Enterolobin, a hemolytic protein from Enterolobium contortisiliquum seeds (Leguminosae - Mimosoideae): purification and characterization

RESUMO

Hemolytic and phospholipase D activities were found in the saline extract of Enterolobium contortisiliquum seeds. The hemolytic activity is due to a protein which was named enterolobin. This protein was highly purified by extraction with 0.15 M NaCl, precipitation with ammonium sulphate from 0 to 33% of highly purified by extraction with 0.15 M NaCl, precipitation with ammonium sulphate from 0 to 33% of saturation, batch separation by adsorption on DEASE - cellulose and gel filtration chromatography on Sephadex G-100 or G-150. In the batch separation the fraction showing hemolytic activity was not adsorbed by the resin while the fraction with phospholipase activity was. In this manner it was shown that those two activities were due to different proteins. Mouse erythrocytes were less susceptible to hemolysis by enterolobin than human and rabbit erythrocytes. The hemolytic activity was rapidly lost at or above 55§C and in extreme acid (1.6) and basic (10.8) pHs. The following characteristics of purified enterolobin were determined molecular weights of 55.000 D (by SDS-PAGE), 59.800 D (by gel filtration) and 51.300 D (by HPLC); pI=7,0; Gln as the N-terminal amino acid residue; high levels of Asp(Asx), Glu(Glx), Ser and Thr residues and low levels of Cys and Met residues. Similarities were noticed between enterolobin and crotin, a hemolytic protein of Croton tiglium seeds