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 N 6 ‐carboxymethyl‐NAD + . Condensation of this analogue with 1,6‐diaminohexane gave the analogue NAD + ‐ N 6 ‐[ 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 + ‐ N 6 ‐[ 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.