Isolamento e caracterização bioquimica de inibidores de proteinases serinicas de sementes de Copaifera Iangsdorffii : estudo da resistencia contra o Callosobruchus maculatus

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Two inhibitors have been isolated de TOI fraction in seeds of Copaifera langsorffii, ordinarily known as Copaíba, one Brazilian native tree of the Caesalpinoideae subfamily of Leguminosae family. The fraction TOI was purified by ammonium sulfate preeipitation, ion-exehange ehromatography on OEAE-Sepharose and affinity ehromatography on trypsin-Sepharose. TOI presented homogeneity in PAGE-SOS geI12,5%, with apparent moleeular mass of 24 and 12 kOa in non-and reduetion condition, respeetively. The relative moleeular mass determined by FPLC gel filtration on a Superdex 75 eolumn, eonfirmed on the results in PAGE-SOS. TOI have Ki of 1,2nM to bovine trypsin enzyme showing high affinity. However, no inhibition was found for quimotrypsin. The stoiehiometrie study of the trypsin-TOI eomplex evideneed that inhibitor is aetive for trypsin at 1: 1 molar ratio. The trypsin- TOI eomplex was eluted on a gel filtration ehromatography with retention time equivalent a relative moleeular mass of the 39kOa. Evidenced too, that TOI is active on enzyme trypsin on its monomerie form and not dimerie one. The isoelectrie foeusing of TOI demonstrated the presenee of two proteie bands, with isoelectrie point between 6.45 e 7,35, indicating two inhibitors in TOI. These inhibitors of TOI, designated for TOI-I and TOI-II, was isolated by reversed - phase HPLC on a J.L-Bondapaek column (Waters System) and was observed in Trieine-gel 16,5% one minimal differenee on apparent moleeular mass, of approximately 12 and 10 kOa for TOI-I and TOI-1I respectively. N-terminal sequeneing of the inhibitors of TOI, demonstrated the presenee of the eonsensus sequenee present in the soybean Kunitz-Type trypsin inhibitor for TOI-I, however, protein database searehes did not reveal any homology to TOI-II. The TOI showed high stability to pH different eonditions, temperature and amount of OTT, indicating one elevated number of disulfides bonds in a moleeular strueture. The TOI was not proteolytically degraded on a in vitro digestibility by midgut proteinases of the gorgulho Callosobruchus maculatus and demonstrated one deleterious effect on a insect s development, suggesting that TOI acts like toxie factor and deereased survival of Callosobruchus maculatus

ASSUNTO(S)

inibidores da tripsina leguminosa tripsina sementes

ACESSO AO ARTIGO

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