David 1. Martin
and James E. Huheey' University of Maryland College Park, 20742
Sickle-Cell Anemia, Hemoglobin Solubility, and Resistance to Malaria
I n a recent contribution to Chemical Principles Exemplified (I), the vital difference in solubility between normal hemoglobin and the abnormal sickle-cell hemoglobin was related to the difference in a single amino acid (valine in place of glutamic acid) in the peptide chain. It is the purpose of this note to expand upon this subject somewhat to illustrate further the interesting consequences of this difference. The phenomenon of sickling of erythrocytes is of considerable pedagogical usefulness since it provides a bridge of "relevance" between chemistry and biology. I n a population illustrating sickle-cell anemia there will be three types of individuals: those with only normal hemoglobin (genotype AA, where A represents the gene coded for the synthesis of normal adult hemoglobin), those with only the abnormal sickling hemoglobin (genotype SS, where S represents the gene coded for the synthesis of the abnormal hemoglobin), and those with both types of hemoglobin (genotype AS). I n a randomly breeding population the frequencies of these three types will be (1 - s)%,s2 and 2(9 - s2), respectively, where s represents the fraction of S genes in the population. Hemoglobin of the S type has decreased solubility as outlined previously (1). However, the cause of the solubility decrease is more subtle than a simple in-
' Drs. Martin and Huheey are members of the Interdisoiplinary Approaches to Chemistry Project at the University of Maryland. This article is related to materials being developed for high school use in the IAC Project.
crease in the hydrophobic character of the protein resulting from the substitution of the nonpolar amino acid valine for the ionic amino acid glutamate. This is clearly demonstrated by the fact that normal hemoglobin and sickle hemoglobin have the same solubility when they are both in the oxygenated form (9). However, the solubility of both normal and sickle hemoglobin decreases when oxygen is removed. The solubility of unoxygenated, normal hemoglobin is one half the solubility of its oxygenated form but the solubility of siclclc hemoglobin decreases by a factor of fifty when oxygen is removed. The explanation for this phenomenon lies in the fact that the protein chains of the hemoglobins undergo a substantial conformational change when they are deoxygenated. I n the case of sickle hemoglobin the presence of valine allows the protein to assume a conformation not possible when the normal amino acid, glutamate, is present. The conformation of the S hemoglobin allows the formation of a sufficient number of intermolecular bonds so that the protein essentially polymerizcs into a microfilament in which the molccules are stacked on each other (3). These microfilaments combine with similar microfilaments to form a rod-like structure which is insoluble and which is responsible for the sickling of the erythrocyte. Filament formation occurs in capillaries where the hemoglobin is deoxygenated by the tissues. The sickled cells clog the capillaries causing localized necrosis and gradual deterioration of vital organs such as the brain, heart, and kidney. An individual possessing nothing but S hemoglobin usually dies from the effects, though not all die in infancy. About half reach re-
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productive age but almost all die before the age of forty. This disease has received recent attention in the national press as a result of the discovery that urea can be used to relieve the painful "crisis" symptoms which periodically appear as a result of blockage of the c a p illaries. The mode of action of urea as a drug in this instance is a matter of speculation but its effects may not be due to a specific interference with bonds formed by the substituted valine. One of the best known properties of urea is its ability to disrupt protein structure by forming hydrogen bonds and intercalating itself into the folds of the protein thereby causing a conformation change. Thus, urea may act by preventing the deoxygenated hemoglobin S from attaining the proper conformation required to form the microfils, ments. It should be pointed out that the urea treatment is not in any sense a cure but merely a palliative. Since the disease is genetic in origin, i t can only be eliminated in future generations by genetic counseling or perhaps by future advances in "genetic engineering." Individuals that are heterozygous (i.e., possess both normal and S hemoglobin) normally do not exhibit the extremely serious effects of sickle-cell anemia. Although part of their hemoglobin is abnormal, the reduced solubility normally does not proceed to a crippling extent. The tendency for the cells to sickle can be seen, however, when the partial pressure of oxygen is extremely low, as at high altitudes or in solutions of sodium metabisulfite (a clinical test for the sickle cell trait). In view of the decreased ability to survive to reproductive age and bear children that may receive the d e fective S genes, how does the trait survive? It is obvious that the strong selection against bearers of two S genes will tend to reduce their frequency in a population. The general expression (4) for the frequency of the S gene in the nth generation, assuming no reproduction by those bearing only S hemoglobin is
Thus if both the A gene and the S gene were to occur initially at equal frequencies the frequency of the S gene would be reduced to 0.1 after only eight generations and to 0.01 after only ninety-eight generations. Although the selection against sickle-cell anemics is less severe than assumed in this model, clearly the mutation would have disappeared long ago were it not favored in some way. The mechanism favoring retention of the S gene in the population is as fascinating as the mechanism that selects against it. As was pointed out, the
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heterozygotes normally do not suffer problems from sickling since the oxygen partial pressure does not get low enough in the body to induce sickliug if only a portion of the hemoglobin is abnormal. The oxygen level becomes sharply reduced, however, in an erythrocyte in which the parasitic protozoan Plasmodium, causative agent for malaria, is developing. Sickling of such a cell occurs and it tends to be destroyed by phagocytes as an "alien particle" (5). The Plasmodium organism is destroyed in the process and the disease, malaria, is controlled. In the tropics where malaria is pandemic, susceptibility to malaria can be just as debilitating as sickle-cell anemia. As a result, both types of homozygotes tend to fail to reproduce: SS because of early death from sickle-cell anemia, AA because of early death from malaria. The heterozygotes survive to reproduce. As a result of recombination of genes, the new generation will contain all three types of individuals again and a balanced system can result. In some areas of Africa which are heavily infested with malaria, the incidence of the S gene has risen to about 25%. Finally, the various aberrant hemoglobins serve as an ideal example of the usefulness of electrophoretic methods of separating and identifying different proteins. The pioneering work of Pauling and co-workers (6) illustrated this technique in the separation of S and A hemoglobins. Again, this diierence can be related directly to the difference in a single amino acid per peptide chain (two per hemoglobin molecule): at pH 6.9 glutamic acid exists as the negative conjugate base but valine is neutral. Normal hemoglobin therefore has a greater tendency to migrate towards the positive electrode. Recently this method has been applied to the routine testing of patients a t a clinic in Chicago. The chemistry of the hemoglobins presents a potential gold mine of material for instructors interested in showing the relationship between chemistry and other disciplines. Over 150 mutant hemoglobins have been discovered. Some are lethal; some are harmless. The data obtained, however, have been more than a chemical curiosity and have been important in fields as diverse as medicine, physiology, genetics, and anthropology (8). Thus, there is material for the interested student no matter what his bent. Literature Cited (1) PATTON,A. R.,J. CHEM.EDUC., 47,691 (18701. H.,A N D HUNTSMAN, R. G., " M d 8 H&em~~lobina," J. B . (2) LEHMANN. Lippinoott Co.,Philadelphia, 1966. (8) M o n h u ~ M.. ~ ~ Clin. . Chen.. 13, 578 (19671. (4) LI, C. C., "Population Genetioa," The University of Chicago Press. Chicago. 1955,p.252. (51 Reference @I, p. 208. 8. J., AND WBLLB,I. C.,Scienes (6) P ~ u L w a .L.. ITANO. H. A,, SINOER, 110,543 (19491.
