Substrate-stat: a method for characterization of enzymes in concentrated solutions at low substrate concentrations

A method is proposed, which is suitable for kinetic studies with concentrated enzyme preparations, and its principal features are described. The method makes it possible to keep constant the concentration of substrate(s) or product(s) during the observation time. For this purpose, the increase or decrease of an indicator extinction signal, dependent on enzyme velocity, is monitored and automatic titration is performed using a suitable titer solution. This latter contains equimolar amounts of indicator and substrate(s). The whole system operates in a similar manner as in pH-stat devices. The technical set-up, model reaction systems and practical applications are shown. Operation problems and possible sources of error are examined. Finally, the main limitations as well as possible improvements are discussed.