Immobilized pyridine nucleotide transhydrogenase from Azotobacter vinelandii: Resolution and some properties

Abstract

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) has been successfully immobilized on Sepharose 4B by means of cyanogen bromide. A method for measuring the enzyme activity by continuously monitoring product formation spectrophotometrically is described. The effects of pH and buffers on the catalytic behavior of the enzyme were examined. The enzyme was deactivated by imidazole buffers at pH values where the enzyme in either potassium phosphate or Tris buffers exhibited close to maximal catalytic activity. The immobilized enzyme is partially inactivated by exposure to 0.1 m potassium phosphate buffer at pH 5.5 in the presence of NADH or NADPH. Phosphate buffer at pH 5.5 alone or in combination with thioNAD +, NAD +, or NADP + was not very effective in inactivating the enzyme. The catalytic activity of the enzyme could be restored by FAD but not FMN at pH 7.0. It appears that the enzyme is a flavoenzyme in which the reduced flavin is much less tightly bound than the oxidized flavin.