The application of the pH-stat to studies of enzymes

Abstract

Many enzyme-catalyzed reactions involve the uptake or release of protons into the aqueous medium and are therefore susceptible to kinetic analysis through the measurement of the rate and extent of appearance of hydrogen ion. Historically, such measurements have taken two general forms: 1. the measurement of the rate of actual change of hydrogen ion concentration and 2. the measurement of the rate of addition of base or acid necessary to maintain a constant hydrogen ion concentration. As both procedures depend on the measurement of the pH of the solution, the continued development of more precise instrumentation for pH measurement has led to a continued improvement in the quantity and quality of kinetic data based on this particular analysis. In several early studies, for example, an acid-base indicator with an appropriate pK′a was added to the enzyme-substrate mixture and the rate of addition of base needed to maintain the color of the indicator constant, by visual inspection, was noted. This constant pH technique, or pH-stat, can be removed from a reliance on visual estimation of color by employing spectrophotometric measurement. In another type of system the actual color change of the indicator, reflecting a change in pH of the solution, was measured spectrophotometrically and used in kinetic analyses of the system. As this latter technique requires an actual pH change during the reaction, it is inherently less desirable than the constant pH technique, particularly if more than initial reaction rates are desired.