Characterization of asclepain cI and asclepain cII by PMF MALDI - TOF analysis

OBREGÓN, D.; LIGGIERI, C.; TREJO, S.; VAIRO CAVALLI, S.; COLOMBO, M. L.; PRIOLO, N.S

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The aim of this work was to obtain the peptide mass fingerprints (PMF) of asclepain cI and asclepain cII, two plant proteases isolated from latex of Asclepias curassavica, by MALDI-TOF MS in order to compare them. The enzymes have similar physical and biochemical features. Purified proteases were separated by SDS PAGE. Selected bands were cut, washed and dried under vacuum. The gel fragments were treated with 0.1M NH4HCO3 and 10 mM DTT, centrifuged, washed with acetonitrile, and then incubated in darkness in 0.1M NH4HCO3 with 50 mM iodoacetamide for 20 min at 25ºC. Digestions were carried out with Trypsin and Lys-C during 12 h at 37ºC. The peptides obtained were recovered by centrifugation, washed with milli Q water and acetonitrile, dried in a SpeedVac, redissolved in 0.1% (v/v) TFA and analyzed by MALDI-TOF MS. PMF analysis showed the enzymes have some equivalent peaks, revealing a high degree of homology among them, which would reflect the presence of conserved domains in these plant peptidases belonging to Asclepiadaceae family. By means of MASCOT tool searches were made in order to identify the studied proteins with these tryptic maps. No identification was possible due to the slight information of plant cysteine proteinases consigned in the available databases. Results obtained represent an interesting contribution for PMF databases containing information on plant endopeptidases.