Affinity Chromatography of Sn-Glycerol-3-Phosphate Dehydrogenase on Immobilized NAD

Three types of potential affinity chromatography columns have been examined for the purification of sn-glycerol-3-phosphate dehydrogenase (EC 1.1.l.R) from rabbit tissues. Each column contained nicotinamide adenine dinucleotide (NAD) covalently attached to an agarose matrix with a different mode of attachment for each column. The most effective column was one in which the NAD was linked to the agarose via the C-8 position of the adenine moiety. Please of the bound enzyme from this column was accomplished by elution with NADH or MAD. The enzymes from brain, heart, kidney, muscle and liver were purified using this procedure with nearly quantitative yields and up to a 90-fold purification. The binding capacity and elution profiles were dependent upon pH, ionic strength and temperature. The capacity was lowest at pH 7 and increased at higher and lower values. Increasing ionic strength and higher temperatures decreased the binding capacities.