Akio Tomoda

Mechanism of o-aminophenol metabolism in human erythrocytes

o‐Aminophenol was found to be rapidly metabolized to a brown compound in the presence of purified human oxy‐ and methemoglobin, coupled with the oxidation and reduction of these hemoglobins by o‐amino‐phenol. The final product of o‐aminophenol was identified as 2‐aminophenoxazine‐3‐one, by using spectrophotometry and HPLC. The metabolism of o‐aminophenol was also observed in human erythrocytes. The production rates of 2‐aminophenoxazine‐3‐one in the cells were very fast, but these were strongly decreased by bubbling carbon monoxide into the cell suspension when intracellular hemoglobin was in the ferrous state. The production of 2‐aminophenoxazine‐3‐one from o‐aminophenol in the cells was completely suppressed by cyanide and azide when intracellular hemoglobin was in the ferric state. These results suggest that oxy‐ and methemoglobin are involved in metabolism of o‐aminophenol to 2‐aminophenoxazine‐3‐one in human erythrocytes.

Isolation of human erythrocyte membranes in glucose solution

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.

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.

Effect of inositol hexaphosphate on hemoglobin oxidation by nitrite and ferricyanide

Abstract In the presence of inositol hexaphosphate (IHP), the rate of hemoglobin oxidation by nitrite was much inhibited; however, that of the hemoglobin oxidation by ferricyanide was much accelerated. The difference in the reaction mode was discussed in relation to the interaction of hemoglobin with IHP. The dissociation constant of IHP to oxyhemoglobin was estimated from the rate of the hemoglobin oxidation by ferricyanide in different concentrations of IHP under oxygen saturated conditions.

On the relationship of hemoglobin oxidation with the conformation of hemoglobin

The rate of hemoglobin oxidation by various oxidants was studied under aerobic and anaerobic conditions, and the mechanism of hemoglobin oxidation was discussed in relation to the conformation of hemoglobin.

Spectroscopic studies of brunescent cataractous lenses

The absorption spectra of brunescent cataractous lenses and their homogenates were analyzed under various conditions by using a double wavelength spectrophotometer. The absorption spectra of the samples were in good agreement with those of synthetic xanthommatin derived from 3‐hydroxykynurenine. The results provided evidence that brown pigment in the brunescent cataractous lenses is mainly composed of xanthommatin.

A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2

Abstract The improved methods for the preparation of valency hybrid hemoglobins, ( α 3+ β 2+ ) 2 and ( α 2+ β 3+ ) 2 were presented. The ( α 3+ β 2+ ) 2 valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The ( α 2+ β 3+ ) 2 valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of ( α 3+ β 2+ ) 2 and ( α 2+ β 3+ ) 2 valency hybrids.

Analysis of met-form hemoglobin in glucose-deleted human red cells

Though many investigations have been reported on the mechanism of methemoglobin formation in human red cells, complete explanation is not available. Methemoglobin accumulates in human red cells as glucose was consumed during a long period incubation of these cells at 37°C [ 1 ]. These glucose-deleted red ceils seem to be suitable to investigate the mechanism of hemoglobin oxidation in intact cells because enzymatic methemoglobin reducing systems including NADH and NADPH diaphorases are suppressed due to the deficiency of reproduction of NADH and NADPH. In this paper, we investigated the methemoglobin formation in intact human red cells, which were incubated without glucose for a long period at various pH values. Especially, we analyzed the nature of methemoglobin derivatives detected in these cells. As a result, fully oxidized hemoglobin (methemoglobin) as scarcely found in these ceils, however, met-form hemoglobin was detected as half-oxidized hemoglobin (as+/32+). On the basis of these results, the mechanism of hemoglobin oxidation and physiological significance of the oxidation process were discussed.

Reactions of Oxy- and Methemoglobin with Tryptophan Metabolites, 3-Hydroxyanthranilic Acid and 3-Hydroxykynurenine

It was found that 3-hydroxyanthranilic acid and 3-hydroxykynurenine have the capacity of oxidizing and reducing oxy- and met-hemoglobin. However, tryptophan metabolites such as kynurenine and anthranilic acid in which hydrxyl group is not involved did not oxidize and reduce these hemoglobins. The oxidation of oxyhemoglobin with 3-hydroxyanthranilic acid and 3-hydroxykynurenine was much accelerated in the presence of myo-inositol hexakisphosphate or superoxide dismutase, but was much suppressed in the presence of catalase. Deoxyhemoglobin was not oxidized by these compounds. On the other hand, the reduction of methemoglobin with 3-hydroxyanthranilic acid and 3-hydroxykynurenine proceeded both under aerobic and anaerobic conditions, though the rate of reduction was much faster under aerobic conditions than under anaerobic conditions. The reduction of methemoglobin with these compounds was accelerated by myo-inositol hexakisphosphate, but was partially suppressed by superoxide dismutase under aerobic conditions. On the basis of these results, the paradoxical effects of 3-hydroxyanthranilic acid and 3-hydroxykynurenine are discussed in relation to the mechanism of oxidation and reduction of hemoglobin.

Analysis of intermediate hemoglobins in solutions of hemoglobin partially oxidized with ferricyanide

Abstract The intermediate hemoglobins which were produced by the partial oxidation of hemoglobin with ferricyanide were clearly separated by preparative isoelectric focusing. Two intermediate hemoglobins (IB I and IB II ) were identified as α 2+ β 3+ and α 3+ β 2+ valency hybrids by the studies of absorption spectra and inositol hexaphosphate-induced difference spectra. The distribution of oxyhemoglobin, intermediate hemoglobin and methemoglobin in the solutions of hemoglobin (0.05 M bis-Tris with 0.1 M NaCl) which were partially oxidized by ferricyanide was analyzed in the presence or absence of inositol hexaphosphate at pH 6.6–7.8. The proportion of the α 2+ β 3+ valency hybrid was always higher than that of α 3+ β 2+ . Inositol hexaphosphate increased this difference, i.e. the level of α 2+ β 3+ rose and that of α 3+ β 2+ fell in the presence of the organic phosphate. At pH regions studied, the proportion of α 2+ β 3+ and α 3+ β 2+ was not significantly altered by a change in pH whether inositol hexaphosphate was present or not. Furthermore, the effects of ionic strength on the proportion of the hemoglobin derivatives were studied in phosphate buffer at pH 7.0. At low ionic strength (10 mM phosphate buffer), the proportion of α 3+ β 2+ was higher than that of α 2+ β 3+ . At higher ionic strength (10 mM phosphate buffer with 0.1 M NaCl or 0.1 M phosphate buffer), the proportion of these two intermediate hemoglobins was nearly equal or the proportion of α 2+ β 3+ was higher than that of α 3+ β 2+ . The heterogeneity of the proportion of the intermediate hemoglobins was explained by the functional differences of α and β chains in tetrameric hemoglobin in the reaction with ferricyanide, which were influenced by the presence of high concentration of inorganic phosphate, chloride and low concentration of inositol hexaphosphate. The effects of inositol hexaphosphate on the oxidation of hemoglobin by ferricyanide was considered to be due to the changes in hemoglobin conformation notably involving the β chains.