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Characterization of intermediate hemoglobin produced during methemoglobin reduction by ascorbic acid

A Tomoda, M Takeshita, Y Yoneyama

The Journal of Biological Chemistry

|October 25, 1978

Summary

Ascorbic acid partially reduces methemoglobin, forming an intermediate alpha3+beta2+ valency hybrid. This suggests beta-methemoglobin chains initiate the reduction process, with minimal involvement of the alpha2+beta3+ hybrid.

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Area of Science:

Biochemistry
Hematology

Background:

Methemoglobin is a form of hemoglobin with oxidized iron, impairing oxygen transport.
Ascorbic acid (vitamin C) is known to reduce methemoglobin, but the mechanism is not fully understood.

Purpose of the Study:

To elucidate the intermediate species formed during methemoglobin reduction by ascorbic acid.
To identify the specific hemoglobin chains involved in the initial reduction steps.

Main Methods:

Isoelectric focusing on Ampholine plate gel to analyze hemoglobin composition.
CM Sephadex C-50 column chromatography for isolating intermediate hemoglobin.
Spectroscopic analysis (absorption and difference spectra) and comparison with reconstituted valency hybrids.

Main Results:

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Methemoglobin reduction by ascorbic acid halts at an intermediate stage, yielding oxyhemoglobin, intermediate hemoglobin, and remaining methemoglobin.
The intermediate hemoglobin was identified as an alpha3+beta2+ valency hybrid, with negligible amounts of the alpha2+beta3+ hybrid.
The reduction pathway appears to be initiated by beta-methemoglobin chains.

Conclusions:

Ascorbic acid-mediated methemoglobin reduction primarily involves beta-methemoglobin chains.
The proposed reduction pathway is Methemoglobin → alpha3+beta2+ valency hybrid → Oxyhemoglobin.
The contribution of the alpha2+beta3+ valency hybrid to this reduction process is minimal.