Thrombin specificity with tripeptide chromogenic substrates: comparison of human and bovine thrombins with and without fibrinogen clotting activities.
To assess the thrombin specificity of tripeptide chromogenic substrates, we determined the Michaelis--Menten (Km), catalytic (kcat), and specificity (kcat/Km) constants for S-2238 (H-D-Phe-Pip-Arg-p-nitroanilide), Chromozym-TH (Tos-Gly-Pro-Arg-p-nitroanilide), and Spectrozyme-TH (H-D-hexahydrotyrosyl-Ala-Arg-p-nitroanilide) with high-purity thrombin preparations. Human and bovine alpha-thrombins, prepared by essentially the same procedure, were each greater than 95% in the form of the enzyme (alpha-thrombin) and had a specific fibrinogen-clotting activity greater than 2000 kilo-clotting units per gram of protein (kcu/g). In contrast, human gamma- and bovine beta-thrombins, made by controlled passage of alpha-thrombin through trypsin agarose, were greater than 98% of their respective forms and had fibrinogen-clotting activity less than 1 kcu/g. With the tripeptide chromogenic substrates these four thrombin forms at pH 7.8 and 23 degrees C had Km values of 1.6 to 16 mol/L, kcat values of 35 to 130 s-1, and kcat/Km ratios of 4.7 to 52 L X mol-1 X s-1. Although Km for an individual substrate was slightly higher for bovine than for human alpha-thrombins and although Km values for the nonclotting forms (human gamma- and bovine beta-thrombins) were higher than for clotting forms (alpha-thrombins), we found no major differences among the kinetic values for the three substrates.
S A
SA
Sonder
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JW
Fenton
D000818:Animals; D001777:Blood Coagulation; D002417:Cattle; D002863:Chromogenic Compounds / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*
D002863:Chromogenic Compounds; D004151:Dipeptides; D009842:Oligopeptides; C014413:chromozym TH; C021129:S 2238; D005340:Fibrinogen; C049024:H-hexahydrotyrosyl-alanyl-arginine-4-nitroanilide; D013917:Thrombin