Wednesday 22 May 1996, 3:30 to 6:30 PM in 101C Pearson Hall

H. B. White Instructor

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. You may refer to your course reader, course notes, course handouts, and homework assignments.

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

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

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

Part I (75 Points) Individual work. Write your answers in the bluebooks provided.

1. Sometimes science is taught and learned as a collection of incontrovertible facts as I if knowing about the natural world applied only to multiple choice tests where every question had a single correct answer. While there is a body of knowledge in science that we feel is certain, the process of discovery in science is driven by uncertainty and the questioning of things thought to be certain. (If one knew the answer, there would be no point in doing an experiment.) The importance of scientific knowledge and understanding is that it helps people evaluate situations, make reasonable predictions, and then make better decisions in the face of uncertainty. Thus uncertainty is very much a part of science both in the discovery and in the use of scientific knowledge.

You have read and discussed in your group the article published in 1954 by Dr. Anthony Allison. Its conclusions appear in virtually every biology textbook. Answer the following questions relating to Allison's experiment and what he knew and didn't know.

A. (5 Points) State clearly and concisely the hypothesis Dr. Allison was testing?

B. (20 Points) What facts and observations did Allison know that contributed to the design of his experiments? Divide your answer into two columns. On the left, list those facts and observations (4 or more) that Allison knew. In the second column, opposite each entry, indicate briefly how each contributed to the design of his experiment.

C. (10 Points) Allison probably designed and conducted his experiments without formal approval by an independent human subjects review board. If you were to serve on such a board, specifically what would you like to know, that was not addressed in Allison's article, before considering whether or not to approve such an experiment today? How would this information help you make a better decision?

2. Pauling et al. (1949) demonstrated that persons with sickle cell disease have a chemically distinct hemoglobin (HbS). A few years later, Ingram identified the difference between HbS and HbA as the replacement of valine for glutamic acid at position 6 of the beta-chain. Subsequently, hundreds of other variant human hemoglobins have been discovered and characterized. Usually the variants differ from HbA by a single amino acid replacement in the alpha or beta-chain.

A. (15 Points) Consider a man who is heterozygous for a mutation affecting his hemoglobin such that HbA and "HbX" are observed. HbX has the same electrophoretic mobility as HbS but does not cause red blood cells to sickle at low oxygen tension. His wife has sickle cell trait. Upon examining the electrophoretic pattern of hemoglobin from their second child, you conclude that HbX results from a mutation affecting the alpha-chain, not the beta-chain. Draw and clearly label a diagram showing the electrophoretic patterns for both parents and the second child. Explain and show how these observations would differ if the mutation affected the alpha-chain.

B. (10 Points) Human HbA contains 12 sulfur atoms and 4 iron atoms for a S to Fe ratio of 3 to 1. How could it be possible for someone in this class to have a hemoglobin with a S to Fe stoichiometry of 11:4 or 13:4? No credit without an explanation that shows a clear understanding.

3. Answer one of the following two questions based on quotations from Stokes's 1864 article.

A. (15 Points) "Very different is the effect of carbonic acid.... I took two portions of defibrinated blood; to one I added a little of the reducing iron solution, and passed carbonic acid into the other, and then compared them. They were as nearly as possible alike. We must not attribute these apparently identical changes to two totally different causes if one will suffice."

Clearly carbonic acid is not a reducing agent. Can you attribute conceptually the "apparently identical changes" to a single process?

Or

B. (15 Points) "When the watery extract from blood clots is left aside in a corked bottle, or even in a tall narrow vessel open at the top, it presently changes in colour from a bright red to a dark red, decidedly purple in small thickness.... The tint agrees with that of purple cruorine obtained immediately by reducing agents.... On shaking the solution with air it immediately becomes bright red, and now presents the characteristics of scarlet cruorine."

Explain Stokes's observations in terms of our current understanding of the chemistry of hemoglobin.

Part II Group work (25 Points) If your group cannot come to consensus, dissenting members may submit a separate answer for separate grading.

On March 21 of this year, the day of your midterm examination, the journal Nature published a paper about hemoglobin and nitric oxide that caused quite a stir.⁽¹⁾ National Public Radio had a program about it and the New York Times described the discovery in a prominent article. Who would have thought that hemoglobin had not yet yielded all of its structural and functional secrets after more than a century of intense study by thousands of researchers?

Nitric oxide (NO), like CO and O₂, reacts with the iron in the heme group of hemoglobin (Hb). When NO reacts with oxyhemoglobin (HbO₂), methemoglobin (metHb) and nitrate form. This chemistry was known in the 1920's at a time when NO was thought to be an inorganic molecule of no biological significance except as a poison akin to CO. Within the past decade, NO became recognized as a very potent vasodilator and messenger molecule⁽²⁾ and was named "Molecule of the Year" by Science magazine in 1992.⁽³⁾ It is involved in activities as diverse as regulation of blood pressure, male erection, and neurotransmission. The plot thickened in March when it was shown that NO in the form of S-nitrosothiols such as S-nitrosoglutathione (G-SNO) transfers its NO group to the reactive sulfur of cysteine 93 on the -chain of hemoglobin to form S-nitrosohemoglobin (Hb-SNO). These S-nitrosothiols do not react with heme iron the way free NO does. In fact, the nitrosylation of Cys 93 occurs more readily with HbO₂ than with Hb and does not form metHb. Conversely, S-nitroso-deoxyhemoglobin transfers NO back to glutathione (GSH), a tripeptide, more rapidly than does S-nitroso-oxyhemoglobin. The fact that hemoglobin in arterial blood is nitrosylated while in venous blood it is not suggests that these reactions are physiologically important and that the NO is produced by the lungs and distributed throughout the body by hemoglobin.

One goals of the CHEM-342 is to encourage you to think chemically about biological systems. Another goal is to be able to apply what you know to new situations. Prepare a diagram (model) that would serve as a figure to illustrate the preceding paragraph. [Note: There is no single right answer. Models will be evaluated and graded on their informativeness, simplicity, clarity of presentation, inclusion of important information, creativity, and lack of extraneous details or misinformation. Please, nothing PG or X-Rated.]

1. Jia, L., Bonaventura, C., Bonaventura, J., & Stamler, J. S. (1996) S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature 380, 221-226.

2. Bredt, D. S. & Snyder, S. H. (1994) Nitric Oxide: A Physiologic Messenger Molecule. Annu. Rev. Biochem. 63, 175-195.

3. Culotta, E. & Koshland, D. E., Jr. (1992) NO News Is Good News. Science 258, 1862- 1865.