Development and Application of Fluorogenic Peptide Substrates for Determination fo Blood Clotting Proteases

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Seventy-four peptide amides of 7-amino-4-methylcounarine (Mec), which contain various Boc-x-y-Arg-NH-Mecs, were newly synthesized and tested to find specific substrates for blood clotting proteases and trypsin, The X and Y residues of these substrates have been replaced by 12 and 15 different samino acids, respectively. Among these peptides, the followings were found to be most sensitive substrates for individual enzymes: Boc-Asp(OBz1)-Pro- Arg-NH-Mec (Kcat = 160 s^<-1> , Km = 11 (micrn)M, Kcat/Km = 15,000,000 M^<-1> s^<-1> ) for human (alpha)-thrombin, Z- Glu-Gly-Arg-NH-Mec (Kcat = 19 s^<-1>, Km = 59 (micrn)M, Kcat/km = 320,000 M^<-1> s^<-1>) for bovine factor Xa, Boc-Ln-Gly-Arg-NH-Mec (Kcat = 5.8 s^<-1>, Km = 140 (micrn)m, Kcat/Km = 42,000) for bovine factor XIIa, Boc-Asp(OBzl)- Ala-Arg-NH-Mec (Kcat = 9.2 9.2 s^<-1> , Km = 120 (micrn)m, Kcat/Km = 77,000 M^<-1>s^<-1>) for bovine activated protein C, and Boc-Gly-Phe-Arg-NH-Mec (Kcat = 29 s^<-1>, Km = 230 (micrn)m, Kcat/Km = 130,000 M^<-1>s^<-1>) for bovine plasma kallikrein. Moreover, Boc-Glu(OBzl)-Ala-Arg-NH-Mec (kcat = 46 s^<-1>, km = 370 (micrn)m, kcat/Km = 120,000 M^<-1>s^<-1>) was newly found as a good substrate for human factor XIa, Bovine trypsin effectively hydrolyzed peptide-NH-Mecs containing Ala and Por at the P2 site. The most reactive substrate was Boc-Gln-Ala-Arg-NH-Mec (kcat = 120 s^<-1>, km = 6.0 (micrn)m, kcat/km = 20,000,000 M^<-1> s^<-1>),