Sh. Ashouri - Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Islamic Republic of Iran.
F. Zihnioglu - Department of Biochemistry, Faculty of Science, Ege University, Izmir, Turkey.
R. Farshbaf Pourabad - Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Islamic Republic of Iran.
E. Kocadag - Department of Biochemistry, Faculty of Science, Ege University, Izmir, Turkey.

The aim of the present work was to purify cysteine protease inhibitors from rapeseed (Brassica napus L.), with potential activity on digestive protease of Colorado Potato Beetle (CPB), Leptinotarsa decemlineata (Say). Ammonium sulfate precipitated proteinaceous fractions; ۳۰, ۵۰, ۷۰, and ۱۰۰% showed ۳۹.۰۷, ۵۷.۰۳, ۵۱.۴۷, and ۲۲.۴۴% inhibition on the fourth instar larval gut general protease activity, respectively. Fraction ۵۰% showed the highest inhibitory effect on digestive general protease activity of all developmental stages. Gel assays approved the inhibition of the enzyme activity. Fraction ۵۰% was purified by using various chromatography techniques; ion-exchange using DEAE, gel filtration and affinity using SiO۲-CPB larval gut homogenate. Three peaks of protein were eluted from ion exchange chromatography using NaCl step gradient, also from gel filtration chromatography. When Z-Ala-Arg-Arg-۴mßNA was used as cysteine protease substrate, the purification fold of second fraction of ion exchange chromatography was obtained ۲۴.۸۰, also the yield was ۵۹.۰۹%, the third fraction of gel permeation resulted in a ۲۵.۶۰ fold purification with ۲۸.۵۳% of recovery, and the fraction of affinity chromatography obtained a ۲۲.۷۲ fold purification and yielded ۳۶.۳۵%. In the SDS-PAGE, apparent molecular mass of purified proteins were ۳۴ and ۳۲ kDa by ion-exchange and ۲۴ and ۲۲ kDa by affinity. However, gel filtration was not an appropriate method in this study, because the purified protein band(s) were not observed on the gel. Consequently, these chromatography methods were appropriate methods to purification of inhibitor cystatins, specially affinity which was prepared by using CPB gut enzyme as ligand and obtained specific inhibitor proteins of CPB gut protease activity.

Chromatography, Colorado potato beetle, Enzyme inhibition, Protein, Rapeseed