1. Alcohol Dehydrogenase
A comprehensive overview and experimental analysis
Elliott Weideman

2. 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.

3. Alcohol dehydrogenase
Chemical Reaction
RCH2OH + NAD+
RCHO + NADH + H+
NAD+ is needed as an important cofactor for ADH
ADH

4. 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.

5. Alcohol dehydrogenase
S. Cerevisiae
Materials
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

6. 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

7. 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

8. 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]

9. Alcohol dehydrogenase
[Ethanol] M
Abs T0
Abs T1
Rate (ΔA340 /min)
Average Rate
0.7 3+ -
0.082+
2.923
0.080+
2.917
0.083+
0.375
2.466
0.534+
0.539+
1.072
1.102
0.030
1.946
1.054+
0.1
1.066
2.182
1.116
1.003
1.215
2.229
1.014
1.283
2.163
0.880
0.05
0.904
1.443
0.539
0.552
0.854
1.47
0.616
0.697
1.197
0.500
0.025
0.651
1.152
0.501
0.435
0.566
0.928
0.362
0.636
1.077
0.441
Data

10. Alcohol dehydrogenase
Results
Lineweaver-Burke Data and Plot
1/[Ethanol]
1/Abs T1
1.4285
2.6667 10 20 40

11. Alcohol dehydrogenase
Results
Vmax – The fastest the enzyme can perform under ideal circumstances
M/min
Km – Michaelis constant (several rate constants) mM
3D rendering of ADH

12. 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

13. 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

14. Alcohol dehydrogenase
Discussion
Application
Cirrhosis of liver – condition marked by chronic liver disease and subsequent degeneration

15. Alcohol dehydrogenase
References
Bendinskas, K., DiJiacomo, C., Krill, A., & Vitz, E Kinetics of alcohol dehydrogenase – catalyzed oxidation of ethanol followed by visible spectroscopy, Journal of Chemical Education, 82(7),
Edenberg, H. J 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 J. Am. Chem. Soc, 130,
Laboratory Manual, 2012.
Dr. Christenson