[77] Lactate dehydrogenase: Specific ligand approach

Publisher Summary In the search for an effective immobilized ligand, attention was concentrated on analogs of pyruvate rather than of lactate, because kinetic studies indicate that pyruvate has considerably higher affinity for the enzyme. As explained in this chapter, the kinetic mechanism of the enzyme requires the addition of NADH to the irrigating buffer during affinity chromatography. Under these conditions catalytically susceptible immobilized analogs (e.g., β-mercaptopyruvate, attached through the β-thiol group) underwent enzymatic reduction when the enzyme was applied, and such analogs proved to be unsatisfactory for affinity chromatography. Oxamate is a structural analog of pyruvate that is not catalytically susceptible, but retains the binding characteristics of pyruvate for lactate dehydrogenase. This analog, immobilized through the amide grouping proved to be a very effective affinity chromatographic ligand. The amide nitrogen group corresponds to the methyl group of pyruvate and was chosen as the point of attachment because available specificity studies indicated that pyruvate analogs in which the methyl group is substituted remain effective as substrates for lactate dehydrogenase.