Sulfotransferase 5 Phenolsulfotransferase

Phenolsulfotransferase catalyzes the transfer of active sulfate from 3 -phosphoadensine 5 -phosphosulfate to various phenols and catechols. Honkasalo and Nissinen (1988) developed an assay that is suitable for measuring both the thermolabile (TL) and thermostable (TS) isoforms of phenol sulfotransferase. Both are active toward phenols, while the TL form also conjugates catechols including dopamine. 

The product, p-nitrophenylsulfate, is separated from p-nitrophenyl by chromatography on a Viosfer-ODS column (4 mm x 150 mm, 5 /xm). The mobile phase contained 25% methanol in 50 mM sodium phosphate (pH 3.0). The flow rate was 1.5 mL/min, and the column effluent was monitored at 300 nm. 

The reaction mixture in a total volume of 250 yL contained 50 /xL of 10 mM sodium phosphate buffer (pH 7.2), 50 /xL of enzyme preparation, and 100 /xL of 50 fiM 3 -phosphoadenosine 5 -phosphosulfate. The reaction was started by adding 50 /xL of 12.5 mM p-nitrophenol (when assaying the thermo-labile form) or 50 /xL of 50 yM p-NP (when determining the thermostable form). After incubation for 30 minutes at 37°C, the reaction was stopped by the addition of 25 /xL of 4 M perchloric acid. After centrifugation, a 20 /xL aliquot was injected into the HPLC system. Formation of product was linear with time for both forms of enzyme for up to 45 minutes, and with protein amount up to 0.5 mg. 

The enzyme source was livers of female rats. Livers that had been kept frozen at -80°C were homogenized in 1 5 (w/v) ice-cold 10 mM sodium phosphate buffer (pH 7.2) containing 10 mAf dithiothreitol. The homogenate was centrifuged at 15,000g for 20 minutes at 4°C, and the supernate was 

In the assay described by Khoo et al. (1990), 7V-acetyldopamine sulfate was separated within 7 minutes from 7V-acetyldopamine by chromatography on a Hypersil-ODS microbore column (2.1 mm x 100 mm, 5 /xm). The mobile phase contained 1.2 acetic acid and 1 mM EDTA at pH 4.4. The flow rate was 0.6 mL/min. An electrochemical detector was set at a sensitivity of 2 nA and a working potential of 0.8 V. The detector response was calibrated by using a standard of 7V-acetyldopamine-[35S]sulfate that had been prepared in the laboratory. The standard curve was linear in the range of 10 to 140 pmol. 

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