Open AccessA New Immobilized NAD + Analogue, Its Application in Affinity Chromatography and as a Functioning Coenzyme
Author(s) -
Lindberg Margareta,
Larsson PerOlof,
Mosbach Klaus
Publication year - 1973
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1973.tb03184.x
Subject(s) - nad+ kinase , cofactor , affinity chromatography , alcohol dehydrogenase , chemistry , lactate dehydrogenase , malate dehydrogenase , dehydrogenase , chromatography , glycerol 3 phosphate dehydrogenase , cyanogen bromide , biochemistry , enzyme , peptide sequence , gene
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.