A New Immobilized NAD+ Analogue, Its Application in Affinity Chromatography and as a Functioning Coenzyme

Abstract

Alkylation of NAD+ with iodoacetic acid followed by alkaline rearrangement gave N6‐carboxymethyl‐NAD+. Condensation of this analogue with 1,6‐diaminohexane gave the analogue NAD+‐ N6‐[N‐(6‐aminohexyl)‐acetamide]. The coenzymic activity of the two derivatives was tested with malate dehydrogenase, alcohol dehydrogenase and lactate dehydrogenase. The efficiency relative to unsubstituted NAD+ was in the range of 50–100%. NAD+‐N6‐[N‐(6‐aminohexyl)‐acetamide] was attached to Sepharose 4B by the cyanogen bromide method. The immobilized NAD+ analogue thus obtained, exhibited cofactor activity when tested in a recycling three‐enzyme system (malate dehydrogenease‐citrate synthase‐lactate dehydrogenase). The immobilized NAD+ analogue proved to be and effective ligand in affinity chromatography. Thus a mixture of albumin, alcohol dehydrogenase and lactate dehydrogenase was resolved with good recovery. The enzymes were eluted with NAD+ and NADH, respectively. Copyright © 1973, Wiley Blackwell. All rights reserved