Crystal structures of unliganded and half-liganded human hemoglobin derivatives cross-linked between Lys 82beta1 and Lys 82beta2

A number of ligand binding studies of human adult hemoglobin (HbA) cross-linked between Lys 82beta(1) and Lys 82beta(2) with bis(3,5-dibromosalicyl)fumarate have been reported. The oxygen binding properties of native HbA, including the cooperativity and Bohr effect, are not substantially changed by the modification, provided care is taken to remove electrophoretically silent impurities arising from side reactions. We have refined the high-resolution structure of this modified Hb and found it adopts the T state when crystallized in the absence of heme ligands, contrary to a previously published structure. These results suggest the slightly altered crystal form determined previously may be due to unremoved side products of the cross-linking reaction with high oxygen affinity. Two nickel-substituted Hbs cross-linked in the same way have also been crystallized in the presence of carbon monoxide, which binds only to the ferrous heme. In the case of the nickel-substituted alpha subunit, the absence of a covalent link between the central metal of the heme and the proximal histidine leads to a new conformation of the histidine stabilized by a water molecule. This structure may mimic that of partially NO-liganded species of HbA; however, overall, the changes are highly localized, and both doubly ligated species are in the T conformation.     

Reaction of haptoglobin with hemoglobin covalently cross-linked between the alpha beta dimers.

Hemoglobin tetramers which cannot split into alphabeta dimers, because they are covalently cross-linked between the beta chains across the polyphosphate binding site, form complexes with haptoglobin. The reaction is biphasic as measured by fluorescence quenching and peroxidase activity. A complex in which one of the alpha beta dimers of the cross-linked hemoglobin is bound to one of the sites in the divalent haptoglobin molecule, is formed reversibly during the initial fast phase. In the subsequent slower step, this product then either polymerizes, adds another cross-linked hemoglobin molecule or, in the presence of excess haptoglobin, combines with a second haptoglobin molecule. This latter complex, in which two haptoglobin molecules are bridged by a cross-linked hemoglobin tetramer, can still combine with normal alpha beta dimers at the vacant haptoglobin combining sites. In spite of the very low oxygen affinity of the cross-linked hemoglobin, combination with haptoglobin shifts if oxygen affinity to the very high value of the normal hemoglobin-haptoglobin complex.     

Carbon dioxide binding to human hemoglobin cross-linked between the alpha chains

The binding of carbon dioxide to human hemoglobin cross-linked between Lys alpha 99 residues with bis(3,5-di-bromosalicyl) fumarate was measured using manometric techniques. The binding of CO2 to unmodified hemoglobin can be described by two classes of sites with high and low affinities corresponding to the amino-terminal valines of the beta and alpha chains, respectively (Perrella, M., Kilmartin, J. V., Fogg, J., and Rossi-Bernardi, L. (1975b) Nature 256, 759-761. The cross-linked hemoglobin bound less CO2 than native hemoglobin at all CO2 concentrations in deoxygenated and liganded conformations, and the ligand-linked effect was reduced. Fitting the data to models of CO2 binding suggests that only half of the expected saturation with CO2 is possible. The remaining binding is described by a single affinity constant that for cross-linked deoxyhemoglobin is about two-thirds of the high affinity constant for deoxyhemoglobin A and that for cross-linked cyanomethemoglobin is equal to the high affinity constant for unmodified cyanomethemoglobin A or carbonmonoxyhemoglobin A. The low affinity binding constant for cross-linked hemoglobin in both the deoxygenated and liganded conformations is close to zero, which is significantly less than the affinity constants for either subunit binding site in unmodified hemoglobin. Comparing the low affinity sites in this modified hemoglobin to native hemoglobin suggests that cross-linking hemoglobin between Lys alpha 99 residues prevents CO2 binding at the alpha-subunit NH2 termini.     

On the oxygen-linked anion-binding sites in human hemoglobin. Functional properties of human hemoglobin reacted with 4-isothiocyanatobenzenesulphonic acid and its hybrids

Human hemoglobin, reacted at the four amino termini with 4-isothiocyanatobenzenesulphonic acid (Hb-ICBS), was separated into its constituent chains. Recombination of the ICBS-reacted chains with the unmodified mate chains produced the hybrid tetramers modified at either the beta or the alpha chains: alpha 2 beta 2ICBS and alpha 2ICBS beta 2. All of the modified tetramers show a reduced oxygen affinity and reduced cooperativity; furthermore the oxygen affinity of the Hb-ICBS and alpha 2 beta 2ICBS is unaffected by 2,3-bisphosphoglycerate while the oxygen affinity of alpha 2ICBS beta 2 is decreased in the presence of this organic phosphate. The oxygen affinity of Hb-ICBS and alpha 2ICBS beta 2 is independent of chloride concentration, while the alpha 2 beta 2ICBS hybrid shows a reduced response to this anion. The tetramers alpha 2ICBS beta 2 and alpha 2ICBS beta 2ICBS show a decreased alkaline Bohr effect, which can be rationalized as being due to disruption of the oxygen-linked chloride-binding sites; in the case of alpha 2 beta 2ICBS the Bohr effect is instead (partially) maintained. The functional properties of artificial tetramers have been studied also from a kinetic point of view by CO combination and the results obtained compare satisfactorily with equilibrium data. The possibility of obtaining selectively modified hemoglobins promises to provide further insight into the properties of the oxygen-linked anion-binding sites in hemoglobin.     

Three-point cross-linking: potential red cell substitutes from the reaction of trimesoyl tris(methyl phosphate) with hemoglobin.

The symmetrical trifunctional cross-linking reagent trimesoyl tris(methyl phosphate) (3), reacts selectively with amino groups (beta 1Val and beta 82Lys) in the diphosphoglycerate binding site of human hemoglobin A, producing cross-linked tetrameric species in good yield. A major species is triply linked, alpha alpha beta 1(82) greater than B beta 82, where B symbolizes benzene-1,3,5-tricarbonyl. Both this triply linked species and the doubly linked species, alpha alpha beta 1B beta 82, produced from deoxyhemoglobin have a considerably lower oxygen affinity than does native hemoglobin while maintaining a high degree of cooperativity (n50 = 2.4), making them potentially useful as red cell substitutes, in principle delivering twice as much oxygen as whole blood between pO2 = 100 and = 40 Torr. The yield of products indicates that triply and doubly linked species form in parallel so that there are independent routes to each. It is proposed that differences in routes are due to stereoisomerism about the amide bonds which form from reaction of the reagent with the protein.     

Crystal structure of Lysbeta(1)82-Lysbeta(2)82 crosslinked hemoglobin: a possible allosteric intermediate

The crystal structure of human hemoglobin crosslinked between the Lysbeta82 residues has been determined at 2.30 A resolution. The crosslinking reaction was performed under oxy conditions using bis(3, 5-dibromosalicyl) fumarate; the modified hemoglobin has increased oxygen affinity and lacks cooperativity. Since the crystallization occurred under deoxy conditions, the resulting structure displays conformational characteristics of both the (oxy) R and the (deoxy) T-states. beta82XLHbA does not fully reach its T-state conformation due to the presence of the crosslink. The R-state-like characteristics of deoxy beta82XLHbA include the position of the distal Hisbeta63 (E7) residue, indicating a possible reason for the high oxygen affinity of this derivative. Other areas of the molecule, particularly those thought to be important in the allosteric transition, such as Tyrbeta145 (HC2) and the switch region involving Proalpha(1)44 (CD2), Thralpha(1)41 (C6) and Hisbeta(2)97 (FG4), are in intermediate positions between the R and T-states. Thus, the structure may represent a stabilized intermediate in the allosteric transition of hemoglobin.     

Site-selective glycosylation of hemoglobin on Cys beta93

In this work, we describe the synthesis and characterization of a novel glycosylated hemoglobin (Hb) with high oxygen affinity as a potential Hb-based oxygen carrier. Site-selective glycosylation of bovine Hb was achieved by conjugating a lactose derivative to Cys 93 on the beta subunit of Hb. LC-MS analysis indicates that the reaction was quantitative, with no unmodified Hb present in the reaction product. The glycosylation site was identified by chymotrypsin digestion of the glycosylated bovine Hb followed with LC-MS/MS and from the X-ray crystal structure of the glycosylated Hb. The chemical conjugation of the lactose derivative at Cys beta93 yields an oxygen carrier with a high oxygen affinity (P(50) of 4.94 mmHg) and low cooperativity coefficient (n) of 1.20. Asymmetric flow field-flow fractionation (AFFFF) coupled with multiangle static light scattering (MASLS) was used to measure the absolute molecular weight of the glycosylated Hb. AFFFF-MASLS analysis indicates that glycosylation of Hb significantly altered the alpha(2)beta(2)-alphabeta equilibrium compared to native Hb. Subsequent X-ray analysis of the glycosylated Hb crystal showed that the covalently linked lactose derivative is sandwiched between the beta(1) and alpha(2) (and hence by symmetry the beta(2) and alpha(1)) subunits of the tetramer, and the interaction between the saccharide and amino acid residues located at the interface is apparently stabilized by hydrogen bonding interactions. The resultant structural analysis of the glycosylated Hb helps to explain the shift in the alpha(2)beta(2)-alphabeta equilibrium in terms of the hydrogen bonding interactions at the beta(1)alpha(2)/beta(2)alpha(1) interface. Taken together, all of these results indicate that it is feasible to site-specifically glycosylate Hb. This work has great potential in developing an oxygen carrier with defined chemistry that can target oxygen delivery to low pO(2) tissues and organs.     

Oxygen equilibrium and EPR studies on alpha1beta1 hemoglobin dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.     

Proton nuclear magnetic resonance studies of hemoglobin Providence (beta82EF6 Lys replaced by Asn or Asp): a residue involved in anion binding

High-resolution proton nuclear magnetic resonance studies of hemoglobins Providence-Asn (beta82EF6 Lys replaced by Asn) and Providence-Asp (beta82EF6 Lys replaced by Asp) show that different amino acid substitutions at the same position in the hemoglobin molecule have different effects on the structure of the protein molecule. Hemoglobin Providence-Asp appears to be in a low-affinity tertiary structure in both the deoxy and carbonmonoxy forms. Deoxyhemoglobin Providence-Asn has its beta heme resonance shifted downfield slightly from its position in normal adult hemoglobin; however, the tertiary structures of the heme pocket of hemoglobins A and Providence-Asn are very similar when both proteins are in the carbonmonoxy form. These results are consistent with the oxygen equilibrium measurements of Bonaventura, J., et al. [(1976) J. Biol. Chem. 251, 7563] which show that both Hb Providence-Asn and Hb Providence-Asp have oxygen affinities lower than normal adult hemoglobin, with Hb Providence-Asp having the lowest. Our studies of the effects of sodium chloride on the hyperfine shifted proton resonances of deoxyhemoglobins A, Providence-Asn, and Providence-Asp indicate that the beta82EF6 lysine is probably one, but not the only binding site for chloride ions.     

Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency

A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined.     

Two mutations in recombinant Hb beta F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinity.

Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb beta YD) in which the beta F41Y substitution is associated with K82D. Functional studies have shown that the Hb alpha 2 beta 2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased autooxidation rate. The oxygen affinity of the natural mutant beta K82D (Hb Providence-Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG-binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb-based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 degrees C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 A) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 degrees C. Nevertheless, the rate of autooxidation, 3.5-fold larger than that of Hb A, remains a problem.     

Influence of ligation state and concentration of hemoglobin A on its cross-linking by glycolaldehyde: functional properties of cross-linked, carboxymethylated hemoglobin

The ligation state of hemoglobin during its cross-linking by glycolaldehyde influences the ultimate oxygen affinity of the cross-linked protein. Thus, if the cross-linking is performed with carbonmonoxy-hemoglobin, the oxygen affinity increases slightly to a P50 of 7 mmHg from a P50 of 9 mmHg for unmodified hemoglobin. In contrast, when deoxyhemoglobin is cross-linked with glycolaldehyde, the oxygen affinity of the product decreases (P50 = 15 mmHg). When deoxyhemoglobin is first carboxymethylated and then cross-linked with glycolaldehyde, an even lower oxygen affinity is achieved (P50 = 23 mmHg). Carboxymethylated hemoglobin is very responsive to the presence of 5% CO2 with a P50 of 33 mmHg, which is lowered further to 42 mmHg when chloride (0.1 M) is also present. Hemoglobin carboxymethylated and cross-linked under anaerobic conditions is also responsive to the modulators CO2 and chloride with a resultant oxygen affinity of 27 mmHg. The type of cross-linking of liganded hemoglobin by the mild reagent glycolaldehyde is dependent upon the initial hemoglobin concentration. Thus, with dilute hemoglobin (45 microM in tetramer), cross-linking by glycolaldehyde (50 mM) results in about 75% of 64,000 molecular weight species (some of which are cross-linked within tetramer) and 25% of intertetrameric cross-linked species with a range of molecular weights averaging 128,000-512,000. With hemoglobin solutions of higher concentration (360 microM), the amount of the higher molecular weight species increases to about 65% with a corresponding reduction to 35% in the 64,000 molecular weight component.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

Hemoglobin providence. Functional consequences of two alterations of the 2,3-diphosphoglycerate binding site at position beta 82.

Position beta 82 in human hemoglobin (Hb) is normally occupied by lysine, a positively charged residue that is involved in the binding of anionic cofactors. This residue is substituted by a neutral residue in Hb Providence Asn and by a negatively charged residue in Hb Providence Asp. Hb Providence Asp shows more differences from Hb A than does Hb Providence Asn in studies of the kinetics and equilibria of ligand binding. For both forms, homotropic (cooperative) interactions are normal with n values of 2.5 to 2.7, while heterotropic (pH and anion) interactions are reduced greatly. The reduction in anion sensitivity is attributed to the absence of a positive residue at position beta 82. Reduction in pH sensitivity may be due to a ligand-linked change in the pK of a neighboring residue, beta 143 histidine, which normally is not a Bohr group. This change in pK would act in opposition to the normal Bohr effect. Reduction in the net positive charge of the central cavity has a further consequence. Relative to Hb A, both Hb Providence Asn and Hb Providence Asp show decreased oxygen affinities at neutral pH in the absence of cofactors. This suggests that in Hb A the binding of anionic cofactors directly influences the oxygen affinity by neutralizing the charged groups of the diphosphoglycerate binding site and thus stabilizing the low affinity (T) conformation. From pH 6 to 9 in the presence of 1 M NaCl, where all the charged groups may be masked, the oxygen-binding properties of Hb A and the Hb Providence mutants are identical. Moreover, subunit dissociation of the liganded Hb Providence mutants appears to be increased, as is known to occur for Hb A in the presence of high salt. The results obtained with Hb Providence Asn and Hb Providence Asp illustrate how single amino acid substitutions can modify hemoglobins' pH and anion interactions without altering cooperative interactions between subunits. The alteration in cofactor effects observed with these mutants also illustrates differences between the allosteric effects induced by organic and inorganic anions.     

Determination of the rate and equilibrium constants for oxygen and carbon monoxide binding to R-state human hemoglobin cross-linked between the alpha subunits at lysine 99

The kinetics of O2 and CO binding to R-state human hemoglobin A0 and human hemoglobin cross-linked between the alpha chains at Lys99 residues were examined using ligand displacement and partial photolysis techniques. Oxygen equilibrium curves were measured by Imai's continuous recording method (Imai, K. (1981) Methods Enzymol. 76, 438-449). The rate of the R to T transition was determined after full laser photolysis of the carbon monoxide derivative by measuring the resultant absorbance changes at an isosbestic point for ligand binding. Chemical cross-linking caused the R-state O2 affinity of alpha subunits to decrease 6-fold compared with unmodified hemoglobin. This inhibition of O2 binding was the result of both a decrease in the rate constant for ligand association and an increase in the rate constant for dissociation. The O2 affinity of R-state beta subunits was reduced 2-fold because of an increase in the O2 dissociation rate constant. These changes were attributed to proximal effects on the R-state hemes as the result of the covalent cross-link between alpha chain G helices. This proximal strain in cross-linked hemoglobin was also expressed as a 5-fold higher rate for the unliganded R to T allosteric transition. The fourth O2 equilibrium binding constant, K4, measured by kinetic techniques, could be used to analyze equilibrium curves for either native or cross-linked hemoglobin. The resultant fitted values of the Adair constants, a1, a2, and a3 were similar to those obtained when K4 was allowed to vary, and the fits were of equal quality. When K4 was fixed to the kinetically determined value, the remaining Adair constants, particularly a3, became better defined.     

Magnesium(II) and zinc(II)-protoporphyrin IX's stabilize the lowest oxygen affinity state of human hemoglobin even more strongly than deoxyheme.

Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha2(Fe)beta2(M) and alpha2(M)beta2(Fe); M=Mg(II), Zn(II) (neither of these closed-shell metal ions binds oxygen or carbon monoxide)) are reported along with the X-ray crystal structures of alpha2(Fe)beta2(Mg) with and without CO bound. We found that Mg(II)-Fe(II) hybrids resemble Zn(II)-Fe(II) hybrids very closely in oxygen equilibrium properties. The Fe(II)-subunits in these hybrids bind oxygen with very low affinities, and the effect of allosteric effectors, such as proton and/or inositol hexaphosphate, is relatively small. We also found a striking similarity in spectrophotometric properties between Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrids, particularly, the large spectral changes that occur specifically in the metal-containing beta subunits upon the R-T transition of the hybrids. In crystals, both alpha2(Fe)beta2(Mg) and alpha2(Fe-CO)beta2(Mg) adopt the quaternary structure of deoxyhemoglobin. These results, combined with the re-evaluation of the oxygen equilibrium properties of normal hemoglobin, low-affinity mutants, and metal substituted hybrids, point to a general tendency of human hemoglobin that when the association equilibrium constant of hemoglobin for the first binding oxygen molecule (K1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effectors is virtually abolished. This is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin. Moreover, excellent agreement between the reported oxygen affinity of deoxyhemoglobin in crystals and the lowest affinity in solution leads us to propose that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution.We also survey the oxygen equilibrium properties of various metal-substituted hybrid hemoglobins studied over the past 20 years in our laboratory. The bulk of these data are consistent with the Perutz's trigger mechanism, in that the affinity of a metal hybrid is determined by the ionic radius of the metal, and also by the steric effect of the distal ligand, if present. However, there remains a fundamental contradiction among the oxygen equilibrium properties of the beta substituted hybrid hemoglobins.     

Cobalt-substituted hemoglobin Zürich (alpha 2 beta 263His leads Arg). Oxygen equilibria and EPR spectra.

Cobalt hemoglobin Zürich (alpha 2 beta 263His leads to Arg) has been successfully reconstituted from the apohemoglobin Zürich and cobaltous protoporphyrin IX. The oxygen affinity of cobalt hemoglobin Zurich, as well as that of iron hemoglobin Zürich, were measured in the absence and presence of organic phosphate and Cl-. The overall oxygen affinity of cobalt hemoglobin Zürich was found to be higher and the cooperativity as measured by the n value was smaller than those of cobalt hemoglobin A. Organic phosphate and Cl- affect the oxygen equilibrium properties of cobalt hemoglobin Zürich in a manner similar to that of cobalt hemoglobin A, but to a lesser extant than cobalt hemoglobin A. The EPR spectrum of oxy cobalt hemoglobin Zürich is less sensitive to the replacement of the buffer system from H2O to 2H2O, indicating that the hydrogen bond between the distal amino acid residue and the bound oxygen is not formed in the abnormal beta subunits. The deoxy EPR spectrum of cobalt hemoglobin Zürich is similar to that of deoxy cobalt hemoglobin A, suggesting that the deoxy cobalt hemoglobin Zürich is predominantly in the deoxy quaternary structure (T state).     

Preparative affinity electrophoresis: application to human erythrocyte carbonic anhydrase

A modification of affinity electrophoresis for preparative purposes is described. This method has been applied to the purification of human erythrocyte carbonic anhydrases B and C. During conventional affinity chromatography some hemoglobin contamination occurs. By introduction of an electrophoretic purification step after the immobilization of carbonic anhydrase to the affinity gel, the hemoglobin impurity is reduced about eight and two times in the preparations of the B and C enzymes, respectively, compared to the enzymes purified by affinity chromatography.     

Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.