KINETIC MONITORING OF ENZYMATIC-REACTIONS IN REAL-TIME BY QUANTITATIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

The study of enzyme kinetics under steady-state conditions represents a common and very useful method for investigating the mechanisms of en zymatic reactions. We report the use of mass spectrometry (MS) coupled with HPLC for the kinetic analysis of enzymatic reactions in real tim e. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A (RNase A) and the substrate-specific hydrolysis of lactose with bet a-galactosidase can be monitored using ion-spray (pneumatically assist ed electrospray) mass spectrometry as a sensitive and specific detecto r for the native substrates. The resulting data can be used to calcula te both K-M and V-max for each system. Kinetic parameters obtained for RNase A and beta-galactosidase paralleled those obtained by conventio nal techniques. These findings suggest the possibility of developing a lternative techniques, based on mass spectrometric detection, for perf orming kinetic analyses of enzymatic processes where no simple spectro photometric assay is feasible. In addition to enabling the determinati on of kinetic parameters for authentic substrates, and not chromogenic analogs, such assays would also be useful in situations where very hi gh sensitivity and specificity are desired. (C) 1995 Academic Press, I nc.