Human thrombins. Group IA and IIA salt-dependent properties of alpha-thrombin.

The biologically functional proteolytic activity of human a-thrombin for the clotting of fibrinogen was not irreversibly altered by brief exposure to high concentrations of Group Ia and IIa salts (-0.6 mg of enzyme/ ml in I 5 3 M salts, pH 8.3, 22 "C for -1 h and subsequently diluted >2000-fold, since I > 0.1 M salts markedly prolong clotting).With increasing salt concentrations, the thermal stability of this activity increased in Group Ia chlorides (Na' > K' 3> Rb+ > Cs', I I 3 M), decreased in Group IIa chlorides ( M g + c Sr2+ c Ba2+ < Ca", I -3 M), and changed only slightly in LiCl or N a 0 3 (50% inactivation T for 5-min heating in the 10 salts extrapolated to -51 "C at Z = 0).The autolytic degradation of a-to nonclotting 8and y-thrombins was retarded by increasing concentrations of Li' , Cs+, and Group IIa chlorides, while it was enhanced by Na' > K' > Rb' chlorides (4 "C for 8 days; NaN03 had little effect).For a-thrombin in 3 M Na' through Cs' chlorides, the degradation rate increased with temperature (4 < 22 < 37 "C) and directly correlated with the cation Iyotropic number (p > 0.98).The synthetic-substrate esterolytic activity of the enzyme determined by potentiometric titration also followed the Na' through Cs' series (C1-versus Nos-had little effect).Both K,(app) and kcst, but not Km(app)/koat (= K,/kz), inversely correlated with the cation lyotropic number for the hydrolysis of Na-tosyl-L-arginine methyl ester.This effect was attributed to decreasing K, and kz, since these salts did not appreciably change kcat for k3-limited substrates.Contrastingly, L i ' and Group IIa chlorides caused nonlinear hydrolysis dependent on the order of reagent addition.Although its aqueous solubility (50.3 mg/ml, Z = 0) * The preceding paper of this series described the production of human a-thrombin from Fraction 111 paste, criteria for purity evaluation, and general properties (I).The present studies describe subsequently found salt effects on the enzyme.