Haemoglobin and cooperativity

91 amplify the double stranded cDNAs. If the restriction sites in primers numbers 1 and 2 are different, 'directional' cloning of PCR products is possible in a suitable vector.

PCR-based cDNA library construction: general cDNA libraries at the level of a few cells, Belyavski, A et al (1989) Nucleic Acids Res 17, 2919-2932

Further reading Many books and reviews are available. The following are useful:

Haemoglobin and Cooperativity

Basic principles and the PCR reaction PCR Protocols. A Guide to Methods and Applications, edited by Innis, M A, Gelfand, D H, Sninsky, J J and White, T J, Academic Press, New York (1990). PCR Technology. Principles and applications for DNA amphfication edited by Erlich, H A, Stockton Press (1989) The polymerase chain reaction, White, T J, Arnheim, N and Erlich, H A (1989) Trends in Genetics 5, 185-189 The polymerase chain reaction, Bell, J (1989) Immunology Today 10, 351-355 Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Saiki, R K et al (1988) Science 239, 487-491 How sensitive is PCR? Avoiding false positives with PCR, Kwok, S and Higuchi, R (1989) Nature 339, 237-238 Human immunodeficiency virus -- infected individuals contain provirus in small numbers of peripheral blood mononuclear cells and at low copy numbers, Simmonds, P et al (1990) J. Virol 64, 864-872 Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro, Abbott, M A et al (1988) J Infect Dis 158, 1158-1169 How accurate is PCR? DNA polymerase fidelity and the polymerase chain reaction, Eckert, K A and Kunkel, T A (1991) PCR Methods & Applications 1, 17-24 Detection of mRNA sequences by PCR Detection of exon-intron structure: a novel appfication of the polymerase chain reaction technique, Bruzdzinski, C J and Gelehrter, T D (1989) DNA 8, 691-696 Applications The polymerase chain reaction: current and future cfinical applications, Lynch, J R and Brown, J M (1990) J Med Genet 27, 2-7 A simple method for site-directed mutagenesis using the polymerase chain reaction, Helmsley, A et al (1989) Nucleic Acids Res 17, 6545-6551 Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction Wilks, A F (1989) Proc Natl Acad Sci USA 86, 1603-1607 Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer, Frohman, M A et al (1988) Proc Natl Acad Sci USA 85, 8998-9002 B I O C H E M I C A L E D U C A T I O N 20(2) 1992

P D JOSEPHY

Guelph-Waterloo Center for Graduate Work in Chemistry Department of Chemistry and Biochemistry University of Guelph Guelph, Canada N1G 2W1 Introduction Several biochemistry textbooks present a separate chapter on the structure and function of myoglobin and haemoglobin, and use this discussion as a starting point for the examination of protein biochemistry. 1'2 There are many pedagogical advantages to this approach: haemoglobin is probably the protein whose structure and function are most comprehensively understood, and its principal biological function, oxygen transport, is intuitive - - even if other functions, such as carbon dioxide transport, are not. The comparison of myoglobin and haemoglobin can serve as a paradigm for the great significance of cooperative behaviour in biochemistry. Cooperative interactions allow proteins to achieve kinetics which approximate 'allor-nothing' behaviour: activity varies drastically over a small range of substrate concentration. This behaviour is crucial to the transport function of haemoglobin. In our undergraduate course Structure and Function in Biochemistry we consider several other examples of cooperativity, such as substrate binding to the enzymes aspartate transcarbamoylase and phosphofructokinase, and, in the retinal rod cell, the opening of cation-specific channels by the cooperative binding of cyclic AMP. The explanation of these systems is based on the understanding of cooperativity, introduced with the case of haemoglobin. However, the chemical mechanism of haemoglobin cooperativity is subtle. It is based upon the delicate interplay between oxygen binding to the haem groups, the motions of the alpha helices adjacent to the haems, and the many contacts at the tx1-132 interface between globin subunits: the 'Perutz mechanism'. 3 In my experience, students can easily become frustrated with the chemical details of these interactions, and fail to grasp the central idea of cooperativity. The textbooks are not very helpful here. Stryer uses the 'postage-stamp' analogy: as each successive stamp is torn from a block of four, the next one can be removed more easily. However, this analogy is rather limited. For example, it fails to explain why the binding of the third oxygen molecule may differ from the binding of the second. Voet and Voet do not use any analogy, and base their discussion of cooperativity on the Hill equation.

92 I have developed several analogies and representations to convey the idea of cooperativity, and I hope that other teachers will find'them appealing.

The railway car analogy Oxygen molecules binding to haemoglobin for transport behave like passengers boarding a railway car. Each compartment has four seats. Boarding passengers prefer to sit in compartments that are already partially occupied. At the destination, some of the passengers depart; passengers are more likely to depart if their compartment-mates have already left. This analogy explains why cooperativity is not significant at the upper and lower extremes of the oxygenbinding curve. At the lower extreme, passengers are unlikely to find a compartment that is not empty; at the upper extreme, passengers are unlikely to find a compartment that is not full. Non-cooperative systems, such as myoglobin, resemble trains with individual seating, in which passengers pay no attention to the presence or absence of others (one observes this sort of behaviour in the London Underground!). The analogy also conveys, at least partially, the critical idea that cooperativity is dependent on the interaction between the ligands and the protein; in contrast, the 'postage-stamp' analogy implies, incorrectly, a direct ligand-ligand contact. The 'Communist Party' analogy Individual oxygen molecules bind to haemoglobin subunits with low affinity, as the Communist Parties of Eastern Europe 'bound' rather weakly to their respective nations (which tried, repeatedly, to rid themselves of their governments). However, when several Communist Parties 'bound' simultaneously to neighbouring countries, their affinities became very much greater, since they could offer one another mutual assistance (eg Hungary, 1956; Prague, 1968). Conversely, the fall from power of one Communist Party weakened the affinities of all the others. Therefore, no biochemist should have been surprised by the tumultuous political events of 1989,1991; they represented a grand example of 'all-or-nothing' binding! The 'distribution of bound oxygens' picture Oddly, none of the textbooks presents a 'snapshot' picture of the distribution of bound oxygens among available haemoglobin molecules, as a complement to the presentation of oxygen-binding curves. This is easily represented, as shown in Fig 1, by a diagram in which squares represent haemoglobin molecules, and filled spots represent oxygenated haems. Forty sites are drawn, and the numbers at right give the total number of occupied sites. This picture shows clearly that the 'cooperative' distribution is 'skewed' towards the n = 0 and n = 4 states, and also that the cooperativity is most manifest in the regime of partial occupancy (around Y = 0.5 on the Hill plot). A useful exercise is to ask students to draw an BIOCHEMICAL EDUCATION 20(2) 1992

N~ooperafivebin~g 35

Cooperativebinding 35

[ ~ [ ' ~ ~ [ ' ~ [ ~ ~

15

Figure 1 Distribution of bound oxygens

analogous set of sketches for the case of negative cooperativity.

The 'TeeteR-TotteR' picture Haemoglobin cooperativity is a consequence of the shift of the protein from T to R conformations upon oxygen binding. Perutz has elucidated the structures of these two states. Haemoglobin is finely balanced or poised between the two conformations, and the small enthalpic Contribution of oxygen binding is sufficient to cause the global transition from T to R. I use the analogy of the TeeteRTotteR (even the name is apt!). In the absence of oxygen, one state (T) is slightly more favourable, but the protein can still flip into the R state occasionally, because the energy difference between the states is small. Nevertheless, the conformations are very different, just as the 'left-up' and 'left-down' states of a teeter-totter are distinct, if the ends are marked somehow. Oxygen binding shifts the T to R equilibrium drastically, just as the addition of a very small weight shifts a teeter-totter (see Fig 2). The effects of allosteric effectors, such as b/s-phosphoglycerate, H ÷, and CO2 can all be viewed as analogous to 'hanging' small additional weights onto the T side. Although their chemical mechanisms are distinct, they all act by shifting the R - T equilibrium. The physiological importance of haemoglobin cooperatively has been underlined by the recent achievement of producing human haemoglobin in transgenic pigs. 4 Purified human haemoglobin cannot be used for transfusion because (among other problems) it loses bisphosphoglycerate, dissociates into monomers, and binds oxygen non-cooperatively, as myoglobin does, and with very high affinity. Researchers at several 'bio-tech' companies are trying to develop cross-linked or polymerized haemoglobin which will display cooperative behaviour.

93

the human body. Of course carbohydrates and fats could

T-state

Oxygenbinding ~ T Oxygenrelease

R-state

Figure 2 The Teeter TotteR An excellent illustration of the importance of the cooperative shift from T to R is provided by the recent crystallization of deoxyhaemoglobin in the presence of polyethylene glycol. Previously-obtained crystals of deoxyhaemoglobin, of course, shattered upon oxygenation, due to the T to R transition. The polyethylene glycol-induced crystals yield a T form which is unable to relax to the R state upon oxygenation, due to the constraints of the crystal lattice, but which remains intact. 5 T state crystals of haemoglobin bind oxygen noncooperatively (Hill coefficient of exactly 1.0).

References 1Stryer, L (1988) Biochemistry, third edition, W H Freeman, New York, chapter 7 2Voet, D and Voet, J G (1990) Biochemistry, Wiley, New York, chapter 9 3perutz, M F (1989) Quart Rev Biophys 22, 139-236 4Moffat, A S (1991) Science 253, 32-34 5Mozzarelli, A, Rivetti, C, Rossi, G L, Henry, E R and Eaton, W A (1991) Nature 351,416-419

Once More Round the Citric Acid Cycle BERNARD S. BROWN

Department of Biochemistry and Molecular Biology Medical School University of Manchester Manchester M13 9PT, UK It was the time of year when you awaken to the sound of birds singing, the bright sunshine streaming through your bedroom window. It was the time of year when the frenzied activity of football is replaced by the more peaceful (dare I say civilised?) pace of cricket. And yes, it was the time of when when hard-working biochemistry lecturers' desks are piled high with examination scripts waiting to be marked!

Essay marking and obesity The essay I was marking was an old favourite of mine: Discuss the interconvertibility of carbohydrates and fats in BIOCHEMICAL EDUCATION 20(2) 1992

be interconverted - - within certain limits! Carbohydrate could be converted to both glycerol and fatty acids, thence to triacylglycerol (fat). Many was the time I had presented this pathway in lectures, sobbing my heart out over the implication that chocolates, cakes, potatoes and - - yes - syrup sponge pudding, if indulged to excess, would end up as fat. But converting fat to carbohydrate was a different matter. Oh, we could break the fat into glycerol and fatty acids. And yes, we could convert the glycerol to glucose by gluconeogenesis. But to make straight-chain, evennumbered fatty acids into glucose was not possible for us, although plant seedlings and some microbes could do it. We converted fatty acids to acetyl CoA, then oxidised this in the citric acid cycle. Well, I was convinced of that - - but some of my students thought otherwise.

Essay marking and depression I perused one of the 44 scripts before me and read: 'Acetyl CoA can be converted through the citric acid cycle to oxaloacetate.' Another script informed me: 'The acetyl CoA can enter the Krebs cycle ... the oxaloacetate formed can be turned into glucose.' And another: 'The acetyl CoA then enters the gluconeogenesis cycle by which it is converted into glucose.' Through tearful eyes I read in another script: 'Glucose can be produced from noncarbohydrate substrates such as fatty acids, glycerol and amino acids by gluconeogenesis.' I felt better when I came upon, 'Acetyl CoA --x--> pyruvate (which can be converted to glucose) because this step is irreversible...'. If only I had stopped reading the script there, but no, I had to read on: ' . . . therefore acetyl CoA must be converted to oxaloacetate by entering the citrate cycle (NB this involves condensing with oxaloacetate which is only regenerated therefore no change really occurs).' Some confusion there since acetyl CoA was not converted to oxaloacetate. A possible solution Of my 44 scripts, 15 (or 34%) informed me with various turns of phrase that acetyl CoA (derived from fatty acids) could be changed into glucose. What can we do to convince them that it can't be done? Suppose after presenting the details of the citric acid cycle 1-5 we invited our students to have one last look at it in a new light. Suppose we then said that the cycle could be viewed as a 'core' of 4-carbon 'bits' to which 2-carbon 'bits' were added then removed, carbon by carbon. One of the 4carbon 'bits', oxaloacetate, could enter gluconeogenesis to form glucose. Could fatty acids give rise to this 4carbon compound? Suppose we then went on to point out that the fatty acid carbons 'stay out of the core'. That although two carbons (from acetyl) enter the cycle, two carbons leave in each 'turn' of the cycle. No new oxaloacetate is formed: the cycle changes fatty acid carbon into carbon dioxide. Would this help? Perhaps, particularly if it were accompanied by a diagram (Fig 1) emphasising the cycle's