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 × 10⁴M⁻¹ for the interaction of enzyme with NADH at 5°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 × 10⁵M⁻¹, 3 × 10⁵M⁻¹, 4 × 10⁵M⁻¹, 7 × 10⁵M⁻¹ and 2 × 10⁶M⁻¹ for the interaction of NADH with the M₄, M₃H, M₂H₂, MH₃ and H₄ isoenzymes respectively of lactate dehydrogenase.