Kinetics of trypsin-catalyzed hydrolysis of some arginine-containing peptide methyl esters

The kinetics of trypsin-catalyzed hydrolysis (at pH 8.5) of methyl esters of some synthetic dipeptides containing the residues of both arginine and L(D)-p-fluorophenylalanine or L(D)-tyrosine has been studied. The digestion of Tos-(pF)Phe-Arg-OMe was shown as unfollowing the Michaelis-Menten kinetic since in the reaction course the substrate activation is observed and while the reaction product is the enzymatic process inhibitor. In contrast to this, the hydrolysis of other substrates studied, follows the normal Michaelis-Menten kinetic.     

Chromogenic and fluorogenic peptide substrates of proteolytic enzymes

The review describes approaches to designing chromogenic and fluorogenic substrates for proteolytic enzymes, mainly for assay of serine proteinases. Principles of substrate polypeptide chain construction and some methods for detection of chromogenic and fluorogenic products of their hydrolysis are considered. The use of these substrates for the study of blood clotting enzymes and for clinical diagnostics is briefly treated. Methodology of chemical synthesis of principal chromogenic and fluorogenic substrates is also discussed.     

Isolation of human thrombin using affinity chromatography on gramicidin C-silochrome C 80

Thrombin purification is conducted by biospecific chromatography on gramicidin C-silochrome C 80. Preparations possessing the fibrinogen-coagulating activity of 2500-3200 NIH units per 1 mg of protein and containing 98% of active sites are obtained. Data obtained from electrophoresis in PAAG with the presence of DS-Na show the alpha-thrombin content to be 96%; the admixture of beta-thrombin possessing no coagulating activity does not exceed 4%. The kinetic constants are presented for thrombin hydrolysis of tosyl-L-arginine methyl ester (TAME), benzoyl-L-arginine ethyl ester (BAEE) and chromogenic substrate S-2238. The addition of isopropanol increases sharply the stability of thrombin when storing it in the aqueous-salt solutions.     

Comparative study of hydrolysis of methyl esters of N-arylsulfonyl-valyl-arginine by thrombin and trypsin]

The esterase action of thrombin and trypsin on N-arylsulfonyl-valyl-arginine methyl esters was studied. The values of Km and kcat under steady-state conditions at pH 8,5 were determined. It was shown that the nature of the arylsulfonyl group does not affect the kinetic parameters of the reactions under study. The Michaelis constants of the thrombin-catalyzed reactions appeared to be one order of magnitude lower than the Km values of the corresponding TAME analogs.     

Effect of isopropanol on the enzymatic activity and stability of thrombin

Isopropanol is shown to affect considerably the thrombin activity. Its low concentrations (5-15%) activate the hydrolysis reaction of benzoyl-1-arginine ethyl ester (BAEE) by thrombin, whereas incomplete uncompetitive inhibition of the enzyme is observed in the presence of 20% isopropanol. Alcohol in the concentration of 25% results in complete reversible inhibition of the clothing and esterase activity of the enzyme. Isopropanol in the concentration of 25% is able to suppress the thrombin autolysis and therefore it may be used as a reagent which stabilizes thrombin during its storage in the aquatic-salt solutions.