Continuous fluorometric assay of phenol sulfotransferase.
Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the sulfation of the fluorescent compound, resorufin, is investigated. Reaction of PAPS with resorufin, catalyzed by bovine lung phenol sulfotransferase, bleaches the emission of this acceptor at the pH of the reaction (pH 6.4 optimum). It is thereby possible to continuously record the sulfation reaction. Analysis of single progress curves by integrated replot can be used to determine the initial velocities and also indicates the formation of a product inhibitor, probably resorufin sulfate ester, with Ki less than Km. Sensitivity of the reaction is less than 1 pmol/min. The maximal rate of resorufin sulfation by the bovine lung enzyme is estimated at 57 nmol/mg/min, which is 10% of the rate with an optimal substrate 2-naphthol. This assay may be most sensitive for phenol sulfotransferases with optimal activities at greater than pH 6, due to the acid-base properties of resorufin (pK alpha 6), which becomes nonfluorescent upon protonation.
Beckmann
J D
JD
Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198.
D000818:Animals; D015239:Arylsulfotransferase / Q000032:analysis*; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000201:enzymology; D009284:Naphthols / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D019800:Phenol; D010636:Phenols / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013455:Sulfur / Q000378:metabolism; D013997:Time Factors
D009284:Naphthols; D010078:Oxazines; D010636:Phenols; D019800:Phenol; D010724:Phosphoadenosine Phosphosulfate; C014180:resorufin; D013455:Sulfur; D015239:Arylsulfotransferase; C028405:2-naphthol