Purification and physicochemical characterization of a novel trypsin inhibitor from Pinus ponderosa seeds

YOSHIZAKI LUCILA; TRONCOSO M. FERNANDA; SÁNCHEZ EDUARDO I.; WOLFENSTEIN-TODEL CARLOTA

Mar del Plata, Buenos Aires

Congreso; Reunión científica de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2007

SAIB

Plants synthesize a variety of molecules including proteinase inhibitors, which play an important role as defense proteins against plant predators. To isolate a trypsin inhibitor from Pinus ponderosa (PPTI) seeds, these were ground and extracted with 150 mM NaCl, 5 mM CaCl2. The extract was submitted to affinity chromatography on a trypsin-agarose column and a purified fraction of PPTI was obtained. SDS-PAGE under non reducing conditions showed two bands of 12,000 and 14,000 Da while in reducing conditions only one band of around 6,000 Da was observed. Only one peak was obtained by HPLC on a C18 column and on a Superdex S75 column, confirming the purity of the protein so that the two bands are considered as isoforms. Further separation of the reduced and modified chains by HPLC revealed two fractions with a mass of 6,800 and 4,600 Da by mass spectrometry analysis, indicating that PPTI is an heterodimer. Two amino-terminal sequences were determined by Edman degradation: SCDPQRLSACRDYLQR and GREEEE showing high homology with a 2 S albumin-like protein from Picea glauca and Pinus strobus. PPTI was able to inhibit trypsin and quimotrypsin. Furthermore, viability of human leukemia Jurkat cells was evaluated in the presence of increasing concentrations of PPTI. Results demonstrated that this protein (640 ug/ml) has a cytotoxic effect on Jurkat cells.