Rapid separation of dehydrogenases by affinity chromatography with new induced specificity phases.

ABSTRACT

We describe a procedure using immobilized nicotinamide as an affinity chromatographic ligand for the binding of NAD(P)+-dependent dehydrogenases. The procedure involves preparation of nicotinamide N1-(N-(6-aminohexyl)-acetamide)-agarose and modification of the immobilized nicotinamide by the addition of a ketone or an aldehyde to form an adduct. The nicotinamide, which has no affinity for dehydrogenase, becomes a very specific ligand of dehydrogenase, which binds the ketone or the aldehyde as substrate or inhibitor. In tests, the adduct prepared with immobilized nicotinamide and sodium pyruvate bound specifically to lactate dehydrogenase (EC 1.1.1.27), whereas the adduct prepared with alpha-ketoglutarate bound to glutamate dehydrogenase (EC 1.4.1.3). This technique enables the rapid isolation of a given dehydrogenase.