The component of Carica papaya seed toxic to A. aegypti and the identification of tegupain, the enzyme that generates it.

Abstract

As Aedes aegypti transmits the etiologic agents of both yellow and dengue fever; vector control is considered essential to minimise their incidence. The aim of this work was to identify the component of Carica papaya seed toxic to A. aegypti, and the identification of tegupain, the enzyme that generates it. Aqueous extracts (1%, w/v) of the seed tegument and cotyledon of C. papaya are not larvicidal isolately. However, a mixture of 17μgmL(-1) tegument extract and 27μgmL(-1) cotyledon extract caused 100% larval mortality in a bioassay. The mixture was no longer larvicidal after the tegument extract was pre-treated at 100°C for 10min. The enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (Km 58.8μM, Kcat 28020s(-1), Kcat/Km 5×10(8)M(-1) s(-1)), and its activity increased with 2mM dithiothreitol (DTT), at 37°C, pH 5.0. The chelating agent EDTA did not modify the enzyme activity. Inhibition of tegupain by cystatin (Kiapp 2.43nM), E64 (3.64nM, 83% inhibition), and the propeptide N-terminal sequence indicate that the toxic activity is due to a novel cysteine proteinase-like enzyme, rendered active upon the hydrolysis of a cotyledon component of C. papaya seeds.