A Continuous Assay for the Spectrophotometric Analysis of Sulfotransferases Using Aryl Sulfotransferase IV

✍ Scribed by Michael D. Burkart; Chi-Huey Wong

• Publisher: Elsevier Science
• Year: 1999
• Tongue: English
• Weight: 87 KB
• Volume: 274
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1999.4264

✦ Synopsis

We have developed a continuous spectrophotometric coupled-enzyme assay for sulfotransferase activity. This assay is based on the regeneration of 3-phosphoadenosine-5-phosphosulfate (PAPS) from the desulfated 3-phosphoadenosine-5-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitrophenyl sulfate as the sulfate donor and visible spectrophotometric indicator of enzyme turnover. Here recombinant rat aryl sulfotransferase IV (AST-IV) is expressed, resolved to the pure ␤-form during purification, and utilized for the regeneration. The activity of ␤AST-IV to catalyze the synthesis of PAPS from PAP and p-nitrophenyl sulfate is demonstrated via capillary zone electrophoresis, and the kinetics of this reverse-physiological reaction are calculated. ␤AST-IV is then applied to the coupled enzyme system, where the steady-state activity of the commercially available Nod factor sulfotransferase is verified with an enzyme concentration study and substrate-specificity assays of N-chitoses. The potential applications of this 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 other tissues for specific sulfotransferase enzyme activity and substrate concentration.