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Biomedical subjects

A Tomoda

Publications and source records attributed to A Tomoda.

At least 91 records · Page 5Linked to original sources
PubMed1981

Kinetic analysis of myoglobin autoxidation by isoelectric-focusing electrophoresis.

The autoxidation of horse myoglobin was studied in the presence or absence of catalase (EC 1.11.1.6) and/or superoxide dismutase (EC 1.15.1.1) at various pH values (6.6-7.8). Changes in the percentages of oxymyoglobin and metmyoglobin during the reaction were analysed by means of isoelectric focusing on Ampholine gel plates. Oxymyoglobin was decreased in a first-order manner, with an accompanying increase in metmyoglobin, under the various conditions studied. The observed reaction rate constants obtained under these conditions were pH-dependent; however, they were also greatly affected by the presence of the enzymes. The pH-dependence of the overall reaction was explained by the acid-base three-state model of myoglobin proposed by Shikama & Sugawara [(1978) Eur. J. Biochem. 91, 407-413]. The reaction process of myoglobin autoxidation was explained by the model suggested by Winterbourn, McGrath & Carrell [(1976) Biochem. J. 155, 493-502], indicating that superoxide anion and hydrogen peroxide are involved in the reaction mechanism.

Animals↗
PubMed1981

Mechanism of hemoglobin oxidation by ferricytochrome c and nitrite.

The mode of hemoglobin oxidation by ferricytochrome c and nitrite was investigated by using isoelectric focus electrophoresis on Ampholine plate gel. It was found that two intermediate hemoglobins, (alpha 2 + beta 3 +)2 and (alpha 3 + beta 2 +)2 valency hybrids, were produced during the reactions, though the modes of these reactions were quite different from each other. The changes in oxyhemoglobin, (alpha 2 + beta 3 +)2, (alpha 3 + beta 2 +)2 and methemoglobin during the oxidation of hemoglobin by ferricytochrome c well fit first order kinetics. On the other hand, the changes in these hemoglobin derivatives during nitrite oxidation of hemoglobin did not fit first order kinetics, but instead were appropriate for the chain reaction models proposed by us. Since we found that superoxide anion may be involved in the reaction of hemoglobin with nitrite, a plausible reaction process was proposed. The differences in the reaction mechanism of hemoglobin oxidation by ferricytochrome c and by nitrite are discussed.

Cytochrome b Group↗
PubMed1980

Reduction of ferricytochrome c by human red cells.

Human red cells reduced extracellular ferricytochrome c to ferrocytochrome c under various conditions, suggesting that ferricytochrome c reducing systems are present at the outer surface of the red cell membrane.

Cytochrome c Group↗
PubMed1980

Mechanism of methaemoglobin reduction by human erythrocytes.

The time course of methaemoglobin reduction in human erythrocytes treated with nitrite was studied at pH 7.4, 37 degrees C, in the presence or absence of Methylene Blue, and the changes in methaemoglobin, intermediate haemoglobins and oxyhaemoglobin during the reaction were analysed by isoelectric-focusing on Ampholine/polyacrylamide-gel plates. In both cases, with or without the dye, the intermediate haemoglobins were found to be present at (alpha 3+beta 2+)2 and (alpha 2+beta 3+)2 valency hybrids from their characteristic position on electrophoresis, but amounts changed consecutively with time. The amount of (alpha 3+beta 2+)2 was always greater than that of the (alpha 2+beta 3+)2 valency hybrid. This result is explained by the differences in redox potentials between alpha- and beta-chains in methaemoglobin tetramer. It was concluded that methaemoglobin was reduced in human erythrocytes through these two different pats: methaemoglobin leads to k+3 (alpha 2+beta 3+)2 leads to k+3 oxyhaemoglobin. The reaction rate constants k'"1 (= k+1+k+3) and k'+2(=k+2+k+4) were estimated from the changes in each component methaemoglobin, intermediate haemoglobins [(alpha 3+beta 2+)2+(alpha 2+beta 3+)2] and oxyhaemoglobin.

Cytochrome-B(5) Reductase↗
PubMed1980

Met-form hemoglobins in long-term stored ACD blood.

On passing ACD blood stored for a year at 4 degrees C through a column of Sephadex G-75, the surviving erythrocytes were separated from hemolyzed cells and plasma. About 18% of stored erythrocytes survived a year of storage, and were spherocytic. The concentration of total ferric hemoglobins in the surviving erythrocytes was about 75% of that in the supernatant. The met-form hemoglobins in the erythrocytes and hemolytic solutions were further analyzed by isoelectric focusing electrophoresis, and (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 valency hybrids were found in addition to methemoglobin. Since the activity of NADH cytochrome b5 reductase was extremely low in the surviving erkythrocytes, the difference in proportion of ferric hemoglobins (valency hjybrids + methemoglobin) in these two fractions might be due simply to the difference in pH between intracellular and extracellular fluids, which is largely reflected in the oxidation rate of hemoglobin. The course of hemoglobin oxidation seems to be through hemoglobins such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 to methemoglobin.

Blood Specimen Collection↗
PubMed1979

Analyis of met-form haemoglobins in human erythrocytes of normal adults and of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase.

Isoelectric-focusing analysis on an Ampholine/polyacrylamide-gel plate revealed that met-form haemoglobins are present as half-oxidized haemoglobins such as the (alpha 2+ beta 3+)2 and (alpha 3+ beta2+)2 forms rather than as methaemoglobin in the erythrocytes of normal human adults and also of a patient with hereditary methaemoglobinaemia due to deficiency of NADH-cytochrome b5 reductase.

Cytochrome Reductases↗
PubMed1979

Kinetic studies on methemoglobin reduction by human red cell NADH cytochrome b5 reductase.

The intermediate hemoglobins which were produced by the partial reduction of methemoglobin with human red cell NADH cytochrome b5 reductase were fractionated by the preparative isoelectric focusing. These were found to be composed of alpha3+beta2+ and alpha2+beta3+ valency hybrids by the studies of absorption spectra and inositol hexaphosphate-induced difference spectra. Furthermore, the changes in these intermediate hemoglobins during reduction of methemoglobin by the enzyme were studied in the presence or absence of inositol hexaphosphate using the isoelectric focusing fractionation on Ampholine plate gel...

Cytochrome Reductases↗
PubMed1979

Changes in intermediate haemoglobins during methaemoglobin reduction by NADPH-flavin reductase.

The changes in intermediate haemoglobins produced during methaemoglobin reduction by NADPH-flavin reductase were analysed by an isoelectric-focusing method. The alpha 3+ beta 2+ and alpha 2+ beta 3+ valency hybrids were observed as intermediate haemoglobins and changed consecutively with time during the reaction. On the basis of the analyses, the course of methaemoglobin reduction was found to involve two different pathways: (1) methaemoglobin kappa+1 leads to alpha 3+ beta 2+ kappa+2 leads to oxyhaemoglobin; (2) methaemoglobin kappa+3 leads to alpha 2+ beta 3+ kappa+4 leads to oxyhaemoglobin. The reaction rate constants of each phase (kappa+1--kappa+4) were also estimated. The addition of inositol hexaphosphate to the reaction mixture did not affect the overall reaction. The mechanism of methaemoglobin reduction by NADPH-flavin reductase is discussed on the basis of these results.

Cytochrome-B(5) Reductase↗
PubMed1978

Mechanism of methemoglobin reduction by ascorbic acid under anaerobic conditions.

The time course of methemoglobin reduction by ascorbic acid under anaerobic conditions was analyzed by using isoelectric focusing on Ampholine plate gel in order to compare results obtained by studies of the changes in absorption during the reaction. The intermediate hemoglobin which appeared all through the reaction was single and identified as the alpha3+beta2+ valency hybrid. In the presence of inositol hexaphosphate, reduction of methemoglobin was considerably accelerated and this acceleration was restricted to the step in which methemoglobin is reduced to the alpha3+beta2+ valency hybrid. The phase containing alpha3+beta2+ valency hybrid reduction to deoxyhemoglobin was not affected by the presence of this organic phosphate. The reaction rate constant of each phase was estimated by the analysis of the changes in three components such as methemoglobin, alpha3+beta2+ valency hybrid, and deoxyhemoglobin. The specific attack of beta-methemoglobin chains in methemoglobin tetramer by ascorbic acid was explained by the functional differences in alpha- and beta-methemoglobin chains in the protein.

Anaerobiosis↗
PubMed1978

Characterization of intermediate hemoglobin produced during methemoglobin reduction by ascorbic acid.

Methemoglobin reduction by ascorbic acid was found apparently to cease halfway without further reduction. Studies by isoelectric focusing on Ampholine plate gel revealed that the solutions of the halfway reduced methemoglobin are composed of about 6% oxyhemoglobin, 59% intermediate hemoglobin, and 35% methemoglobin. The intermediate hemoglobin was isolated by CM Sephadex C-50 column chromatography and identified as alpha3+beta2+ valency hybrid by studies using the pattern of isoelectric focusing of p-chloromercuribenzoate-treated intermediate hemoglobin on Ampholine plate gel, absorption spectra, and difference spectra induced by the addition of inositol hexaphosphate in comparison with the reconstituted valency hybrids, alpha3+beta2+ and alpha2+beta3+. Essentially no alpha2+beta3+ valency hybrid was included in the intermediate hemoglobin solutions. These results suggest that methemoglobin reduction by ascorbic acid is mainly initiated by the attack of beta-methemoglobin chains accompanied by the following scheme. Methemoglobin leads to alpha3+beta2+ valency hybrid leads to oxyhemoglobin. The course of methemoglobin reduction by ascorbic acid through alpha2+beta3+ is likely to be small.

Ascorbic Acid↗
PubMed1978

Haemichrome formation from haemoglobin subunits by hydrogen peroxide.

The effect of H2O2 on ferrous human haemoglobin subunits (alphash-, betash-, alphapmb- and betapmb-chains) was studied. These chains were easily transformed to haemichrome by the addition of H2O2 or H2O2-generating systems, including glucose oxidase (EC 1.1.3.4) AND XANTHINE OXIDASE (EC 1.2.3.2), and this was ascertained by e.p.r. measurements and by absorption spectra. The changes in these haemoglobin subunits were not inhibited by superoxide dismutase (EC 1.15.1.1), but were decreased by catalase (EC 1.11.1.6). The rate of oxidation of alphapmb-chains was higher than that of alphash-chains, and the rate of oxidation of betapmb-chains was higher than that of betash-chains. Haemichrome was demonstrated to be formed directly from these ferrous chains by the attack by H2O2, and this process did not involve formation of methaemoglobin. On the basis of these findings the kinetics of the reaction between the haemoglobin subunits and H2O2 was studied, and the pathological significance of H2O2 in disorders of erythrocytes such as thalassaemia was discussed.

Electron Spin Resonance Spectroscopy↗
PubMed1978

Acceleration of methaemoglobin reduction by riboflavin in human erythrocytes.

The effect of riboflavin on nitrite treated erythrocytes from normal subjects and patients with hereditary methaemoglobinaemia due to the deficiency of NADH-cytochrome b5 reductase was studied in the presence of glucose, 2-deoxy-D-glucose or lactate. When glucose or 2-deoxy-D-glucose was used as a substrate for these erythrocytes, the rate of methaemoglobin reduction in these cells was accelerated more than two-fold in the presence of riboflavin. The acceleration was dependent on the concentration of riboflavin and was suppressed by the addition of atebrin. The stimulative effect of riboflavin was, however, not observed when lactate was used in place of glucose or 2-deoxy-D-glucose. On the basis of these results, the acceleration of methaemoglobin reduction by riboflavin was considered to be due to the activation of NADPH-flavin reductase (Yubisui et al, 1977) in erythrocytes by the reagent. The availability of riboflavin for patients with methaemoglobinaemia due to the deficiency of NADH-cytochrome b5 reductase and for those with toxic methaemoglobinaemia is discussed in relation to methaemoglobin reducing systems in erythrocytes.

Cytochrome-B(5) Reductase↗