Direct Enzyme Titration Curve of NADH: Cytochrome b5 Reductase by Combined Isoelectric Focusing/Electrophoresis

Publisher Website
Google Scholar
Abstract
Methemoglobin reduction in human red cells involves successively an electron transport from NADH to a soluble form of cytochrome b5 (step 1) and from cytochrome b5 to methemoglobin (step 2). Step 1 is catalyzed by an enzyme, soluble NADH:cytochrome b5 reductase (EC 1.6.2.2). Step 2 is non-enzymatic and involves complementary electrostatic interactions between acidic residues of cytochrome b5 and basic residues of Hb. Data indicating a similar mode of interactions occurring in step 1 between cytochrome b5 reductase and cytochrome b5 are presented. These results were obtained by using the combined isoelectric focusing/electrophoresis method allowing a direct titration of both entities either separately or in a mixture. This is the 1st report on the obtention of a direct titration curve of an enzyme visualized after specific staining (zymogram). The pH dependence of Km for cytochrome b5 is also in agreement with the hypothesis that electrostatic charges, which are maximal below pH 7.0, are essential in the interaction between cytochrome b5 and its reductase.