Proteolytic enzymes are involved in several physiological processes and therefore are maintained under tight control, mainly by inactivation by inhibitors. In plants, some serine proteinase inhibitors are particularly abundant and used as tools in studies of animal enzymes. Caesalpinia echinata (brazil‑wood) belongs to the Leguminosae family (Caesalpinoidae sub-family) from where we have already identified human plasma kallikrein (HuPK) and cathepsin B inhibitors.
The aim of this study was to purify and characterize an elastase inhibitor isolated from Caesalpinia echinata seeds.
An initial saline extraction of ground seeds and acetone fractionation were followed by protein purification using an ion exchange chromatography (Resource Q column in 0.1 M ammonium acetate buffer, pH 8.3, 0.03 M NaCl and developed with NaCl gradient - 0.03 to 0.5 M), a gel filtration chromatography (Superdex 200 column in 0.1 M sodium phosphate buffer, pH 7.0, 0.15 M NaCl) and an hydrophobic chromatography (C18 column in 0.1% trifluoroacetic acid and developed with acetonitrile gradient - 0 to 95%). All purification steps were evaluated by electrophoresis in polyacrilamide gel (SDS-PAGE). The inhibitory activity was followed on chromogenic substrate Suc-Ala-Ala-Ala-pNan (0.5 mM) hydrolysis by porcine pancreatic elastase (85 nM) and on pyro-Glu-Pro-Val-pNan (0.2 mM) hydrolysis by human neutrophil elastase (1.0 nM). Protein N-terminal sequence was determined by Edman degradation.
The purified inhibitor (23 kDa) presented only one aminoacid chain and inhibitory constants (Ki) of 24 nM for porcine pancreatic elastase and 1.9 nM for human neutrophil elastase. N-terminal sequence indicated that CeEI belongs to the Kunitz family of vegetal inhibitors (FAPESP, CNPq, CAPES, FADA‑UNIFESP).