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 that overcomes the main drawbacks encountered with the usual methods for sulfate determination in complex biological medium. Conditions are described allowing direct measurement of inorganic sulfate that is enzymatically produced in the reaction mixture. The main features of this method are electrokinetic sample introduction, which allows selective extraction of sulfate from the matrix into the separation capillary, counter-electroosmotic flow migration 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 quantification 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 spectrophotometric assay. The method was applied to the kinetic monitoring of the activity 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 effective on such sulfated polysaccharides.
Saillard
S
S
Laboratoire d'Electrochimie et Chimie Analytique, UMR CNRS 7575, ENSCP, 11, rue Pierre et Marie Curie, Paris cedex 05, 75231, France.
Gareil
P
P
Jozefonvicz
J
J
Daniel
R
R
D000818:Animals; D004573:Electrolytes; D019075:Electrophoresis, Capillary / Q000379:methods*; D008974:Mollusca / Q000201:enzymology; D011134:Polysaccharides / Q000378:metabolism; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013431:Sulfates / Q000032:analysis*; D015238:Sulfotransferases / Q000378:metabolism*
D004573:Electrolytes; D011134:Polysaccharides; D013431:Sulfates; D015238:Sulfotransferases