Quantitative screening method for hydrolases in microplates using pH indicators: determination of kinetic parameters by dynamic pH monitoring.
The presented pH-dyn assay serves as a versatile tool for screening enzymatically catalyzed reactions consuming or producing acids. The method is based on material balances of substrates and products. Ion balances relate concentrations of acids and bases to pH. pH-changes caused by the enzymatically catalyzed reaction in a well-defined buffer system are recorded by light-absorption measurements of a pH-indicator. Kinetic parameters are estimated by fitting the modeled pH changes to the experimentally observed ones. The enzymatically catalyzed hydrolysis of 4-nitrophenol is used as a model system. A pH indicator, bromothymol blue, is used to monitor the reaction progress. The reaction is monitored until the limiting substrate is completely consumed. This allows the estimation of the parameters of the Michaelis-Menten kinetics, K(M) and k(cat), in a single run. The results agree well with conventional spectrophotometric experiments and values reported in literature. Around pH 7, environmental CO(2) influences pH. Carbon dioxide influence was included in the model. Thus it was possible to estimate initial CO(2) concentrations as a model parameter, and therefore automatic correction for the CO(2) disturbances was achieved. This was important to detect low conversions at low buffer concentrations.
John
G T
GT
Department of Biochemical Engineering, University of Saarland, P.O. BOX 151150, D-66041 Saarbrücken, Germany.
Heinzle
E
E
D002138:Calibration; D002245:Carbon Dioxide / Q000737:chemistry; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000032:analysis* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009596:Nitrophenols / Q000378:metabolism*
D009596:Nitrophenols; D002245:Carbon Dioxide; C008642:4-nitrophenyl acetate; D006867:Hydrolases