On Dielectric Constant and Enzymatic Kinetics III. Interrelationships of dielectric constant and pH

The dielectric effects on trypsin and a-chymotrypsin activities have revealed that at pH 7.8 the active species of the former is the cation while that of the latter is the anion. The present study on the dielectric effects along the pH-activity curves shows that trypsin remains positive within the pH range of 5.5 to 8.5. Conversely, a-chymotrypsin is positive from pH 5.5 to 6.6, negative from 6.6 to about 8.1, and at pH 8.25 becomes positive again. The first point of inversion in charge sign shifts from 6.6 to 7.15 with the addition of 0.05 M phosphate buffer. The point of inversion does not seem to be modified significantly by changes in the substrate structure. At pH values near the point of inversion the plots of rate log vs. 100 /D are broken lines formed by various straight portions, the slope of each varying progressively from a maximum positive to a maximum negative value. This suggests an effect of resonance possibly attributable to an imidazole group. As an attempt to explain the two observed points of sign inversion in a-chymotrypsin, the possibility is suggested that different enzyme configurations are disclosed by the combined action of pH and dielectric constant. On this theoretical basis, it is feasible that more than one isoionic point exists. I N T R O D U C T I O N It was reported previously that the rate of ester hydrolysis by trypsin (5) or a-chymotrypsin (6) is modified by changes in the medium dielectric strength. By comparison with non-enzymatic reactions, it might be inferred from the behavior of these two enzymes that at pH 7.8 the active f o r m of trypsin is the cation and that of ot-chymotrypsin is the anion. These observations are in harmony with two earlier findings : (a) Northrop (16) deduced, by Donnan equilibrium considerations, that trypsin behaves like a univalent positive Received for publication, January 18, 1960.