IMPROVED HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF BIOTINIDASE ACTIVITY WITH BIOTINYL-6-AMINOQUINOLINE AS SUBSTRATE

An unproved high-performance liquid chromatographic assay method for b iotinidase activity was developed using the fluorimetric substrate bio tinyl-6-aminoquinoline, which was found to be more specific than the b iotinyl-4-aminobenzoate previously used. The new method measures the i ntensity of the fluorescent signal at wavelengths (excitation 350 nm; emission 550 nm) longer than those (excitation 276 nm); emission 340 n m) for 4-aminobenzoate. The analysis of fluorescence in the visible sp ectrum reduced considerably the number of interfering peaks compared w ith analysis in the ultraviolet region. This method also made it possi ble to measure the biotinidase activity directly in samples usually di fficult to calculate, such as human and bovine milk or porcine serum; the use of biotinyl-6-aminoquinoline allowed the analysis of the enzym e reaction in milk and porcine serum without pretreatment or dialysis. Stoichiometric increase and decrease of the substrate and product, re spectively, were demonstrated. Michaelis constants for biotinyl-6-amin oquinoline were measured at various stages of partial purification. Be cause the solubility of these synthetic substrates in the aqueous phos phate buffer is limited, the determination of both Michaelis constant and maximum velocity by extrapolation may be helpful for the character ization of the kinetics of biotinidase.