Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa

• Published in: Phytochemistry (Oxford) 2009-05, Vol.70 (7), p.871-879
• Main Authors: Lopes, J.L.S., Valadares, N.F., Moraes, D.I., Rosa, J.C., Araújo, H.S.S., Beltramini, L.M.
• Format: Article
• Language: English
• Subjects: Acacia - chemistry; Acacia plumosa; Amino Acid Sequence; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antifungal activity; Antifungal agents; Antifungal Agents - chemistry; Antifungal Agents - isolation & purification; Antifungal Agents - pharmacology; Aprotinin - chemistry; Aprotinin - isolation & purification; Aprotinin - pharmacology; Aspergillus niger - drug effects; Biological and medical sciences; Chemical constitution; Chymotrypsin - drug effects; Fundamental and applied biological sciences. Psychology; Kunitz-type protease inhibitor; Leguminosae; Medical sciences; Microbial Sensitivity Tests; Pharmacology. Drug treatments; Plant physiology and development; Plants, Medicinal - chemistry; Sequence Homology, Amino Acid; Surface plasmon resonance; Trypsin - drug effects
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2009.04.009

Three protease isoinhibitors from Acacia plumosa are characterized in terms of biochemical properties, kinetic binding constants to trypsin and α-chymotripsin by SPR, and antifungi activity against Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp. This study was aimed at investigating the purification, biological activity, and some structural properties of three serine protease inhibitors isoforms, denoted ApTIA, ApTIB, and ApTIC from Acacia plumosa Lowe seeds. They were purified from the saline extract of the seeds, using Superdex-75 gel filtration and Mono-S ion exchange chromatography. They were further investigated by mass spectrometry, spectroscopic measurements, surface plasmon resonance, and inhibition assays with proteases and phytopathogenic fungi. The molecular mass of each isoform was estimated at ca. 20 kDa. Each contained two polypeptide chains linked by a disulfide bridge, with different isoelectric points that are acidic in nature. The N-terminal sequences of both chains indicated that they were Kunitz-type inhibitors. Circular dichroism (CD) analyses suggested the predominance of both disordered and beta-strands on ApTI isoforms secondary structure, as expected for β-II proteins. In addition, it was observed that the proteins were very stable, even at either extreme pH values or at high temperature, with denaturation midpoints close to 75 °C. The isoinhibitors could delay, up to 10 times, the blood coagulation time in vitro and inhibited action of trypsin (Ki 1.8 nM), α-chymotrypsin (Ki 10.3 nM) and kallikrein (Ki 0.58 μM). The binding of ApTIA, ApTIB, and ApTIC to trypsin and α-chymotrypsin, was investigated by surface plasmon resonance (SPR), this giving dissociation constants of 0.39, 0.56 and 0.56 nM with trypsin and 7.5, 6.9 and 3.5 nM with α-chymotrypsin, respectively. The growth profiles of Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp. P10 were also inhibited by each isoforms. These three potent inhibitors from A. plumosa may therefore be of great interest as specific inhibitors to regulate proteolytic processes.