Action of urea and certain other amide reagents on crystalline porcine pancreatic amylase

Abstract

The inactivation of porcine pancreatic amylase with various concentrations of urea with a fixed concentration of pancreatic amylase was investigated in one series of experiments. The changes in viscosity, absorbance in the ultraviolet, and optical rotation accompanying the loss in amylase activity were likewise determined. It was observed that the rate of inactivation increased rapidly with an increase in the urea concentration. When the enzyme concentration is 1.5 mg/ml or less, the inactivation process follows first-order kinetics. This loss in activity is accompanied by an increase in reduced viscosity and in levorotation of the amylase. A very slight decrease in ultraviolet absorbance intensity was observed. In a second series of experiments, the urea concentration was fixed while the concentration of the amylase was varied. The rate of inactivation increases with a decrease in enzyme concentration. For concentrations greater than 1.5 mg/ml of amylase, the urea-amylase reaction does not follow first-order kinetics. The inactivation by urea is irreversible. Comparative inactivation studies of equimolar solutions of urea and other amide reagents show that the effectiveness in inactivating pancreatic amylase decreases in this order: guanidine > urea > 1,3-dimethylurea > tetramethylurea.