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Measurement of specific protease activity utilizing fluorescence polarization
L M Levine1, M L Michener, M V Toth
1Monsanto Corporate Research, St. Louis, Missouri, USA.
Analytical Biochemistry
|April 5, 1997
Summary
A novel fluorescence polarization assay quantifies human cytomegalovirus protease activity by measuring peptide substrate cleavage. This high-throughput screening method efficiently detects protease inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- Human cytomegalovirus (HCMV) protease is a key target for antiviral therapies.
- Accurate and efficient methods are needed to screen for HCMV protease inhibitors.
Purpose of the Study:
- To develop a novel, homogenous fluorescence polarization assay for measuring HCMV protease activity.
- To validate the assay's utility for high-throughput screening of potential inhibitors.
Main Methods:
- A dual-labeled peptide substrate (biotinylated and fluorescein-labeled) was synthesized.
- Proteolytic cleavage by HCMV protease was detected by measuring changes in fluorescence polarization upon avidin binding.
- The assay was used to evaluate the inhibitory activity of 3,4-dichloroisocoumarin.
Main Results:
- The assay generated a polarization signal proportional to the extent of substrate cleavage.
- Cleaved substrate yielded low polarization, while uncleaved substrate bound to avidin produced high polarization.
- The assay successfully detected inhibition by 3,4-dichloroisocoumarin.
Conclusions:
- A robust and convenient fluorescence polarization assay for HCMV protease activity has been established.
- The assay is suitable for high-throughput screening due to its homogenous nature and lack of interference from absorptive compounds.