Assay of nicotinamide deamidase activity using high-performance liquid chromatography

A rapid, simple and reproducible method has been developed for the determination of nicotinamide deamidase activity using high-performance liquid chromatography (HPLC). Nicotinic acid (NA) liberated from nicotinamide (NAA) after a 15-min enzyme reaction was determined directly by HPLC without further separation steps. Both NA, the product, and NAA, the substrate were separated by reversed-phase ion-pair isocratic chromatography and detected at 261 nm. The present method could be applied to the measurement of deamidase activity in crude cell extracts prepared from several bacterial strains. The Michaelis constant of nicotinamide deamidase in Enterobacter agglomerans was 36 microM for NAA. This method is useful for the measurement of nicotinamide deamidase from various sources.