CHEM-342 Introduction to Biochemistry            Name ________________________
Final Examination - Individual Part
Tuesday, 21 May 2002
7:00 - 8:45 PM
H. B. White - Instructor

Important - Please read this before you turn the page.

        There are 7 pages to this part of the examination. Please write your name on each page.

        Part I (75 points) This individual part of the examination, includes 9 problems and short essay

1.    1. (5 points) What is the significance of the increased number of nucleated red blood cells (reticulocytes) that Herrick saw in his patient with sickle-cell disease, a hemolytic anemia? Your answer needs to make a logical connection between the phenomenon and the disease.

2. (5 points) Linus Pauling advocated consumption of megadoses of Vitamin C to combat the common cold and other ailments. He took 10 g/day and lived to 93. Ascorbic acid (aka Vitamin C) has a pKa of 4.17. Put a circle around the dissociable proton. Explain your choice.

3. (5 points) Ingram used 2,4-dinitrofluorobenzene for determination of the amino terminal amino acid in a peptide. Later he used phenylisothiocyanate (Edman's Reagent), which is now preferred.  What are the merits of Edman's reagent compared to DNFB.

4.    (5 points) Bilirubin and biliverdin, drawn below, are breakdown products of heme. They provide the array of colors seen in a bruise. Structurally, they differ by the reduction of a double bond. Their two carboxyl groups appear virtually identical. However, the pKa values for them are different. In one case the values are 4.2 and 4.9 while in the other they are 3.9 and 5.3 [J. Biol. Chem. 271, 2397 (1996)]. Predict which set of values goes with which compound. State the basis for your prediction.
5. (5 points) The function of hemoglobin is tightly coupled to its structure. The effects of mutation affecting a single amino acid can range from benign to lethal as is seen in the variety of hemoglobinopathies studied by you and other students in the class. List 5 conceptually different ways a structural change in hemoglobin can affect its function. Start with the variant you studied.






6.    Fig 1 is from Ingram's first article. It monitors the time course for the digestion of HbA and HbS by trypsin at pH 8. NaOH is added to maintain the pH.


A. (5 points) Chemically describe in words and structures the reaction catalyzed by trypsin.

B. (5 points) Explain how there is a net production of protons (H+) in this reaction

C. (5 Points) What would the curve look like (sketch it in above) if the reaction in Figure 1 were carried out at pH 7.0 or 10, rather than 8.0? Explain your answers.

D. (5 points) How does the number of equivalents of hydroxide compare to the number of peptide bonds hydrolysed by trypsin?

7.   (10 points) Professor Essigsaure has discovered an electrophoretic variant of human hemoglobin that he wants to call HbGore Hall. On electrophoresis at pH 8, its mobility suggests that it has two more negative charges than HbA. Before he can publish anything about it, he needs to show that it hasn't been described previously. First, he wants to determine whether the mutation affects the ? or ? globin subunit. One morning, as he was taking a shower, he had a Eureka! moment and almost forgot to get dressed. Skipping breakfast, he ran most of the mile from his home to his lab. From the freezer, he took a small sample of his carefully purified HbGore Hall, thawed it, then incubated it with an equal amount of HbS, and ran the mixture on electrophoresis. By noon jubilant Prof. Essigsaure proclaimed that the mutation in HbGore Hall affected the ?-chain. What did the electrophoretic pattern look like for him to make his conclusion? You must draw a picture to receive credit. [Based on Itano & Robinson, Nature 184, 1468 (1959)]
Part III - Short Essays

8. (10 points) While we know of hundreds of human hemoglobin variants generated by single amino acid replacement, this is a small portion of the number possible. Consider all the possible single amino acid replacements that might occur. How large is that number? Make an estimate. Was Pauling lucky that HbS had a charge difference? Explain.

9. (10 points)  The fact that HbS confers resistance to malaria appears in textbooks everywhere. It represents a classic demonstration of natural selection operating on the human population and provides an example of a heterozygote being fitter than either homozygote where malaria occurs. When students read about the experiments reported by Allison (1954) to test the hypothesis, they often are shocked. What did Allison do to test the hypothesis? What about his experiment is shocking? Evaluate student reaction in the context of: ethical standards have changed, the risks of doing these experiments have changed, the details of the experiment are not understood.

Bonus Question
10. (5 points) An adult human body contains about 2 grams of iron of which about 70% is associated with hemoglobin. Estimate to within an order of magnitude the number of hemoglobin molecules in your body. (i. e. your answer needs only to be expressed as a power of 10.)

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Posted: 23 May 2002 by Hal White
Copyright 2002, Harold B. White, Department of Chemistry and Biochemistry, University of Delaware