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A new protease activity assay using fluorescence polarization

R Bolger1, W Checovich

  • 1PanVera Corporation, Madison, WI.

Biotechniques
|September 1, 1994
PubMed
Summary
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Fluorescence polarization (FP) technology offers a highly sensitive, real-time assay for protease activity. This novel method requires no sample manipulation, outperforming traditional fluorescent assays for various protease classes.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Assay Development

Background:

  • Protease activity assays are crucial in biological research and drug discovery.
  • Existing nonradioactive assays often lack sensitivity or require complex procedures.
  • Fluorescence polarization (FP) measures changes in molecular volume, offering a potential improvement.

Purpose of the Study:

  • To develop a novel, highly sensitive protease activity assay using FP technology.
  • To compare the sensitivity of the FP-based assay with a conventional non-FP fluorescent assay.
  • To demonstrate the assay's utility for different protease classes.

Main Methods:

  • Utilized fluorescence polarization (FP) to detect changes in molecular volume.
  • Employed fluorescein thiocarbamoyl (FTC)-casein as a substrate, measuring cleavage into smaller peptides.

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  • Performed real-time kinetic and single-time-point measurements.

    • Main Results:

    • The FP protease assay demonstrated significantly higher sensitivity (twofold to twentyfold) compared to a non-FP fluorescent assay.
    • The assay allowed for real-time monitoring of protease activity, enabling kinetic analysis.
    • Successfully measured the activity of serine proteases, sulfhydryl proteases, and acid proteases.

    Conclusions:

    • FP technology provides a sensitive, separation-free method for protease activity assessment.
    • This assay is versatile, applicable to common protease classes.
    • The developed FP assay offers advantages in speed, sensitivity, and simplicity over existing methods.