The effects of divalent cations on trypsin.

Although the activating effect of metal ions, such as Co++ and Mn*, on a variety of aminopeptidases has long been known (see Smith (2)), no requirements of the pancreatic proteinases for these ions have been demonstrated. It has been found, however, that calcium salts improve the yield of trypsin from trypsinogen (3) and that they increase the stability of trypsin at alkaline pH (4, 5). Earlier reports contain conflicting data on the effects of ions on crude pancreatic enzyme preparations and, apart from the work of Sugai (6) on the activation of crude trypsin preparations by some metal ions (Mn++, Co”, Ni++, Fe++, and Fe-) and the inhibition by others (Ag+, Hg++, Zn*, and Pb++), these data appear to be of questionable significance. In recent work in this laboratory it was noted that the addition of 0.01 M Ca* to the reaction mixture increased the zero order rate constant of the tryptic hydrolysis of benzoyl-n-arginine ethyl ester (BAEE) by approximately 25 per cent and at the same time improved the reproducibility of the rate measurements (1). Since, in comparison to typical metal ion activation (2, 7), the increase in activity was small, it was thought to represent an effect of the divalent cation on the stability of the enzyme, similar to that suggested by Gorini (4) and by Bier and Nord (5). In addition to calcium ions, Mn++, Cd*, Co++ and, to a lesser extent, Mg++and Ba* were found to increase the activity of trypsin toward BAEE, whereas metal ions such as Hg++, Cu ++, Ag+, and, to a lesser extent, Zn* inhibited trypsin at low concentrations (1). The metal ion-inhibited enzyme could be partially reactivated by calcium and other inorganic salts, as well as by the complex-forming agent Versene (ethylenediaminetetraacetic acid) and by thioglycolate. The results of these studies are the subject of the present paper.