Applicability of esterase activity change analysis using a combined method of non-denaturing two-dimensional electrophoresis and reverse staining

Abstract

By using a combinational method of non-denaturing two-dimensional electrophoresis (2DE) and reverse staining, various water-soluble esterases in the mouse liver were separated and detected. When the effects of Zn²⁺ and imidazole were investigated, Zn²⁺ increased esterase activity while imidazole had no effect on esterase activity. After separation using non-denaturing 2DE and reverse staining, esterase activity on the 2DE gel was also increased by Zn²⁺, whereas there was no effect on esterase activity by the treatment of imidazole. In addition, the activity of various esterases was inhibited by 1 mM Fe²⁺ using esterase activity staining after the combination of non-denaturing 2DE and reverse staining. Furthermore, by 1 mM Fe²⁺, the activities of various esterases separated and detected using the combined method were found to be suppressed to 34–42% of that in the absence of Fe²⁺ after analyzing substrate changes using fluorometry. Based on the above, the combination of non-denaturing 2DE and reverse staining is applicable to search for substances that inhibit various esterase activities.