“Purification and Partial Characterization of Philibertain I, a Cysteine Protease from Latex of Philibertia gilliessi Hook. Et arn. Asclepiadaceae”

SEQUEIROS, C.; FERNÁNDEZ TEYELDÍN, M.; TORRES, M. J.; TREJO S. A.; LÓPEZ, L. M. I., AND NATALUCCI, C. L.

Bariloche, Río Negro, Argentina

Congreso; XXXIX Reunión Anual de SAIB y I Simposio de Proteinas; 2003

SAIB

The crude extract obtained from latex of Philibertia gilliesii was partially purified by acetone fractionation as a first purification step that deprived the enzyme preparation of most soluble sugars, gums and low molecular weight peptides, retaining 73% of protein with 90% of proteolytic activity. Isoelectric focusing followed by zymogram analysis revealed the presence of several basic bands showing caseinolytic activity. According to pI values of the proteolytic components (8.75, 8.9, 9.8, and >10.25), the crude extract was purified by FPLC using a cation exchanger (SP-Sepharose Fast Flow equilibrated with 50 mM Tris-Gly buffer). To improve resolution, several saline gradients and different pH values were tested and the best resolution was obtained when a 0.1-0.4 M sodium chloride gradient at pH 9.0 was applied. The highest basic active fraction (philibertain I) was homogeneous by IEF, SDS-PAGE and mass spectrometry (Mr 23530) .