Isolation of plant lactate dehydrogenase by affinity chromatography and role of histidine in the molecule of the enzyme

Abstract

Lactate dehydrogenase (EC 1.1.1.27) was isolated from soybean seedlings (Glycine max. L.) by affinity chromatography on an AMP-Sepharose 4B column. The enzyme obtained was inactivated by treatment with diethyl pyrocarbonate; the inactivation rate was proportional to the molar ratio of the enzyme to the reagent. The plot of the inactivation rate versus pH shows that of all the functional groups of the protein the imidazole groups of histidine only were modified by diethyl pyrocarbonate. By this procedure 20 histidine residues were ethoxyformylated in the molecule of soybean lactate dehydrogenase yet 8 only, i.e. two in every subunit were essential for thae activity of the enzyme. A comparison of the effect of diethyl pyrocarbonate on the lactate dehydrogenase apoenzyme with its effect on the binary complexes of the enzyme with coenzymes or on ternary complexes with its both substrates permits the conclusion that histidine is involved not only in the proton transfer during the redox reaction but also in the coenzyme-binding site.