Direct enzyme titration curve of NADH: cytochrome b5 reductase by combined isoelectric focusing/electrophoresis. Interactions between enzyme and cytochrome b5.

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 catalysed 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 hemoglobin [Gacon et al. (1980) Proc. Natl Acad. Sci. USA, 77, 1917-1921]. Here we present data indicating a similar mode of interactions occurring in step 1 between cytochrome b5 reductase and cytochrome b5. These results have been obtained by using the combined isoelectric focusing/electrophoresis method [Righetti et al. (1978) J. Chromatogr. 166, 455-460] allowing a direct titration of both entities either separately or in a mixture. This is the first report on the obtention of a direct titration curve of an enzyme visualized after specific staining (zymogram). The pH dependence of the Michaelis constant 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.