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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
RCH2OH + NAD+
RCHO + NADH + H+
Chemical Reaction
ADH
NAD+ is needed as an important cofactor for ADH
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 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
S. Cerevisiae
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
Data
ALCOHOL DEHYDROGENASE
[Ethanol] M Abs T0 Abs T1
Rate (ΔA340
/min)
Average Rate
0.73+ 3+ -
0.082+2.923 3+ 0.080+2.917 3+ 0.083+
0.3752.466 3+ 0.534+
0.539+1.072 1.102 0.0301.946 3+ 1.054+
0.11.066 2.182 1.116
1.0031.215 2.229 1.0141.283 2.163 0.880
0.050.904 1.443 0.539
0.5520.854 1.47 0.6160.697 1.197 0.500
0.0250.651 1.152 0.501
0.4350.566 0.928 0.3620.636 1.077 0.441
Results Lineweaver-Burke Data and Plot
ALCOHOL DEHYDROGENASE
1/[Ethanol] 1/Abs T1
1.4285 0.539539
2.6667 0.617408
10 0.84685
20 1.13729
40 1.62507
0 5 10 15 20 25 30 35 40 450
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
f(x) = 0.0276960846636675 x + 0.542802013525725R² = 0.993762636749275
1/[Ethanol] M
1/A
bsorb
an
ce
34
0n
m
Results Vmax – The fastest the enzyme can perform
under ideal circumstances 0.0511 M/min
Km – Michaelis constant (several rate constants) 1.8423 mM
ALCOHOL DEHYDROGENASE
3D rendering of ADH
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
ReferencesBendinskas, 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
ALCOHOL DEHYDROGENASE