Coenzyme properties of NAD+ bound to different matrices through the amino group in the 6-position.
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.
D001665:Binding Sites; D002846:Chromatography, Affinity; D004355:Drug Stability; D005021:Ethylamines; D009243:NAD / Q000031:analogs & derivatives; D010088:Oxidoreductases / Q000378:metabolism; D012685:Sepharose; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D005021:Ethylamines; D009243:NAD; D012685:Sepharose; D010088:Oxidoreductases