Simultaneous Hydrolyses of Esters and Proteins at Saturation Levels

A direct titration method for the determination of proteolytic activity is discussed. This involves the potentiometric measurement of the volume of 0.08 N NaOH required to maintain a constant pH (8.0) during the time of the hydrolysis. I t is a sensitive method which presents several advantages; ds., it measures simultaneonsly protease and esterase activity, it follows the hydrolysis very dosdy and from the first stages; the titration is continuous and on the same sample. This method determines a constant fraction of the groups titratable by formol titration. The ratio formol:direct titration is represented by a factor '~" which is presumed to be distinct for each protein-enzyme system. Kinetic studies, using this method, revealed that the rates of hydrolysis of mixtures casein-gelatin on one hand, casein-BAEE or gelatin-BAEE on the other, are always larger than those of the corresponding isolated substrates. In many cases the resulting rates are equal or nearly equal to the sum of the individual rates, even though the mentioned rates have been determined within the saturation zones for every substrate. The former observations are inconsistent with the theory of the formation of an intermediary enzyme-substrate compound, unless it is assumed that the enzyme has a specific active group for each substrate. In a previous paper (1), evidence was presented to show that Northrop's phenomenon, an anomaly in enzymatic kinetics first described by Northrop (5, 6), holds good for enzymatic systems other than trypsin-casein-gelatin. I t was demonstrated that the addition of gelatin does not affect the rate of hydrolysis of casein or hemoglobin by trypsin, papain, or pepsin. I t was then thought of interest to investigate whether such independent hydrolysis also occurs in protein and ester systems and in the presence of substrate-saturated enzymes. The need to determine esterase and protease activity gave rise to a search for adequate methods. The usual procedures for the measurement of proteolyric activity do not determine initial steps of hydrolysis, and these are not the best conditions for comparison with ester hydrolysis which is a very simple