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 enzymat ically catalyzed reactions consuming or producing acids. The method is base d on material balances of substrates and products. ion balances relate conc entrations of acids and bases to pH. pH-changes caused by the enzymatically catalyzed reaction in a well-defined buffer system are recorded by light-a bsorption 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 p rogress. The reaction is monitored until the limiting substrate is complete ly 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 wi th conventional spectrophotometric experiments and values reported in liter ature. Around pH 7, environmental CO2 influences pH. Carbon dioxide influence was included in the model. Thus it was possible to estimate initial CO2 concent rations as a model parameter, and therefore automatic correction for the CO 2 disturbances was achieved. This was important to detect low conversions a t low buffer concentrations. (C) 2001 John Wiley & Sons, Inc.