Dye—ligand affinity partitioning of lactate dehydrogenase isoenzymes

Abstract

Aqueous two-phase systems consisting of dextran and polyethylene glycol (PEG) were used to study the partition behaviour of isoenzymes of lactate dehydrogenase (LDH; E.C. 1.1.1.27) from rabbit tissues in the presence and absence of a series of triazine dyes covalently coupled to PEG. The variations in the primary structures of LDH1(H 4) and LDH5(M 4) are reflected by significantly different partition coefficients. A class of dyes exhibiting defined structural elements is able to distinguish between both of these isoenzymes. This may be based on differences in the binding affinity to the catalytic site of the enzyme. The difference in the relative affinities of LDH1 and LDH5 to Procion Blue H-5R, as estimated by affinity partitioning, were corroborated by chromatographic experiments. Affinity partitioning in aqueous two-phase systems can be used to predict and to optimize conditions for the fast and simple chromatographic separation of isoenzymes.