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THE STRUCTURAL BASIS OF DIFFERENCES IN ELECTROPHORETIC BEHAVIOR OF HUMAN HEMOGLOBINS I. HERBERT SCHEINBERG Sickle-cell hemoglobin, hemoglobin C, and several other variants of human hemoglobin differ in electrophoretic mobility from normal human adult hemo- globin (hemoglobin A).1 2 Since all of these hemoglobins probably have the same size and shape,3 their differences in electrophoretic mobility are pro- portional to differences in the net electrical charge of the molecules. The present paper is an attempt to relate these differences in charge to differences in chemical structure. HEMOGLOBIN A ( Total Charge 186 Net Charge 14 Total Charge 189 Net Charge 1 1 HEMOGLOBIN S - - - - ...... )~\ ~+~+++~ )~> K+++++++ I- )_41 w---- HEMOGLOBIN S - Total Charge 183 Net Charge 11 FIG. 1. Total and net charges of hemoglobins A and S. including two different total charges of S with the same net charge (pH 8.6). Figure 1 shows that two hemoglobins can have the same difference in net charge with more than one total charge. In both cases illustrated, sickle-cell hemoglobin, or hemoglobin S. is shown at pH 8.6 with a net charge which is three units more positive than normal hemoglobin. In one case, however, this is the result of three more positive charges on hemoglobin S than on normal hemoglobin. In the other case it is the result of three less negative charges on hemoglobin S than on normal hemoglobin. Electrophoretically, there should be no difference between both of these hypothetical hemoglobin S molecules but, as will be clear below, both represent different chemical structures. Near the physiological pH probably all of the charges on hemoglobins come from their constituent amino acids. Neutral amino acids contribute one terminal carboxyl and one terminal amino group to each polypeptide chain which is not a ring. Additional positive charges are contributed by the nitro- genous groups of the three basic amino acids, Rb (fig. 2~. Additional negative charges are contributed by the second carboxyl group of aspartic and glutamic acids, Ra. Within the pH range of about 6.5 to 9.0 all of these groups (except the imidazole of histidine above pH 8.0) generally exist in their charged forms4 (fig. 31. Thus, in this pH range a greater net positive charge on sickle- cell hemoglobin may be the consequence of a greater number of positively 227

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228 H3N~ - Rb - COO- basic amino acid PART III. ABNORMAL HEMOGLOBINS lysine, if -Rb- is-CH- (CH-~) 4 arginine, if-RI,- NH;3+ is -CH- (CH .), AH NH3+ histidine, if -Ret,- is -CH- \C NTH (AH.,- C - CH 1 1 HN NH+ C H FIG. 2. Structure of the positively charged forms of the three basic amino acids. Rb-NHm = Rb-N pH 9.5 = pH 11.5 R., is lysine or arginine ;Rb-NH~ = Rb-N pH 6.0 = pH 8.0 Rb is histidine RaCOO = RaCOOH pH 5.0 = pH 3.0 Ra is asp.artic or glutamic acid FIG. 3.The approximate ranges of pH in which various charged groups of amino acids ionize. charged nitrogenous groups, or a smaller number of carboxyl groups on this molecule in comparison with the normal. Figure 4 shows the result of a paper electrophoresis experiment performed in a buffer of pH 8.6 with hemoglobins A, S and C. The vertical line is the locus of the points of application of the hemoglobins, and the anode, as indi- cated at the top of the figure, is at the right. All three hemoglobins moved toward the anode indicating that, at this pH, all three have a net negative charge. This charge is obviously least negative, or most positive, on hemoglobin C and most positive, or least negative, on hemoglobin A. Since all of the groups mentioned above, except the imidazole of histidine, are charged at this pH, the observed differences in net charge may be due to differences in the numbers of free carboxyl, amino, or guanidinium groups on these hemoglobins (fig. 3~. This experiment suggests that the differences in charge between the three hemoglobins are not due to differences in their content of histidine since

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STRUCTURE AND ELECTROPHORETIC BEHAVIORSCHEINBERG 229 ::~::~ ::: :~ .~.~ ~ I. .A , ~ s ~ ~-~ FIG. 4. Paper electrophoresis experiment in a veronal buffer of pH 8.6. In this and succeeding figures the letters to the left of the starting points denote the type of hemo- globin applied, and the + and signs in- dicate the sides on which the anode and cathode, respectively, were placed. charge differences persist at pH 8.6 where imidazole groups are largely un- charged.4 If electrophoretic mobilities are determined at other than near neutral pHs it is possible to observe the effect of selectively making some groups electrically neutral. If we raise the pH of the buffer used for electrophoresis to about 12.0, almost all of the lysine and some of the arginine residues of hemoglobin will have lost their electrical charger (fig. 3~. An experiment performed at pH 11.7 is shown in figure 5 and it is apparent that differences in mobility, and therefore in charge, persist at this pH. It is true that the difference are less A. .~ ,^ FIG. 5.- Paper electrophoresis experi- :3: : ment in a phosphate buffer of pH 11.7. :::

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230 PART ITI. ABNORMAL HEMOGLOBINS marked than those seen at pH 8.6 but this is due to the fact that the net charge of hemoglobin A rises from about -14 at pH 8.6 to about -67 at pH 12 so that a difference of about three in the net charge is relatively less at the higher pH. This result suggests that the differences in charge between these hemo- giobins are not due to differences in lysine or, probably, arginine since the differences in charge remain even when most lysine and arginine residues have become electrically neutral. If we now lower the pH at which the electrophoresis is carried out we first notice that the relative Nobilities change in sign. Thus, at HI 5.25 hemoglobin C now moves fastest, and hemoglobin A, slowest (fig. 6 ). This is, calf course, a consequence of being below the isolectric point of all three forms of hemoglobin so that each now has a net positive charge, with hemoglobin still possessing a greater net positivity than hemoglobin S. and hemoglobin S possessing greater net positivity than the normal form. ........ a . ~. ,,., , >,.,.,< ~ .. ... . , ~ FIG. 6. Paper electrophoresis experiment in an acetate buffer of pH 5.25. FIG. 7. Paper electrophoresis experiment in an acetate buffer of plI 4.01. F`IG. 8.- Paper electrophoresis experiment in a glycine buffer of pH 3.35. Figure 3 shows that carboxyl groups lose their charges between about pH 5 and 3. If we perform electrophoresis experiments at about pH 4 and below, the carboxyl groups of the hemoglobins should be uncharged. Figure 7 shows that at pH 4.01 there is essentially no difference in mobility between the three hemoglobins. This is also true at pH 3.3i in a glycine buffer (fig. 8~. These results indicate that the differences in net charge between the three hemoglobins are due to differences in their content of free carboxyl groups because the differences in charge are abolished at a pH at which only the carboxyl groups of the proteins' amino acid residues are uncharged. That this disappearance of the differences in Nobilities between the hemo- globins is not due to irreversible denaturation at acid pH is shown by the following experiment. One set of the three hemoglobins was dialyzed against a buffer of pH 4.01, and another set was dialyzed against a buffer of pH 3.35

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STRUCTURE AND ELECTROPHORETIC BEHAVIOR SCHEINBERG 231 for the length of time that the electrophoretic runs were carried out at these pHs. Some hemoglobin precipitated, but the supernatant solutions were re- turned to pH 8.6 and subjected to electrophoresis. Figures 9 and 10 show that the differences in mobility characteristically observed at pH 8.6 are still present in these supernatant hemoglobin solutions. Even the presumably de- natured and precipitated hemoglobins retained these differences when dissolved in sodium hydroxide and subjected to electrophoresis in a buffer of pH 10.8 (fig. 11~. FIG. 9. Paper electrophoresis experiment in a veronal buffer of pH 8.6 with hemo- globins which had been dialyzed against an acetate buffer of pH 4.01 for 88 minutes, and then against veronal for 16 hours. (Supernatants) FIG. 10. - Paper electrophoresis experiment in a veronal buffer of pH 8.7 with hemo- g;obins which had been dialyzed against a glycine buffer of pH 3.35 for 74 minutes, and then against veronal for 16 hours. (Supernatants) FIG. 11.- Same as Fig. 9, but using precipitated hemoglobins dissolved in NaOH. The observed differences in net charge between these hemoglobins do not depend on differences in small-ion binding since complete removal of small- ions by ion-exchange resins results in different isotonic points for the three proteins.5 Differences in tyrosine or sulfhydryl content should not contribute to differences in charge except at pH values of about 10 and higher.4 The phosphorus content of hemoglobins A and S is not large enough, according to Havinga, to account for the difference in charge.6 Pauling and his co-workers calculated, on the basis of the difference in isoelectric points between hemoglobins A and S. that hemoglobin S possessed two to four more net positive charges per molecule than hemoglobin A.i On the basis of that calculation, and the results reported above, it appears that hemoglobin S possesses about two to four less free carboxyl groups, and hemo- globin C possesses perhaps five or six less free carboxyl groups than normal adult human hemoglobin.7 Several possible structures could account for these differences as shown below. The abnormal hemoglobins may have fewer