Horse liver aldehyde dehydrogenase. I. Purification and characterization.

Horse liver aldehyde: NAD oxidoreductase (EC 1.2.1.3) has been purified to homogeneity by a procedure consisting of salt fractionation, ion exchange chromatography, and isoelectric focusing. The purif?ed material has a turnover number of 1.85 pmoles of NADH per min per mg of protein when assayed at pH 9.0 with propionaldehyde as substrate. Values obtained for the molecular weight of the native enzyme by sucrose density centrifugation, sedimentation equilibrium, and multiple porosity disc gel electrophoresis were 220,000, 260,000, and 252,000, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated a subunit molecular weight of 57,000, suggesting a tetrameric structure for the native enzyme. Specificity studies indicated that both aromatic and aliphatic aldehydes were oxidized. For most aldehydes tested, the Michaelis constants were between 0.1 to 1 PM when corrected for equilibrium concentrations of inactive hydrated aldehyde. Chloral, which is completely hydrated, was an inhibitor of the dehydrogenase reaction. Despite the broad aldehyde specificity, substrate analogues in which the aldehydic hydrogen of RCHO was replaced by NH2, CH, or even OH were not found to be inhibitors.