Purification of a novel trypsin inhibitor from Peltophorum dubium seeds, with capacity to trigger lymphoma cell apoptosis”

M. FERNANDA TRONCOSO; PAULA CERDÁ ZOLEZZI; ULF HELLMAN; CARLOTA WOLFENSTEIN-TODEL

Carlos Paz, Córdoba

Congreso; XXXVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).; 2002

  The initial purpose of this study was to isolate a lectin from Peltophoum dubium seeds. An extract of the seeds was submitted to affinity chromatography on a thyroglobulin-agarose column, followed by reverse phase HPLC. Two bands corresponding to Mr 20,000 and 22,000 were detected by SDS-PAGE, which were not resolved by any of the chromatographic techniques assayed. The amino-terminal sequence of both bands was identical and displayed 73 % identity with Kunitz-type soybean trypsin inhibitor (SBTI). The same bands were obtained by affinity chromatography on a trypsin-agarose column. Mass spectrometry analysis of tryptic digests of the two bands showed coincidence of 16 peaks, suggesting that they are closely related. Trypsin and chymotrypsin inhibitory activities of PDTI were measured and the respective Ki values were 1.6 x 10-7 M and 1.3 x 10-5 M. Lectin-like activity of PDTI and SBTI was detected by hemagglutination of trypsin-treated rabbit erythrocytes in presence of Ca2+, and this activity was inhibited by sialic acid containing compounds. PDTI and SBTI caused apoptosis of Nb2 lymphoma cells, as demonstrated by a decrease of viability, assessment of  DNA hypodiploidy, electrophoretic analysis of DNA fragmentation and detection of caspase-3-like activity.