Alcohol Dehydrogenase

Report
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

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