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PART III. ABNORMAL HEMOGLOBINS ELECTROPHORETIC ANALYSES OF THE ABNORMAL HUMAN HEMOGLOBINS HARVEY A. ITANO Although other methods have been introduced in recent years for the separation of hemoglobin components, electrophoretic analysis remains the most effective and widely used technique. The moving boundary method, which was used exclusively in the early studies of the abnormal forms of human hemoglobin, 223 retains its advantage for certain purposes, for example, electrophoresis in acid buffers, determination of absolute mobilities and isoelectric points, and separation of components of similar mobilities. However, zone electrophoresis is more readily available and is better adapted for the screening of large populations. The accelerated rate of discovery of new forms in recent years is largely the result of the widespread use of zone electrophoresis. This method is also a more effective tool for the separation and purification of components in useful quantities. Other investigators will discuss the application of zone electrophoresis elsewhere in these Proceedings, and I shall confine my remarks to practical applications of the moving boundary method in the study of human hemoglobin. In order to facilitate further discussion and clarify the notation on the figures which accompany this presentation, I shall review briefly the nomen- clature of the human hemoglobins as agreed upon by interested investi- gators.4 5 Normal adult hemoglobin is the form found in most humans and is called hemoglobin A. Since minor electrophoretic components are present in the hemoglobin prepared from the red cells of normal adults,6 the designa- tion Al has been given to the major electrophoretic component, which com- prises 90-95 per cent of the total and which is electrophoretically homog- eneous.7 The minor components will be discussed elsewhere in these Pro- ceedings. The component designated hemoglobin A in the present discussion and figures is, strictly speaking, the Al component of the most recent nomen- clature.' Fetal hemoglobin is hemoglobin F. and the abnormal hemoglobin characteristic of sickling cells is hemoglobin S. Since sickle cell hemoglobin was also designated hemoglobin ~ at one time,3 the letter B has been omitted in naming the other abnormal forms, which are called hemoglobins C, D, E, G. etc., in the order of their discovery. In the initial studies of hemoglobins A and S,~ it was shown that hemo- globin A migrates more rapidly at alkaline pH and that hemoglobin S migrates more rapidly at acid pH (fig. 1~. At pH 6.9 the two forms migrate in opposite directions (fig. 2~. These observations led to the conclusion that ~ 1 1 A 144

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ELECTROPHORETIC ANALYSES ITANO 145 the difference in the molecules is in their net charge, for if one of the mole- cules has a greater frictional resistance to transport than the other, its mo- bility would be lower on both sides of the isoelectric point. X-ray diffraction studies supported this conclusion by show-in" that molecules of hemoglobins A and S are identical in shape.S 43.0 o -1.0 _ -2.0 -3.0 =50 ~ _~ ~ , __- ~C~. ~v I ] - emle I'm\\ 8.0 TO S.0 6.0 70 pH FIG. 2. ( below ) Electrophoretic pat- terns of carbonmonoxyhemoglobins A and S in nhosohate huger of nH ~S.9. ionic strength 0.1. Hemoglobins A and S migrate in opposite directions as anion and cation, respectively, in this buffer. r----r~ rip 7 From Pauling, L., Itano, H. A., Singer, S. J., and ~'ells, I. C.: Science 110: 54~, 1949. by Sickle Anemia FIG. 1. ( above ) Relation- ship of mobility to pH for carbonmonoxyhemoglobins A and S in phosphate buf- fers of 0.1 ionic strength. From Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Science 110: 543, 1949. a) Normal Sicftis Trait d) Mixture crf a) and b) The net charge of a protein molecule depends, not only on the number and identity of ionizable groups, but also on the pK of the groups and on the affinity of the molecules for ions other than hydrogen ion. Since the relative mobilities of the various forms of human hemoglobin at a given pH do not depend upon their ionic environment, the actual basis of their mobility dif- ferences in all probability lies in the numbers and relative positions of their acidic and basic amino acid residues. Although the pK of each type of ion- izable group lies within a characteristic range, the exact value varies with structure, charge, number, and proximity of neighboring groups. It is there- fore conceivable that two forms of human hemoglobin have the same amino acid composition and yet have different net charges at the same pH. It is also possible for two hemoglobins to differ in their content of charged

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146 PART III. ABNORMAL HEMOGLOBINS amino acid residues and have the same electrophoretic mobility at a given pH if their net charges are equal. Hemoglobins which have the same electro- phoretic behavior may be regarded as belonging; to an "electrophoretic pheno- type," and the detection of genetically-determined alterations that do not affect net charge requires the use of an independent method. Hemoglobins S and D, for example, are indistinguishable by electrophoresis (fig. 3 ); how- ever, solubility measurements and sickling tests indicate that they are dif- ferent molecular species. 1 3, 9 Normal (A/A) Hi_ In_ Sickle Trait (A/S) Sickle Cell Anemia (S/S) Slekle Cell Anem i a plus Fetal tS/S,f) M. H. (A/O) R. H. (S/D) B. H. (S./ D, F) FIG. 3. Electrophoretic pat- terns showing the similarity of carbonmonoxyhemoglobins S and D in cacodylate buffer of pH 6.5, ionic strength 0.1. From Stu rgeon, P., Itano, H. A., and Bergren, W. R.: Blood 10: 389, 1955. Hemoglobins A and S were resolved in phosphate buffers of ionic strength 0.1, and their charge difference was estimated to be two to four units from titration and electrophoretic data.t These hemoglobins are also well resolved in cacodylate buffer of pH 6.5 and ionic strength 0.1. Hemoglobin S and C are also well resolved in the cacodylate buffer although their mobility dif- ference is less than that between A and S.' However, a mixture of hemo- globins A and F or of hemoglobins S and E does not resolve in this buffer.~ The components in the respective mixtures are so closely similar in mobility that separation of their boundaries is obscured by diffusion. The absolute mobility of each component can be determined separately; however, it is a difficult task technically to obtain sufficient accuracy to establish the small differences in mobility encountered in the study of the

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ELECTROPHORETIC ANALYSES ITANO 147 human hemoglobins. Such small differences can be demonstrated in other ways. If the pH-mobility curves of the two components are not parallel, a different pH which results in better separation can be found. This is true of hemoglobins S and E (Eg. 4), which differ more in mobility in alkaline buffers than in acid buffers.9 ii Components of very similar mobility may be H E M O G LO B I N E Acid ~ , ti Sicl~lc Trait (AJS) L ~ I ~ a I I n 0 Norma ~ (~/A) Control Slel~l. Ccil-~/omogiobin C Disease (S/C) Sicille Call-~halesesmia Dieso.e (A,f/S) ad, H. (A,f/E) FIG. 4. Comparison of moving boundary patterns of carbonmonoxyhemoglobin in cacodylate buffer of pH 6.5, ionic strength 0.1, with the results of paper electro- phoresis in barbital buffer of pH 9.2, ionic strength 0.01. The close similarity of hemoglobins S and E in the acid louder is contrasted with their difference in the alkaline buyer. The preponderance of hemoglobin E and the presence of hemoglobin associated with the thalassemia gene is also shown. From Sturgeon, P., Itano, H. A., and Bergren, W. R.: Blood 10: 389, 1955. resolved with use of very dilute buffers,7 it in which continuous sharpening of the boundaries of one limb overcomes the effect of diffusion ~ fig. j ~ . Finally, each of the two similar components can be mixed with a known hemo- globin which differs appreciably in mobility from the components to be com- pared. The mixtures are analyzed under identical conditions, and the rela-

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log PART. III. ABNORMAL HEMOGLOBINS Modification ~ pH 65 pil 8.8 1 Sickle Cell si F Anemia Sickle Cell st A F S Thalassemia Sickle Cell CtS sit Hb-C Disease ~ _i Sickle Cell l Hb-D Disease si A Ei ~ IF (a) sl A,F - (e) (b) (f ) Si ~ jA F (C) (d) . (h) FIG. 5. ( left) Comparison of the components of carbonmonoxyhemoglobin in three types of sickle cell disease. To the left, analyses in cacodylate buffer of pH 6.5, ionic strength 0.1. To the right, analyses in 0.01 M Na.,HPO4. Hemoglobins A and F are resolved in the dilute phosphate buffer and are not resolved in the cacodylate buffer. Hemoglobins S and D are not resolved in either buffer and are distinguished from each other by solubility measurements.3 FIG. 6. ( right) Ascending boundary patterns of various mixtures of human car- bonmonoxyhemoglobin in cacodylate buffer of plI 6.5, ionic strength 0.1. These ex- periments show the slight but discernible differences in mobility between hemoglobins A and F and between hemoglobins S and E at this pH. Hemoglobin F is the reference component in the comparison of hemoglobins S and E. Hemoglobin S is the reference component in the comparison of hemoglobins ~ and F. The difference between E and F is virtually the same as that between A and S. From Itano, PI. A., Bergren, W. R., and Sturgeon, P.: Medicine 35: 121, 1956. tive mobilities of the two components in each mixture is observed (fig. 6~. Comparison of the patterns obtained in this manner will permit the detection of very small differences in mobility. A reference mixture that contains two known hemoglobins which migrate more rapidly and more slowly, respectively, than the two similar hemoglobins can also be used, and the relative position of the boundary of each of the two hemoglobins can be compared with those of the reference components (fig. 7~. The proportion of the reference compo- nent or components should be the same in both mixtures of a pair of compara- tive analyses since the apparent relative Nobilities vary with composition. Ci. F Go S F FIG. 7.Ascending boundary patterns of two mixtures of car- bonmonoxyhemoglobin in caco- dylate buffer of pH 6.5, ionic strength 0.~. Hemoglobins F and C were used as reference com- ponents to compare hemoglob- ins E and S. The difference in net charge between hemoglob- ins E and S in this buffer ap- pears to be less than one.

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ELECTROPHORETIC ANALYSES ITANO 149 The greater reliability of the mixture method over the determination of absolute mobilities rests upon the fact that differences in mobility are less sensitive to slight changes in pH than are absolute mobilities. Electrophoretic analyses not only detect the components in a mixture but also show the relative amounts of each of the components. Because of the diagnostic and genetic significance of the composition of the hemoglobin mixture in an individual, samples used in electrophoretic analyses of hemo- globin are not pooled. In order to avoid alteration of composition that might result from crystallization or other procedures for fractionation, samples are analyzed as obtained from hemolyzates of washed red cells, and preparative procedures other than removal of insoluble stroma material by centrifugation is avoided. Aside from the disappearance of fetal hemoglobin in infants and its appearance in some forms of chronic anemia,~3 the composition in a given individual probably remains constant with time.~4 At 0.1 ionic strength the apparent composition of a mixture indicated by the schlieren pattern is close to the true composition,~4 but at low ionic strength the disparity between apparent and true composition may become large. Although it is possible to compute the deviation from true composition,7 it is usually more practicable to obtain an empirical relationship between apparent and true compositions by analyzing known mixtures in the same buffer. Both inherited and acquired characteristics of the hemoglobin molecule are demonstrable by electrophoresis. Inherited characteristics are presumably in- corporated into the structure of the molecule at the time of its biosynthesis under genetic controls Acquired characteristics result from alterations that take place after completion of biosynthesis and include denaturation of A X~ (a) fib Fib ~ SEA (c) HbCf~b: | (e) HbAHbs (b) fib sHbs I (d) Nb5Ht: I (f) Nba~bC FIG. 8. Hemoglobins A, S. and C are synthesized under the control of allelic genes at the locus designated Hb.'3 24 The electrophoretic patterns of carbonmonoxyhemo- globin in cacodylate buffer of pH 6.5, ionic strength 0.1 corresponding to the six possible combinations of the three genes are shown. The X component is probably the same as hemoglobin A.,.5' 6 The presence of fetal in homozygosity for the sickle cell gene is shown. From Itano, H. A., and Pauling, L.: Svensk. Kem. Tidskr., in press.

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150 PART III. ABNORMAL HEMOGLOBINS globin and both reversible and irreversible reactions involving the heme groups. In general, an individual has one or two major components in his red cells, depending upon his genotype (fig. 8~. These are the adult hemo- globins, normal (A) and abnormal ~ S. C, D, E, etc. ~ . In addition, fetal hemoglobin (F) occurs in severe anemia,: and minor components are also present in anemic as well as non-anemic individuals.6 The proportion of the major components as well as the type appears to be an inherited characteristic. i' In most cases of heterozygosity for the genes for hemoglobin A and for an abnormal hemoglobin, hemoglobin A comprises more than 50 per cent of the total. However, in the simultaneous presence of one gene for thalassemia and one gene for an abnormal hemoglobin,9 i& the abnormal hemoglobin is usually the preponderant species (figs. 4, 94. In other words, the net effect of the thalassemia gene, according to the results of electrophoretic studies, is the relative suppression of the synthesis of hemo- globin A or of an electrophoretic phenotype of hemoglobin A. (a) Normal . ~ c ) (e) Sickle Cell ~ ~ Father BG Trait (b) Sickle Cel' Anemia (d) l Patient LO(F) _ (f) Mother E FG ~ F`IG. 9. Electrophoretic patterns of carbonmonoxyhemoglobin in cacodylate buffer of pH 6.5, ionic strength 0.1, showing the effect of a thalassemia gene on electrophor- etic composition. Since the mother is heterozygous for the sickle cell gene and the father does not have this gene, the patient cannot be homozygous in the sickle cell gene However, the pattern closely resembles that seen in sickle cell anemia. The relative increase in hemoglobin S can be ascribed to the thalassemia gene, which was inherited from the father and which inhibits the synthesis of hemoglobin A. The slow boundary in the patient's pattern represents both hemoglobins A and F. which are not resolved in the buffer used. Art elevated proportion of hemoglobin A., is evident in the pattern of thalassemia minor. From Sturgeon, P., Itano, H. A., and Valentine, W. N.: Blood 7: 350, 1952. _, ~ Inherited differences are demonstrated on preparations . in which all or nearly all of the molecules of hemoglobin are present in the native state and as the same compound, such as carbonmonoxyhemoglobin or ferrohemoglobin. Each of the inherited hemoglobin components in a preparation may become heterogeneous if the molecules are altered after synthesis, either within the circulating red cell or during preparation, storage, or analysis. If the degree of heterogeneity changes during storage or in the course of an analytical

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ELECTROPHORETIC ANALYSES ITANO 151 procedure, it is likely that alterations are taking, place in the state of the molecule. An example of a reaction which changes the net charge of a molecule of hemoglobin is the oxidation of ferrohemoglobin or one of its compounds to ferrihemoglobin, which results in an increase of one unit in the net positive charge of each heme iron. In order to study the effect of this reaction on electrophoretic homogeneity, partially oxidized mixtures of carbonmonoxy- hemc~globin were used since the components of such mixtures, carbonmonoxy- hemoglobin and ferrihemoglobin, are stable compounds. Analyses of partially oxidized preparations of carbonmonoxyhemoglobin in phosphate buffer of pH 6.85 and ionic strength 0.01 revealed a mixture of components, including some with mobilities between those of carbonmonoxyLemoglobin and ferri- hemoglobin ~ fig. 10 ~ . Comparison of the composition of such mixtures as determined by electrophoretic arid spectrophotometric analyses indicated the intermediate components are molecular species in which one to three of the four heme irons are oxidized. FIG. TO.Separation of intermediate compounds of carbonmonoxyhemoglobin and ferrihemoglobin by electrophoresis in phosphate buffer of pH 6.85, ionic strength 0.01. The major components correspond to molecules in which 0, 1, and 2 of the four hemes of carbonmonoxyhemoglobin have dissociated carbon monoxide and acquired r positive charges by oxidation lo rom ltano, H. A., and Robinson, E.: J. Am. Chem. Soc. 78: 6415, 1956. terrihemoglobin combines with cyanide ion at each of the hemes to form ferrihemoglobin cyanide, a stable compound which dissociates very slowly. The cyanide ion neutralizes The positive charge on the heme iron of ferri- hemoglobin and diminishes the electrophoretic mobility of the latter com- pound at acid plI. Partial saturation of ferrihemoglobin with cyanide re- sulted in the appearance of components identifiable as molecules in which one to three of the ferrihemes were combined with cyanide.~9 Ferribemoglobin cyanide, produced by the complete saturation of the ferriDemes with cyanide, was found to have the same mobility as carbonmonoxybemoglobin. Other alterations that can affect the electrophoretic mobility of hemo- globin no doubt occur. Loss of amide groups from the globin, configurational changes of globin that alter the pK of dissociating groups, and degradation o the hemes are possible examples. The occurrence of an alteration of this type is to be suspected if electrophoretic data cannot be related to genetic data. Or if the degree of heterogeneity in a given sample changes with time. The buffer used in the separation of intermediate compounds can be applied to other separations. Since intermediate compounds that differ by a

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152 PART III. ABNORMAL HEMOGLOBINS single charge can be separated, these components can be used as markers to estimate the charge difference between the normal and abnormal he~no- globins. Results to date indicate that difference in charge between hemo- globins S and C in phosphate buffer of pH 6.85 is less than that between hemoglobins A and S. As noted earlier, a similar observation was made in cacodylate buffer of pH 6.5. A problem that interests both biochemists and physical chemists is the homogeneity of a protein synthesized under the control of a single gene. If random errors occur in the incorporation of charged amino acids during the biosynthetic process, components that differ from the principal component by multiples of a single charge would be produced, and such components would be distributed symmetrically about the major component. While minor com- ponents do occur, these are not distributed symmetrically and do not appear as discrete components that differ in multiples of a single charge. Therefore such minor components do not arise from random errors in incorporation and their presence suggests either the action of a different gene or alterations that occur after synthesis. The results summarized above indicate the reliability and relative sim- plicity of the electrophoretic method for the detection of small differences in the net charge of hemoglobin molecules. The fact that all of the known abnormal forms of human hemoglobin are electrophoretically abnormal is partially a consequence of the almost exclusive reliance of investigators on eliectrophoresis for the initial detection of abnormal forms. However, it is also true that no unambiguous physical method is yet available for the de- tection of slight alterations that do not affect the net charge of a protein. Other methods that have been successful in the separation of similar proteins also appear to depend upon differences in net charge. The chromatographic separation of the human hemoglobins is one example of such a separation,'' and the countercurrent distribution of insulin components that differ by an amide group is another.' ''- On the other hand, insulin molecules that have the same net charge and differ only in their content of uncharged amino acid residues are indistinguishable by physical methods, including countercurrent distribution.22 Thus, while it is likely that mutations which result in the alteration of content of uncharged residues occur, there exists no satisfactory physical method for the detection of such events. The electrophoretically normal hemoglobin associated with the thalassemia allele may indeed be an abnormal hemoglobin in which the net charge is normal and which is synthesized at a subnormal rate. A complete amino acid analysis may provide evidence for a net change in composition but does not detect differences in position of residues. An alteration such as the transposition of two similar uncharged amino acid residues within a polypeptide chain, for example, can be detected only by complete sequential analyses. Until less laborious procedures for the

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ELECTROPHORETIC ANALYSES ITANO 153 detection of abnormal molecules with normal reset charge are developed, experimental studies of the effect of mutations on the structure of hemo- globin will depend on the use of electrophoretically abnormal forms. REFERENCES 1. Pauling, L., Itano, H. A., Singer, S. J., and Wells, I. C.: Sickle cell anemia, a molecular disease, Science 110: 543 (25 Nov.) 1949. 2. Itano, H. A., and Neel, J. V.: A new inherited abnormality of human hemoglobin, Proc. Nat'l. Acad. Sci., IJ. S. 36: 613 (Nov.) 1950. 3. Itano, H. A.: A third abnormal hemoglobin associated with hereditary hemo- lytic anemia, Proc. Nat'l. Acad. Sci., U. S. 37: 775 (Dec.) 1951. . Chernoff, A. I., et al.: Statement concerning a system of nomenclature for the varieties of human hemoglobin, Blood 8: 386, April 1953; Science ll8: 116 (July) 1953. 5. Lehmann, H.: International Society of Hematology: The hemoglobinopathies, Blood I2: 90 (Jan.) 1957. 6. Kunkel, H. G., and Wallenius, G.: New hemoglobin in normal adult blood, Sci- ence 122: 288 (12 Aug.) 1955. 7. Hoch, H.: The steady state, a test for electrophoretic homogeneity, B~ochem. I. (London) 46: 199 (Feb.) 1950. 8. Perutz, M. F., Liquori, A. M., and Eirich, F.: X-ray and solubility studies of haemoglobin of sickle-cell anemia patients, Nature 167: 929 (9 June) 1951. 9. Sturgeon, P., Itano, H. A., and Bergren, W. R.: Clinical manifestations of in- herited abnormal hemoglobins. I. The interaction of hemoglobin S with hemo- globin D. II. Interaction of hemoglobin E and thalassemia trait, Blood 10: 389 (May) 1955. 10. Itano, H. A., Bergren, W. R., and Sturgeon, P.: The abnormal human hemo- globins, Medicine 35: 121 (May) 1956. 11. Itano, H. A., Bergren, W. R., and Sturgeon, P.: Identification of a fourth ab- normal human hemoglobin, J. Am. Chem. Soc. 76: 2278 (20 April) 1954. 12. Itano, H. A., and Robinson, E.: Demonstration of intermediate forms of carbon- monoxy- and ferrihemoglobin by moving boundary electrophoresis, J. Am. Chem. Soc. 78: 6415 (20 Dec.) 1956. 13. Singer, K., Chernoff, A. I., and Singer, L.: Studies on abnormal hemoglobins. I. Their demonstration in sickle cell anemia and other hematolo'~ric disorders by means of alkali denaturation. II. Their identification by means of the method of fractional denaturation, Blood 6: 413, 429 (May) 1951. 14. Wells, I. C., and Itano, lI A.: Ratio of sickle-cell anemia hemoglobin to normal hemoglobin in sicklemics, J. Biol. Chem. 188: 65 (Jan.) 1951. 1 5. Itano, PI. A.: The human hemoglobins: Their properties and genetic control, Advances in Protein Chem. 12) in press. 16. Neel, l. V., Wells, I. C., and Itano, H. A.: Familial differences in the propor- tion of abnormal hemoglobin present in the sickle cell trait, J. Clin. Invest. 30: 1120 (Oct.) 1951. 17. Itano, H. A.: Qualitative and quantitative control of adult hemoglobin syn- thesis a multiple allele hypothesis, Am. J. Human Genetics 5: 34 (March) 1953. 18. Sturgeon, P., Itano, H. A., and Valentine, W. N.: Chronic hemolytic anemia associated with thalassemia and sickling traits, Blood 7: 350 (March) 1952.