Isolation of a trypsin inhibitor from Peltophorum dubium seeds with lectin-like activity

TRONCOSO, MARÍA FERNANDA; CERDÁ ZOLEZZI, PAULA; WOLFENSTEIN-TODEL, CARLOTA

La Haya, Holanda.

Congreso; XVIth International Symposium on Glycoconjugates (GLYCO XVI); 2001

Lectins and proteinase inhibitors are frequently found in Leguminosae seeds. Proteinase inhibitors are classified in several families, including Kunitz-type inhibitors which are Mr 20000 proteins. They are useful tools in the study of different biochemical processes, such as platelet aggregation, blood coagulation, fibrinolysis and inflammation. The initial aim of this work was to isolate a lectin. An extract of Peltophorum dubium seeds was submitted to affinity chromatography on a tyroglobulin-agarose column. The fraction with hemagglutinating activity on rabbit erythrocytes was further purified on a Superdex-75 column. Two bands were detected on SDS-PAGE, corresponding to 20 and 22 kDa, both in presence or absence of reducing agent. Nevertheless, only one peak was obtained by HPLC on a C4 column. The amino-terminal sequence of both bands was DFVLDVEGIFLR NGGIYYILPD... This sequence has 73 % identity with Kunitz-type soybean trypsin inhibitor. Therefore, trypsin inhibitory activity of the isolated protein was determined using BAEE as substrate and the Ki obtained was 1.6 x 10-7 M. Hemagglutinating activity was Ca2+ dependent and hemagglutination inhibition assays showed that neuraminic acid, tyroglobulin, colominic acid, triganglioside GT1b and heparin  were potent inhibitors. These results encourage future studies about the effects of this protein on different biological systems, in view of its dual lectin and protease inhibitor properties.