Digestibilidade in vitro e toxicidade de lectinas vegetais para náuplios de Artemia sp.

Abstract

The toxicity to Artemia sp. nauplius and the in vitro digestibility of lectins isolated from Canavalia ensiformis, C. brasiliensis, Cratylia floribunda, Dioclea guianensis, D. virgata and Artocarpus integrifolia seeds were studied. The results showed that the lectins isolated from Cratylia floribunda, Dioclea guianensis and D. virgata were the most toxic, leading to LD50 of 400, 450 and 450 µg/ml, respectively. The degree of toxicity decreased in presence of glucose, showing that the carbohydrate-specific binding sites were involved in the toxic mechanism. Direct immunofluorescence of the artemia nauplius pretreated with glucose/mannose lectins and FITC-antibody against Con Br revealed that these lectins attached to the nauplius surface. Nevertheless, no correlation was observed between the degree of lectin toxicity and the fluorescence intensity. The in vitro lectin digestibility was conducted with pepsin (2 hours) + trypsin and chymotrypsin (3 hours), both treatments in presence or absence of glucose, Ca++ and Mn++. The degree of digestibility was initially measured by SDS-PAGE but, because of the low resolution of this technique on quantifying the susceptibility of proteins to enzymatic treatments, it was replaced by molecular exclusion chromatography on Superose 12 HR 10/30 column, carried out on a FPLC system. The degree of digestibility was established by comparing the areas of the chromatograms obtained from untreated and hydrolysed lectins. The areas were calculated by trapezoidal proximation of the plotted chromatograms. The results show that glucose/mannose lectins were 53 to 84% digested. Contrary, jacalin showed to have a much high degree of resistance (> 90%). The tridimensional structure of jacalin was essentialy tetrameric (91%) at pH 8,1, while the glucose/mannose lectins predominated as dimers (72 - 96%). The presence of glucose Ca++ and Mn++ in the digestibility assay showed little importance on increasing the stability of lectins to enzymatic treatments. Dot-blotting analysis of glucose/mannose lectins demonstrated that many peptides fractions resulting from hydrolysis of these proteins were still recognized by the antibody raised against the native lectin from Canavalia brasiliensis (Con Br). No clear correlation was observed between the lectin digestibility and the toxicity to Artemia sp. nauplius.