Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa.
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 beta-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 degrees C. The isoinhibitors could delay, up to 10 times, the blood coagulation time in vitro and inhibited action of trypsin (Ki 1.8 nM), alpha-chymotrypsin (Ki 10.3 nM) and kallikrein (Ki 0.58 microM). The binding of ApTIA, ApTIB, and ApTIC to trypsin and alpha-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 alpha-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.
Lopes
J L S
JL
Instituto de Física de São Carlos (IFSC), Universidade de São Paulo (USP), SP, Brazil.
Valadares
N F
NF
Moraes
D I
DI
Rosa
J C
JC
Araújo
H S S
HS
Beltramini
L M
LM
D000045:Acacia / Q000737:chemistry*; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D007611:Aprotinin / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D001234:Aspergillus niger / Q000187:drug effects; D002918:Chymotrypsin / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D010946:Plants, Medicinal / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000187:drug effects
D000935:Antifungal Agents; D007611:Aprotinin; D002918:Chymotrypsin; D014357:Trypsin