Analysis of recovery from enzyme activity inhibition by Fe²⁺ using nondenaturing two-dimensional electrophoresis

Abstract

Enzymes retain their activity even after separation by nondenaturing two-dimensional electrophoresis (2DE), along with fluorescent substances that can associate and dissociate from the enzymes. Esterase and sorbitol dehydrogenase, which retain their activity, can be reseparated by nondenaturing electrophoresis following the separation of mouse liver cytosolic proteins by nondenaturing 2DE. Fluorescent detection was achieved using enzymatic products such as 4-methylumbelliferone and the reduced form of nicotinamide adenine dinucleotide (NADH). Moreover, the activities of these two enzymes could be quantitatively reanalyzed even after separation via nondenaturing 2DE and fluorescent detection. The activities of esterase and sorbitol dehydrogenase were considerably suppressed by 1 mM Fe²⁺, reducing to 0.24-fold and 0.28-fold of their original levels, respectively. After washing out of Fe²⁺, esterase activity remained suppressed, further decreasing to 0.14-fold, whereas sorbitol dehydrogenase activity increased to 1.31-fold of its original suppressed level. These findings demonstrate that the current method of reseparating and reanalyzing enzymes is suitable for investigating the reversibility or irreversibility of enzyme activity in response to factors such as metal ions.