Measurement of specific protease activity utilizing fluorescence polarization

Anal Biochem. 1997 Apr 5;247(1):83-8. doi: 10.1006/abio.1997.2047.

Abstract

A fluorescence polarization assay was designed to measure proteolytic cleavage of a specific peptide substrate for human cytomegalovirus protease. The peptide substrate was derivatized by biotinylation of a gamma-aminobutyric acid-modified amino-terminus and labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein at the carboxy-terminus. Incubation of this substrate with recombinant human cytomegalovirus protease and subsequent addition of egg white avidin produced a polarization signal that was proportional to the relative amounts of cleaved and uncleaved substrate. The uncleaved substrate produced a high polarization value upon binding to avidin, whereas the cleaved, low-molecular-weight fluorescently tagged peptide that cannot bind to avidin produced a low polarization value. The inhibitory activity of a 3,4-dichloroisocoumarin against the protease was evaluated by comparing the change in polarization with a noninhibited control. The fluorescence polarization protease assay does not suffer from interference due to the presence of absorptive interferants making this a convenient, homogenous assay for high throughput screening.

MeSH terms

  • Amino Acid Sequence
  • Avidin
  • Biotin
  • Cytomegalovirus / enzymology
  • Endopeptidases / analysis*
  • Endopeptidases / metabolism
  • Evaluation Studies as Topic
  • Fluorescein
  • Fluoresceins
  • Fluorescence Polarization / methods*
  • Humans
  • Kinetics
  • Molecular Structure
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism
  • Substrate Specificity

Substances

  • Fluoresceins
  • Oligopeptides
  • Avidin
  • Biotin
  • Endopeptidases
  • Fluorescein