A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV

We have developed a continuous spectrophotometric coupled-enzyme assay for sulfotransferase activity. This assay is based on the regeneration of 3'-ph osphoadenosine-5'-phosphosulfate (PAPS) from the desulfated 3'-phosphoadeno sine-5'-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitr ophenyl sulfate as the sulfate donor and visible spectrophotometric indicat or of enzyme turnover. Here recombinant rat aryl sulfotransferase IV (AST-I V) is expressed, resolved to the pure beta-form during purification, and ut ilized for the regeneration. The activity of beta AST-IV to catalyze the sy nthesis of PAPS from PAP and p-nitrophenyl sulfate is demonstrated via capi llary zone electrophoresis, and the kinetics of this reverse-physiological reaction are calculated. beta AST-IV is then applied to the coupled enzyme system, where the steady-state activity of the commercially available Nod f actor sulfotransferase is verified with an enzyme concentration study and s ubstrate-specificity assays of N-chitoses. The potential applications of th is assay include rapid kinetic determinations for carbohydrate and protein sulfotransferases, high-throughput screening of potential sulfotransferase substrates and inhibitors, and biomedical screening of blood samples and ot her tissues for specific sulfotransferase enzyme activity and substrate con centration. (C) 1999 Academic Press.