Name ___________________________



Monday, 24 May 1999, 7:00 - 9:00 p. m., 208 Gore Hall

H. B. White - Instructor

Important - Please read this before you turn the page.

A. (15 points) One-word Answer Questions

B. (15 points) Short Summary Statements

C. (10 points) Problem to Solve

D. (8 points) Essay Question about the seminar you attended

E. (12 points) Application of principles to a new situation

F. (15 points) Essay

Part II, the group part of the examination, has one problem worth 25 points.

Name __________________________
A. One-Word Answer Questions (15 points) These questions address factual information that you are likely to have encountered this semester.

_______________________ 1. Color of a solution of oxyhemoglobin.

_______________________ 2. Color of a solution of oxyhemoglobin after it has reacted with dithionite, S2O4=.

_______________________ 3. Color of a solution of methemoglobin.

_______________________ 4. Color of a solution of methemoglobin after it has reacted with dithionite, S2O4=.

_______________________ 5. Ionizable functional group associated with histidine.

_______________________ 6. Addictive alkaloid that can be chemically converted into the vitamin niacin.

_______________________ 7. Bilirubin is associated with this symptom of sickle cell anemia as well as with hepatitis.

_______________________ 8. Sulfur-containing amino acid in proteins.

_______________________ 9. Number of heme groups in Hb S.

_______________________ 10. South American country closest to Grenada.

_______________________ 11. Bond hydrolyzed by trypsin.

_______________________ 12. Principle on which Longsworth Scanning is based.

_______________________ 13. The guanido groups is associated with this amino acid.

_______________________ 14. Functional group in proteins that reacts with Sanger's Reagent.

_______________________ 15. In the one-letter abbreviations, "E" refers to this amino acid.

Name __________________________
B. Summary Statements (3 points each). In a single sentence, state concisely and clearly the hypothesis (or purpose) for each of the articles you have read since Spring Break.

1. Herrick (1910)

2. Diggs et al. (1933)

3. Pauling et al. (1949)

4. Ingram (1959)

5. Allison (1954)

C. Problem (10 points) Consider the figure below from Svedberg and Fåhraeus (1926) which shows the concentration of hemoglobin as a function of the distance from the center of rotation in a centrifuge. What would the profile look like if hemoglobin had a molecular weight of 16,700 rather than 66,800 and the rotational velocity were doubled? Draw a line on the figure representing your conclusion and justify it in words.


Name _________________________________
D. Biochemistry Seminar (8 Points) In lieu of the final two class meetings, you were asked to attend at least one of four biochemistry seminars. I suspect that all were in some way difficult to understand and I did not expect you to understand everything. Please identify the seminar(s) you attended and, using your own words, state in a short paragraph what you learned from the seminar. Then write two well-focused learning issues generated by the seminar.

Name _________________________________
E. Application to New Situations (12 points) Lactate dehydrogenase (LDH) catalyzes the reversible NAD-dependent interconversion of lactate and pyruvate. Like hemoglobin, active LDH contains four subunits. Unlike hemoglobin, the subunits are interchangeable and can be identical. For example, in heart muscle the H subunit is produced giving H4 molecules and in the liver L4 predominates. H4 and L4 have distinct electrophoretic mobilities. Other tissues produce both H and L LDH subunits that can generate hybrid tetramers.

From the information provided, predict the number of bands and the relative amounts of each band in an electrophoretic separation of LDH from a tissue producing equal amounts of H and L subunits. Assume the subunits randomly associate to form active, non-dissociating tetramers. Describe your reasoning and then draw a figure depicting the appearance of an electrophoretic separation of LDH from this hypothetical tissue.Predict the relative amounts of LDH in each band?

Name ___________________________
F. Essay (15 points) One of the goals of Introduction to Biochemistry is to enable you to reflect on and make sense of new information on your own. This involves relating new information to things you already know, identifying learning issues, and cultivating your curiosity about the world of biochemistry and associated disciplines.

As you read the two abstracts of recent articles on the following page, keep track of the things you don't understand and the thoughts you have. (Use the margins for notes, if you want.) Collect your thoughts and write in the space below a one-page narrative that integrates the new information with things you know and the things you would like to know relating to the topic. I am looking for a well organized and well articulated presentation of your personal reflections rather than a summary or paraphrasing of information in the abstracts. You may continue on the back of this page, if you need more space.

Blood 85(4): 1111-1117 (1995)

An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta globin haplotypes, alpha-globin gene number, gender, and age.

Chang YC, Smith K, Moore RD, Serjeant GR and Dover GJ.

Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD.

Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analysis showed that each variable, when analyzed alone, had a significant association with Hb F levels (P<.05). Multiple regression analysis, including all variables, showed that the FCP locus is he strongest predictor, accounting for 40% of Hb F variation. Beta-globin haplotypes, alpha-globin genes, and age account for less han 10% of Hb F variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus s removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other yet identified factor(s)

Blood 93(6): 1790-1797 (1999)

Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease

Atweh GF, Sutton M, Nassif I, Boosalis V, Dover GJ, Wallenstein S, Wright E, McMahon L, Stamatoyannopoulos G, Faller DV and Perrine SP.

Departments of Medicine, Pediatrics and Biomathematical Sciences, Mount Sinai School of Medicine, New York, NY

High levels of fetal hemoglobin (Hb F) protect from many of the complications of sickle cell disease and lead to improved survival. Butyrate and other short chain fatty acids were previously shown to increase Hb F production in erythroid cells in vitro and in animal models in vivo. However, butyrates are also known to inhibit the proliferation of many cell types, including erythroid cells. Experience with the use of butyrate in animal models and in early clinical trials demonstrated that the Hb F response may be lost after prolonged administration of high doses of butyrate. We designed a regimen consisting of intermittent or pulse therapy in which butyrate was administered for 4 days followed by 10 to 24 days with no drug exposure. This pulse regimen induced fetal hemoglobin expression in 9 of 11 patients. The mean Hb F in this group increased from 7.2% to 21.0% (P<.002) after intermittent butyrate therapy for a mean duration of 29.9 weeks. This was associated with a parallel increase in the number of F cells and F reticulocytes. The total hemoglobin levels also increased from a mean of 7.8 g/dL to a mean of 8.8 g/dL (P<.006). The increased levels of Hb F were sustained in all responders, including 1 patient who has been on butyrate therapy for more than 28 months. This regimen, which resulted in a marked and sustained increase in Hb F levels in more than two thirds of the adult sickle cell patients enrolled in this study, was well tolerated without adverse side effects. These encouraging results require confirmation along with appropriate evaluation of clinical outcomes in a larger number of patients with sickle cell disease.

Group Number_________________________
Group Member Signatures _________________________


Monday 24 May 1999, 9:00 - 10:00 PM

H. B. White - Instructor

Group Number _______


(25 points total) The abstract below accompanied an article published six months ago in the British Journal of Hematology. Read it carefully and then answer the questions that follow.

Br J Haematol 103(4): 950-956 (1998)

A new sickle cell disease phenotype associating Hb S trait, severe pyruvate kinase deficiency (PK Conakry), and an alpha2 globin gene variant (Hb Conakry).

Cohen-Solal M, Prehu C, Wajcman H, Poyart C, Bardakdjian-Michau J, Kister J, Prome D, Valentin C, Bachir D, and Galacteros F. INSERM U474, Hopital Henri Mondor, Creteil, France

Guinean woman, heterozygous for hemoglobin (Hb) S, was studied because of episodes of marked anaemia, repeated typical metaphyseal painful crises and hemosiderosis. Her sickling syndrome resulted from the association of Hb S trait with severe pyruvate kinase deficiency leading to a 2,3-DPG concentration twice normal levels. Sequence of the PK-R gene revealed an undescribed mutation in the homozygous or hemizygous state within exon 5 (nucleotide 2670 C A), leading to the interchange of Ser 130 into Tyr (PK Conakry). In addition, the patient carried a new hemoglobin variant, Hb Conakry [alpha80(F1) Leu Val], which seemed to have a mild effect. The high intraerythrocytic 2,3-DPG concentration induced by the PK deficiency resulted in a decreased oxygen affinity which favored sickling to a level almost similar to that of Hb S/C compound heterozygous patients. This was confirmed by oxygen binding measurements of Hb A/Hb S erythrocytes in which 2,3-DPG content was modified in vitro. Hysteresis between deoxy- and reoxygenation curves, as well as increase in the n(max) value, demonstrated that the extent of Hb S polymerization in the propositus was almost the same as that of RBCs from a homozygous sickle cell patent or those of an A/S heterozygous patient with an artificial increase of 2,3-DPG concentration.

A. (10 points) Predict the appearance of the Hb electrophoretic pattern from the patient compared to normal Hb A and people with classical sickle cell anemia Hb S. Draw a picture and explain your reasoning.

B. (15 points) Based on the information provided in the abstract, draw oxygen-binding curves (% Hb saturation vs pO2) for erythrocytes from individuals with Hb S, Hb A/S, Hb S/C, and the patient. Indicate the normal range of pO2 in the body and, where relevant, note where sickling begins to occur. Explain your reasoning. Conceptual understanding and relationships here are more important than specific numbers.