Unmodified Hemoglobin Research Articles

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)

Inhibition of the gelation of extracellular and intracellular hemoglobin S by selective acetylation with methyl acetyl phosphate.

Methyl acetyl phosphate binds to the 2,3-diphosphoglycerate (2,3-DPG) binding site of hemoglobin and selectively acetylates three amino groups at or near that site. The subsequent binding of 2,3-DPG is thus impeded. When intact sickle cells are exposed to methyl acetyl phosphate, their abnormally high density under anaerobic conditions is reduced to the density range of oxygenated, nonsickling erythrocytes. This change is probably due to a combination of direct and indirect effects induced by the specific acetylation. The direct effect is on the solubility of deoxyhemoglobin S, which is increased from 17 g/dL for unmodified hemoglobin S to 22 g/dL for acetylated hemoglobin S at pH 6.8. Acetylated hemoglobin S does not gel at pH 7.4, up to a concentration of 32 g/dL. The indirect effect could be due to the decreased binding of 2,3-DPG to deoxyhemoglobin S within the sickle erythrocyte, thus hindering the conversion of oxyhemoglobin S to the gelling form, deoxyhemoglobin S.

Sulfhemoglobin. Properties of partially sulfurated tetramers.

The properties of sulfhemoglobin (sulfHb) were investigated using disc gel isoelectric focusing and optical spectrophotometry. Laboratory-prepared samples, which contained a high yield of sulfHb (70-85%), and a patient-derived sample, which contained a low yield (12%), contain a tetrameric population that reflects a random distribution of modified (sulfurated) subunits. Hybrid tetramers, i.e. those containing sulfurated and unmodified subunits, were resolved from fully sulfurated and unmodified hemoglobin upon electrofocusing of ferric or ferrous sulHb samples. The electrophoretic differences between ferric sulfHb and metHb arise from a difference in the pK for the met to hydroxymet conversion of sulfurated subunits (Carrico, R., Peisach, J., and Alben, J. O. (1978) J. Biol. Chem. 253, 2386-2391). Both partially and fully sulfurated tetramers, when in the deoxy form and when in the fully ligated CO form, co-focus with their unmodified deoxy-Hb and Hb CO counterparts. Thus, these sulfurated tetramers exhibit normal ligand-dependent ionization of Bohr protons. In air-equilibrated samples, hybrid tetramers are partially ligated as a result of the reduced O2 affinity of the sulfurated subunits. These partially ligated tetramers exhibit a pI intermediate between oxy-Hb and deoxy-Hb due to the fractional ionization of Bohr protons. The partially sulfurated, partially ligated tetramers as well as deoxy-sulfHb tetramers were found to bind 2,3-diphosphoglycerate in disc gels. Despite the preservation of these heterotropic effects, which are confirmed in CO binding studies in solution, fully sulfurated tetramers recovered using preparative isoelectric focusing exhibit little or no cooperativity.

A high resolution NMR study of localized dynamic and structural perturbations in human hemoglobin modified with thiol reagents.

The hydrogen exchange kinetics of the N delta H proton in His F8 of iodoacetamide- and N-ethylmaleimide-treated human deoxyhemoglobins were studied using a NMR method. Comparison with unmodified hemoglobin shows that the reagents, covalently bound to Cys beta 93, significantly increase (about one order of magnitude) the exchange kinetics in beta chains only. This effect was partially reversed by the strong allosteric effector inositol hexaphosphate. Study of the high resolution 400-MHz NMR spectra of modified oxy- and deoxy-hemoglobins permitted localization of the extent of chemically induced structural perturbations. The resonances corresponding to hydrogen bonds specific to the deoxy conformation are not changed, in accord with the preserved cooperativity. Under the experimental conditions (0.1 M bis-Tris, 10 mM Cl-, pH 7.2), the salt bridge at the C terminus of the beta chain in the deoxy state (His beta 146-Asp beta 94) is perturbed by both modifications. The His beta 146 appears to be rendered more immobilized by the reagents in the oxy conformation. From the resonances corresponding to heme pocket protons of oxyhemoglobin it is deduced that the perturbations do not extend over the distal side of the heme pocket but are limited to the FG, F, and HC segments of the beta chain.

Reductive hydroxyethylation of the α-amino groups of amidated hemoglobin S

Val-6(β) of hemoglobin S forms the primary site of intertetrameric interaction in the polymerization of deoxy hemoglobin S. However, a number of other intermolecular interactions contribute significantly to the polymerization process as well as to the stability of the polymerized gel. The strong stabilizing influence of Val-6(β) in the polymerization process is reflected in the fact that although a number of mutations at any one of the intermolecular contact regions (or perturbation of these contact regions by chemical modification) result in some increase in the solubility of deoxy hemoglobin S, none of these mutations and/or chemical modifications completely neutralize the polymerizing influence of Val-6(β), i.e., restores the solubility to that of hemoglobin A. Additivity and/or synergy of the solubilizing influence of two or more chemical modification reactions each of which independently increases the solubility may be considered as a possible strategy to restore the solubility of deoxy hemoglobin S to that of hemoglobin A. In the present study, the cumulative solubilizing influence of amidation of Glu-43(β) and hydroxyethylation of α-amino groups of hemoglobin S has been investigated by preparing hemoglobin S with double modification. Modification of Glu-43(β) by amidation with glycine ethyl ester did not influence the reactivity of the α-amino groups of hemoglobin S toward reductive hydroxyethylation, thus permitting the preparation of hemoglobin S with the two modifications. The reductive hydroxyethylation increased the oxygen affinity of amidated hemoglobin S to nearly the same degree as it does on modification of unmodified hemoglobin. In addition, hemoglobin S with double modification has a Hill coefficient that is the same as that of unmodified hemoglobin S, suggesting that the overall quaternary interaction of hemoglobin S with a double modification is nearly the same as the unmodified protein. However, the reductive hydroxyethylation of the amidated hemoglobin S increased the solubility of the protein further. The solubility of hemoglobin S with a double modification is nearly twice that of the unmodified protein and is close to that of 1:1 mixture of hemoglobin S and hemoglobin F. The results demonstrate the additivity of the solubilizing influence of perturbing the quinary interactions at the intermolecular contact regions of deoxy hemoglobin S.

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

The reversibility of the ketoamine linkages of aldoses with proteins.

The reaction of glyceraldehyde (aldotriose) with hemoglobin A is analogous to the nonenzymic glycosylation of the protein with glucose in that the initial reversible Schiff base adduct (aldimine) of aldotriose undergoes Amadori rearrangement as does that of aldohexose to form the more stable ketoamine adduct. The modification of the alpha-amino group on Val-1(beta) of hemoglobin A as a ketoamine (2-oxo-3-hydroxypropyl group) apparently lowers the pKa of the alpha-amino group of the protein, since this derivative of hemoglobin elutes earlier on carboxymethylcellulose columns than the derivatives containing 2-oxo-3-hydroxypropyl groups on the epsilon-amino groups, and unmodified hemoglobin A. Similar chromatographic behavior has been reported for hemoglobin A1c which contains glucose at its Val-1(beta) as the ketoamine adduct. This suggests the similarity in the chemical consequences of having the ketoamine adduct of an aldose at Val-1(beta) of hemoglobin A under physiological conditions. The formation of the 2-oxo-3-hydroxypropyl groups on Val-1(beta) is nearly irreversible as has been suggested for similar adducts of glucose. On the other hand, the 2-oxo-3-hydroxypropyl groups on the epsilon-amino groups appear to be labile. The buffer conditions considerably influence the reversibility of the ketoamine adducts of aldotriose on the epsilon-amino groups; the reversibility is significantly higher in Tris buffers as compared with that in phosphate buffers. It is suggested that under physiological conditions the ketoamine adducts of aldotriose on the epsilon-amino groups exist in equilibrium with the aldimine, the equilibrium being favored toward the ketoamine. The enhanced release of the 2-oxo-3-hydroxypropyl groups in Tris buffers is probably a reflection of the trans-Schiff base reaction of aldimine with Tris. In support of this hypothesis, sodium cyanoborohydride, a reagent selective for the reduction of the aldimine linkages, inhibited the labilizing influence of Tris. A similar reversibility appears to be true of the ketoamine adducts of aldohexoses at the epsilon-amino groups of hemoglobin A, and ribonuclease A as well. This information on the reversibility of the ketoamine adducts of glucose on the epsilon-amino groups of proteins should be useful in understanding the details of the mechanism of nonenzymic glycosylation. It is also expected to influence the interpretation of the measurements of nonenzymically glucosylated hemoglobin A in diabetic patients.

Selective carboxymethylation of the alpha-amino groups of hemoglobin. Effect on functional properties.

Hemoglobin A hybrids with carboxymethyl groups at the alpha-NH2 termini of the alpha-chains or the beta-chains or at the termini of both chains have been prepared by reductive alkylation of the protein with glyoxylate and NaCNBH3 under controlled conditions. A hemoglobin derivative, which was selectively carboxymethylated at the NH2-terminal residues, was separated into its alpha- and beta-chains. These derivatized chains were recombined to yield alpha 2 Cm beta 2 Cm or were combined with unmodified beta- or alpha-chains, respectively, and purified to yield alpha 2 Cm beta 2 or alpha 2 beta 2 Cm. These hybrid tetramers retained their cooperativity and function (average n = 2.4). The hybrid alpha 2 Cm beta 2 had a lower oxygen affinity (p50 = 12 mm) than unmodified hemoglobin (p50 = 7 mm) and was reactive with 2,3-diphosphoglycerate (p50 = 48 mm). The oxygen affinity of the derivative alpha 2 beta 2 Cm was lower (p50 = 17 mm) than unmodified hemoglobin and was affected only slightly by 2,3-diphosphoglycerate (p50 = 25 mm). The tetramer carboxymethylated at all 4 NH2-terminal residues, alpha 2 Cm beta 2 Cm, exhibited a very low intrinsic oxygen affinity (p50 = 37 mm) that was further lowered to a limiting value of 50 mm by saturating amounts of 2,3-diphosphoglycerate. Each carboxymethyl tetramer, except alpha 2 Cm beta 2 Cm, was reactive with chloride to lead to a lower oxygen affinity. These carboxymethylated hybrids (Hb-NH-CH2COO-) may provide a useful model system for studies on the binding of anions to hemoglobin or on the interaction of CO2 with hemoglobin to form the carbamate Hb-NH-COO-.

An equilibrium model of the oxygen association curve of normal human erythrocytes under standardized conditions.

Hemoglobin-oxygen association curves of human erythrocytes were measured in metabolically stable cells under the standardized conditions of extracellular pH 7.400, carbon dioxide tension 38 mmHg (approximately 5 kPa) and temperature 37 degrees C. A model of the oxygen association curve of normal erythrocytes was subsequently developed, based on assumed equilibrium reactions between hemoglobins (Hb), oxygen, and 2,3-diphosphoglycerate (DPG). Conditional equilibrium constants were determined for reactions leading to the formation of species designated Hb-O2 (K = 0.14 mmHg-1), Hb-(O2)4 (K = 0.44 X 10(-4) mmHg-4), Hb-DPG (K = 0.30 X 10(4) L mol-1) and Hb-O2-DPG (K = 0.61 X 10(2) L mol-1 mmHg-1) for unmodified hemoglobin, and Hb-O2 (K = 0.14 mmHg-1) and Hb-(O2)4 (K = 0.44 X 10(-4) mmHg-4) for glycosylated hemoglobin. This model provides a practical means of calculating the expected oxygen association curve for a sample of erythrocytes under standardized conditions, given the intracellular concentrations of hemoglobins and 2,3-diphosphoglycerate and the assumption that the hemoglobins in those cells function normally.

Preparation and evaluation of pyridoxalated-polymerized human hemoglobin

A solution of hemoglobin has several potential applications as a blood substitute. However, because of high oxygen affinity ( P 50 ∼ 14 mm Hg) and short vascular retention time of hemoglobin (plasma half-disappearance time ∼ 3.5 hr), a solution of hemoglobin presents limitations for its general use in blood replacement therapy. To overcome these limitations crystalline hemoglobin was modified by pyridoxalation and subsequent polymerization. Pyridoxalation yielded a product with a P 50 ranging from 23 to 26 mm Hg. The pyridoxalated hemoglobin was then polymerized with glutaraldehyde and the final modified hemoglobin showed a P 50 of 19 to 22 mm Hg. The modified hemoglobin was tested in vitro for coagulation activities. The results indicated that no adverse coagulant activity was demonstrated by the modified products. In vivo studies in the rat have shown that pyridoxalated-polymerized hemoglobin has a plasma half-disappearance time of about 25 hr. The data demonstrated that a solution of pyridoxalated-polymerized hemoglobin, because of its lower oxygen affinity and longer vascular retention than unmodified hemoglobin, has significant potential as a basis for an efficient resuscitation solution.

Molecular modifications of hemoglobin.

There are two major limitations on the use of acellular hemoglobin solutions in vivo: rapid renal clearance and increased oxygen affinity. A series of chemical modifications of hemoglobin are presented in an attempt to address these problems. Reactions of hemoglobin with phosphorylated sugars and gluteraldehyde under a number of conditions, resulted in either hemoglobin denaturation or production of multiple hemoglobin polymers. In contrast, phosphorylated cyclic nucleotides upon ring opening with sodium periodate resulted in high-yield crosslinking of hemoglobin to exclusively form species with a molecular weight of 68,000. Because yield of modified hemoglobin increased with degree of phosphorylation of the cyclic nucleotide, this reaction is most consistent with formation of a crosslinked product within the 2,3-diphosphoglycerate pocket. Resultant oxygen affinities of such modified hemoglobins were variable and believed to be due to residual nonspecific sodium periodate oxidation. Affinity difunctional crosslinking agents like the phosphorylated dialdehydes seem useful in overcoming the major limitations of unmodified hemoglobin while maintaining basic hemoglobin structure.

Labeling of hemoglobin with pyridoxal phosphate.

The reaction of pyridoxal 5'-phosphate (PLP) with deoxyhemoglobin is confined to 2 residues in the beta chains, i.e. the alpha-amino group of valine 1 and the epsilon-amino group of lysine 82, both of which are located in the polyphosphate binding site. The major product is a hemoglobin in which only the two NH2-terminal amino groups are substituted (symmetric diPLPHb). It is formed by subunit rearrangement of monoPLPHb which is the initial product of the pyridoxylation under anaerobic conditions. TetraPLPHb, with substitutions at lysine 82 and valine 1 of both beta chains is found as a minor component. It results from subunit exchange of asymmetric diPLPHb consisting of one unmodified alpha beta dimer and one which is pyridoxylated at both sites. Anaerobic electrophoresis and oxygenation curves show that this reaction is readily reversed by mixing the tetrasubstituted derivative with unmodified hemoglobin.

The carbamate equilibrium of alpha- and epsilon-amino groups of human hemoglobin at 37 degrees C.

We have investigated the carbamate equilibrium of human adult hemoglobin, human cord blood hemoglobin, methemoglobin, and carbamylated hemoglobin using a stopped flow, rapid reaction pH apparatus described previously. The carbamate formation of human adult hemoglobin at 37 degrees C and ionic strength 0.15 was measured at pH values ranging from 6.2 to 8.8 and at CO2 partial pressures between 15 and 140 Torr. From experiments with unmodified hemoglobin as well as with hemoglobin specifically carbamylated at the four NH2 termini, it was found that already at pH 8, the epsilon-amino groups contribute significantly to carbamate formation in addition to the alpha-amino groups. At pH 8.5, about 70% of the total carbamate is due to epsilon-amino groups. The carbamate formation of alpha- and epsilon-amino groups can be suppressed by complete carbamylation of the hemoglobin. The results obtained from human adult deoxy- and oxyhemoglobin were used to calculate the equilibrium constants governing carbamate formation of these hemoglobins: Kc, the carbamate equilibrium constant, Kz, the R-NH2 ionization constant, and n, the number of binding sites per hemoglobin tetramer. Accordingly, two types of alpha-amino groups, each comprising two groups per tetramer, participate in carbamate formation of deoxyhemoglobin, one of low CO2 affinity (pKc = 5.2; pKz = 7.1; n = 2) and one of high CO2 affinity (pKc = 4.4; pKz = 6.1; n = 2). The pKz values derived from carbamate measurements agree within experimental error with figures obtained by difference titration of unmodified and specifically carbamylated hemoglobin. In addition to the alpha-amino groups, 15 epsilon-amino groups with pKc = 5.0 and pKz = 9.8 form carbamate in deoxyhemoglobin. In oxyhemoglobin, the carbamate data could be fitted with only two similar alpha-amino groups per tetramer in addition to 15 epsilon-amino groups, the latter with pKc = 4.7 and pKz = 10.2. The difference titration of the alpha-amino groups of oxyhemoglobin showed abnormal titration behavior of the beta-chain alpha-NH2. The pKc and pKz values obtained for the epsilon-amino groups of unmodified hemoglobin also provide a good description of the carbamate formed by hemoglobin specifically carbamylated at the four alpha-amino groups. The oxylabile carbamate, according to these results and in agreement with earlier reports, is only formed by alpha-amino groups; it amounts to 0.18 mol/mol of hemoglobin monomer at physiological conditions of pH 7.2 and pCO2 = 40 Torr. The amount of CO2 bound by methemoglobin equals that of oxyhemoglobin. Experiments carried out in the presence of 2,3-diphosphoglycerate provided evidence for competitive binding of CO2 and diphosphoglycerate to adult and cord blood oxy- as well as deoxyhemoglobin, the sites of competition probably being alpha-amino groups.

Large-volume preparation of pyridoxylated hemoglobin with high P50

A technique is described for the preparation of a stroma-free pyridoxylated hemoglobin solution in volumes of up to 20 liters and hemoglobin concentrations of 18–20 g%. The methemoglobin concentration is 3–7%; the phospholipid content is 5–10% of lysate; and normal values for prothrombin time and activated partial thromboplastin time are obtained. Electrophoresis on cellulose acetate strips shows that 70% of the hemoglobin is covalently linked to pyridoxal-5′-phosphate. This proportion can be raised to greater than 85% (PLP fraction) by batch purification with DEAE-Sephadex. The final solution has an in vitro P 50 of 22–26 mm Hg (pH = 7.40, pCO 2 = 40 mm Hg, temperature = 37°C). Both the proportion of pyridoxylated to unmodified hemoglobin and the P 50 remain unchanged throughout the course of total exchange transfusion in the baboon.

Polyvinylpyrolidone-fixed hemoglobin as an oxygen carrying blood substitute (author's transl)

The development of blood substitute oxygen carriers has branched off into several directions: stroma-free hemoglobin solutions, modelling of hemoglobin by fixation of heme or hemopeptides to polymers, filling of microcapsules with hemoglobin solution, crosslinking of hemoglobin. However, the application as blood replacement fluids in patients has not been possible so far because of several disadvantages. In this paper synthesis and properties of an oxygen carrying blood substitute are described in which the hemoglobin molecule is covalently bound to polyvinylpyrrolidone. In aqueous solution the preparation fulfills some basic requirements such as high solubility, only slightly increased oxygen affinity, and low viscosity. Normothermic perfusion experiments with isolated pig kidneys demonstrate that the solution provides an adequate oxygen supply to the organ, has no toxic side effects on kidney function, and has a significantly lower elimination rate compared to an unmodified hemoglobin solution.

The acetylation of hemoglobin by aspirin. In vitro and in vivo.

The chemical modification of hemoglobin by aspirin (ASA) has been studied, both in intact human red cells and in purified hemoglobin solutions. After incubation of red cells with 20 mM [acetyl-1minus14C]ASA, incorporation of radioactivity into hemoglobin was observed in agreement with the results of Klotz and Tam (1973. Proc. Natl. Acad. Sci. U. S. A. 70: 1313-1315). In contrast, no labeling of hemoglobin was seen when [carbosyl-14-C]ASA was used. These results indicate that ASA acetylates hemoglobin. The acetylated hemoglobin was readily separated from unmodified hemoglobin by both gel electrofocusing and by column chromatography. Quantitation of the extent of acetylation by densitometric scanning of gels agreed very well with estimates obtained from radioactivity measurements. Hemolysates prepared from red cells incubated with ASA showed normal oxygen affinity and heme-heme interaction. Purified acetylated hemoglobin had a slightly increased oxygen affinity and decreased heme-heme interaction. There was no difference in the rate of acetylation of oxy- and deoxyhemoglobin. ASA acetylated column-purified hemoglobin A more readily than hemoglobin in crude hemolysate, but less rapidly than purified human serum albumin. The rate of acetylation of hemoglobulin increased with pH up to approximately pH 8,5. Structural studies were done on hemoglobin incubated with 2.0 mM and 20 mM [acetyl-1-14-C]ASA. Alpha- and beta-chains were acetylated almost equally. Tryptic digests of purified acetylated subunits were fingerprinted on cellulose thin layer plates and autoradiographed. Both alpha- and beta-chains showed a number of radioactive spots that were either ninhydrin negative or weakly ninhydrin positive. These results indicate that hemoglobin is acetylated at a number of sites, probably at the epislon-amino group of lysine residues. To determine whether ASA acetylates hemoglobin in vivo, hemolysates of 14 patients on long-term high-dose ASA therapy were analyzed by gel electrofocusing and compared to specimens of individuals not receiving ASA. The ASA-treated group had a twofold increase in a minor hemoglobin component having an isoelectric point lower than that of hemoglobin A, and indistinguishable from the minoe component which appears when hemoglobin is incubated with ASA in vitro.

Partial restoration of normal functional properties in carboxypeptidase A-digested hemoglobin.

In the absence of organic phosphates human hemoglobin A digested with carboxypeptidase A (des His, Tyr beta) has high ligand affinity, a greatly reduced Bohr effect, and no heme-heme interaction. Under these conditions, it shows the simple, homogeneous ligand-binding kinetics characteristic of noncooperative heme proteins in which the high combination velocity for both O(2) and CO accounts, to a larger extent, for the increased affinity for both these ligands. Addition of inositol hexaphosphate dramatically alters the functional properties of this digested hemoglobin. The Bohr effect is greatly increased, and at neutral pH the protein shows significant, though still reduced, heme-heme interaction, together with a 5-fold decrease in affinity. In the presence of saturating amounts of the organic phosphate, the value of n is pH dependent, dropping from 1.9 at pH 5.8 to 1.3 at pH 8.6. After inositol hexaphosphate addition, the combination of the deoxy form of the digested hemoglobin with CO is 10-times slower than that observed in the absence of the inorganic phosphate; also the combination with CO after flash photolysis is biphasic and is similar, in many respects, to that observed for unmodified hemoglobin. Besides these functional changes, addition of inositol hexaphosphate to the modified deoxyhemoglobin results in an increase in the extinction coefficient at 430 nm similar to that observed on mixing the isolated alpha and beta chains of normal hemoglobin. The results are consistent with the idea that inositol hexaphosphate shifts an equilibrium between high- and low-affinity forms of the protein.

The Interactions between α and β Chains of Human Hemoglobin

Abstract This paper describes experiments designed to show the linkage of the two chains in the αβ dimer. The approach has been a kinetic one. If modification of one chain leads to conformational change in the other, this should be detectable as a difference in the reaction velocities of functional groups on the unsubstituted chain. We studied three derivatives of human hemoglobin; one was modified in the α chain and two in the β chain. Substitution at the NH2 terminus of the α chain with 1-fluoro-2,4-dinitrobenzene leads to a total loss of cooperative interactions as judged by oxygenation parameters. In this derivative, the —SH group of cysteine 93β reacts more rapidly with N-ethylmaleimide than does the —SH group of unmodified hemoglobin. The effect of deoxygenation upon the reactivity of the 93β —SH is abolished. There is no change in the reactivity of cysteine 93β with iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid). The COOH termini of the α and β chains of the dinitrophenyl-carbonmonoxyhemoglobin are digested more slowly with carboxypeptidase B and A, respectively. When the primary substitution is made on the β chain at cysteine 93, the effects upon the α chain are less marked and depend on the substituent: N-ethylmaleimide has more potent effects than iodoacetamide. The N-ethylmaleimide derivative has a partial loss of cooperative interactions, as determined by oxygenation properties, while the iodoacetamide derivative shows normal oxygen binding except for an increased oxygen affinity. N-ethylmaleimide substitution at —SH 93β allows faster digestion of the α chain COOH terminus, but alkylation with iodoacetamide has no effect. Neither modification alters the reactivity of the NH2 terminus of the α chain to 1-fluoro-2,4-dinitrobenzene. Intrachain effects are produced by both substituents: substitution with N-ethylmaleimide slows and with iodoacetamide speeds the digestion of the β chain by carboxypeptidase A. Since modification of either chain of the dimer changes the reactivity of widely separated groups, a general conformational change must occur. The degree of change in the kinetics of the groups tested correlates with the degree of disruption of functional properties. These experiments show the close interaction of the two chains in the αβ dimer and are further evidence that the αβ dimer is the fundamental unit of both structure and function.