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

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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.