COFACTOR-SUBSTRATE ELUTION OF ENZYMES BOUND TO THE IMMOBILIZED NUCLEOTIDES ADENOSINE 5'-MONOPHOSPHATE AND NICOTINAMIDE-ADENINE DINUCLEOTIDE By K. MOSBACH, H. GUILFORD, R. OHLSSON and M. SCOTT

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD+-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD+-Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NADI was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP-Sepharose, N6-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD+ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity ofthe reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMPSepharose, is strong enough to resist elution by gradients of KCI of up to at least 0.5M. A 0.0-0.15M gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP-Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5nM-NAD+); 80 (lactate+NAD+); 95 (lactate+semicarbazide+NAD+); 100 (0.5mM-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a singlestep procedure by using AMP-Sepharose.