Involvement of Glu G3(101)@ in the Function of Hemoglobin COMPARATIVE 0 2 EQUILIBRIUM STUDIES OF HUMAN MUTANT HEMOGLOBINS*

The glutamyl residue at G3(101)8 of normal hemo- globin (Hb A) is one of the a& subunit contacts which are vital to O2 binding properties of the molecule. The O2 equilibrium properties of the four mutants with different substitutions at this site are studied in order to elucidate the role of this residue. Under stripped conditions with minimum chloride the order of 0 2 affinity is: Hb A (Glu) << Hb Rush (Gln) Hb British Columbia (Lys) c Hb Potomac (Asp) c Hb Alberta (Gly). The first Adair constants, K1, for the mutant hemoglobins are greater than that for Hb A whereas the fourth, K,, are similar, indicating that the allo- steric constants (L) of these mutants are greatly re-duced. Therefore, the G3(101)0 residue contributes intrinsically to the strengthening of the structural constraints that are imposed upon the deoxy (T) forms but not the oxy (R) form. On addition of 0.1 M C1- and further addition of 2,3-diphosphoglycerate or inositol hexaphosphate, their 0 2 affinities and cooperativities are altered, reflecting different responses to anionic ligands. H b Rush exhibits a stronger chloride effect than Hb A and the other variants and, as a result, an increased Bohr

Columbia (Lys) c Hb Potomac (Asp) c Hb Alberta (Gly). The first Adair constants, K1, for the mutant hemoglobins are greater than that for Hb A whereas the fourth, K,, are similar, indicating that the allosteric constants (L) of these mutants are greatly reduced. Therefore, the G3(101)0 residue contributes intrinsically to the strengthening of the structural constraints that are imposed upon the deoxy (T) forms but not the oxy (R) form. On addition of 0.1 M C1-and further addition of 2,3-diphosphoglycerate or inositol hexaphosphate, their 0 2 affinities and cooperativities are altered, reflecting different responses to anionic ligands. H b Rush exhibits a stronger chloride effect than Hb A and the other variants and, as a result, an increased Bohr effect and a smaller heat of oxygenation at pH 6.5. These changes are consistent with an increased positive net charge in the central cavity of Hb Rush and subsequent extra anion binding in the deoxy form. The tetramer to dimer dissociation constants are estimated to be greater than normal for W British Columbia and Iess than normal for Hb Alberta.
This comparative study of the G3(101)B mutants indicates that the size and the charge of this residue may influence the switching of two neighboring interchain hydrogen bonds that occurs during oxygenation of normal hemoglobin.
A structure-based understanding of the Os binding mechanism of the hemoglobin tetramers has been developed by Perutz and his co-workers (1-4) using x-ray crystallography.
According to these studies structural sites for Os-linked allosterism are grouped into three specific regions: 1) Os binding sites (heme and its contacts), 2 ) allosteric binding sites (H+, Cos, 2,3-diphosphoglycerate, C1-binding sites), and 3) subunit interfaces (mainly the a&). Simultaneously with the xray crystallographic investigations, extensive studies of the functional properties of abnormal hemoglobins as well as * This study was supported in part by United States Public Health Service Research Grants HL20142 and AM17850 (D. T.-b. S. and R. T. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. chemically modified hemoglobins in solution state have been carried out by other techniques. Conclusions from these results are complementary to those drawn from x-ray crystallographic studies in many ways, but they are not entirely in agreement. Therefore, further studies of abnormal and chemically modified hemoglobins are continuing in order to gain a better understanding of the ligand binding mechanism of the hemoglobin tetramer.
According to the Monod, Wyman, and Changuex two-state theory (5)  These changes in the allosteric equilibrium are due to alterations of some of the contacts of these residues which stabilize either the T form or the R form of the quaternary structure of normal hemoglobin. In this paper we describe a comprehensive, comparative study of four mutant hemoglobins that affect the residue 101 (G3) of the @ chain. This is located between Gl(99)B and G4(102)@ at the a& subunit interface. Earlier reports of G3(101)@ hemoglobin mutants indicate that substitution for the glutamyl residue in normal hemoglobin results in variable changes of the functional property of the hemoglobin molecule. Hb British Columbia (Lys (l0l)p) (8) and Hb Alberta (Gly (1Ol)p) (9) were found to have high O2 affinities while Hb Rush (Gln (l0l)P) (10) was reported to exhibit a normal function. The remaining mutant, Hb Potomac (Asp (lOl)p), had been detected because of an abnormal O2 affinity of the blood of the affected individuals rather than by electrophoresis because its amino acid substitution does not involve a charge difference (11). Explanations of these abnormal functional properties based upon x-ray crystallographic data are not yet available (12). Furthermore, the earlier reports were individual studies that were carried out under different experimental conditions and therefore cannot be compared easily. To elucidate the functional differences of these mutants as well as to evaluate the importance of the glutamyl residue of G3(101)0 in the normal allosteric oxygen binding, we have done comparative studies of these four mutants under the same experimental conditions.

MATERIALS AND METHODS
Heparinized blood was obtained from the propositus or affected relative for whom the abnormal hemoglobins were first described. In 5919 the case of Hb British Columbia, a sample was also obtained from a second apparently unrelated person from Alaska. Unfractionated hemolysates obtained from washed, lysed erythrocytes were then freed from organic phosphates by passage through an ion-exchange (Dintzis) column at 4 "C (13). These were stored in the CO form on ice. The CO derivative of abnormal hemoglobin was isolated from normal CO hemoglobins on a DEAE-Sephadex (Pharmacia) column at 4 "C. A nine-chamber pH gradient system from pH 8.1 to 7.4 of 0.05 M Tris-HC1 was used for the chromatography. Hb Potomac was isolated using an anaerobic chromatography system as described earlier (14). The bound CO was removed by a photoirradiation method (15). Tris, bis-Tris,' inositol hexaphosphate, 2,3-diphosphoglycerate, and all the components of a methemoglobin reducing system (16) were purchased from Sigma. Reagent grades of NaCI, HCl, K2HP04, and KH,P04 were products of J. T. Baker Chemical Co. Oxygen equilibrium curves were measured with an automatic recording apparatus of Imai et al. (17)(18)(19). A Gilford 250 spectrophotometer and a polarographic oxygen electrode of Beckman Instruments (No. 39065) were employed in this apparatus. Data acquisition and reduction were accomplished using a PDP ll/V03 computer (Digital Equipment Corp.), as previously reported (19-21). The concentration of hemoglobin samples was 60 /IM on a heme basis. To maintain the methemoglobin formation at minimum levels, the methemoglobin reducing system (16) was added to each sample before measurement. The Adair constants (Kl to K,), i.e. the intrinsic association equilibrium constants for four-step oxygen binding, were evaluated by a least-squares curve-fitting method (19, 21). Overall oxygen affinity and cooperativity were measured by median oxygen pressure (P,) and maximal slope of the Hill plot (n.,,,J, respectively, which were calculated from the Adair constants. Oxygen equilibrium data obtained for various hemoglobin concentrations were analyzed in terms of the Hill scheme as described by Imai and Yonetani (22) to evaluate the tetramer-dimer dissociation constant and other related parameters.

RESULTS
Oxygen equilibrium curves for Hb A isolated from the four hemolysate samples showed good agreement. The percentage of methemoglobin was less than 3% before the equilibrium measurements and not more than 7% after. At pH 7.4 in the presence of minimum amounts of anions (approximately 7 mM C1-, phosphate free) all of the abnormal hemoglobins presented intrinsic properties of high O2 affinity (l/P,,,) and impaired cooperativity (nmax) (Fig. lA). The order of O2 affinity is: Hb A << Hb Rush Hb British Columbia < Hb Potomac s Hb Alberta. The differences in oxygen affinity among the abnormal hemoglobins are amplified to different degrees on the addition of 0.1 M chloride ( Fig. 1B). At this condition the O2 binding properties of Hb Rush resemble those of Hb A. On the addition of stronger allosteric effectors such as 2 mM 2,3-diphosphoglycerate or 2 mM inositol hexaphosphate, the O2 binding of these abnormal hemoglobins is partially restored to normal in varying degrees (Table I). The order of O2 affinity becomes: Hb Rush < Hb A < Hb British Columbia < Hb Potomac < Hb Alberta. Table I    for Hb A, Hb British Columbia, Hb Potomac, and Hb Alberta, but is significantly larger for Hb Rush (Fig. 2). Interestingly    be neglected as will be described later. The pH dependences of for the mutant hemoglobins except for Hb Alberta are similar to that for Hb A while Hb Alberta shows a somewhat larger pH dependence of rima.
Heats of oxygenation ( A H ) were obtained by using the Van't Hoff equation from oxygen equilibrium curves determined at different temperatures (20, 25, and 30 "C) in the presence of 0.1 M C1-. All of the hemoglobins studied show similar AH values at pH 9.0, while only Hh Rush shows a smaller AH value at pH 6.5 (Table 11).
Oxygen equilibrium curves were determined at different hemoglobin concentrations ranging from 2 to 120 MM in 0.05 M bis-Tris buffer (pH 7.4) containing 0.1 M C1-at 25 "C. Values of log P50 and n, (the Hill coefficient at half-saturation) are plotted against the inverse of protein concentration in Fig. 4. Hb British Columbia shows greater concentration dependences than the other hemoglobins. The decrease in both parameters upon going to low protein concentrations is attributed to partial dissociation of the tetramer to the (./I dimers that bind oxygen with a high affinity but without cooperativity. The concentration-dependent oxygenation data were analyzed by the method of Imai and Yonetani (22) in which oxygen saturation of a given hemoglobin solution is expressed by a linear combination of Hill equations for the tetramer and for the dimer. The fractions of the tetramer and dimer are treated as functions of oxygen saturation. Table I11 summarizes values of the parameters which are contained in the scheme described above. Lines drawn in Fig. 4 were calculated from these parameter values. The fits of the calculated lines to the experimental plots are good except for the log P50 data of Hb Rush and the n50 data of Hb Alberta. The parameter values indicate that: the free cub dimers of all the hemoglobins listed in Table I11 exhibit the same oxygen affinity; the order of oxygen affinity for the tetramer is Hb Rush < Hb A < Hb British Columbia < Hb Alberta; the cooperativity of Hb Alberta is lower than those of the other hemoglobins; and the tetramer-dimer dissociation constant of Hb Rush is normal, but the subunit dissociations of Hb Alberta and Hb British Columbia are less and more, respectively, compared to that of Hb A. From the present analysis, the P, and n, values for purely tetrameric hemoglobin can be inferred. The parameter values calculated for 60 PM hemoglobin solutions (P50 (60 p~) ) are somewhat different from the inferred values for the tetramer (p60 ( t ) ) , being most marked in Hb British Columbia (Table 111). However, these differences are not so great that the qualitative conclusions drawn in the present study are seriously affected. al. (27) indicated that the loss of the H bonds that occur in Asp G1(99)/3 mutants causes a drastic R shift which is greater than that observed for the substitutions occurring at the switching point, His FG4(97)B or at the carboxyl terminus His HC3(146)/3. These results indicate that the H bond tradeoffs along the ala2 interfaces may serve as key factors in the quarternary structural switching from the T to the R during ligation which is responsible for the cooperativity of hemoglobin. In normal hemoglobin A, the glutamyl residue G3(101)P is located between these two H-bonds listed under a above. Results of the present comparative study of BlOl mutants demonstrate the consequence of substituting for Glu G3(101)/3 on the affinity and cooperativity of the mutant hemoglobins.
Comparison of the Adair constants, ICl and K4, for the normal and mutant hemoglobins in Table I indicates that the oxy conformation is similar for these hemoglobins while the deoxy conformation is not, and hence the Glu G3(101)B residue of Hb A contributes to maintaining the structural constraints imposed upon the deoxy form and stabilizing the deoxy tense structure relative to the oxy relaxed structure.
The relatively large decreases of O2 affinity for Hb Rush on the addition of anions, such as C1-, 2,3-diphosphoglycerate, or inositol hexaphosphate, indicate an increased Oz-linked anion effect in this variant. Dr. Perutz      prevent the neutralization of the positive Arg G6(104)/3. According to this explanation we could account for the extra C1binding by deoxy-Hb Rush by postulating the attraction of the C1-by a positively charged cluster formed along the a& contact in the cavity, Following from this postulation, one would expect the chloride effect on the function of the 101 mutants to be in the order of Hb British Columbia > Hb Rush = Hb Alberta > Hb A = Hb Potomac. However, the chloride effect on oxygen affinity (expressed by A log Pm/A log [Cl-1) for these hemoglobins was found to be in the order of Hb Rush > Hb A = Hb British Columbia = Hb Potomac = Hb Alberta. Tentative explanations for the apparent normal effect for Hbs British Columbia and Alberta could be due to an enhanced dimer dissociation and a relatively large R shift of the allosteric equilibrium, respectively. Both of these properties, therefore, would cancel out the extra C1-binding.
The Bohr effect of hemoglobin is a typical heterotropic effect that arises from differences in the proton binding due to changes of the pK of specific ionizable groups that occur during oxygenation. Alteration of the alkaline Bohr effect of hemoglobin can be expected in cases of 1) direct or indirect modification of the Bohr groups, 2) modification of the binding sites of cofactors (28)(29)(30), and 3) extreme R-shift of the allosteric equilibrium with essential loss of subunit cooperativity (27). Due to the low pK and negative charge of the Glu G3(101)/3, this residue is not likely to be a binding site for a Bohr proton or for other anion cofactors. Although increases to the 0, affinity are found in most of the 101 mutants, the subunit interactions remain in cooperative modes. Therefore, with the exception of Hb Rush it is not surprising that 6101 mutants display a normal Bohr effect under the experimental conditions used. The increase of the Bohr effect in Hb Rush can be explained by its enhanced chloride binding because part of Bohr proton binding is associated with anion binding during deoxygenation.
Besides the non-heme ligands, there are other factors which can affect the oxygen equilibrium of hemoglobin such as temperature and hemoglobin concentration. As the concentration of hemoglobin decreases the 0 2 equilibrium curve shifts to the left and the Hill coefficient decreases. This effect is attributed to an increased dissociation of the hemoglobin into its dimers in the oxy state (22,31). The 0 2 equilibrium of Hb British Columbia exhibits a higher dependency on the protein concentration than the other 101 mutants and Hb A, indicating a relatively greater dissociation into its dimer in the oxy form (Table 111).
The average heat of oxygenation of hemoglobin is pH dependent and can be determined indirectly by using the Van't Hoff equation. The difference of the heat of oxygenation, AH, between neutral pH and alkaline pH has been attributed to the heat of release of Bohr protons and anions upon oxygenation (32). In the present study, all of the @lo1 mutants except Hb Rush exhibit a normal heat of oxygenation at both pH 6.5 and 9.0. Hb Rush exhibits a smaller than normal heat of oxygenation at pH 6.5. This can be explained by the greater extent of cancellations of the average heat of 0 2 binding to heme by the greater heat of 02-linked proton and anion release of Hb Rush due to its excess binding of anion and proton.
It is clear that various amino acid residue substitutions that occur at the Glu G3(101)/3 site result in dramatic changes in the functional properties of hemoglobin. This presents a question of what factor contributes most towards maintenance of normal hemoglobin function: charge, hydrophilicity, or size of this amino acid residue. The O2 affinities of the Dl01 mutants relative to Hb A are in increasing order: Hb A (Glu) Hb Rush (Gln) < Hb British Columbia (Lys) < Hb Potomac (Asp) < Hb Alberta (Gly), when measured under the stripped condition with minimal chloride. The data suggest that the size of the amino acid residue is the single most critical factor in determining the relative oxygen affinities of this set of hemoglobins. However, the negative charge of the normal /3101 Glu is also important in maintaining normal function. In the case of Hb British Columbia, the increase in 0 2 affinity is amplified by its enhanced dimer dissociation which is probably the result of the extra lysyl positive charges along the a& interfaces. The substantial increase in the O2 affinity of Hb Potomac (Asp), which has similar charge and hydrophilic properties as Hb A (Glu), can only be explained by the change in size of the residue side chain or location of its carboxyl group. Hb Rush (Gln), which has the least change in size but has lost the negatively charged carboxyl group, shows the least change in O2 binding properties compared to Hb A (Glu). The increased chloride effect of Hb Rush is most Involvement of Glu G3(101)/3 likely due to the extra allosteric binding of chloride anion due to the increase in the net positive charge in the central cavity along the alp2 interface due to the loss of the negative charges of the ,8101 Glu. Thus, the size and charge of Glu pl01 apparently are both involved in maintaining normal O2 affnity and allosteric properties, respectively. Therefore, the contribution of the ,8101 site to the mechanism underlying the molecular function does not seem to be the result of any single factor alone.
Although the complete role of Glu G3(101)/3 in the functional properties of hemoglobin remains uncertain, the data presented here do provide further insight into the involvement of this important residue in the allosterism of hemoglobin. As mentioned in the Introduction, mutant hemoglobins which have substitutions at the alp2 interface manifest dramatic changes in their functional properties. The most drastic changes in both homotropic and heterotropic properties of ala2 mutants yet found occur from substitutions for the normal 899 residue (27). All of the p99 mutants appear to disrupt a critical hydrogen bond which stabilizes the normal T conformation (7). This leads us to believe that the H bonds at the alp2 subunit interface are more important than other electrostatic or van der Waals forces in maintaining the normal T conformation of the molecule. The @lo1 residue is located between this critical p99 site and the p102 residue that also forms a H bond which stabilizes the normal R conformation. We postulate that any change in the size or charge of the normal Dl01 residue adversely affects the switching of the molecule between its two normal quaternary structures (T and R).