Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography

Abstract

Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase.