Characterization of proteases from latex of Vasconcellea quercifolia using proteomics tools

TORRES M.J.; OBREGÓN W.D.; TREJO S.A.; NATALUCCI C.L. Y LÓPEZ L.M.I

Carlos Paz

Congreso; XLIV Reunión Anual de la SAIB; 2008

Sociedad Argentina de Investigaciones Bioquímica y Biología Molecular

The latex of Vasconcellea quercifolia (Caricaceae) contain several cysteine endopeptidases with very high proteolytic activity, which are the active compounds used by the plant as a defence mechanism against herbivorous insects. The crude extract presents several basic proteins with proteolytic activity and also other acid fractions without activity. The specific proteolytic activity, measured by BAPNA degradation, is 2.35 times higher in latex of V.quercifolia that in latex of Carica papaya. This higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. The crude extract was purified by cation exchange chromatography (SP-Sepharose HR, Tris-HCl 50mM, pH 7.5 and sodium tetrathionate 1mM, using a linear gradient of 0.15-0.40M NaCl) on FPLC system. Following SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequence of polypeptides was determined using automated chemical Edman degradation. The Peptide Mass Fingerprinting (PMF) of the fractions was achieved by using a trypsin (0,4 µg/μl) for 12 h at 37ºC and analyzed by MALDI-TOF TOF/MS. The PMF allowed their characterization and their comparison with other proteases. These analysis reveals the presence of several distinct cysteine proteinase isoforms in the latex of V. quercifolia.