PRIMARY STRUCTURE OF TRYPSIN-INHIBITORS FROM SICYOS-AUSTRALIS

Three trypsin inhibitors from Sicyos australis, have been isolated, pu rified and sequenced. Following protein extraction with ammonium sulph ate, the mixture of inhibitors was separated from other proteins by tr ypsin-affinity chromatography. Subsequent purification of the individu al inhibitors was accomplished by reversed-phase HPLC. The primary str uctures of each inhibitor were elucidated by a combination of protein sequencing and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) on both the untreated and the reduced and S-carboxymethylated inhibitors. All three inhibitors show extensi ve sequence similarity with inhibitors from cultivated Cucurbitaceae s pecies, although there are a number of novel residues present. One of the inhibitors has a blocked N-terminus (pyroglutamic acid) and the us e of MS-MS was crucial to the elucidation of its primary structure. ES I-MS was further used to characterize the non-covalent complex between one of the inhibitors and trypsin.