.102

0.150

0.188

0.239

Table 1: Molar concentration of L-Abrine in 60uL reaction and absorbance measured at 450nm in 1cm
path length of oxidized TMB in varies with time.
Here are the steps in order to find Vmax and KM.
1. Find the concentration of H2O2 by using yellow TMB substrate in the 240uL samples by using
Beer-Lambert law.

A
=[S ]
l

A: Absorbance measured at 450nm in 1cm path length of oxidized TMB (240uL) = Table 1
: molar absorptivity of yellow TMB at 450nm = 5.9 x 104 M-1 cm-1
l : path length of the sample = 1 cm
[S]: Concentration of substrate, yellow TMB
* We will multiply the concentration of yellow TMB substrate in the 240uL by 4 in order to get
the concentration of H2O2 because the actual concentration of H2O2 is 4 times greater than that of the
oxidized TMB observed during absorbance measurement since the sample has been diluted by a factor
of 4 due to addition of H2SO4.

Concentration of H2O2 (240uL) x 105 (mol L-1)


[S], Molar concentration of L-Abrine in
60uL reaction catalyzed by MTOX
enzyme (units are in moles/L)

5 minute

10 minutes

20 minutes

30 minutes

0.000164

2.820

3.647

5.553

7.200

0.000082

2.129

2.807

3.722

4.712

0.0000328

1.302

1.647

2.061

2.908

0.0000164

0.692

1.017

1.275

1.620

2. Plot the concentration of H2O2 generated vs. time and fit a line to the curve in order to find
the slope V in units of moles/(L*min)

3. Use a Lineweaver-Burk plot to determine what is the KM (mole/L) and Vmax (moles/(L*min)) of
MTOX enzyme for the biotoxin marker L-abrine.

[L-abrine Substrate]
(moles/L)

1
[S]

(L/moles)

V {moles/(L min)}

1
V

{(L

min)/moles}
0.000164

6097.56

1.771 x 10-6

564652.74

0.000082

12195.12

1.010 x 10-6

990099.10

0.0000328

30487.80

6.18 x 10-7

1618122.98

0.0000164

60975.61

3.50 x 10-7

2857142.86

4. Find KM (moles/L) and Vmax (moles/(L*min)).


Vmax= 1 / 3.98868
= 2.5 x 10-6 moles/(L min)

Km= 40.404 x Vmax


= 1.013 x 10-4 moles/L