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담당교수: Justyn Jaworski 교수님 학 과: 화학공학과 학 번: 2012007558 이 름: 라우 (8 조) 실험일자: 2014 년 11 월 25 일
담당교수: Justyn Jaworski 교수님 학 과: 화학공학과 학 번: 2012007558 이 름: 라우 (8 조) 실험일자: 2014 년 11 월 25 일
담당교수: Justyn Jaworski 교수님 학 과: 화학공학과 학 번: 2012007558 이 름: 라우 (8 조) 실험일자: 2014 년 11 월 25 일
: 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:
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
1
[S]
1
Vmax
Color change
L-abrine + MTOX
Colorless
Light blue
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.
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