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'-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 beta-form during purification, and utilized for the regeneration. The activity of betaAST-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. betaAST-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.
Burkart
M D
MD
Skaggs Institute for Chemical Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.
Wong
C H
CH
D000818:Animals; D001322:Autoanalysis / Q000379:methods; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D019075:Electrophoresis, Capillary / Q000379:methods; D015723:Gene Library; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009578:Nitrobenzenes; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000032:analysis* / Q000378:metabolism
D009578:Nitrobenzenes; D009844:Oligosaccharides; D011994:Recombinant Proteins; C023379:4-nitrophenyl sulfate; D015238:Sulfotransferases; C077557:tyrosine-ester sulfotransferase