Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A.
Further development of our recently published Glu(pNA)-containing peptides (Anal. Biochem. 428 (2012) 73-80) provided new fluorogenic substrates for the activated blood coagulation factor XIII. A first series was designed by incorporation of Glu(AMC) at the penultimate position from the N terminus. For the best derivative H-Tyr-Glu(AMC)-Val-Lys-Val-Ile-NH2, a moderate kcat/Km value of 34s(-1)M(-1) was determined, which is more than 100-fold reduced compared with the previously reported Glu(pNA) substrates. Furthermore, two fluorescence resonance energy transfer (FRET) substrates were prepared by incorporation of an N-methyl-anthraniloyl fluorophore and a 2,4-dinitrophenyl quencher. Both substrates were excellently cleaved by FXIII-A2(∗), which is generated from its zymogen by activation of thrombin in the presence of calcium ions. In the absence and presence of H-Gly-ethyl ester, kcat/Km values of 8010 and 8660s(-)(1)M(-)(1), respectively, were found for the conversion of H-Lys(N(Me)Abz)-Glu(NH-(CH2)4-NH-Dnp)-Val-Lys-Val-Ile-Gly-NH2 (substrate 8). These values are more than 200-fold improved compared with the Glu(AMC) substrates. Substrate 8 is suitable for the measurement of FXIII-A2(∗) activities in plasma samples as well as for in vitro measurements. Furthermore, it was used for the determination of the inhibitory potency of a newly synthesized chloromethyl ketone derivative, Cbz-Phe-Glu(CMK)-Val-Lys-Val-Ile-Gly-NH2, which was found to be a potent irreversible inhibitor of FXIII-A2(∗).
Institute of Pharmaceutical Chemistry, Philipps University Marburg, D-35032 Marburg, Germany.
Kornelia
K
Hardes
M
M
Zouhir Hammamy
Torsten
T
Steinmetzer
D000595:Amino Acid Sequence; D060326:Chemistry Techniques, Synthetic; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D026122:Factor XIIIa / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009842:Oligopeptides; D026122:Factor XIIIa
Chloromethyl ketone; Enzyme kinetic; Factor XIII; Fluorometric assay; Peptide synthesis