Plateau absorbance measurements: an alternative approach to enzyme activity determination illustrated by the example of alkaline phosphatase.

Abstract

A simple and reproducible method was used for the cytophotometric assay of alkaline phosphatase activity by end point measurements after incubation at 70 degrees C. Alkaline phosphatase was incorporated in polyacrylamide gel model films and its activity was demonstrated with a simultaneous coupling method. The initial reaction rate was 4.7 times faster than at 37 degrees C. At 37 degrees C, linear reaction rates were obtained up to 90 min incubation. Deviation from linearity occurred only when the amount of final reaction product precipitated inside the films was too high to be measured cytophotometrically. In that case, levelling off of the reaction rate was due to the out-of-range error of the cytophotometer. At 70 degrees C, reaction rates were distinctly non-linear from the onset of incubation. This was due to heat inactivation of the enzyme molecules. A plateau level was reached after approximately 60 min incubation, irrespective of the amount of enzyme incorporated, indicating that all enzyme molecules had become inactivated after this incubation period. The inactivation process followed first-order kinetics. The plateau value as well as the slope of the initial reaction were found to be linearly related to the amount of enzyme incorporated. Therefore, plateau absorbance values can be used as a relative measure of enzyme activity instead of initial reaction rates. This type of measurement could be valuable for routine applications of enzyme cytochemistry in diagnostic pathology, or when cytochemical reaction products are used as markers in immunocytochemistry or hybridocytochemistry. Precise control of incubation time is not necessary once the plateau value has been reached and preparations can be mounted and measured later.