INSTRUCTIONS READ BEFORE YOU GO ON

1. There are two parts to this examination. Each will take about 90 minutes.

2. There are 3 questions in Part I. Answer any combination of questions that totals 75 points. You may refer to your course reader, course notes, course handouts, and homework assignments.

3. While this examination emphasizes work done in this course since Spring Break, knowledge is not so conveniently compartmentalized. Therefore, you should feel free to use any relevant example from your experience, if it is appropriate.

4. Please write legibly and compose your answers so that you say what you mean.

5. If you finish Part I early, you may leave the room and relax until Part II begins around 5 P.M.

6. P.S. Don't forget to put your name on your blue book.

ABSTRACT 1: Science 247, 566-568 (1990)

DNA molecules that contain the beta- and beta^S-globin genes inserted downstream of erythroid-specific, deoxynuclease I super-hypersensitive sites were coinjected into fertilized mouse eggs and a transgenic mouse line established that synthesizes human sickle hemoglobin (HbS). These animals were bred to beta-thalassemic mice to reduce endogenous mouse hemoglobin levels. When erythrocytes from these mice were deoxygenated, greater than 90 percent of the cells displayed the same characteristic shapes as erythrocytes from human cells with sickle cell disease. Compared to controls the mice have decreased hematocrits, elevated reticulocyte counts, lower hemoglobin concentrations, and splenomegaly, which are indications of the anemia associated with human sickle cell disease.

[Note: An article describing very similar work by another research group appeared in Nature 343, 183-185 (1990) within a month after this article.]

1a. (10 points) What don't you understand about the work described? Make a list of clearly expressed learning issues that define what it is that you would need to look up and learn in order to understand the article. From your list, identify and justify the one learning issue that you feel is the most important.
 

1b. (10 points) In a clearly written paragraph or two, discuss the importance of the work described in the above abstract and relate it to one or more of the articles you have read this semester.

1c. (5 points) What do you think researchers would do with a strain of mice in which the red blood cells sickled?

1d. (5 points) If you were a member of an animal welfare review committee, what assurances, if any, would you want to have before you would approve the work described?

ABSTRACT 2: J. Biol. Chem. 269, 9562-9567 (1994)

Previous studies suggested that 87 Gln in hemoglobin (Hb) F is an important site for promoting inhibition of Hb S (alpha ₂ beta₂^{6 Glu Val}) polymerization by Hb F. We engineered and isolated the double mutant (Hb alpha ₂ beta₂^{6 Glu Val, 87 Thr Gln}) using a yeast expression system and characterized polymerization properties of this modified tetramer in an effort to clarify the role of Gln at position 87 in inhibiting Hb S polymerization. Electrophoretic mobility and absorption spectra of this double mutant were the same as that of Hb S, while oxygen affinity was higher, and the effects of organic phosphates on oxygen affinity were reduced. The deoxy form of the double mutant showed a characteristic delay time prior to polymerization in vitro. The critical concentration for the double mutant was about 1.5 times higher than Hb S, and the delay and polymerization times were much longer than Hb S at the same hemoglobin concentrations. The logarithmic plot of delay time versus hemoglobin concentration for the double mutant showed a straight line that was intermediate between lines for AS and FS mixtures. These results and those of kinetics of polymerization of Hb S/double mutant mixtures indicate that substitution of Gln for Thr at 87 in Hb S prolongs delay time and inhibits polymerization, although the double mutant forms polymers like Hb S.

2a. (10 points) Draw a model of hemoglobin that conceptualizes what the Hb double mutant is. Use that representation to illustrate the phenomenon that the authors of this paper studied. You may provide a short narrative that explains your model.

2b. (10 points) In a clearly written paragraph or two, discuss the importance of the work described in the above abstract and relate it to one or more of the articles you have read this semester.

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ABSTRACT 3: FEBS Letters 315, 47 50 (1993)

The relationship between different amino acids at the 6 position of hemoglobin and tetramer stability was addressed by a site-directed mutagenesis approach. Precipitation rates during mechanical agitation of oxyhemoglobins with Gln, Ala, Val, Leu, and Trp at the position increased 2, 5, 13, 21 and 53 time, respectively, compared with that for Hb A. There was a linear relationship between the log of the precipitation rate constant and amino acid hydrophobicity at the 6 position, suggesting that enhanced precipitation of oxy Hb S during mechanical agitation results in part from increased hydrophobicity of Val. Deoxyhemoglobin solubility in creased in the order of 6 Ile, Leu, Val, Trp, Gln, Ala, and Glu suggesting that hydrophobic interactions between 6 Val and the acceptor site of another hemoglobin molecule during deoxy-Hb S polymerization not only depend on hydrophobicity but also on stereospecificity of the amino acid side chain at the 6 position. Furthermore, our results indicate that hydrophobic amino acids at the 6 position which promote tetramer instability in the oxy form do not necessarily promote polymerization in the deoxy form.

3a. (10 points) There are twenty different amino acids normally found in proteins. Site-directed mutagenesis enables biochemists to create protein molecules that might never be found in nature. For the chain of hemoglobin there are 19x146 different single amino acid replacements that could be made by site directed mutagenesis. Why did the authors of this paper select the ones they did out of that enormous number of possibilities.

3b. (10 points) In a clearly written paragraph or two, discuss the importance of the work described in the above abstract and relate it to one or more of the articles you have read this semester.

3c. (5 points) If you were doing the experiments described above, how would you go about measuring precipitation of hemoglobin in a continuously stirred solution?

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Part II (Group Work, 25 points total)

A major objective of CHEM-342 is to have you learn to recognize what you don't know and empower you to fill those gaps in your knowledge. The following question deals with the application of concepts normally covered in introductory chemistry and organic chemistry but often not understood well by students. As with the midterm examination, a group response is expected but not required. You may hand in a separate answer for separate credit if there is not consensus within your group.

4. (25 points) In the Pauling et al. paper on page 546, it states,

According to titration data obtained by us, the acid-base titration curve of normal human carbonmonoxy hemoglobin is nearly linear in the neighborhood of the isoelectric point of the protein, and a change of one pH unit in the hemoglobin solution in this region is associated with a change in the net charge on the hemoglobin molecule of about 13 charges per molecule.

In Fig. 1 of Ingram's 1958 paper, about 16 moles of NaOH are consumed per mole of hemoglobin when it is digested with trypsin.

a. Both of these statements concern the titration of similar numbers of protons associated with hemoglobin at or near neutral pH. Show, with chemically relevant models of hemoglobin, where these protons are coming from in each case.

b. Assume that trypsin catalyzes the digestion of hemoglobin fastest at pH 8, where Ingram did his experiment, but can catalyze the same reactions more slowly at pH 7 and 9. Based on your analysis in part "a," what would Ingram's Figure 1 look like at pH 7 and at 9 compared to pH 8?