Stopped flow studies on the nonenzymatic reduction of methemoglobin by reduced flavin mononucleotide.

T Yubisui,S Matsukawa,Y Yoneyama

doi:10.1016/s0021-9258(19)70188-4

Abstract

The nonenzymatic reduction of methemoglobin by the reduced form of flavin mononucleotide was studied under various conditions by following the reaction with a stopped flow apparatus. The reaction was very fast, compared with the reduction of the flavin by NADPH-flavin reductase of human erythrocytes, and followed a second order rate law: the rate constant (K) for the reduction of methemoglobin by reduced flavin mononucleotide was determined to be 5.5 X 10(6) M-1 S-1 in 50 mM phosphate buffer (pH 7.0) at 25 degrees C. The reaction was not influenced by changing phosphate buffer concentration from 10 to 100 mM. The rate of reduction at the physiological pH, 7.0, was about 95% of the maximal value that observed at around pH 6.4. Formation of deoxyhemoglobin and oxidized form of flavin mononucleotide by the reaction proceeded stoichiometrically in a ratio of unity. These results apparently indicate that the limiting step for the reduction of methemoglobin by the NADPH-flavin reductase system in human erythrocytes is the enzymatic reduction of flavin.

Highlights

Stopped Flow Measurements-Stopped flow measurements were nucleotideby the reaction proceeded stoichiometricallyperformed with a Union stopped flow apparatus
These results apparently indicate Japan), model RA 401 fitted with a computer system 71
Maximal activity for flavin reduction by the purified only about 50% of FAD added to the reaction mixture was NADPH-flavin reductase of human erythrocyteswas 1pmol/ reduced after 30 min, and the flavin was still continuously min/mg of enzyme protein (2).Present results revealed that the nonenzymatic reduction of methemoglobin by the reduced form of flavin is a very fast reaction with a second order rate constant of 5.5 X lo6 M" s" (3.3 X lo8 M" min") at 25°C in

Summary

Introduction

The maximal reaction mixture in one side of the reservoirs in the apparatus contained 50 m~ phosphate buffer (pH 7.0), 40 mg of glucose, 40 pg of glucose oxidase(4.2units), 20pg of catalase (1300units), 5 mM EDTA, and 40to 90p~ methemoglobin (on heme basis). Oxidation ofFMNH2-0xidation of FMNH2 by the reaction with under various conditions.From the second order rateconstant methemoglobin was determined by following the absorbance change for the nonenzymaticreduction of methemoglobin by FMNHz at 445 nm, an isosbestic point for deoxyhemoglobinand methemogloobtained in this study,and the maximal enzymatic activity of the reduction of FMN by the NADPH-flavin reductase (2), the enzymatic reduction of flavin is suggested to be the ratelimiting step for the reduction of methemoglobin by the bin.

Results

Conclusion