Fundamental Laboratory Approaches
for Biochemistry and Biotechnology

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by Alex J. Ninfa and David P. Ballou

Notes on Experiments Homework Answers Book Revisions Contact Us Authors Home Links		Chapter 9, p228. Question 9-1. The largest source of error will be due to the inaccuracy of the instrument in measuring absorbance at 340 nm. In an instrument such as the Spec 20, a tungsten lamp gives very little intensity at 340 nm. In addition, the 20 nm bandpass may influence the apparent extinction coefficient. The round test tubes are not ideal for reproducibly passing light through the sample. Question 9-2. Start with an excess of NADP and measure the appearance of NADPH.
		Question 9-3. Couple the glucose 6-phosphate dehydrogenase reaction to another reaction that converts glucose to glucose-6-P, such as the hexokinase reaction. Then, measure glucose-6-phosphate by measuring the formation of NADPH from NADP in the glucose-6-P dehydrogenase reaction. If the coupling system is in excess and the reaction can be driven to 6-phosphogluconate, the amount of NADPH formed will reflect the amount of glucose in the original sample. Question 9-4. We added NAD(P) in several aliquots to ensure that a linear response was obtained. This ensures that all other components were present in excess and that under the conditions used the only limiting factor was the concentration of NAD(P). Chapter 9, p231. Question 9-5. You should observe very little effect upon adding more enzyme. The enzyme allows equilibrium to be reached faster, but does not alter the equilibrium distribution of substrates and products, except as noted above for question 9-3. Question 9-6. No, see above. Question 9-7. Yes, within experimental uncertainty. The values you obtain for equillibria will be different, but the Keq calculated should be the same. Question 9-8. The values you obtain should be the same. Try and calculate the experimental uncertainties and how they combine to contribute to the calculated Keq values. Chapter 9, p233. Question 9-9. The molecular mass of ethanol is 46. Therefore a solution containing 0.05% (0.5 mg/mL) is about 11 mM. Assume that the extinction coefficient for NADH at 340 nm is 6.2/mM/cm, the path is 1 cm, and the final volume is 4 mL. 0.3 = (6.2/mM/cm)(11 mM)(x/4 mL) x = volume needed = .018 mL of sample Question 9-10. To determine NAD, use excess ethanol and measure the appearance of NADH. To measure acetaldehyde, omit the semicarbazide and start with excess NADH at low pH. That is, drive the reaction to the left and measure the appearance of NAD. The inclusion of another enzyme that converts ethanol to another product would facilitate driving the reaction to completion.