Friday 30 May 1997, 8:00 - 11:00 a. m., 101C Pearson Hall

H. B. White - Instructor

Important - Please read this before you turn the page.

Answer each of the following three:

1. (15 points) Three short answer questions

2. (20 points) Dynamic equilibria and hemoglobin electrophoresis

3. (20 points) Characterization of HbC

Answer one of the following:

4. (20 points) Correcting inaccuracies and misconceptions about hemoglobin

5. (20 points) Shemin and Rittenberg learning issues

1. Short Answer Questions (5 points each, 15 points total)

A. Shemin and Rittenberg (1946) do not say how they measured the amount of 15N in the hemin (hematin) and red cell protein. In what measurable properties would heme synthesized from 15N-glycine differ from heme synthesized in Shemin's body before the experiment? How would this be measured?

B. The genetics of the sickle cell gene confused scientists for almost three decades. Little wonder students are perplexed by scientists who classify it as recessive, dominant, or co-dominant. In fact, all of the classifications can be correct. Explain.

C. Pauling et al. (1949) estimated the isoelectric point of normal hemoglobin A to be 0.23 units lower than hemoglobin S and that difference corresponded to a net charge difference of 2 to 4. As we know from Ingram's work, the actual difference is 2. Consider two hemoglobin solutions, one at its pI and the other at a pH one unit above (or below) the pI. What is the difference in net charge on hemoglobin in the two cases?

Geography Bonus Questions (One point each)

A. Herrick's patient was from Genada. What South American country is closest to Grenada?

B. In what African country did Allison (1954) conduct his experiments?

2. (20 points total) Dynamic equilibria and hemoglobin electrophoresis.

A. (8 points) Hemoglobin in the red blood cells of a person with sickle cell trait is now known to exist in a dynamic equilibrium among the tetrameric forms a2b2, a2BBS, and a2BS2 and small amounts of the heterodimers. Provide a diagram (no words, please) that clearly represents the dynamic equilibrium described.

B. (4 points) If the relative amounts of the tetrameric species of hemoglobin approximate a binomial distribution, what is the approximate proportion of a2BBS in red cells from a person with sickle cell trait?

C. (8 points) Pauling et al. (1949) and we in class observed only two electrophoretic forms of hemoglobin in the blood from people with sickle cell trait - HbA and HbS. The two forms accounted for all of the hemoglobin present. Provide a reasonable explanation for the absence of the hybrid tetramer (a2BBS) halfway between HbA and HbS on electrophoresis.

3. (20 points) Characterization of HbC peptides. In 1958, Hunt and Allison (Nature 181, 1062) showed that HbC, like HbS, differs from HbA by a single amino acid replacement at position 6 of the -chain (see below). As with HbA and HbS, they denatured HbC, digested it with trypsin, and separated and purified the resulting peptides by a combination of paper chromatography and paper electrophoresis. They determined the amino acid sequence of the distinctive peptides by Edman degradation as described in the Ingram papers you read. Figure 3 from Ingram's paper (below) shows how "peptide 4" changed its position on the two-dimensional peptide map.

A. (8 points) Based on the amino-terminal sequences of the beta-chains of HbC and HbS, analyze the mobility behavior of the two peptides 4 on the peptide maps in terms of their structure and the principles of chromatography and electrophoresis. (Use the back of this page, if you need more room.)

B. (8 points) How would the products of trypsin digestion of HbC differ from those of HbA and HbS? Using the sequence information above, analyze the expected chromatographic and electrophoretic behavior of the unique peptide(s).

C. (4 points) Based on your analysis, predict (draw) on the peptide map for HbA above how the peptide map for HbC would differ.

Answer either question 4 or 5.

4. (20 points, 5 points each part) On the initial survey on 12 February, members of this class provided responses to the question, "Describe as concisely as you can what you know about the chemistry and biology of hemoglobin." Four responses are shown below. Each displays imprecision, inaccuracies, and/or misconceptions. Take each one, identify its problems and rewrite it so that it accurately and unambiguously reflects our knowledge.

A. Hemoglobin is a transport protein that deals with the clotting of the blood. When you have sickle cell anemia, your hemoglobin is in the wrong shape.

B. An iron atom bonded to several (4?) polypeptides.

C. I know its a molecule of iron which binds to 4 oxygens.

D. Hemoglobin is capable of carrying billions of oxygen molecules at a time. The heme itself is responsible for this. Without the heme there is only myoglobin. Fe+3 is bound in the middle of the hemoglobin which give RBC's their red color when it is oxidized.

5. (20 points)

A. (5 points) Of the learning issues you and your group generated for the Shemin and Rittenberg (1946) paper, state concisely the one learning issue that you think was the most substantive in terms of what it helped you to learn.

B. (15 points) Write a short clear paragraph or two that reflects what of importance you learned in the pursuit of that learning issue and why it was important.

Group Number_________________________
Group Member Signatures _________________________



Friday 30 May 1997

H. B. White - Instructor

"Myself as a Guinea Pig. . . in 1944 I undertook, together with David Rittenberg, an investigation on the turnover of blood proteins in man. To this end I synthesized 66 g of glycine labeled with 35% 15N at a cost of $1000 for the 15N . On 12 February 1945, I started the ingestion of the labeled glycine. Since we did not know the effect of relatively large doses of the stable isotope of nitrogen and since we believed that the maximum incorporation into the proteins could be achieved by the administration of glycine in some continual manner, I ingested 1 g samples of glycine at hourly intervals for the next 66 hours. . . . At stated intervals, blood was withdrawn and after proper preparation the 15N concentrations of different blood proteins were determined." David Shemin in BioEssays 10, 30 (1989)

1. A. (5 points) Figure 1 from Shemin and Rittenberg's paper shows the atom percent excess of 15N in hemin (hematin) and in the red cell proteins (mostly hemoglobin) as a function of time after ingestion of 15N-glycine. The profile is atypical of "pulse-chase" experiments. Superimpose on the figure below what Shemin and Rittenberg would have observed had they analyzed hemin and protein from cells in other tissues such as liver where steady state concentrations of proteins result from a balance of synthesis and degradation. Here the general shape of your graph is most important.

B. (8 points) The experiment described by Shemin and Rittenberg has never been done on a patient with sickle cell anemia. However, based on what is known about the disease, you can predict qualitatively what the graph in figure 1 would have looked like if David Shemin had had sickle cell anemia. On the figure below, draw such a graph and explain any substantive differences from the original.

2. Shemin and Rittenberg (1946) showed that the average lifespan of a red blood cell in Shemin's body was about 127 days, a value similar to that estimated by other methods on other people.

A. (8 points) If you were going to estimate within one order of magnitude the number of hemoglobin molecules your body synthesizes every second, what additional information would you need to make the calculation?

B. (4 points) Estimate to within one order of magnitude the number of hemoglobin molecules your body synthesizes per second.