Action of Bauhinia bauhinioides synthetic peptides on serine proteinases

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Abstract

The kallikrein inhibitor found in Bauhinia bauhinioides seeds (BbKI) differs from classical Kunitz plant inhibitors in the lack of disulfide bridges in its structure [Biochim. Biophys. Acta 1477 (2000) 64–74]. In this study, we examined whether structural properties may be involved in inhibitory specificity and, if so, whether those properties might be useful tools in designing compounds that interfere with enzyme activity. Peptides structurally related to the BbKI (RPGLPVRFESPLRINIIKE-NH2) reactive site were synthesized by solid-phase method and assayed for serine proteinase activity. The peptides RPGLPVRFESPLRINIIKE-NH2, RPGLPVRFESPL-NH2, and GLPVRFES-NH2 were efficient tissue kallikrein inhibitors, with I50 values of 0.54 μM, 0.87 μM, and 0.5 mM, respectively. The lasting inhibitory effect was observed in incubation periods of up to 120 min. None of the studied peptides interfere with the activity of thrombin, factor Xa or trypsin, although the native protein BbKI is a potent trypsin inhibitor.

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Materials and methods

Enzymes, substrates, and peptides. Human plasma kallikrein (HuPK) (EC 3.4.21.34) was purified by a previously described procedure [18]. Porcine pancreatic kallikrein (PoPK) (EC 3.4.21.35), trypsin (EC 3.4.21.4), human thrombin (EC 3.4.21.5), and plasmin (EC 3.4.21.7) were purchased from Sigma Chemical Company. Human neutrophil elastase (EC 3.4.21.37) and porcine pancreatic elastase (PPE) (EC 3.4.21.36) were purchased from Calbiochem. The H-d-Pro-Phe-Arg-AMC, Suc-Ala-Ala-Pro-Val-AMC,

Results and discussion

Previously published results have shown that fluorogenic substrate derived from the BbKI reactive site and hydrolyzed by bovine trypsin or trypsin-like enzymes from insect sources displays resistance to hydrolysis by other serine proteinases [12], [13], [15]. This led us to investigate the reactivity of BbKI-derived synthetic peptides to target and non-target proteinases (Fig. 1A).

The peptides ESPLRINI-NH2 (P2) and RFESPLRINII-NH2 (P4), containing the amino acid Arg at the P1 position of the

Acknowledgements

We would like to gratefully acknowledge the skilled technical assistance given by Lucimeire A. Santana. This work was partially supported by CAPES, CNPq, FAPESP, and SPDM.

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