Parameters of oxygen-binding function of human hemoglobin modified by carbon oxide and UV-light

The parameters of oxygen-binding function of human hemoglobin, modified by carbon oxide and UV-radiation: the pressure of half-saturation with the ligand (P50), Hill's constant (n), and arterial-venous difference of HbO2 concentration in the sample were studied. The presence of carboxyform in blood in boundaries of admissible values (lower than 10 per cents) did not noticeably influence to the oxygenation parameters. Functional properties of hemoproteid were substantially modified in case of HbCO concentration increasing from 30 up to 80 percent. It has been discovered, that the leading mechanism of protection from hemic hypoxia in case of poisoning with CO is decreasing of degree of cooperative interactions and relative affinity of hemoglobin for ligands. The stimulating influence of UV-light to the functional properties of modified with carbon oxide human hemoglobin observed in case carboxyform hemoprotein concentration in solution was lower than 10 percent. The disturbance of oxygen-binding ability of hemoglobin at the influence of higher concentrations of Hb-CO was inconvertible and was not correct with UV-radiation.     

UV-sensitivity of hemoglobin dimers in free state and in valency hybrids: modification by serotonin]

Changes of oxygen-binding activity of hemoglobin dimers modified by the therapeutical doses of UV-light and serotonin in free state and in valency hybrids are analyzed. The prior role of photodissociation to dimers at the UV-radiation action on heme-protein molecules has been shown. It has been observed that the complex between hemoglobin and serotonin is formed in fields of alpha beta-dimers contacts.     

Acylation of human hemoglobin with polyoxyethylene derivatives

Monomethoxypolyoxyethylene (Mw = 5000) was covalently linked to human hemoglobin via an amide bond formed between amino groups of the protein and a carboxylic group introduced onto the polymer. The conjugates thus obtained have a molecular size corresponding to that of a globular protein with a molecular weight of about 190 000. Their oxygen-binding properties depend upon the initial conformation of the hemoglobin and reaction pH: hemoglobin modified in the deoxy state exhibited a lower oxygen affinity than that modified in the oxy state, and the lower the reaction pH, the lower the oxygen affinity of polymer-linked hemoglobin. However, the affinity of modified hemoglobin is always higher than that of native hemoglobin. On the other hand, when deoxyHb was complexed with organic phosphates during the condensation reaction, the resulting conjugates exhibited oxygen-binding characteristics quite similar to those of native hemoglobin, i.e., the same oxygen affinity, modified cooperativity and the same alkaline Bohr effect. Finally, in order to decrease the oxygen affinity of hemoglobin conjugates, the polymer was coupled to deoxy hemoglobin previously covalently modified with pyridoxal phosphate. The oxygen affinity of such conjugates was in fact as low as that of the initial pyridoxylated hemoglobin.     

Influence of physical conditions on the oxidation of hemoglobin during freeze-drying

The potential influence of some physical conditions--dialysis, crystallization, concentration, pH, and temperature--on the amount of methemoglobin obtained after freeze-drying of hemoglobin has been studied. Among these parameters, pH and crystallization influence the oxidation. In acid medium (pH 5), the oxygen saturation is better than that obtained for pH 8. Crystalline hemoglobin leads to a methemoglobin rate significantly lower (29% versus 49%) than untreated hemoglobin. Methemoglobin is continuously formed during desiccation even at the lowest temperature. Although it has been possible to lessen the denaturation of hemoglobin by the choice of a definite preliminary treatment of the samples, we were not able to reduce methemoglobin to low and physiological values. However, the results obtained with crystalline hemoglobin make it possible to propose mechanisms for the oxidation of the hemoprotein.     

Effect of NaBH4 concentration and reaction time on physical properties of glutaraldehyde-polymerized hemoglobin

The US is about 1.5 days away from exhausting its entire blood supply. Hence, there is an urgent need for the development of universal blood substitutes. One such blood substitute is glutaraldehyde-polymerized bovine hemoglobin. Despite the commercial development of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes, there has been little published work on the effect of reaction conditions on the physical properties of the polymerized hemoglobin dispersion. In this study, the effect of varying the concentration of the quenching agent NaBH(4), glutaraldehyde concentration, and reaction time on the physical properties of PolyHb dispersions was studied by measuring the absolute molecular weight distribution, as well as oxygen-binding properties such as P(50), Hill coefficient, and methemoglobin level (MetHb) of these dispersions. Bovine hemoglobin was polymerized with glutaraldehyde using a parallel synthetic approach. Asymmetric flow field-flow fractionation (AFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight distribution of the PolyHb dispersions. In general, high glutaraldehyde concentrations (20-30 times the molar concentration of hemoglobin) adversely affected the oxygen-binding properties of PolyHb dispersions, while NaBH(4) concentrations (up to 300 times the molar concentration of hemoglobin) and reaction times (up to 24 h) did not appear to have any effect on the oxygen-binding properties of PolyHb dispersions.     

The role of oxygen-binding properties of hemoglobin in the development of oxygen deficiency in acute exogenous hyperthermia

Acid-base balance and oxygen-binding hemoglobin properties in mixed venous blood have been studied in 25 mongrel rabbits with acute environmental hyperthermia. As oxygen-hemoglobin affinity at standard pH, pCO2 and temperature increases, the effect of heat on oxygen-hemoglobin interaction is considerably attenuated. The Bohr effect increases. The mechanisms of changes in oxygen-binding properties of hemoglobin and their role in development of oxygen deficiency are discussed.     

Structure of nitric oxide hemoglobin

We have compared the structure of horse nitric oxide hemoglobin (HbNO) and methemoglobin in the oxy quaternary structure by difference Fourier analysis at 2.8 Å resolution. Both nitric oxide and oxygen assume bent co-ordination geometry and form low-spin complexes in binding to heme; on the basis of preferred ligand and heme stereochemistry, HbNO is the closest analog of HbO₂ (oxyhemoglobin) examined to date. To the resolution of the X-ray data, the stereochemistry of the heme-NO complex in hemoglobin and the corresponding free heme complex appears similar. In contrast, the ligand pockets in hemoglobin hinder binding of cyanide and carbon monoxide in their preferred linear axial co-ordination modes and force them to assume a strained off-axis binding stereochemistry. The structural similarity between HbNO and HbO₂ is reflected in their kinetic behavior, which is similar, and distinct from that of carboxyhemoglobin.     

The involvement of nitric oxide in formation of hemoglobin oxygen-binding properties

The analysis of literature and results of our investigations indicate the possible involvement of L-arginine-nitric oxide (NO) system in formation of blood oxygen-carrying capacity. In reaction with hemoglobin NO forms methemoglobin, nitrosyl-hemoglobin (HbFe2+NO) and S-nitrosohemoglobin (SNO-Hb). The NO-hemoglobin derivatives have the various biological functions (NO transport, storage, elimination etc.) and are involved in the genesis of different pathologic conditions. The presence of different NO-hemoglobin derivatives can differently influence on the whole blood hemoglobin-oxygen affinity (HOA): methemoglobin and SNO-Hb increases, and HbFe2+NO decreases it. Their effect on the blood oxygen-binding properties may be important for the gas exchange processes. At the level of lung capillaries such effect may be the additional mechanism promoting a blood oxygenation, and in the systemic microcirculation it may optimize blood desaturation and hence the tissue oxygen delivery. Blood oxygen-binding properties affect the state of L-arginine-NO system, however this system also may determine HOA through the intraerythrocytic regulatory mechanisms, oxygen-dependent nature of NO generation, regulation of vascular tone and effect of peroxynitrite.     

On the identity of the dissociation-linked chloride binding sites in human hemoglobin. Studies with hemoglobin modified with 4-isothiocyanatobenzenesulfonic acid and 4-isothiocyanatobenzenesulfonamide

The binding of chloride ions to specific sites on the human hemoglobin molecule has well-known effects on the oxygen equilibrium and on the stability of the tetrameric structure. Several lines of evidence suggest that the oxygen-linked and the dissociation-linked chloride binding sites differ. Direct evidence for this difference has been obtained from the chloride dependence of the dimer-tetramer equilibrium of oxyhemoglobin modified with 4-isothiocyanatobenzenesulfonic acid, in which all the oxygen-linked chloride binding sites are blocked, or with 4-isothiocyanatobenzenesulfonamide, in which the linkage between chloride and oxygen is unperturbed. Thus, the chloride dependence of the dimer-tetramer assembly is unaffected by the chemical modification in both proteins and resembles that of unreacted hemoglobin. It is suggested that histidines alpha-103, alpha-122 and beta-97 may constitute, at least in part, the dissociation-linked chloride binding sites.     

Iron nitrosyl hemoglobin formation from the reactions of hemoglobin and hydroxyurea

Hydroxyurea represents an approved treatment for sickle cell anemia and acts as a nitric oxide donor under oxidative conditions in vitro. Electron paramagnetic resonance spectroscopy shows that hydroxyurea reacts with oxy-, deoxy-, and methemoglobin to produce 2-6% of iron nitrosyl hemoglobin. No S-nitrosohemoglobin forms during these reactions. Cyanide and carbon monoxide trapping studies reveal that hydroxyurea oxidizes deoxyhemoglobin to methemoglobin and reduces methemoglobin to deoxyhemoglobin. Similar experiments reveal that iron nitrosyl hemoglobin formation specifically occurs during the reaction of hydroxyurea and methemoglobin. Experiments with hydroxyurea analogues indicate that nitric oxide transfer requires an unsubstituted acylhydroxylamine group and that the reactions of hydroxyurea and deoxy- and methemoglobin likely proceed by inner-sphere mechanisms. The formation of nitrate during the reaction of hydroxyurea and oxyhemoglobin and the lack of nitrous oxide production in these reactions suggest the intermediacy of nitric oxide as opposed to its redox form nitroxyl. A mechanistic model that includes a redox cycle between deoxyhemoglobin and methemoglobin has been forwarded to explain these results that define the reactivity of hydroxyurea and hemoglobin. These direct nitric oxide producing reactions of hydroxyurea and hemoglobin may contribute to the overall pathophysiological properties of this drug.     

The interaction of organic phosphates with human and chicken hemoglobin.

In this study of the binding properties of inositol hexaphosphate and 2,3-bisphosphoglycerate to chicken and human deoxyhemoglobin and carboxyhemoglobin were compared. It appeared that in all cases the binding to chicken hemoglobin is much stronger than to human hemoglobin. This is very probably due to the fact that 4 out of the 12 residues, responsible for the binding of phosphates in chicken hemoglobin, are arginines. These are absent in human hemoglobin, where the binding site is made up to only 8 residues. For chicken hemoglobin one strong binding site could be observed in both unliganded and liganded hemoglobin. From these observations we conclude that the same binding site is involved in both the oxy- and deoxy structure showing different affinity to phosphates in the two conformational states. For human hemoglobin we reached the same conclusion.     

Low oxygen affinity derivatives of human hemoglobin by fixation of polycarboxylic dextran to the oxyform

Solutions of modified adult human hemoglobin (Hb) have potential applications as physiological oxygen carriers. The chemical modification that has been the most studied during the last few years is the cross-linking of the protein between its two αβ dimers, in order, first, to hamper their diffusion through the kidney and therefore increase the plasma persistence of Hb, and second, to decrease its oxygen affinity. However, despite the cross-linking, the vascular retention time is only increased by a factor of three, and a supplementary modification of cross-linked Hb is needed in order to further improve its in vivo half-life. The Hb derivatives described in this paper were obtained by the covalent fixation of benzene tetracarboxylate-substituted dextran onto oxyHb. The resulting conjugates all exhibited a higher P₅₀ than native Hb. The experiments carried out in the presence of inositolhexaphosphate showed that the allosteric sites of Hb molecules were occupied by the polymeric reagent. The important decrease in the Bohr effect and the lack of the Cl^? effect on the oxygen-binding properties proved that the Val 1α residue was also substituted. Finally, the ability of some conjugates to unload as much O₂ as blood, together with their other properties, make them quite promising candidates as red cell substitutes. © 1993 John Wiley & Sons, Inc.     

Discontinuous release of heat at successive steps of oxygenation in human and bovine hemoglobin at pH 9.0

We have measured the temperature dependence of the oxygen-binding isotherms of human and bovine hemoglobin at pH 9.0 in 0.1 M borate buffer. In both hemoglobins the ionization of the Bohr protons is finished at this pH; therefore, their heat does not interfere with the measurements. Two sets of curves have been obtained, which have been analyzed by either singular or global procedures for estimating the enthalpy changes of subsequent steps of oxygenation. The data indicate that in human hemoglobin the reaction with oxygen is enthalpy driven for steps 1, 2, and 4 while it is entropy driven for step 3. In bovine hemoglobin this phenomenon is even more evident: steps 2 and 4 are enthalpy driven while steps 1 and 3 are entropy driven. The discontinuous distribution of heat at subsequent steps of oxygenation suggests that the T to R transition in hemoglobin is not a monotonic process and involves conformations with novel characteristics.     

Oxygen binding and other physical properties of human hemoglobin made in yeast.

Wagenbach et al. (1991, BioTechnology, 9, 57-61) have recently developed a system for producing soluble recombinant tetrameric hemoglobin in yeast: hemoglobin begins to appear 4-5 h after induction with galactose, alpha- and beta-globin chains fold in vivo and endogeneously produced heme is incorporated into hemoglobin tetramers. We have further characterized the oxygen-binding properties, as well as the tetramer stability, of recombinant human Hb A made in yeast. After purification by ion-exchange chromatography, a single band at the same position as normal human Hb A was obtained using cellulose acetate electrophoresis. Although the oxy and deoxy forms of purified recombinant Hb A made in yeast were spectrophotometrically identical to native human Hb A, the oxygen-binding curve was shifted slightly left of that for native human Hb A. Further purification of recombinant hemoglobin by FPLC revealed two fractions: one (fraction B) with low cooperativity and high oxygen affinity, and the other (fraction A) with almost identical cooperativity and oxygen affinity compared with native human Hb A. The Bohr effect of fraction A was also identical to native human Hb A. Hemoglobin in fraction B with lowered cooperativity precipitated approximately 1.5 times faster than normal human Hb A during mechanical agitation, while hemoglobin in fraction A with normal cooperativity precipitated with kinetics identical to native human Hb A. These results suggest that some of the recombinant molecules made in yeast fold improperly, and that these molecules may exhibit decreased cooperativity for oxygen binding and decreased stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon monoxide binding to human hemoglobin A0

The carbon monoxide binding curve to human hemoglobin A0 has been measured to high precision in experimental conditions of 600 microM heme, 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, 0.1 M NaCl, 10 mM inositol hexaphosphate, 1 mM disodium ethylenediaminetetraacetic acid, pH 6.94, and 25 degrees C. Comparison to the oxygen binding curve in the same experimental conditions demonstrates that the two curves are not parallel. This result invalidates Haldane's two laws for the partitioning between carbon monoxide and oxygen to human hemoglobin. The partition coefficient is found to be 263 +/- 27 at high saturation, in agreement with previous studies, but is lowered substantially at low saturation. Although the oxygen and carbon monoxide binding curves are not parallel, both show the population of the triply ligated species to be negligible. The molecular mechanism underlying carbon monoxide binding to hemoglobin is consistent with the allosteric model [Di Cera, E., Robert, C. H., & Gill, S. J. (1987) Biochemistry 26, 4003-4008], which accounts for the negligible contribution of the triply ligated species in the oxygen binding reaction to hemoglobin [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry 26, 3995-4002]. The nature of the different binding properties of carbon monoxide stems largely from the lower partition coefficient of the T state (123 +/- 34), relative to the R state (241 +/- 19).(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of unaltered hemoglobin from human placentas for possible use in blood substitutes

Hemoglobin extracted from human placentas could be used as the basis of blood substitutes provided it could be prepared on a large scale with appropriate oxygen-binding properties. Unfortunately, the industrial conditions under which it is extracted, produce hemoglobin with high oxygen affinity and which is no longer influenced by the classical effectors. These characteristics were shown to be caused by a degradation of the alpha-chain brought about by an arginine carboxypeptidase present in the placental tissues and leading to the disappearance of the C-terminal arginine residue. This carboxypeptidase which is released from the tissues during the process of crushing the frozen placentas, degrades the protein during the chromatographic purification procedure. The addition of an inhibitor of this carboxypeptidase (for example, arginine) as soon as the placentas are thawed and during the chromatographic process, makes it possible to obtain placental hemoglobin with oxygen-binding properties quite similar to those of HbA prepared from peripheral venous blood.     

Unique features of the hemoglobin system of the Antarctic notothenioid fish Gobionotothen gibberifrons.

The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae.     

Alteration of T-state binding properties of naturally glycated hemoglobin, HbA1c

The thermodynamic and kinetic properties of the most abundant glycated hemoglobin in human blood, HbA1c, have been studied in detail. They display significant differences as compared to normal hemoglobin, HbA0, in that (1) the shape of the oxygen binding curve of HbA1c in the Hill plot is markedly asymmetrical, with a lower asymptote extending up to approximately 40% oxygen saturation, and the oxygen affinity of the T state being tenfold higher than in HbA0; (2) oxygen pulse experiments on HbA1c show a slower rate of ligand dissociation (k = 25 s-1) even at low levels of oxygen saturation, where the T state is largely predominant; (3) kinetics of CO combination to deoxy HbA1c followed by means of stopped-flow experiments reveal the presence of a quickly reacting component, whose fraction increases upon dilution of hemoglobin. These results show that in contrast to what has been stated by other authors, HbA1c displays functional properties markedly different from HbA0. Analysis indicates that glycation of human hemoglobin affects the T quaternary structure, bringing about a more "relaxed" T state and leading to preferential binding to one type of chain (which is unaffected by chloride ions).     

Effect of oxygen binding on the dielectric properties of hemoglobin

The dielectric properties of horse hemoglobin have been investigated in the frequency range for 100 kcps to 15 Mcps at varying degrees of oxygenation. A linear dependence of the specific increment on the degree of oxygenation was found under a variety of experimental conditions, the increment of oxygenated hemoglobin being about 10% larger than that of deoxygenated hemoglobin. A similar difference was obtained with human adult and fetal hemoglobin. No variation of the dielectric parameters as reported by Takashima and Lumry could be detected.     

Application of 19F NMR spectroscopy to a study of carbon monoxide binding to human hemoglobin modified at Cys-beta 93 with the S-trifluoroethyl residue

The S-2,2,2-trifluoroethyl residue (-SCH2CF3) has been incorporated into human hemoglobin, Hb4(SH)2, as a reporter group at Cys-beta 93 using a sequence of disulfide interchange reactions [F. C. Knowles (1981) Anal. Biochem. 110, 19-26]. The 19F NMR spectrum at 235.2 MHz of carboxyhemoglobin (COHb)4(SSCH2CF3)2 was a band 50 Hz wide at half peak height. Conversion of the carbon monoxide derivative to the ligand-free form was accompanied by a downfield shift of 125 Hz (0.53 ppm). Weighed aliquots of solutions of Hb4(SSCH2CF3)2 and (COHb)4(SSCH2CF3)2 were mixed, yielding solutions of known fractional saturation with carbon monoxide. An independent estimate of F of these samples was derived from the amplitudes of the resonance intensities in the 19F NMR spectra. These independent methods for determination of the value of F were not uniformly in agreement. In the presence of inositol hexaphosphate the estimate of F derived from 19F NMR spectra was considerably less than the actual value. The discrepancies between the two independent methods for determining F can be explained by a preferred order of binding of carbon monoxide to the alpha-chains. The preference for binding to the alpha-chains was abolished by removing phosphates. A model for transmission of cooperative effects in hemoglobin was presented which accounted for the characteristic shape of the hemoglobin-oxygen dissociation curves as well as the chain heterogeneity revealed by 19F NMR experiments.