Effect of accessible immobilized NAD(+) concentration on the bioaffinity chromatographic behavior of NAD(+)- dependent dehydrogenases using the kinetic locking-on strategy

preparation for studies aimed at establishing the relationship between immo bilized NAD(+) concentration and the concentration of soluble locking-on li gand required to promote biospecific adsorption of NAD(+)-dependent dehydro genases to immobilized NAD(+) derivatives (the "locking-on" strategy), two approaches were evaluated for varying substitution levels: (i) suitable dil ution of the affinity matrix with unsubstituted Sepharose 4B and (ii) direc t coupling of the required ligand concentration to the inert matrix. The la tter approach was found to be the preferable strategy for evaluation of the locking-on tactic because it produced a more homogeneous distribution of i mmobilized NAD(+) concentration. Affinity chromatographic studies using S-6 -linked NAD(+) derivatives synthesized to various substitution levels showe d that the total accessible immobilized NAD(+) concentration has a direct e ffect on the locking-on behavior of pyridine nucleotide-dependent dehydroge nases, The one-chromatographic-step bioaffinity purification of L-lactate d ehydrogenase (L-LDH, EC 1.1.1.27) from bovine heart illustrates the potenti al of the locking-on strategy for protein purification applications. (C) 19 99 Academic Press.