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.

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

Biochemical Journal ◽

10.1042/bj1790227 ◽

1979 ◽

Vol 179 (1) ◽

pp. 227-231 ◽

Cited By ~ 2

Author(s):

A Tomoda ◽

T Yubisui ◽

A Tsuji ◽

Y Yoneyama

Keyword(s):

Isoelectric Focusing ◽

Rate Constants ◽

Reaction Rate ◽

Flavin Reductase ◽

Inositol Hexaphosphate ◽

Reaction Rate Constants ◽

Beta 2

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.

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Changes in intermediate haemoglobins during autoxidation of haemoglobin

Biochemical Journal ◽

10.1042/bj1950485 ◽

1981 ◽

Vol 195 (2) ◽

pp. 485-492 ◽

Cited By ~ 19

Author(s):

A Tomoda ◽

Y Yoneyama ◽

A Tsuji

Keyword(s):

Isoelectric Focusing ◽

Phosphate Buffer ◽

Time Course ◽

Inositol Hexaphosphate ◽

Reaction Rate Constants ◽

The Media ◽

Beta 2 ◽

Quantitative Changes ◽

Ph Range ◽

Experimental Values

The time course of haemoglobin autoxidation was studied under various conditions at 37 degrees C, and the changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin during the reaction were analysed by isoelectric focusing on Ampholine/polyacrylamide-gel plates. Under various conditions (10 mM-phosphate buffer, 10 mM-phosphate buffer with 0.1 M-phosphate buffer, 10 mM-phosphate buffer with 0.1 M-NaCl, and 10 mM-phosphate buffer with 0.5 mM-inositol hexaphosphate; pH range 6.6-7.8 each case), the intermediate haemoglobins were found to be present as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 valency hybrids from their characteristic positions on electrophoresis. Oxyhaemoglobin changed consecutively to (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2, which were further oxidized to methaemoglobin, and the amounts of (alpha 3+beta 2+)2 were greater than those of (alpha 2+ beta 3+)2 during the reaction. The modes of the quantitative changes in oxyhaemoglobin, intermediate haemoglobins, and methaemoglobin were very similar in all the media used except for the inositol hexaphosphate addition. In the presence of inositol hexaphosphate, the autoxidation rates were considerably accelerated, and the modes of the changes in the haemoglobin derivatives were also considerably altered; the effects of this organic phosphate were maximal at acidic pH and minimal at alkaline pH. It was concluded that haemoglobin autoxidation proceeds by first-order kinetics through two paths: and (formula: see text). The reaction rate constants (k+1-k+4) best fitting all experimental values obtained by the isoelectric-focusing analysis were evaluated. By using these values, the mechanism of haemoglobin autoxidation is discussed.

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