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Alcohol Dehydrogenase

Alcohol Dehydrogenase . A comprehensive overview and experimental analysis. Elliott Weideman. A lcohol d ehydrogenase. 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.

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Alcohol Dehydrogenase

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

  2. Alcoholdehydrogenase • 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. Alcoholdehydrogenase Chemical Reaction RCH2OH + NAD+ RCHO + NADH + H+ NAD+ is needed as an important cofactor for ADH ADH

  4. Alcoholdehydrogenase 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. Alcoholdehydrogenase 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. Alcoholdehydrogenase 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. Alcoholdehydrogenase 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. Alcoholdehydrogenase 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. Alcoholdehydrogenase Data

  10. Alcoholdehydrogenase Results • Lineweaver-Burke Data and Plot

  11. Alcoholdehydrogenase 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

  12. Alcoholdehydrogenase 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. Alcoholdehydrogenase 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. Alcoholdehydrogenase Discussion • Application • Cirrhosis of liver – condition marked by chronic liver disease and subsequent degeneration

  15. Alcoholdehydrogenase 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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