Characterization of a new cysteine protease from latex of Vasconcellea quercifolia (Caricaceae)

TORRES M.J., TREJO S., NATALUCCI C. AND LÓPEZ L.M.I.

Mar del Plata

Congreso; XLIII Reunión Anual de la SAIB; 2007

Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular

Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. The crude extract of Vasconcellea quercifolia was characterized by SDS-PAGE and isoelectrofocusing-zimogram revealing the presence of several basic proteins with high proteolytic activity and also other acid fractions without proteolytic activity. The most basic component (quercifoliain II) present in latex extracts was purified by cation exchange chromatography (SP-Sepharose HR, Tris-HCl 50mM, pH 7.5 and sodium tetrathionate 1mM) using FPLC system. Homogeneity was evaluated by 2D-PAGE and mass spectroscopy. Molecular mass of the enzyme was 23.984 Da (MALDI-TOF TOF/MS) and its isoelectric point was > 10.25. The N-terminal sequence of quercifoliain II (IPASIDWRQKGAVTPIRLQG) by BLAST analysis showed a great deal of sequence similarity to the other cysteine endopeptidases belong to Vasconcellea species, being 90% identical to proteases from Vasconcellea heilbornii and Vasconcellea stipulata . The Peptide Mass Fingerprinting (PMF) of the enzyme 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 differentiation of other proteases.