Purification and biological effects of l-amino acid oxidase isolated from Bothrops insularis venom

Abstract

Bothrops insularis is a snake from Ilha da Queimada Grande, an island located about 20 miles away from the Southeastern coast of Brazil. Compared with other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood, and so far, no fraction from this venom with amino acid oxidase activity had been isolated or its biological activity tested. We investigated the biochemical and biological effects of one l-amino acid oxidase enzyme isolated from B. insularis snake venom (BiLAO), which was purified using HPLC and sequence grade. We also evaluated the renal effects induced by BiLAO. Chromatographic profile of B. insularis whole venom disclosed seven main fractions (I, II, III, IV, V, VI and VII) and the main LAO enzymatic activity was detected in fraction II. The group treated with BiLAO showed a decrease in perfusion pressure ( C 120=110.28±3.69; BiLAO 120=82.2±5.6 mmHg*); renal vascular resistance ( C 120=5.48±0.53; BiLAO 120=4.12±0.42 mmHg/mL/g/min*), urinary flow ( C 120=0.160±0.020; BiLAO 120=0.064±0.012 mL/g/min*), glomerular filtration rate ( C 120=0.697±0.084; BiLAO 120=0.176±0.017 mL/g/min*), sodium ( C 120=79.76±0.56; BiLAO 120=65.39±6.19%*), potassium ( C 120=69.94±6.86; BiLAO 120=60.26±2.24%*) and chloride tubular reabsortion ( C 120=78.53±2.33; BiLAO 120=64.58±6.68%*). Acute tubular necrosis foci were observed in the group treated with the LAO fraction of the B. insularis snake venom. Some findings have the same morphological aspect of apoptosis, more evident cortically; otherwise, reversible degenerative phenomena represented by hydropic ballooning with extensive cytoplasmic vacuolization and discontinuity of the cell brush borders in the proximal tubular epithelium were observed; furthermore, necrotic detachment of these cells into the tubular lumina, and increased amount of protein deposits in the distal and proximal tubules were observed. In conclusion, the slowness of blood flow and of glomerular filtration resulted in more time for filtration and tubular reabsorption, with elevation of the total percentage of sodium and chlorine reabsorption. The maintenance of the decrease in glomerular filtration rate would determine the subsequent decreases, which were noticed in these parameters. The necrosis observed was the result of damage cell induced by l-amino acid oxidase isolated from B. insularis venom.