Development of a Sensitive Spectrofluorometric-Multivariate Calibration Method for Enzyme Kinetic of Aldehyde Oxidase

Attempts to obtain experimental values for the kinetic parameters of phenanthridine oxidation by guinea pig or rabbit liver aldehyde oxidase using common spectrophotometric methods have not been successful due to a lower limit of detection. In the present study, a new spectrofluorimetric assay in combination with a multivariate calibration method for enzymatic kinetic study of aldehyde oxidase activity, using phenanthridine as the substrate, has been developed and validated. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.025-1 µM and the emission flourimetric spectra of the solutions recorded at the excitation and emission wavelengths of 236 and 320-450 nm, respectively. The optimized calibration model of partial least squares (PLS) method was applied for the simultaneous determination of the concentration of each chemical in the prediction set. The limits of detection for phenanthridine and phenanthridinone were found to be 2.13 ± 0.33 and 3.41 ± 0.34 nM (mean ± SD, n = 5), respectively. This method was then used for kinetic study of phenanthridine oxidation using guinea pig and rat hepatic aldehyde oxidase. The results were compared with those obtained from a univariate spectroscopic method. Using this new spectrofluorimetric-multivariate calibration method, the Km value for the oxidation of phenanthridine with guinea pig and rat liver aldehyde oxidase were obtained as 0.83 ± 0.08 and 2.20 ± 0.40, µM (mean ± SD, n = 3), respectively.