Itiro Tyuma

Stoichiometry of the reaction of oxyhemoglobin with nitrite

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.

Effect of 2,3-diphosphoglycerate on the cooperativity in oxygen binding of human adult hemoglobin.

Abstract Oxygen equilibrium curves of human adult hemoglobin (Hb A) have been analysed by the Scatchard and Hill plots and four successive association constants for the binding of oxygen have been determined by fitting the polts with simulated curves calculated by a digital computer. 2,3-Diphosphoglycerate (DPG) markedly reduces the affinity of Hb A to the 1st, 2nd, and 3rd molecules of oxygen without affecting the affinity to the 4th molecule, which is similar to the oxygen affinity of the isolated β chains. Thus, the over-all free energy of interaction increases by about 1400 cal per site and the maximum slope of the Hill plot, n, increases significantly in the presence of 2 × 10 −3 M DPG.

Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite an intermediate detected by electron spin resonance

Oxidation of oxyhemoglobin by nitrite is characterized by the presence of a lag phase followed by the autocatalysis. Just before the autocatalysis begins, an asymmetric ESR signal is detected which is similar to that of the methemoglobin radical generated from methemoglobin and H2O2 in shape, g value (2.005), peak-to-peak width (18 G) and other properties, except the difference in the dependence on temperature. Generation of H2O2 is indicated by the prolongation of the lag phase by the addition of catalase. On the other hand, the oxidation is modified by neither superoxide dismutase nor Nitroblue tetrazolium. The oxidation is prolonged in the presence of KCN. The present results indicate a free-radical mechanism for the oxidation in which the asymmetric radical catalyzes the formation of NO2 from NO2- by a peroxidase action and NO2 oxidizes oxyhemoglobin in the autocatalytic phase.

The interaction between nitrogen oxides and hemoglobin and endothelium-derived relaxing factor

Among nitrogen oxides, NO and NO2 are free radicals and show a variety of biological effects. NO2 is a strongly oxidizing toxicant, although NO, not oxidizing as NO2, is toxic in that it interacts with hemoglobin to form nitrosyl- and methemoglobin. Nitrosylhemoglobin shows a characteristic electron spin resonance (ESR) signal due to an odd electron localized on the nitrogen atom of NO and reacts with oxygen to yield nitrate and methemoglobin, which is rapidly reduced by methemoglobin reductase in red cells. NO was found to inhibit the reductase activity. Part of NO inhaled in the body is oxidized by oxygen to NO2, which easily dissolves in water and converts to nitrite and nitrate. The nitrite oxidizes oxyhemoglobin autocatalytically after a lag. The mechanism of the oxidation, particularly the involvement of superoxide, was controversial. The stoichiometry of the reaction has now been established using nitrate ion electrode and a methemoglobin free radical was detected by ESR during the oxidation. Complete inhibition of the autocatalysis by aniline or aminopyrine suggests that the radical catalyzes conversion of nitrite to NO2, which oxidizes oxyhemoglobin. Recently NO was shown to be one of endothelium-derived relaxing factors and the relaxation induced by the factor was inhibited by hemoglobin and potentiated by superoxide dismutase.

In vivo studies on methemoglobin formation by sodium nitrite

SummaryThe oral LD5o of NaNO2 for rats was found to be 0.15 g per kg body weight. The methemoglobin level increased to 45–80%, 1 h after the administration of the LD5o dose and returned to the normal level after 24 h. The dose-maximum methemoglobin concentration curve was found to be Sshaped. Formation of nitrosyl hemoglobin preceded that of methemoglobin, its maximum concentration being a quarter of that of the latter derivative. In rats receiving 0.5% NaNO2 as drinking water, the concentration of methemoglobin showed a characteristic daily change (4 to 88%) due to the circadian rhythm of the animal in drinking. After 6 months, slight nitrosyl hemoglobin production, Heinz body formation, anisocytosis, and hypohemoglobinemia were observed.

pH dependence of the shape of the hemoglobin-oxygen equilibrium curve

Abstract The shape of the hemoglobin-oxygen equilibrium curve evidently depends upon pH; the maximum slope of the Hill plot significantly decreases with increasing pH. Moreover, the magnitude of the shift of the equilibrium curve due to pH change depends upon the oxygen saturation, markedly decreasing when the latter is above 80%.

Thermodynamical analysis of oxygen equilibrium of stripped hemoglobin

Precise oxygen equilibrium curves of hemoglobin stripped of phosphates were determined at pH 7.4 and five different temperatures. The data were thermodynamically analyzed according to Adairs stepwise oxygenation theory and the allosteric model of Monod et al. Heat of oxygenation of Hb(O2)3 was significantly larger than that of Hb, indicating that the shape of oxygen equilibrium curve is not invariant with the change of temperature. The results do not support the idea that the cooperative effects are essentially entropic in nature, suggesting that the allosteric transition from the unliganded T-state to the unliganded R-state is an endothermic process, during which the hemoglobin molecule gains entropy.

Effect of inositol hexaphosphate and other organic phosphates on the cooperativity in oxygen binding of human hemoglobins

Summary On the contrary to 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP), inositol hexaphosphate (IHP) reduces the affinity of human adult hemoglobin (Hb A) to the 4th oxygen molecule as well as the affinity to the 1st, 2nd, and 3rd molecules. Thus, the over-all free energy of interaction (ΔF I ) increases only by about 600 cal per site and the maximum slope of the Hill plot, n, decreases significantly in the presence of 1.7 mM IHP. Therefore, the mechanism of action of IHP in lowering the oxygen affinity of hemoglobin seems to be quite different from that of DPG or ATP. The effect of DPG on the oxygen equilibrium function of human fetal hemoglobin (Hb F) is similar to but less than that on Hb A.

Production of superoxide anion by N,N-bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane buffer during oxidation of oxyhemoglobin by nitrite and effect of inositol hexaphosphate on the oxidation

Oxidation of oxyhemoglobin by nitrite is characterized by the presence of a lag phase followed by an autocatalysis. As reported previously (Kosaka, H., Imaizumi, K. and Tyuma, I. (1982) Biochim. Biophys. Acta 702, 237-241), in phosphate buffer nitrite produced an ESR signal at g 2.005 (hereafter referred to as the g 2 radical). The g 2 radical produced NO.2 from NO-2, then NO.2 oxidized oxyhemoglobin. Superoxide dismutase did not modify the oxidation. On the other hand in N,N-bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bistris) buffer, superoxide dismutase markedly elongated the lag phase and accelerated the autocatalysis, indicating O-2 production. Bistris scavenged the g 2 radical. O-2 was generated by the reduction of O2 by a radical derived from bistris. Inositol hexaphosphate inhibited the oxidation by decreasing H2O2 production from oxyhemoglobin.

Spectroscopic properties of the hemes of human adult hemoglobin and its subunits

Abstract The circular dichroism (CD) and difference spectra of human adult hemoglobin (Hb A) and its subunits in the deoxy-, oxy- and CO-forms are presented. All four absorption bands of the hemes were optically active in the hemoglobins. The positions of the CD bands observed roughly agreed with those of the corresponding absorption bands. Generally, the α-subunit had more positive ellipticity than the β-subunit, and the CD spectrum of Hb A could be roughly composed by taking the arithmetic mean of those of the subunits. Similarly, the difference spectra of the subunits, between oxy- or CO- and deoxy-forms, differed from each other. These facts suggest that the local milieu around the hemes was not the same in the subunits. Human fetal hemoglobin showed the same CD spectrum as Hb A. In the visible region, two additional bands around 520 and 580 mμ were observed in the CD spectra, but these bands were indiscernible in the absorption spectra. A positive CD band around 580 mμ of the deoxy-form appeared in the α-subunit and Hb A but not in the β-subunit. On the basis of the difference spectra, the difference in the absorption of the deoxy-hemes in the separated subunits and in the tetrameric Hb A was confirmed. In the ultraviolet region, the CD peak positions of a band around 310 mμ differed markedly from each other in the subunits, while those of a band around 260 mμ did not. The CD band around 260 mμ of the deoxy-forms was probably degenerated. A negative CD band around 280 mμ was assigned to the aromatic amino acid residues of the protein moiety, and the band was indiscernible in the α-subunit.