Quantitative determination of carbamino adducts of alpha and beta chains in human adult hemoglobin in presence and absence of carbon monoxide and 2,3-diphosphoglycerate.

The principal component of normal adult human hemoglobin was equilibrated under various conditions with 13CO2. Quantitative analysis of the carbamino resonance intensities over the pH range of 6.5 to 9.0 shows that the effects of conversion from the deoxy to the liganded state in reducing the carbamino adduct formation occur predominantly at Val-1beta. Analysis of the pH dependence of carbamino formation at constant total carbonates yields values of pKz and pKc for Val-1beta and Val-1alpha in the deoxy and liganded conditions. In contrast to the Val-1beta as the allosteric site for CO2, the Val-1alpha site is shown to be primarily an alkaline Bohr group. 2,3-Diphosphoglycerate is shown to reduce substantially the Val-1beta carbamino resonance intensity in deoxyhemoglobin. Evidence for 2,3-diphosphoglycerate effects in carbon monoxide hemoglobin at both Val-1alpha and Val-1beta sites is presented. Enhanced carbamino formation in carbon monoxide hemoglobin at Val-1beta is observed at pH values less than 7.8. Finally, chemical exchange analysis of the spectra shows the release rate of the deoxy Val-1alpha carbamino adduct to be greater than that for deoxy Val-1beta. At pH 7.47 k-1obs,beta congruent to 1.0 and k-1obs, alpha congruent to 11.0 s-1.     

Properties of carboxymethylated cross-linked hemoglobin A

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic effects in hemoglobin: electrostatic energy associated with allosteric transition and effector binding

The pH dependence of the summed electrostatic stabilization for deoxy- and liganded hemoglobin was computed for several ionic strength values. The computed contribution to the stabilization of deoxyhemoglobin by binding of 2,3-diphosphoglycerate in the beta cleft compared well with experimental binding behavior for human hemoglobin A0 and hemoglobin F. The contribution of diphosphoglycerate binding to the alkaline Bohr effect was computed correctly for both hemoglobins A0 and F. The computed effects of simultaneous binding of diphosphoglycerate and formation of Val-1 beta carbamino adducts suggested a competition between these effectors. A direct competition was formulated between these two effectors, with extension to include a simple anion such as chloride or bicarbonate binding in competition with diphosphoglycerate but not with Val-1 beta carbamino formation. This model was found to hold at pH 7.3-7.4 over a range of concentrations of the effectors involved and to predict the pH dependence of Val-1 beta carbamino formation over the pH range 7.0-8.0. The pH dependence of the computed differential stability of liganded vs. unliganded hemoglobin A compared well with observation.     

Carbon 13 resonances of 13CO2 carbamino adducts of alpha and beta chains in human adult hemoglobin.

The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains.     

The interaction of anions with hemoglobin carbamylated on specific NH2-terminal residues.

Carbamylation of the NH2-terminal residues of the beta chains on hemoglobin (alpha2beta2c) leads to a reduced but still significant binding of 2,3-diphosphoglycerate, but has no effect on the oxygen-linked binding of chloride or phosphate, both of which are thought to bind to some of the same residues as the organic phosphate. Studies by others have shown that the binding of inorganic anions is not diminished in either horse hemoglobin or in hemoglobin Little Rock, in which four of the six other binding sites (histidine residues) for organic phosphates are replaced by glutamine residues. We suggest, therefore, that lysines 82 of the beta chains, which are the remaining 2 residues in the binding crevice for the organic phosphate, and which are invariant in the known sequences of mammalian hemoglobins, may be the primary binding site for inorganic anions. The extent of inhibition of gelation by increasing ionic strength is identical for the hybrids alpha2beta2, alpha2cbeta2, and alpha2beta2c of hemoglobin S. These results indicate the NH2-terminal residues of the chains are not involved in primary electrostatic interactions during aggregation of deoxyhemoglobin S.     

Reactivity of cyanate with valine-1 (alpha) of hemoglobin. A probe of conformational change and anion binding.

The 3-fold increase in the carbamylation rate of Val-1 (alpha) of hemoglobin upon deoxygenation described earlier is now shown to be a sensitive probe of conformational change. Thus, whereas this residue in methemoglobin A is carbamylated at the same rate as in liganded hemoglobin, upon addition of inositol hexaphosphate its carbamylation rate is enhanced 30% as much as the total change in the rate between the CO and deoxy states. For CO-hemoglobin Kansas in the presence of the organic phosphate, the relative increase in the carbamylation rate of this residue is about 50%. These results indicate that methemoglobin A and hemoglobin Kansas in the presence of inositol hexaphosphate do not assume a conformation identical with deoxyhemoglobin but rather form either a mixture of R and T states or an intermediate conformation in the region around Val-1 (alpha). Studies on the mechanism for the rate enhancement in deoxyhemoglobin suggest that the cyanate anion binds to groups in the vicinity of Val-1 (alpha) prior to proton transfer and carbamylation of this NH2-terminal residue. Thus, specific removal with carboxypeptidase B of Arg-141 (alpha), which is close to Val-1 (alpha) in deoxyhemoglobin, abolishes the enhancement in carbamylation. Chloride, which has the same valency as cyanate, is a better competitive inhibitor of the carbamylation of deoxyhemoglobin (Ki = 50 mM) compared with liganded hemoglobin. Nitrate and iodide are also effective inhibitors of the carbamylation of Val-1 (alpha) of deoxyhemoglobin (Ki = 35 mM); inorganic phosphate, sulfate, and fluoride are poor competitive inhibitors. The change in pKa of Val-1 (alpha) upon deoxygenation may be due to its differential interaction with chloride.     

The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect.

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.     

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.     

Oxygen-linked CO2 binding to isolated beta subunits of human hemoglobin.

It is known that most of the oxygen-linked carbamate which is formed in normal adult human hemoglobin (Hb A) is confined to the beta subunits rather than to the alpha subunits. In order to find out if similar differences exist in the isolated protomers of Hb A we have measured the effect of various pressures of carbon dioxide (pCO2) on the oxygen affinity in the following heme pigments: isolated alpha and beta subunits with free --SH groups (alphaSH, betaSH), mercurated beta subunits (betaPMB), myoglobin (Mb), and betaSH/PLP in which the terminal alpha-amino group of betaSH was irreversibly blocked with pyridoxal phosphate (PLP). Similar measurements were done on Hb A and the fraction of oxygen-linked carbamate calculated from the effect of pCO2 (at constant pH) on the oxygen half-saturation pressure (p50). A distinct influence of CO2 on p50 was observed in betaSH which was absent in betaSH/PLP and thus indicates that the terminal alpha-amino group mediates the oxygen-linked binding of CO2 in betaSH as it does in the beta subunits of Hb A. However, the fraction of oxygen-linked carbamate was much less dependent on pH and pCO2 in betaSH than in Hb A. Neither alphaSH, betaPMB, or Mb, all of which are known to exist largely or wholly as monomers but have free terminal alpha-amino groups, showed a shift of p50 upon addition of CO2. As both betaSH and betaSH/PLP were shown to be tetrameric molecules, we conclude from this study that homotetramers composed of isolated beta subunits do exhibit a reciprocal interaction between the binding of O2 and CO2.     

Selectivity in the modification of the alpha-amino groups of hemoglobin on reductive alkylation with aliphatic carbonyl compounds. Influence of derivatization on the polymerization of hemoglobin S

The reactivity of the alpha-amino groups of the alpha- and beta-chains of hemoglobn toward reductive alkylation using limiting concentrations of the aliphatic carbonyl compounds, acetaldehyde (ethylation), glyoxylic acid (carboxymethylation), glycolaldehyde (hydroxyethylation), glyceraldehyde (dihydroxypropylation), and dihydroxyacetone (dihydroxyisopropylation) has been investigated. Hemoglobin A reductively ethylated at the alpha-amino groups eluted on CM-52 ahead of unmodified hemoglobin A, and hemoglobin A reductively ethylated at the epsilon-amino groups. This observation is similar to that seen on hydroxyethylation and dihydroxypropylation of the alpha-amino group of hemoglobin A. The presence of the alpha-hydroxyl or the carboxyl group in the carbonyl component used in the reductive alkylation influences considerably the selectivity pattern during the derivatization. The alpha-amino groups of the alpha- and beta-chains are modified to nearly the same degree during reductive hydroxyethylation as well as during reductive dihydroxypropylation. Reductive ethylation (aldehyde lacking the alpha-hydroxyl group) exhibited a slight preferential reaction at Val-1(beta). The presence of a negatively charged carboxyl group in the carbonyl component, i.e. glyoxylic acid, made this preferential reaction at Val-1(beta) even more pronounced. When the reductive alkylation is carried out with dihydroxyacetone (a ketone instead of an aldehyde), the dihydroxyisopropylation occurred at a slower rate and exclusively at Val-1(beta). The ethylation, hydroxyethylation, carboxymethylation, and dihydroxypropylation of the alpha-amino groups of hemoglobin S increased its solubility from the value of 16 g/dl for the unmodified protein to about 25 g/dl for the modified protein. Thus, the alkyl chains on the alpha-amino groups on the polymerization have a strong inhibitory influence. In order to determine the influence of the alkyl chains at the alpha-amino groups of alpha- and beta-chains on polymerization, hybrid hemoglobin S tetramers with hydroxyethylation either at Val-1(alpha) or at Val-1(beta) have been prepared. The solubility of each hybrid is about 26 g/dl. Thus, the hydroxyethyl group either on the alpha- or the beta-chain appears to interfere with the polymerization of deoxygenated HbS to the same degree. The inhibitory influence of the hydroxyethyl chain at Val-1(alpha) on the polymerization, compared with the lack of such an influence when this alpha-amino group is modified by cyanate, suggests that a carbamoyl group on Val-1(alpha) can be accommodated in the intermolecular contact region involving this segment of the molecule without seriously perturbing the mo     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

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.     

Determination of the pK values for the alpha-amino groups of human hemoglobin.

The rate of reaction between alpha-amino groups and cyanic acid was followed at 26 degrees and ionic strength 0.2 M as a function of pH of human hemoglobin Ao solutions to determine the pK and the pH-independent second order rate constant, kappa, for these groups in the alpha and beta chains. At a given point in time, the extent of the reaction was determined by employing the Beckmann Sequencer as a quantitative tool in which the yields of leucine and histidine in the second Edman degradation cycle were used to define the rates of reaction of the alpha and beta chains, respectively. From these results, the individual were evaluated (Garner, M.H., Garner, W.H., and Gurd, F. R.N. (1973) J. Biol. Chem. 248, 5451-5455). Values for pK for the alpha and beta chains were, respectively, 6.74 and 6.93 for cyanoferrihemoglobin, 6.95 and 7.05 for carboxyhemoglobin, and 7.79 and 6.84 for deoxyhemoglobin. Values for kappa, M- minus 1 S-minus 1, for the alpha and beta chains were, respectively, 12.5 and 17 for cyanoferrihemoglobin, 12 and 18 for carboxyhemoglobin, and 91 and 24 for deoxyhemoglobin. Limits of significance were estimated for both variables in each case. The pK results for valine 1alpha agree well with the value obtained by Hill and Davis (1967) J. Biol. Chem. 242, 2005-2012) for carboxyhemoglobin and with that of Kilmartin and Rossi-Bernardi ((1971) Biochem. J. 124, 31-45) for deoxyhemoglobin. Values obtained for sperm whale myoglobin were 7.77 for pK and 7.4 for kappa. The results are useful for the interpretation of the allosteric interactions of hemoglobin with hydrogen ions, with CO2, and with phosphate.     

X-ray diffraction studies of a partially liganded hemoglobin, [alpha(FeII-CO)beta(MnII)]2

We have applied single-crystal X-ray diffraction methods to analyze the structure of [alpha(FeII-CO)beta(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3.0 A, shows that (1) the Mn(II)-substituted beta subunits are structurally isomorphous with normal deoxy beta subunits, and (2) CO binding to the alpha subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the alpha heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the alpha 1 beta 2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.     

Hemoglobin New Mexico: beta 100 (G2) Pro----Arg. A variant hemoglobin associated with erythrocytosis

Hemoglobin New Mexico beta 100 Pro----Arg was found in a 4-year-old black male and represents a new mutation. The propositus is also heterozygous for Hb S. The variant shows high oxygen affinity, reduced cooperatively, and a lowered alkaline Bohr effect. Addition of allosteric effectors leads to improved cooperativity and a Bohr effect that is similar to that of Hb A. The high percentage of the variant (53.5%) and its increased oxygen affinity result in erythrocytosis in this patient. The hemoglobin level and packed cell volume values are elevated. In spite of these factors the patient appears healthy and shows no discomfort. The altered oxygen-linked properties of this variant can be related to the fact that the substituted residue contributes to the alpha 2 beta 1/alpha 1 beta 2 subunit interface, an area that is critical not only to the allosteric transitions between the oxy and deoxy states but also to stabilizing the hemoglobin tetrameer.     

X-ray diffraction study of di and tetra-ligated T-state hemoglobin from high salt crystals.

X-ray diffraction difference electron density maps at 3 A resolution obtained from di and tetra-ligated T-state hemoglobin (Hb) crystals are reported. Crystals isomorphous with native deoxyhemoglobin were obtained from ammonium sulfate solutions incubated with the synthetic allosteric effector RSR-56. RSR-56 binds at two symmetry-related Hb central water cavity sites and each molecule has major interactions with three different subunit side-chains; one effector with Arg141 alpha 2 HC3, Lys99 alpha 1 G6 and Asn108 beta 1 and the other with the symmetry related residues, Arg141 alpha 1 Lys99 alpha 2 and Asn108 beta 2. Crystals mounted in a nitrogen filled glove box were di-ligated as previously found with polyethyleneglycol Hb crystals. Crystals mounted in air under a layer of mother liquor were bright red and showed all four heme groups ligated. The difference electron density from the di-ligated crystals showed atomic movements to be restricted to the immediate neighborhood of the heme groups and the allosteric effector. By contrast, the tetra-ligated structure showed extended difference electron density near amino acid residues around both alpha and beta heme groups and along the alpha 1/beta 2 interface. Ligation of the beta heme group appears to magnify the difference density around the alpha heme groups. There is no evidence of breakage of the Bohr salt bridge, His146 beta HC3----Asp94 beta FG1, in the crystal. The observed difference electron density maps may help to clarify the way the allosteric mechanism is triggered.     

Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2)

Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.     

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...     

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.