Coenzyme Properties of NAD+ Bound to Different Matrices through the Amino Group in the 6‐Position

Abstract

A method for the synthesis of N⁶-(2-aminoethyl)-NAD⁺ is given. The binding of this NAD⁺ derivative to different soluble and insoluble supports and the direct coupling of NAD⁺ to epoxyactivated Sepharose are described. Proofs are given that NAD⁺ is bound through the amino group in 6-position and the NAD⁺ derivative through the aliphatic amino group of the side chain. Non-enzymic reduction of the bound coenzyme to an almost quantitative extent is possible in all cases, but the enzymic reduction is largely influenced by the support. While N⁶-(2-aminoethyl)NAD⁺ coupled to soluble dextran is nearly completely reducible by different dehydrogenases with a velocity of about 40% of that for free NAD⁺, the coenzyme bound to different insoluble matrices is very slowly reduced. Only 5% of the coenzyme derivative bound to BrCN-activated Sepharose are reducible, but 40% when it is bound through a spacer. From capacity determinations evidence is given that, even in this coenzyme gel, only those coenzyme molecules are useful in affinity chromatography which are on the surface of the gel grains; it is supposed that this may be due to the slow diffusion of an enzyme into the inner parts of an affinity gel.