A series of chemically-defined adenosine phosphate ligands attached to Sepharose 4B were used as active-site probes in studying the interaction of enzymes with their coenzymes and substrates and to test the suitability of these matrices for ‘general ligand’ affinity chromatography. Nicotinamide nucleotide-dependent dehydrogenases were used as models to test this methodology. Elution from these columns by NAD+and/or AMP gradients (in the presence or the absence of substrates and/or nicotinamide mononucleotide) was consistent with: (1) the compulsory ordered addition of substrates to lactate and malate dehydrogenase; (2) the necessity for the NMN moiety of NAD+to bind to these enzymes before the substrate; and illustrated: (3) that the binding of these two hydrogenases to these columns compared very well with the published three-dimensional models for these enzymes and (4) that separation of mixtures of dehydrogenases depended on the choice of matrix and displacing ion and whether any additions (e.g. substrates) were made to the gradients used. These techniques were used to purify UDP-glucose dehydrogenase from a crude starting material on a phosphate-linked UDP (or ADP) matrix. The binding of this enzyme to these two columns was not consistent with either an ordered or random addition of substrates and suggested a more complex mechanism.
Affinity chromatography of nicotinamide nucleotide-dependent dehydrogenases on immobilized nucleotide derivatives