A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases

Abstract

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.