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Provide a 7 pages analysis while answering the following question: Determination of the Kinetic Constants With Three Alcohol Substrates. Prepare this assignment according to the guidelines found in th
Provide a 7 pages analysis while answering the following question: Determination of the Kinetic Constants With Three Alcohol Substrates. Prepare this assignment according to the guidelines found in the APA Style Guide. An abstract is required. The objective of this experiment was to compare the catalytic rate constant, kcat, the Michaelis constant, Km and the apparent second-order rate constant, kcat/ Km , for ethanol, propane-1-ol and butane-1-ol.
The various reagents prepared and used in the experiment included the reaction buffer, 0.15 M sodium pyrophosphate with 1mM dithiothreitol, adjusted to pH 8.5 and room temperature. the three substrate solutions, ethanol (0.24M), propane-1-ol (0.24M) and butane-1-ol (0.24M) all adjusted to room temperature. coenzyme NAD+ solution (15mM). yeast alcohol dehydrogenase ( 58 U/mg. Sigma) prepared as a stock solution containing 0.1 mg protein/ml in distilled water and the protein content was accurately determined by measuring the absorbance at 280nm in comparison with a standard solution (1mg/ml) of yeast enolase having A280 value of 0.894. The stock solution of yeast ADH was suitably diluted with 10 mM potassium phosphate buffer, pH 7.4 containing 1 mg/mL of bovine serum albumin to produce A340 /10s of between 0.015 and 0.025 in the ADH assay with an ethanol concentration of 80 mM. The absorbance readings were taken using two 3-ml plastic cuvettes in a Novaspec spectrophotometer adjusted to 340nm. The instrument was adjusted to zero absorbance with a cuvette containing 3000 l of distilled water prior to taking the test readings.
The assay mixture (total volume, 3000 μl) in the cuvette consisted of 1000μl 0.15 M sodium pyrophosphate buffer with mM dithiothreitol, pH 8. 1000 l of 0.24 M ethanol/propane-1-ol/butane-1-ol (yielding the equivalent of 0.08M ethanol in 3 ml of the assay mixture) or suitable dilutions thereof (yielding 0.04M, 0.02M, 0.01M and 0.005M alcohol substrate, Table 1). 100 l 15 mM NAD+ and the contents mixed well after covering the mouth of the cuvette with parafilm. Lastly, after wiping the optical surfaces of the cuvette, 100 l yeast alcohol dehydrogenase were added and the clock started.
