Toxina da salsa (Ipomoea asarifolia R. et Schult.): aspectos bioquímicos, estruturais, funcionais e potencial biotecnológico

Abstract

1pomoea asarifolia (salsa) is a member of the family Convolvulaceae with wide distribution throughout Northern Brazil and represents an alternative food for ruminant animals during drought periods. However some toxic symptoms have been observed in goats after feeding the leaves of this plant. In a search for a substance that could be responsible for these adverse effects we have purified a toxic and hemagglutinating protein from salsa. Fresh leaves were ground to a fine powder in liquid nitrogen, extracted by treatment with 25 mM Tris HCI, pH 7.5, containing 5 mM DTT and 3% PVP (1:3, w/v) and stirred for 2 h, at 4 °C. The suspension was filtered through a nylon cloth and then centrifuged at 10,000 x g, for 30 min, at 4 °C. The toxic lectin, named LTS, was purified from the crude extract by ammonium sulfate fractionation, ion exchange (DEAE-cellulose) and affinity (fetuin Sepharose-4B) chromatographies. The apparent molecular mass determined by gel filtration in Sephadex G-100, at pH 7.5, was 40.5 kDa, whereas by SDS-PAGE it was 44.0 kDa. Mass spectroscopy analysis resolved LTS in two peaks of 38.5 kDa and 19.3 kDa. LTS is a glycoprotein (4.4% carbohydrates) composed of mannose (29.2%), fucose (27.0%), N-acetyl-D-glucosamine (23.6%), and xylose (20.2%). The amino acid composition revealed to be LTS rich in Asp/Asn and Glu/GIn. The pl found for LTS was 5.7, and NGXIATLSLIPY was its NH2-terminal sequence. The toxic lectin was relatively heat stable, requiring about 93.36 kJ/mol of free energy for initiation of the denaturating process. The absorption spectrum showed maximum at 280 nm, with ci%i cm of 4.8. The lectin was a potent agglutinin for rabbit erythrocytes, reacted against horse and ovine red cells, but it was inactive against cow, sheep, goat, dog, and human erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by fetuin (39.1 µg/mI), avidin (312.5 µg/ml), and N-acetyl-D-neuraminic acid (965.6 p.g/ml). This lectin was also highly toxic to mice upon intravenous injection (LD50 0.22 mgP/kg body weight) producing dyspnoea, tonic-clonic convulsions and flaccid paralysis prior to death, being this toxic activity dependent on free sulfydryl groups. The feeding trials conducted with female mice intubated with the fraction (Faso%) obtained by fractionation of the leaf crude extract with 0-30% solid ammonium sulfate, which is LTS rich, induced stunted growth of the animals compared to those intubated with 150 mM NaCI (control). The litter was similarly affected by drinking the milk, which apparently carries the toxic principle. The FO-30% had insecticidal action against the larvae of the bruchid beetle (Callosobruchus maculates), which spoiled the storage cowpea [Vigna unguiculata (L.) Walp.], suggesting a defensive physiological role for LTS.