CHARACTERIZATION OF THE PLASMODICIDAL ACTIVITY OF BASIC PHOSPHOLIPASES FROM BOTHROPS jararacussu VENOM

Snake venom, Phospholipases A2, Plasmodium falciparum.

Malaria is a neglected and endemic disease in underdeveloped countries. The infection is caused by parasites of the genus Plasmodium, annually, thousands of people are infected and many of these die as a result of complications caused by this pathology. With this information, we emphasize the importance of prospecting new biomolecules that are effective for the treatment of this disease. The objective of the present work was to isolate and purify two molecules, namely: BthTX I and II (two basic phospholipase A2) from the venom of the Bothrops jararacussu snake. The purification of PLA2 was carried out in 2 chromatographic steps, the first being ion exchange chromatography on CM-Sepharose resin, followed by reverse phase chromatography on a C-18 column, in the first step the basic fractions of interest were extracted and these were rechromatographed in reverse phase chromatography. On SDS-PAGE, molecules with an apparent molecular mass of 15 kDa, compatible with snake phospholipases A2, were observed. Enzyme activity was evaluated using the chromogenic substrate 4N3OBA and demonstrated that BthTX II is an enzymatically active phospholipase A2. Then, the antiparasitic potential of the isolated proteins was evaluated in vitro against intraerythrocytic forms of Plasmodium falciparum by the cyber green method and cytotoxicity assay against THP-1 and HepG2 cells by the MTT method. The results of antiparasitic inhibition against P. falciparum of PLA2 did not show inhibition at the highest concentration tested (IC50>100 μg/mL). When evaluating the cytotoxicity against the HepG2 and THP-1 strains, it was observed that the molecules do not present cytotoxicity. The results of the work show that the methodologies used for the fractionation and purification of the molecules were effective, as for the biological activity, the two phospholipases A2 need an amount above (100 μg/mL) to reach the (IC50), when compared to other results in the literature with snake venom molecules, this concentration can be considered moderately high. As a conclusion, we have the two basic phospholipase A2 proteins, BthTX-I and BthTX-II isolated in a high degree of purity, and an alternative to improve the specificity of the activity of these molecules would be to make a rational design of peptides from these A2 phospholipases.