A trypsin inhibitor purified from Cassia leiandra seeds has insecticidal activity against Aedes aegypti

A trypsin inhibitor from Cassia leiandra seeds, named ClTI, was purified, characterized, and its insecticidal activity against Aedes aegypti evaluated. ClTI was purified by DEAE-Cellulose and trypsin-Sepharose 4B chromatography, with a 15.5-fold purification and 2.4% yield. ClTI is composed of a 19,484 Da polypeptide chain as revealed by mass spectrometry, it is not a glycoprotein, its amino acid sequence is similar to other Kunitz-type inhibitors, and it comprises 35% beta-sheets, 14% beta-turns, and 50% disordered secondary structures. ClTI is an uncompetitive inhibitor of bovine trypsin (IC50 of 33.81 x 10(-8)M, Ki of 6.25 x 10(-8) M) stable over a broad range of pHs (2.2-10.0) and temperatures (30-70 degrees C), but dithiothreitol led to a partial loss of the inhibitory activity. ClTI, at 4.65 x 10(-6) M, reduced in 50% the activity of the Ae. aegypti midgut proteases. ClTI also promoted acute toxicity on the 3rd instar larvae of Ae. aegypti, with an LC50 of 2.28 x 10(-2) M. Moreover, it caused a 24-h delay of the larvae development and 44% mortality after ten days of exposure. Altogether, these results suggest that ClTI has potential as a natural compound to control Ae. aegypti, a vector of several infection diseases.