title: Methodological aspects of aldehyde dehydrogenase assay by spectrophotometric technique.
abstract: Aldehyde dehydrogenase (ALDH) activity was assayed spectrophotometrically by measuring the increase in delta A at 340 nm, as a criteria of NAD conversion to NADH in the presence of propionaldehyde. The effect of pH and substrate(s) concentration of nonenzymatic increase in absorbance at 340 nm was studied. Results indicate that the increase in absorbance at 340 nm is not entirely due to NAD conversion to NADH. It was observed that nonenzymatic interaction of NAD and aldehyde could as well result in increase in absorbance at 340 nm. The magnitude of the nonenzymatic contribution towards increase in absorbance at 340 nm is found to be pH, substrate(s) conc., and time dependent. Further, the observed nonenzymatic reaction product was found to be different from that of NADH as confirmed by u.v. spectral characteristics (lambda max. 346 nm) and its inability to activate NADH/NADPH-dependent glutathione reductase. Based on these findings, a final assay method comprising a substrate blank consisting of NAD and aldehyde, and the assay pH of 7.4 is recommended for measuring the ALDH activity. Further, under these experimental conditions the Km value of human RBC ALDH was found to be 0.59 mM for propionaldehyde substrate.
authors.affiliation: Department of Neurochemistry, National Institute of Mental Health and Neurosciences, Bangalore, India.
authors.forename: S C
authors.initials: SC
authors.lastname: Guru
authors.forename: K T
authors.initials: KT
authors.lastname: Shetty
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000032:analysis*; D000447:Aldehydes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009243:NAD / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
chemical_list: D000447:Aldehydes; D009243:NAD; C005556:propionaldehyde; D000444:Aldehyde Dehydrogenase