Abstract
A soluble erythrocyte cytochrome b5 was purified as the substrate of methemoglobin reductase and an electron carrier to methemoglobin. The isoelectric point of this protein was at pH 4.3, and E0' was -0.010 at pH 7.0.. The Km value of the enzyme for this protein was 1 x 10(-4) M, and the turnover number (k5) was 3.4 x 10(4) min-1, with NADH as an electron donor at pH 7.0. The optimum pH of the enzyme was pH 4.6 for ferricyanide and pH 5.5 for cytochrome b5, with a shoulder of activity at pH 7 to 9 for both substrates. The rate equation which represents the reduction of either methemoglobin or cytochrome c was obtained as a function of methemoglobin or cytochrome c, methemoglobin reductase, and cytochrome b5 by considering the E . S complex for both reductase and cytochrome b5, and the rate constants involved were determined. The rate constants between methemoglobin and reduced cytochrome b5 (k1, M-1 min-1) were 1.6 x 10(4), 3.1 x 10(6), and 4.1 x 10(6) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constants between the reduced enzyme and oxidized cytochrome b5 (k'3, M-1 min-1) were 4.3 x 10(8), 12 x 10(8), and 9.3 x 10(8) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constant between reduced hemoglobin and oxidized cytochrome b5 (k2) was 35 M-1 min-1 at pH 7.0. The theoretical Km for methemoglobin was 2.1 M at an infinite enzyme concentration at pH 7.0
Highlights
X iob4M, and the turnover number (k6)was 3.4 X lo4 humanerythrocytes as thetruesubstrate of the enzyme, which has become an object of the study
Hemoglobin reduction by the use of the homogeneousenzyme and erythrocyte cytochromebs.a theoretical rate equation is applied to solve the complex effectsof the components of a sequentialelectrontransportreaction of: NADH methemoglobin reductase erythrocyte cytochrome br, methemoglobin, and the rate constants involved are detercomplex for both reductase and cytochrobmse, and the mined by a graphical method
The preparationof erythrocyte extractwhich catalyzes the Methemoglobin reductase which is responsible for the re- reduction of methemoglobin in a quantitative and preparative duction of methemoglobin in erythrocyetes has beepnurified way and the concomitant purification of methemoglobin reto ahomogeneous form by Kuma et al [1, 2], who have ductase and cytochrome bs from human erythrocytes have demonstrated that theenzyme is a flavoprotein, having flavin made itpossible to elucidate the mechanismof the reduction adenine dinucleotide as a prosthetic group, and that its mo- of methemoglobin in uitro
Summary
From the Division of Internal Medicine, Saiseikai Karatsu Hospital, Karatsu, Saga847, Japan. A soluble erythrocyte cytochrome b6 was purified as of methemoglobin ina reconstituted system has not been the substrate of methemoglobin reductase and aenlec- studied quantitatively. Under these circumstances, onoef the tron carrierto methemolgobin. The reductase using erythrocyte cytochrome bs and dyes as the rate constants between the reduced enzyme and oxi- substrates, and 4) the presentation of a rate equation which dized cytochrome bs (k;,M” min”) were 4.3 X lo represents the reductionof methemoglobin ina reconstituted x lo, and9.3 X lo at pH 7.0, pH5.2,andpH5.0, system, and the determinatioofnrate constantsin the neutral respectively. The theoretical K,,, for methemoglobin was 2.1 M at an infinite enzyme concentratiaotnpH 7.0
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