Kazuhiko Imaizumi

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.

Electron spin resonance study on peroxidase- and oxidase-reactions of horse radish peroxidase and methemoglobin

The intermediate free radicals generated from phenols, naphthols and benzoate, in the peroxidase- and oxidase-reactions of horse radish peroxidase and in the peroxidase-reaction of methemoglobin, were studied by electron spin resonance spectroscopy. The difference between the peroxidase- and oxidase-reactions of HRP are demonstrated, i.e., the ferro horse radish peroxidase-O2 system attacks both phenols and benzoate yielding unidentified radicals, which may be hydroxy-cyclohexadienyl radicals, while the horse radish peroxidase-H2O2 system reacts only with phenols and naphthols producing the phenoxy-and naphthoxy-radicals. Phenoxy-radical formation from phenols, in the reactions horse radish peroxidase-H2O2 and methemoglobin-H2O2, occurs independently of the molecular sizes of phenols but dependently on their redox-potentials. On the basis of kinetic studies on methemoglobin-H2O2 system, the existence of a reactive intermediate complex between methemoglobin and H2O2 is proposed, which may be similar to compound-I or -II of horse radish peroxidase and which further degenerates to MetHb radical. The oxidation of phenols and naphthols takes place outside of the hemepocket of methemoglobin.

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.

Generation of phenoxy radicals by methemoglobin-hydrogen peroxide studied by electron paramagnetic resonance

Abstract The free radical intermediates of phenol derivatives, produced by the methemoglobin-hydrogen peroxide system at pH 5 and 7, are detected by electron paramagnetic resonance equipped with a continuous-flow apparatus. All the radicals from phenols are the phenoxy radicals, as identified by analyzing the observed hyperfine structures of the spectra with the aid of SCF-LCAO MO calculations. Comparing with the reaction of Fentons reagent, it is concluded that free OH radical, even if it exists, is not liberated into the solution in the methemoglobin-hydrogen peroxide system.

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.

Linkage between carbon dioxide binding and four-step oxygen binding to hemoglobin☆

Abstract In order to elucidate the molecular mechanism of the effect of carbon dioxide on the four-step oxygenation equilibria of hemoglobin, accurate oxygen equilibrium curves of human adult hemoglobin were determined at different concentrations of CO 2 and in the presence and absence of chloride (Cl − ), 2,3-diphosphoglycerate (P 2 G), and/or inositol hexaphosphate (IHP) and were analyzed according to Adairs stepwise oxygenation scheme to evaluate the four Adair constants, k i ( i = 1 to 4). The effects of CO 2 on oxygen affinity and co-operativity are influenced by H + , Cl − , P 2 G and IHP. The shape of the oxygen equilibrium curve varies with changes of CO 2 concentration; the four Adair constants are affected by CO 2 non-uniformly. Hence, the number of CO 2 molecules released upon oxygenation is not the same in the individual oxygenation steps. In the absence of added Cl − , CO 2 lowers the overall oxygen affinity expressed by median oxygen pressure ( p m ) and increases the co-operativity expressed by Hills coefficient ( n max ) by reducing k 1 , k 2 and k 3 without changing k 4 . significantly. The effect of CO 2 on oxygen affinity becomes smaller with decrease in pH, disappearing below pH 6.5. The alkaline Bohr effect is reduced by CO 2 . The first oxygenation step contributes to the reduction of the Bohr effect more than the fourth step. When log p m is plotted against log [CO 2 ] at several constant Cl − concentrations, the plots converge to a common point that is named “iso-effective point”. When log p p is plotted against log [Cl − ] at several constant CO 2 concentrations, the plots also converge to an iso-effective point. This phenomenon can be explained in terms of linkage relations in oxygen-linked competitive binding of CO 2 and Cl − . It was found to be useful to consider in this analysis that the bicarbonate ion introduced by added CO 2 exerts a heterotropic effect equivalent to that of Cl − . The combined effects of Cl − , CO 2 and IHP were not explained satisfactorily by the present analysis using linkage relations.

Erythrocyte Aggregation Induced by Immunoglobulin G and Related Macromolecules Studied with Rheoscope-Image Analyzer-Computer System

Erythrocyte aggregation is induced in blood flowing at low shear rates, especially in the stasis of blood, by macromolecular bridging between adjacent erythrocytes [1]. The process of erythrocyte aggregation is reversible; aggregates disintegrate at high shear rates. Since the facilitation of erythrocyte aggregation reduces the blood flow, the kinetic analysis of erythrocyte aggregation is very important for understanding the control of blood flow, rheologically. This paper describes a method for measuring the velocity of erythrocyte aggregation and the effect of immunoglobulin G and related macromolecules on the velocity.