Isolation of a trypsin inhibitor from Pterogyne nitens seeds

LUCILA YOSHIZAKI; M. FERNANDA TRONCOSO; CARLOTA WOLFENSTEIN-TODEL

San Carlos de Bariloche, Río Negro

Congreso; XXXIX Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) y XXXII Reunión Anual de la Sociedad Argentina de Biofísica (SAB).; 2003

 Protease inhibitors are pseudosubstrates with variable affinity for enzyme catalytic sites. Many inhibitors of vegetal origin have been studied, especially those belonging to the Leguminosae family because of their economic importance. With the purpose of isolating a trypsin inhibitor from the legume Pterogyne nitens seeds, these were ground and extracted for 18 h with 150 mM NaCl, 5 mM CaCl2. After centrifugation, the extract was submitted to affinity chromatography on a trypsin-agarose column, equilibrated with 20 mM Tris-HCl buffer, pH 8.2, 20 mM CaCl2, washed thoroughly with the same buffer and eluted with 100 mM glycine-HCl buffer, pH 2.6, 100 mM NaCl. The protein was further purified by gel filtration on a Superdex G-75 column, equilibrated and eluted with 150 mM NaCl, 5 mM CaCl2. A single band corresponding to a Mr of approximately 24 kDa was detected by SDS-PAGE. Tryptic inhibitory activity was measued using BAEE (N-Benzoyl-L-arginine ethyl ester) as a substrate, and chymotryptic inhibitory activity using BTEE (N-Benzoyl-L-tyrosine ethyl ester). The protein obtained was able to inhibit the activity of both trypsin and chymotrypsin.