Alcohol Dehydrogenase
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Transcript Alcohol Dehydrogenase
ALCOHOL DEHYDROGENASE
A comprehensive overview and experimental analysis
Elliott Weideman
ALCOHOL DEHYDROGENASE
What is it?
ADH
Enzyme that breaks down alcohols
Why is it important?
In most animals it is used to metabolize alcohols that
have been ingested.
Yeasts and bacteria use ADH in reverse to convert
sugars into ethanol via fermentation.
ALCOHOL DEHYDROGENASE
Chemical Reaction
RCH2OH + NAD+
RCHO + NADH + H+
NAD+ is needed as an important cofactor for ADH
ADH
ALCOHOL DEHYDROGENASE
Experimental Question
What is the rate of reaction that ADH is capable of
maintaining?
Experimental Design Overview
Chemical assay techniques using spectrophotometry to
measure the amount of product synthesized from ADH.
ALCOHOL DEHYDROGENASE
Materials
S. Cerevisiae
Yeast ADH - Provided from Sigma
NAD+ - Provided from Sigma
95% Ethanol - Provided from Sigma
Tris Buffer pH 8 - Provided from Acros
Equipment
Genesys 10 UV/vis Spectrophotometer - Provided from
ThermoSpectronic
Cuvettes - Provided from Fisher Scientific
ALCOHOL DEHYDROGENASE
Procedure
Obtain Tris buffer - pH 8
Prepare ADH enzyme by adding dry reagent with water to
equal 20 units/mL
Dilute Ethanol to concentrations of
0.7M, 0.375M, 0.1M, 0.5M and 0.25M
C1 V1 =C2 V2
Prepare NAD+ to final concentration of
15mM
ALCOHOL DEHYDROGENASE
Procedure
Enzyme assays by combining the following
900uL Tris buffer
33uL NAD+
33uL ethanol (0.7M, 0.375M, 0.1M, 0.05M, and 0.025M)
33uL ADH
The solution was mixed thoroughly and gently before
quickly being measured in the spectrophotometer at 340nm
ALCOHOL DEHYDROGENASE
Procedure
Assays were measured for absorbance at T0 and T1
Average rate (Abs340 T1 – Abs340T0/min) was recorded
Three trials were run for each [ethanol]
ALCOHOL DEHYDROGENASE
[Ethanol]
Data M Abs T0 Abs T1
0.7
0.375
0.1
0.05
0.025
3+
2.923
2.917
2.466
1.072
1.946
1.066
1.215
1.283
0.904
0.854
0.697
0.651
0.566
0.636
3+
3+
3+
3+
1.102
3+
2.182
2.229
2.163
1.443
1.47
1.197
1.152
0.928
1.077
Rate (ΔA340 /min) Average Rate
0.080+
0.083+
0.534+
0.030
1.054+
1.116
1.014
0.880
0.539
0.616
0.500
0.501
0.362
0.441
0.082+
0.539+
1.003
0.552
0.435
ALCOHOL DEHYDROGENASE
Results
1.8
y = 0.0277x + 0.5428
Lineweaver-Burke Data and Plot
R² = 0.9938
1.6
1/Absorbance 340nm
1/[Ethanol]
1.4
1/Abs T1
1.4285
0.539539 1.2
2.6667
0.617408
1
10
0.84685 0.8
20
1.13729 0.6
40
1.62507 0.4
0.2
0
-30
-20
-10
-0.2
0
10
1/[Ethanol] M
20
30
40
50
ALCOHOL DEHYDROGENASE
Results
Vmax – The fastest the enzyme can perform under ideal
circumstances
0.0511 M/min
Km – Michaelis constant (several rate constants)
1.8423 mM
3D rendering of ADH
ALCOHOL DEHYDROGENASE
Discussion
Drawbacks
Incorrect preparation of trial 2 assay for 0.375M Ethanol
Possible contamination when preparing NAD+
Inconsistent handling of cuvettes when loading into
spectrophotometer
Uniform mixing
Time loading
Use less ADH enzyme to get a more accurate reading of the rate of
reaction
Positive control – Lacking an inhibited version of the enzyme
ALCOHOL DEHYDROGENASE
Discussion
Future Research
Investigate performance of ADH
with other concentrations of ethanol
under different temperatures or levels of pH
Run the reaction backwards by manipulating the equilibrium level
Use other versions of ADH and or types of alcohols
Applications to medical industry involving alcoholism and genetics
ALCOHOL DEHYDROGENASE
Discussion
Application
Cirrhosis of liver – condition marked by chronic liver disease and
subsequent degeneration
ALCOHOL DEHYDROGENASE
References
Bendinskas, K., DiJiacomo, C., Krill, A., & Vitz, E. 2005. Kinetics of alcohol
dehydrogenase – catalyzed oxidation of ethanol followed by visible
spectroscopy, Journal of Chemical Education, 82(7), 1068-1070.
Edenberg, H. J. 2007. The genetics of alcohol metabolism: Role of alcohol
dehydrogenase and aldehyde dehydrogenase variants. Alcohol Research
& Health, 30(1), 5-13.
Voss, C. Gruber, K. Faber, T. Knaus, P. Macheroux, W. Kroutil. 2008. J. Am.
Chem. Soc, 130, 13969-13972.
Laboratory Manual, 2012.
Dr. Christenson