The Precipitation of Gelatin by Ethanol, and Its Use in the Estimation of Proteolytic Activity

Abstract

Within certain limits of gelatin concn., pH, temp., salt concn., and ethanol concn. the percentage light treansmitted through precipitated gelatin solns. was an exponential function of the gelatin concn. It was concluded that the mean micelle size of the precipitated gelatin is independent of the gelatin concn. within these prescribed limits. Two methods for estimating proteolytic activity were based on the precipitation of gelatin by ethanol. In the 1st method buffered solns. of gelatin, digested by the protease under standard conditions, were precipitated by an ethanol-buffer mixture, the decrease in turbidity as the result of proteolysis providing a measure of its activity. The relationship between the logarithm of the percentage transmitted light and the enzyme concn. approximated to a straight line over a considerable range of enzyme concns. The 2d method depended on the gravimetric detn. of the amount of isoelectric gelatin rendered soluble in 80% ethanol by the action of the enzyme. Using solns. of protease from Aspergillus oryzae, this value was found to be proportional to the amt. of enzyme present if dilute solns. of enzyme were employed. The relationship between the amt. of gelatin made soluble and the enzyme concn. varied according to the type of protease used. These methods were compared with other conventional methods for estimating proteolytic activity and for a number of proteases provided equal or greater sensitivity.