Analytical Chemistry Related to Biofunctional Research. Enzyme Activity Analysis of Soluble Proteins Using Non-Denaturing Two-Dimensional Electrophoresis.

Non-denaturing two-dimensional electrophoresis (2-DE) can be applied to the separation of soluble proteins with high resolution. The separated proteins retain their physiological functions. The present study indicates that soluble proteins of the bovine retina were separated by non-denaturing 2-DE, and the enzyme activities of esterase, dehydrogenase, superoxide dismutase and transferase were detected in the presence of each enzyme-specific substrate and chromophore. While the activities of esterase and dehydrogenase were inhibited by a cholinesterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), the transferase activity was not affected. The enzymes separated by non-denaturing 2-DE were analyzed by matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS). These results indicate that non-denaturing 2-DE is applicable to analyzing of the functions and structures of soluble proteins.