Spectrofluorometric assay for monoamine-preferring phenol sulfotransferase (SULT1A3).
A continuous and real-time fluorometric assay for monoamine-preferring phenol sulfotransferase (SULT1A3) was developed. The methodology was based on the coupling of SULT1A1 to regenerate 3'-phosphoadenosine-5'-phosphosulfate (PAPS) using 4-methylumbelliferyl sulfate (MUS) as a sulfuryl group donor. The fluorophore product (4-methylumbelliferone, MU) was continuously produced and monitored when SULT1A3 catalyzed dopamine sulfation with PAPS. The optimal conditions of this turnover reaction and substrate inhibition of SULT1A3 were also determined. This coupled-enzyme assay allows the continuous measurement of initial reaction velocity and the sensitivity is comparable to that of end-point radioactive isotope assay.
Lu
Lu-Yi
LY
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC.
Hsu
Yen-Chang
YC
Yang
Yuh-Shyong
YS
D000818:Animals; D015239:Arylsulfotransferase / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*; D015238:Sulfotransferases / Q000378:metabolism*
C019492:4-methylumbelliferyl sulfate; D006923:Hymecromone; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; D015239:Arylsulfotransferase; C403170:monoamine-sulfating phenol sulfotransferase