Related Experiment Videos
Fluorometric assay for alcohol sulfotransferase.
Wei-Ti Chen1, Ming-Chih Liu, Yuh-Shyong Yang
1Institute of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC.
Analytical Biochemistry
|March 16, 2005
Summary
A new fluorometric assay for alcohol sulfotransferase (AST) offers high sensitivity, enabling the detection of nanogram or picomole enzyme amounts. This method is valuable for enzyme studies and potential high-throughput screening.
Area of Science:
- Biochemistry
- Enzymology
- Analytical Chemistry
Background:
- Alcohol sulfotransferase (AST) plays a crucial role in various biological processes.
- Existing methods for AST activity measurement often lack sensitivity or are cumbersome.
- Development of a sensitive and efficient assay is needed for detailed kinetic studies and enzyme characterization.
Purpose of the Study:
- To develop and validate a novel, continuous fluorometric assay for alcohol sulfotransferase (AST).
- To assess the sensitivity and applicability of the assay for purified enzymes and crude biological extracts.
- To explore the potential of this assay for high-throughput screening.
Main Methods:
- A continuous fluorometric assay was established using a regenerated sulfuryl donor system.
- Recombinant phenol sulfotransferase (PST) was employed to regenerate 3'-phosphoadenosine 5'-phosphosulfate.
- Fluorescence intensity changes of 4-methylumbelliferone were monitored to quantify AST activity.
Main Results:
- The developed fluorometric assay demonstrated sensitivity comparable to radioactive assays for AST.
- The assay successfully measured AST activity in purified forms and crude extracts from pig liver, rat liver, and E. coli.
- The method accurately determined properties of human dehydroepiandrosterone sulfotransferase and arylsulfatase contamination.
Conclusions:
- A sensitive and reliable continuous fluorometric assay for alcohol sulfotransferase (AST) has been successfully developed.
- This assay is suitable for routine kinetic studies, enzyme characterization, and analysis of AST in various biological samples.
- The method holds promise for adaptation into a high-throughput screening procedure.