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In the conversion of pyruvate to lactate, NADH is required. The oxidized NAD+ is later reduced by FADH2.
In the conversion of pyruvate to lactate, NADH is required. The oxidized NAD+ is later reduced by FADH2. The reaction can be monitored by UV spectroscopy since the oxidized form FAD is an intense yellow and the reduced from FADH2 is pale yellow. The reaction is catalyzed by lactate dehydrogenase. Pyruvate + NADH -lactate dehydrogenase----> Lactate + NAD+ 2NAD+ + FADH2 -----> 2NADH + FAD A student needed to collect data to determine the Michaelis -Menten constant for lactate dehydrogenase under physiological conditions. Ten data points should be collected. The protocol calls for the following FINAL conditions and concentations of reactants: Reaction volume 1 mL Acetate buffer - pH=6 Pyruvate 10mM --to -- 100 mM FADH2 15 mM NAD+ 30 mM Enzyme - 50uM The following is available in the laboratory: Acetic acid - 6 M Sodium Acetate solid Pyruvate solid FADH2 solid NAD+ solid Lactate dehydrogenase 0.1mM distilled water Prepare a table noting the contents of each of the appropriate 10 assay tubes. (Explain how to prepare each sample) Your final concentrations must match those required. Your solutions made must be practical and cost effective. You must prepare the buffer properly. (note - your answer must be practical - i.e., you must be able to accomplish the task in the laboratory as described.)
