The separation of partially modified lactate dehydrogenase by affinity chromatography. The specific activity of protomers?

Abstract

Pig heart lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) was partially inactivated by reaction with phenylmaleimide. The resulting protein mixture was separated by affinity chromatography on a Sepharose-linked oxamate column yielding three distinct enzyme fractions with one, two and three out of four subunits being modified. The specific activities of these samples were lower than expected from the degree of modification, while the maximum binding capacity of NADH-oxamate related well to the number of catalytic centers modified. A possible cooperative effect in the native enzyme is discussed.