Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Kinetics of ligand binding to ferrous heme groups of hemoglobin MSaskatoon.

Hemoglobin M_Saskatoon (α₂^Aβ₂^63tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin M_Saskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin M_Saskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin M_Saskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin M_Saskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.     

Chlamydomonas chloroplast ferrous hemoglobin. Heme pocket structure and reactions with ligands

We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>10(7) microM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.     

Kinetics of oxygen and carbon monoxide binding to liver fluke (Dicrocoelium dendriticum) hemoglobin. An extreme case?

The kinetics of oxygen and carbon monoxide binding to the monomeric liver fluke (Dicrocoelium dendriticum) hemoglobin have been studied. The ligand association rates are approximately 1 X 10(8) and approximately 3 X 10(8) M-1 s-1, respectively, for CO and O2 and show no pH dependence. On the contrary the ligand dissociation rates decrease by lowering the pH below 7, the pK of the transition being around 5.5. These findings, together with spectroscopic properties of the protein, are discussed in relation to the fact that, in this hemoglobin, the distal histidine is replaced by a glycine.     

Hemoglobins of the Lucina pectinata/bacteria symbiosis. I. Molecular properties, kinetics and equilibria of reactions with ligands

Three hemoglobins have been isolated from the symbiont-harboring gill of the bivalve mollusc Lucina pectinata. Oxyhemoglobin I (Hb I), which may be called sulfide-reactive hemoglobin, reacts with hydrogen sulfide to form ferric hemoglobin sulfide in a reaction that may proceed by nucleophilic displacement of bound superoxide anion by hydrosulfide anion. Hemoglobins II and II, called oxygen-reactive hemoglobins, remain oxygenated in the presence of hydrogen sulfide. Hemoglobin I is monomeric; Hb II and Hb III self-associate in a concentration-dependent manner and form a tetramer when mixed. Oxygen binding is not cooperative. Oxygen affinities are all nearly the same, P50 = 0.1 to 0.2 Torr, and are independent of pH. Combination of Hb I with oxygen is fast; k'on = (estimated) 100-200 x 10(6) M-1 s-1. Combination of Hb II and Hb III with oxygen is slow: k'on = 0.4 and 0.3 x 10(6) M-1 s-1, respectively. Dissociation of oxygen from Hb I is fast relative to myoglobin: koff = 61 s-1. Dissociation from Hb II and Hb III is slow: koff = 0.11 and 0.08 s-1, respectively. These large differences in rates of reaction together with differences in the reactions of carbon monoxide suggest differences in configuration of the distal heme pocket. The fast reactions of Hb I are comparable to those of hemoglobins that lack distal histidine residues. Slow dissociation of oxygen from Hb II and Hb III suggest that a distal residue may interact strongly with the bound ligand. We infer that Hb I may facilitate delivery of hydrogen sulfide to the chemoautotrophic bacterial symbiont and Hb II and Hb III may facilitate delivery of oxygen. The midpoint oxidation-reduction potential of the ferrous/ferric couple of Hb I, 103 +/- 8 mV, was independent of pH. Potentials of Hb II and Hb III were pH-dependent. At neutral pH all three hemoglobins have similar midpoint potentials. The rate constant for combination of ferric Hb I with hydrogen sulfide increases 3000-fold from pH 10.5 to 5.5, with apparent pK 7.0, suggesting that undissociated hydrogen sulfide is the attacking ligand. At the acid limit combination of ferric Hb I with hydrogen sulfide, k'on = 2.3 x 10(5) M-1 s-1, is 40-fold faster than combination with ferric Hb II or myoglobin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin.

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M.     

Kinetic and equilibrium studies of the reactions of heme-substituted horse heart myoglobins with oxygen and carbon monoxide.

In order to study the effects of chemical modifications of the vinyl groups of heme on oxygen and carbon monoxide binding to myoglobin, apomyoglobins from horse heart were reconstituted with six different hemins with various side chains. Laser flash photolysis experiments of these reconstituted myoglobins showed that the combination rate constants for oxygen (k') and carbon monoxide (l') were closely related to the electron-attractive properties of the side chains. The k' values obtained in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees were 0.83 (meso-), 2.4 (deutero-), 1.1 (reconstituted proto-), 1.2 (native proto-), 1.5 (2-formyl-4-vinyl-), 1.9 (2-vinyl-4-formyl-), and 2.7 X 10(7) M-1 S-1 (2,4-diformylmyoglobins), and the corresponding l' values were 2.8, 18, 4.8, 5.1, 7.1, 15, and 35 X 10(5) M-1 S-1, respectively. These rate constants tend to increase as the electron-withdrawing power of the side chains increases, indicating that reduced electron density of the iron atom of heme in myoglobin favors the combination reaction for both oxygen and carbon monoxide. Equilibrium constants (L) between carbon monoxide and various myoglobins were also determined by measuring the partition coefficients (M) between oxygen and carbon monoxide for the myoglobins, and were also found to be closely related to the electronic properties (pK3 of porphyrin) of the heme side chains. The equilibrium association constants for carbon monoxide thus obtained increased with a decrease in pK3 value of the porphyrin. This order was completely opposite to the case of the oxygen binding reaction. The dissociation rate constants for oxygen (k) and carbon monoxide (l) were calculated from the equilibrium and the combination rate constants. The dissociation rate constants showed a similar characteristic to the combination rate constants and increased with the increase in electron attractivity of heme side chains. The concomitant increase in both the combination and dissociation rate constants with increase in electronegativity of the iron atom suggests that these reactions have different rate determining steps, although such a reaction process is contradictory to the generally accepted concept that in a reversible reaction, both on and off reactions proceed through the same transition state. In the on reaction sigma bond formation appears to be dominant, while in the off reaction eta bond break-up is more important.     

Relative stabilities of the two quaternary conformations of human fetal hemoglobin.

The pH dependence of several functional properties of human fetal and adult hemoglobins have been studied to determine the relative stabilities of the high and low affinity (R and T) quaternary conformations of the two proteins under different conditions. Fetal aqumethemoglobin undergoes changes in sulfhydryl reactivity, absorption spectrum, and circular dichroism in the presence of insitol hexaphospahte which are consistent with a transition from the R to T quaternary state, but only at pH values below 6.8. In adult hemoglobin this transition can be induced pH values below 7.2. Even in the absence of phosphates, the ultraviolet (uv) circular dichroism spectrum of fetal aquomethemoglobin at low pH indicates the presence of some T conformation. The initial value for the second-order rate constant for combination of fetal deoxyhemoglobin with carbon monoxide is comparable to that for adult hemoglobin in the absence of organic phosphates and is not reduced by organic phosphates as much as that for the adult protein. The apparent first-order rate constant for dissociation of CO from fully liganded fetal hemoglobin, measured by replacement with NO, increases threefold in the absence of organic phosphates, and fourfold in the presence of organic phosphates, with decreasing pH; the midpoint of the pH dependent transition occurs around 6.8. A similar increase in the apparent first-order rate constant for O2 dissociation as measured by replacement with CO, can also be seen with decreasing pH. NO-hemoglobin F can be converted to the T state even when fully liganded simply by lowering the pH, as judged by uv circular dichroism, visible difference spectrum in the region of the alpha and beta bands, and a dramatic increase in the rate of NO dissociation, measured by replacement with CO in the presence of dithionite. These results are all consistent with a model for fetal hemoglobin in which the organic phosphate site may be functionally weakened by replacement of a residue involved in ionic interactions with the negatively charged phosphate groups, but in which the low affinity T conformation is intrinsically more stable than that of adllt hemoglobin. According to this model,the differences between fetal and adult hemoglobin can be accounted for primarily in terms of the relative stabilities of R and T conformations in each of the proteins with differences in the intrinsic properties of the individual conformations contributing effects of only secondary importance.     

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.     

Structural, functional, and subunit assembly properties of hemoglobin Attleboro [alpha 138 (H21) Ser----Pro], a variant possessing a site maturation at a critical C-terminal residue

Hemoglobin Attleboro, a new alpha-chain variant with a substitution of proline for serine at position 138 (H21), was found to be a noncooperative high-affinity hemoglobin (P50 = 0.26 mmHg at pH 7 and 20 degrees C) which lacked an alkaline Bohr effect. Addition of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP) led to a decrease in oxygen affinity but to no alteration in either Bohr effect or cooperativity. Ligand binding kinetics studies revealed an overall rate of oxygen dissociation at pH 7.0 and 20 degrees C that was 2.7-fold slower than that for Hb A. At pH 8.5, the kinetic profile was identical with that at pH 7, confirming the absence of a Bohr effect for this variant hemoglobin. Measurement of the rate of oxygen dissociation with carbon monoxide replacement indicated a lack of cooperativity. Sedimentation velocity experiments yielded s20,w values of 2.8 and 4.3 for 65 microM solutions of oxyhemoglobins Attleboro and A, respectively (indicating an enhancement in the oxy dimer population of this variant). Studies of the carbon monoxide combination of this variant revealed an association rate 20-fold faster than that for Hb A; only in the presence of a 1000-fold molar excess of IHP was there a significant reduction in the overall rate. Rapid-scan and traditional stopped-flow experiments conducted in the Soret Soret region demonstrated an alteration in the structure and rate of assembly of the deoxy tetramer of Hb Attleboro relative to that of Hb A. The abnormal properties of this hemoglobin variant can be attributed to major perturbations in the C-terminal region.     

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.     

The Binding of Asparagine,Glutamine and Homoserine to Human Erythrocytes Containing Hemoglobins S and CS and to Soluble Hemoglobins S and CS

Abstract

Tritium labeled asparagine binds to oxyhemoglobin S and to a mixture of hemoglobins C and S in the molar ratio of 3.38:1 and 8.2:1 respectively. From the dialysis equilibrium studies it appears that labeled asparagine does not bind to oxy- or deoxy- hemoglobin A nor to deoxyhemoglobin S. The constant for equilibrium association of asparagine for oxyhemoglobin S is 7.38 × 10⁷ M^?1 and for'oxyhemoglobin CS 4.8 × 10⁴ M^?1 at 23°C. Tritium labeled asparagine is bound to oxyhemoglobin S and CS sufficiently strongly to prevent dissociation under the conditions of gel electrophoresis at pH 9.50. The protein with and without bound asparagine, gluta-mine or homoserine, is indistinguishable in molecular net charge and size by the criteria of quantitative polyacrylamide gel electrophoresis (PAGE). Also there were no significant differences in mobility between hemoglobin S and hemoglobin C in the presence and absence of asparagine, glutamine and homoserine as detectable in agar coated cellulose acetate electrophoresis at pH 6.3. Erythrocytes containing hemoglobin S and CS, after incubation with tritium labeled asparagine and lysis under the conditions of gel electrophoresis at pH 9.5, release hemoglobin S and C with bound tritiated asparagine. No tritiated asparagine remains bound to the ghost.     

Varying apparent rate constant: determination of uptake and release of protons during tetramer-dimer dissociation in human hemoglobin A

The effect of association-dissociation on the sulphydryl reactivity of human hemoglobin A is reported. The reactivity of CysF9(93)beta towards the sulphydryl reagent, 5,5'-dithiobis(2-nitrobenzoate), is higher at lower concentrations of hemoglobin at all pH values. This is because hemoglobin dimers have higher sulphydryl reactivity than tetramers and it is known that the proportion of dimers increases as the hemoglobin concentration decreases. This study takes advantage of this observation to determine the tetramer-dimer dissociation constant, K(4,2), of hemoglobin A and subsequently the proton uptake and the proton release during this process. The concentration dependence profiles of the apparent second-order rate constants, k(app), show that (between 2 and 20 microM heme) k(app) decreases with increasing hemoglobin concentration. Above 30 M heme k(app) remains fairly constant for all hemoglobin derivatives (oxy, carbonmonoxy and aquomethemoglobin) used. The pH dependence of the negative logarithm of tetramer-dimer dissociation constant, pK(4,2), for oxy- (and for carbonmonoxy-) hemoglobin exhibits a biphasic character with a maximum near pH 7.4 (and 6.6). For aquomethemoglobin, pK(4,20 decreases with increasing pH. The tetramer-dimer dissociation of human oxyhemoglobin A at an ionic strength of 200 mM uptakes 0.87 +/- 0.09 mole of protons between pH 6.2 to 7.4 phase and releases 0.84 0.09 mole of protons between pH 7.4 and 9.0 phase. Under a similar condition carbonmonoxyhemoglobin uptakes 0.54 +/- 0.05 mole of protons between pH 5.8 and 6.6 phase and releases 0.48 +/- 0.05 mole of protons between pH 6.6 and 9.0 phase. Aquomethemoglobin has only a single phase, it releases 0.39 +/- 0.05 mole of protons during tetramer-dimer dissociation.     

Reversible oxygenation of protoheme-imidazole complex in aqueous solution (1,2)

Solutions of protohemin in aqueous buffer containing imidazole were reduced and exposed to carbon monoxide forming the carbon monoxide-imidazole complex similar to that in carboxyhemoglobin. This complex is stable for long periods in the presence of low pressures of oxygen and thus the standard flash photolysis methods can be used to determine rates of combination of the heme-imidazole complex with oxygen. Combination rates for both carbon monoxide and oxygen are faster than any on rates for hemoglobin and oxygen dissociation rates are also faster. But the equilibrium constant for binding of this isolated site is larger than that for hemoglobin.     

Tetramer-dimer dissociation in homoglobin and the Bohr effect.

The pH dependence of the apparent tetramer to dimer dissociation constant has been determined at 20 degrees for both oxy- and deoxyhemoglobins A and Kansas. These measurements were made by three different procedures: gel chromatography, sedimentation velocity, and kinetic methods in either of three buffer systems: 0.05 M cacodylate, Tris, or glycine with 1 mM EDTA and 0.1 M NaCl between pH 6.5 and 11. The tetramer-dimer dissociation constant of human oxyhemoglobin A decreases from about 3.2 X 10(-6) M at pH 6.0 to about 3.2 X 10(-8) M at pH 8.5. The slope of this line indicates that the dissociation of tetramer to dimer is accompanied by the uptake of about 0.6 protons per mol of tetramer in this region. The corresponding dissociation constant for deoxyhemoglobin in the same pH region increases apparently almost linearly from 1.0 x 10(-12) M at pH 6.5 to about 1.0 x 10(-5) M at pH 11. To dimer is associated with the release of about 1.6 protons per mol of tetramer. Comparison of these data with the known proton release accompanying the oxygenation of tetramers confirms that the pH dependence of oxygen binding by dimers must be very small. The present data predict that the overall proton release or uptake per oxygen bound by dimer should be less than 0.1. The tetramer-dimer dissociation equilibria of oxy- and deoxyhemoglobins above pH 8.5 have identical pH dependences. In this range the dissociation constant of deoxy-Hb is about one-tenth that of oxyhemoglobin. Human oxyhemoglobin Kansas is known to have an enhanced tetramer-dimer dissociation compared with that of hemoglobin A. Below pH 8.5 the tetramer-dimer dissociation constant of Hb Kansas is about 400 times greater than that of HbA in the absence of phosphate buffers. In contrast, the tetramer-dimer dissociation constants of deoxyhemoglobins A and Kansas appear to be identical. These findings are consistent with previous structural observations on these hemoglobins. The data on the tetramer-dimer dissociation of human hemoglobin were used to calculate the total free energy of binding of oxygen to the tetramer and the median oxygen pressure on the basis of fundamental linkage relations and a pH-independent estimate of the total free energy of binding oxygen to dimer. Simulated oxygen binding curves were generated with the equations of Ackers and Halvorson (Ackers, G. K., and Halvorson, H. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4312-4316) by making two assumptions: (a) that the dimers are noncooperative and pH-independent in O2 binding and (b) that the distribution of cooperative energy in the oxygenation of tetramers is independent of pH. We have compared these simulations with experimental data obtained at low protein concentrations (30 to 124 muM heme) to show that the variation in oxygen affinity with pH can be described in terms of the subunit equilibria. We conclude that an accurate analysis of the contributions of individual oxygen binding steps to the Bohr effect cannot be made without considering the contributions of the dimers to oxygen binding...     

Mechanism of the antihypoxic action of zinc compounds

It was established in experimental normobaric and hypobaric hypoxia and hemic hypoxia induced by carbon monoxide poisoning that zinc compounds administered in a dose of 0.15 mA/kg have a marked prophylactic protective effect. The mechanism of action of zinc compounds consists in changes of oxygen transport blood function. It was shown that interaction of the hemoglobin molecule with zinc ion brings about an increase in Hb affinity for O2 (the left drive of the oxyhemoglobin dissociation curve), a reduction in cooperative interaction of hemoglobin subunits, and a relative decrease in hemoglobin affinity for carbon monoxide. The leading defence mechanism against hypoxic hypoxia is the left drive, the mechanism of defence against carbon monoxide protection consists in the lowering of the "hem-hem" cooperation and of the relative hemoglobin affinity for carbon monoxide.     

Interactions of adenosine triphosphate with snake hemoglobins. Studies in Liophis miliaris,Boa constrictor and Bothrops alternatus

The hemoglobins of three snake species: Liophis miliaris, Bothrops alternatus and Boa constrictor present a single ATP binding site per tetramer. The ATP association constant values for the deoxyhemoglobins at pH 7.5 were about K_D ≅ 10⁶ M⁻¹ (10⁷ M⁻¹ for B. contrictor), three to four orders of magnitude higher than the respective values for oxyhemoglobin of about K_O ≅ 10² M⁻¹. The deoxyhemoglobin constant values markedly decrease as a function of pH, becoming, at pH 8.5, about K_D ≅ 10³ M⁻¹ whereas for the oxyhemoglobin the constants remain of about the same, K_O ≅ 10² M⁻¹, at the pH range studied. The high ATP binding affinity constants, compared to those of human hemoglobin A, were explained from a molecular structural standpoint, considering L. miliaris hemoglobin, whose complete primary sequence is known. Two distinct amino acid residue differences were found in the β-chain, one being Trp (NA3) (more hydrophobic) in the snake hemoglobin which substitutes the Leu (NA3) in human hemoglobin, and the second being Val 101 β (G3) instead of Glu 101 β (G3). The substitutions could provide an un-neutralized, positively charged, residue Lys-104β and, taking into account its high pK value, the pH dependence of ATP binding affinity for the snake hemoglobin would originate from pH-dependent ionization of phosphate groups of the allosteric effector. The physiological implications of the high ATP binding constant, as well as the possible protective role of the nucleotide binding against the effect of high environmental temperatures on the oxygen dissociation curves, are discussed.     

The kinetics of the reaction between NO and O2 as studied by a novel approach

The kinetics of the reaction between NO and O2 was determined by measuring the time course of the decrease in the concentration of NO with a quench-flow technique. NO and O2 were mixed rapidly and reacted for periods of time varying from 10 to 50 s. A second rapid mixing with a solution containing an excess of deoxyhemoglobin and sodium hydrosulfite trapped free NO as nitrosylhemoglobin and reduced O2. The spectrum of the mixture of deoxy- and nitrosylhemoglobin was recorded within 30 s from the second mixing, before any appreciable dissociation of NO from the protein, by means of a flow-cell mounted on-line with the quench-flow apparatus. The amount of NO not consumed in the auto-oxidation reaction was calculated from the proportion of nitrosylhemoglobin in the mixture. As NO and O2 bind deoxyhemoglobin at comparable rates and NO is oxidized to nitrate by oxyhemoglobin, the ratio of hemoglobin/(NO + O2) had to be optimized to avoid the interference of this oxidation reaction. The kinetics was first and second order with respect to O2 and NO, respectively and third order overall with a rate constant k = 4 x kaq = 4 x 2.23 (+/- 0.26) x 10(6) M-2 s-1 at 20 degrees C, invariant in the pH range 7-9, in agreement with published values obtained by different methodologies.     

The kinetics of assembly of normal and variant human oxyhemoglobins

The kinetics of assembly have been monitored spectrophotometrically for normal and variant human oxyhemoglobins in 0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, at 21.5 degrees C. Oxyhemoglobin versus oxy chain static difference spectra were performed and revealed subtle but significant absorption changes in both the visible and Soret regions. Kinetic experiments were performed by rapidly mixing equivalent (in heme) concentrations of alpha and beta A chains and following the change in absorbance at 583 nm with time. Over a protein concentration range of 10-100 microM in heme prior to mixing, these time courses were homogeneous and followed first-order kinetics, yielding a value of 0.069 s-1 for the apparent rate constant of dissociation of oxygenated beta A chain tetramers. Under these conditions, the overall assembly of oxyhemoglobins S (beta 6Glu----Val) and N-Baltimore (beta 95Lys----Glu) were also governed by the rates of dissociation of their respective oxygenated beta S and beta N-Baltimore chain tetramers with the apparent first-order rate constants of 0.044 and 0.15 s-1, respectively. In the Soret region, the alpha, beta monomer combination reaction could be observed if the protein concentration (heme basis) was lowered and if protein nonequivalency (beta chain exceeded alpha chain concentration) mixing experiments were performed. A kinetic oxyhemoglobin A, oxy-alpha, oxy-beta A monomer difference spectrum could be generated, and simple second-order kinetics were observed (415 nm) yielding rate constants of 2.3, 3.3, and 4.8 X 10(5) M-1 s-1 for the assembly of oxyhemoglobins S, A, and N-Baltimore, respectively. To our knowledge, this is the first kinetic study to reveal significant differences between the rate of association of alpha and beta monomers of hemoglobin A and those of two distinctly charged hemoglobin variants.     

Nitric oxide formation from the reaction of nitrite with carp and rabbit hemoglobin at intermediate oxygen saturations

The nitrite reductase activity of deoxyhemoglobin has received much recent interest because the nitric oxide produced in this reaction may participate in blood flow regulation during hypoxia. The present study used spectral deconvolution to characterize the reaction of nitrite with carp and rabbit hemoglobin at different constant oxygen tensions that generate the full range of physiological relevant oxygen saturations. Carp is a hypoxia-tolerant species with very high hemoglobin oxygen affinity, and the high R-state character and low redox potential of the hemoglobin is hypothesized to promote NO generation from nitrite. The reaction of nitrite with deoxyhemoglobin leads to a 1 : 1 formation of nitrosylhemoglobin and methemoglobin in both species. At intermediate oxygen saturations, the reaction with deoxyhemoglobin is clearly favored over that with oxyhemoglobin, and the oxyhemoglobin reaction and its autocatalysis are inhibited by nitrosylhemoglobin from the deoxyhemoglobin reaction. The production of NO and nitrosylhemoglobin is faster and higher in carp hemoglobin with high O(2) affinity than in rabbit hemoglobin with lower O(2) affinity, and it correlates inversely with oxygen saturation. In carp, NO formation remains substantial even at high oxygen saturations. When oxygen affinity is decreased by T-state stabilization of carp hemoglobin with ATP, the reaction rates decrease and NO production is lowered, but the deoxyhemoglobin reaction continues to dominate. The data show that the reaction of nitrite with hemoglobin is dynamically influenced by oxygen affinity and the allosteric equilibrium between the T and R states, and that a high O(2) affinity increases the nitrite reductase capability of hemoglobin.