A capillary zone electrophoretic method for the study of formation of a covalent conjugate between microcystin LR and protein phosphatase 2A.

Abstract

The study of conjugate formation between microcystin (MCYST)-LR and protein phosphatase (PP) 2A, which was isolated from bovine kidney and mouse brain, was achieved by using a highly efficient capillary zone electrophoretic (CZE) separation method. The MCYST-LR-PP2A conjugate (from bovine kidney) was resolved from its precursor after just 15 min of incubation. Moreover, the migration time, and, hence, the total analysis time, was less than 5 min. While the present findings of the time lag between conjugate formation and full inhibition are not novel, the CZE method does provide an alternative tool to HPLC with a higher separation efficiency to yield data for kinetic and mechanistic studies of the enzyme-toxin interaction. The CZE data reported here were found not to be adequately described by a first-order kinetic model. Moreover, the CZE method, which involves the use of a low ionic strength aqueous buffer, does not suffer from the drawback of the use of denaturing organic solvents such as those used in HPLC.