Factors influencing the recovery of dopamine sulfate in the assay of phenol sulfotransferase.
Phenol sulfotransferase activity is often measured by incubating dopamine with a source of the enzyme and the sulfate donor 35S-3'-phosphoadensine-5'-phosphosulfate and then separating the product, 35S-dopamine sulfate, from the substrates using precipitation with barium hydroxide and zinc sulfate. Using dopamine sulfate standards and high performance liquid chromatography with dual-electrode electrochemical detection, we investigated the effects of several parameters on product recovery obtained with this procedure. Amounts of precipitants needed to produce maximal sample-to-blank ratios were determined using crude enzyme preparations from rat brain and human platelets incubated under conditions generating approximately 3 nM dopamine sulfate. Under these conditions, recovery of 3H-DAS standards was 70-80%. Increasing either the concentration of dopamine sulfate or the amounts of precipitants used resulted in concentration-dependent decreases in dopamine sulfate recovery. These data indicate that the recovery of product in this assay may vary with assay conditions, and should be carefully monitored and optimized. Caution should be exercised when using substrates other than dopamine in this assay procedure, since similar factors might also contribute to variable recovery of other products.
Toth
L A
LA
Kao
G
G
Elchisak
M A
MA
D000818:Animals; D015239:Arylsulfotransferase; D011232:Chemical Precipitation; D004298:Dopamine / Q000031:analogs & derivatives* / Q000032:analysis; D006801:Humans; D051381:Rats; D013466:Sulfurtransferases / Q000032:analysis*
C022807:dopamine 4-O-sulfate; C007558:dopamine 3-O-sulfate; D013466:Sulfurtransferases; D015239:Arylsulfotransferase; D004298:Dopamine