Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin

Abstract

Seventy-four peptide amides of 7-amino-4-methylcoumarine (Mec) of the type Boc-Xaa-Yaa-Arg-NH-Mec were newly synthesized and tested to find specific substrates for blood-clotting proteases and trypsin. The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively. Among these peptides, the followings were found to be most sensitive substrates for individual enzymes: Boc-Asp(OBz1)-Pro-Arg-NH-Mec (k,,, = 160 s-', K, = 11 pM, k,,,/K, = 15000000 M-' s-') for human a-thrombin, Z-< Glu-Gly-Arg-NH-Mec (k,,, = 19 s-', K, = 59 pM, k,,,/K, = 320000 M-' s-') for bovine factor Xa, BocGln-Gly-Arg-NH-Mec (k,,, = 5.8 s-', K, = 140 pM, k,,,/K, = 42000) for bovine factor XIIa, Boc-Asp(OBz1)-Ala-Arg-NH-Mec (k,,, = 9.2 s-', K,,, = 120 pM, k,,,/K, = 77000 M-' s -l ) for bovine activated protein C, and Boc-Gly-Phe-Arg-NH-Mec (k,,, = 29 s-', K, = 230 pM, kcat/Km = 130000 M-' s-') for bovine plasma kallikrein. Moreover, Boc-Glu(OBz1)-Ala-Arg-NH-Mec (k,,, = 46 s-', K, = 370 pM, kcat/Km = 120000 M-' s-'> was newly found as a good substrate for human factor XIa. Bovine trypsin effectively hydrolyzed peptide-NH-Mec substrates containing Ala and Pro at the P2 site. The most reactive substrate was Boc-Gln-Ala-Arg-NH-Mec (k,,, = 120 sK1, K, = 6.0 pM, k,,,/K, = 20000000 M-' s-').Peptide amides of 7-amino-4-methylcoumarine (Mec) originally developed for the sensitive assay for a-chymotrypsin [l] [7], and horseshoe crab clotting factors [12]. Most of these substrates were designed based on the information from the COOH-terminal sequences of the 'activation peptides', which are liberated during the conversions of plasma serine protease zymogens to their active enzymes. The utility of these fluorogenic peptide substrates has been established for the assay of trace amounts of enzymes because of their high sensitivities. Moreover, the specific substrates for a-thrombin, factor Xa, plasma kallikrein and urokinase so far developed by our group [7] are now commercially available.

Calibrating Thrombin Generation in Different Samples: Less Effort with a Less Efficient Substrate

“…prothrombin complex concentrates [10,21]. We took advantage of the work of Kawabata et al [22], who have characterized a large panel of AMC-peptides with respect to their use by several serine proteases. Because the physiological thrombin generation should ideally not be influenced at all (in practice: minimally), we chose the substrate with highest specificity and a low affinity as possible for thrombin.…”

“…Both peptide substrates were originally developed and characterized by Kawabata et al [22]. In this work, they compared a panel of 74 substrates with respect to their conversion by several serine proteases.…”

Biomimetic Hybrid Nanocontainers with Selective Permeability

“…The overall kinetics for signal generation hence comprise 1) the rate‐defining transport of substrate through the DNA nanopores and to a minor extent across the polymersome wall, and 2) the fast and non‐rate‐limiting turnover of the substrate by the encapsulated trypsin. A high catalytic efficiency of 2.9×10 7   m −1  s −1 and a high k cat value of 120 s −1 have been reported for trypsin with the peptide substrate,10 but the values can vary depending on the source of trypsin.…”

“…The assay relied on the transport of fluorogenic enzyme substrate B‐NAR‐AMC through the DNA pores and its cleavage to the fluorescent product AMC by polymersome‐encapsulated trypsin (Figure 3 A). The enzyme substrate has a maximum length of 1.5 nm calculated for an energy‐minimized structure10 and features a positive charge (Figure 3 B). The substrate was deliberately chosen to probe whether it can pass the 2 nm DNA nanopore.…”

Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity

“…The enzyme activity was assayed according to the method described by Kawabata et al [13]. Typically, 2 gl of tert-butoxycarbonyl (Boc)-Gln-Ala-Arg-methylcoumarin (MCA) in dimethyl sulfoxide (DMSO) was added to a 0.2 ml solution containing 10 mM HEPES buffer, pH 7.5, 5 mM octaethyleneglycol dodecylether (C12Es) (Buffer A) and appropriate amount of protein D2, then the mixture was incubated at 37°C.…”