The Effects of Salts and of Ionic Strength on the Hydrolysis of TAME (p-toluenesulfonyl-L-arginine methyl ester) by Thrombin and Thrombokinase

Abstract

SummaryThe effects of varying concentrations of alkali chlorides (LiCl, NaCl, KC1, RbCl and CsCl), alkaline earth chlorides (BeCl2, MgCl2, CaCl2, SrCl2, and BaCl2), choline chloride and Tris,HCl (pH 8.0 at 37°) on the rates of hydrolysis of TAME by a purified preparation of human thrombin and of bovine thrombokinase were determined in 0.25 M Tris.HCl buffer, pH 8.0 at 37°. Each salt had its own individual effects on the reactions and these effects were completely different when thrombin instead of thrombokinase was used. Each salt, however, had the same qualitative effects on TAME hydrolysis by crude bovine thrombin as by purified human thrombin, but only minor effects on the hydrolysis of TAME by trypsin.With the exception of LiCl, low concentrations of alkali chlorides had inhibitory and high concentrations had acceleratory effects on both the thrombin-TAME and the thrombokinase-TAME reactions. LiCl had no accelerating effects on either reaction and it was a stronger inhibitor of thrombokinase than of thrombin. In contrast to the alkali chlorides, the alkaline earth chlorides had no acceleratory effects but had inhibitory effects on both reactions. MgCl2, however, was an exception. It weakly accelerated the thrombokinase -TAME and weakly inhibited the thrombin -TAME reaction. When comparing 0.15 M concentrations of the salts (with the exception of BeCl2 which was the strongest inhibitor of both reactions), NaCl was the strongest inhibitor of the thrombin - TAME reaction, followed by CaCl2, but BaCl2 was the strongest inhibitor of the thrombokinase -TAME reaction, followed by KC1 and LiCl.Choline chloride and Tris.HCl in concentrations up to 3 M had no significant effects on the rate of hydrolysis of TAME by either human or bovine thrombin. Increasing the ionic strength above 0.16 (lower values were not tested), therefore, may have no effect on this reaction, and all of the inhibitions and accelerations found in the presence of the alkali chlorides or the alkaline earth chlorides may be entirely due to the individual cations. On the other hand, increasing the ionic strength may produce a small inhibitory effect, as found in the presence of LiCl or MgCl2, and the lack of any effect in presence of even large concentrations of Tris.HCl or choline chloride may be due to a fortuitous balancing of the weak inhibitory effect of ionic strength by the weak acceleratory effect of the choline and Tris cations.Choline chloride and Tris.HCl had no inhibitory effects but accelerated the thrombokinase -TAME reaction. Choline chloride was the strongest accelerator tested. Rates in the presence of 0.15 and 3 M choline chloride were respectively 204 and 601 % of the controls. Although the effects of ionic strength changes on this reaction could not be established, the data indicate that increasing the ionic strength has at most only a small effect, and the inhibitions and accelerations found are due primarily to the specific cations present.