Development of a capillary electrophoresis assay based on free sulfate determination for the direct monitoring of sulfoesterase activity

A capillary electrophoresis assay of sulfoesterase activity was developed t hat overcomes the main drawbacks encountered with the usual methods for sul fate determination in complex biological medium. Conditions are described a llowing direct measurement of inorganic sulfate that is enzymatically produ ced in the reaction mixture. The main features of this method are electroki netic sample introduction, which allows selective extraction of sulfate fro m the matrix into the separation capillary, counter-electroosmotic flow mig ration mode, indirect absorbance detection and use of an internal standard for quantitative performances. Likewise, perfect linearity was obtained for concentrations of sulfate up to 40 ppm. The limits of detection and quanti fication were 0.2 and 0.6 ppm, respectively. The run-to-run and day-to-day precision are 1 and 4.5%, respectively, for sulfate concentrations varying from 35 ppm down to 1 ppm. The accuracy was established for the synthetic p -nitrocatechol sulfate substrate by comparison with the classical spectroph otometric assay. The method was applied to the kinetic monitoring of the ac tivity of a sulfoesterase extracted from the marine mollusc Pecten maximus on fucoidan, a bioactive sulfated fucose-based polysaccharide derived from brown algae. For the first time, a sulfoesterase activity was shown to be e ffective on such sulfated polysaccharides. (C) 1999 Academic Press.