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Continuous fluorometric assay of phenol sulfotransferase
1Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198.
Analytical Biochemistry
|September 2, 1991
Summary
A new assay continuously monitors phenol sulfotransferase activity by observing the fluorescence quenching of resorufin during sulfation. This method is sensitive and reveals product inhibition, aiding enzyme characterization.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Phenol sulfotransferases (PSTs) catalyze crucial sulfation reactions.
- Existing PST assays require reaction termination and product isolation, limiting real-time analysis.
- PSTs exhibit diverse substrate specificities, necessitating versatile assay development.
Purpose of the Study:
- To develop a continuous, sensitive assay for phenol sulfotransferase activity.
- To investigate the sulfation of the fluorescent substrate, resorufin.
- To characterize kinetic parameters and product inhibition of PSTs using this novel assay.
Main Methods:
- Utilized resorufin as a fluorescent acceptor substrate for PSTs.
- Employed 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor.
- Monitored the decrease in resorufin fluorescence to track sulfation in real-time.
- Analyzed reaction progress curves to determine kinetic parameters and identify product inhibition.
Main Results:
- Resorufin sulfation by bovine lung PST resulted in a measurable decrease in fluorescence.
- The assay demonstrated high sensitivity (< 1 pmol/min) and allowed continuous recording.
- Product inhibition by the likely resorufin sulfate ester was observed (Ki < Km).
- Maximal rate for resorufin sulfation was estimated at 57 nmol/mg/min.
Conclusions:
- Continuous monitoring of PST activity is feasible using resorufin fluorescence quenching.
- This assay provides a sensitive and efficient method for enzyme characterization.
- The assay is particularly suitable for PSTs with optimal activity above pH 6.