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Detection of Protease Activity by Fluorescent Peptide Zymography
Published on: January 20, 2019
Fluorescence detection of hyaluronidase
Rafal Fudala1, Mark E Mummert, Zygmunt Gryczynski
1Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA. Rafal.Fudala@unthsc.edu
Journal of Photochemistry and Photobiology. B, Biology
|June 28, 2011
Summary
We developed a novel hyaluronan (HA) sensor using fluorescence resonance energy transfer (FRET). This HA-FRET sensor effectively detects the presence and activity of hyaluronidase (HA-ase) enzyme.
Area of Science:
- Biochemistry
- Biophysics
- Materials Science
Background:
- Hyaluronan (HA) is a crucial glycosaminoglycan involved in various biological processes.
- HA degradation by hyaluronidase (HA-ase) plays a role in physiological and pathological conditions.
- Developing sensitive and reliable methods for HA-ase detection is important for biological research and diagnostics.
Purpose of the Study:
- To develop a fluorescence resonance energy transfer (FRET) based sensor for hyaluronan (HA).
- To investigate the properties of the HA-FRET sensor in detecting hyaluronidase (HA-ase) activity.
- To establish a ratiometric detection method for HA-ase using the HA-FRET sensor.
Main Methods:
- Labeling hyaluronan (HA) with two spectrally distinct fluorophores, fluorescein amine and rhodamine B amine.
- Utilizing Förster resonance energy transfer (FRET) between the two fluorophores for sensing.
- Measuring time-resolved fluorescence properties in the presence and absence of hyaluronidase (HA-ase).
- Analyzing changes in fluorescein and rhodamine emission intensities for ratiometric detection.
Main Results:
- The labeled HA (HA-FRET) exhibited significant FRET (>50%) in the absence of HA-ase.
- HA-ase activity led to a time-dependent decrease in FRET, indicated by fluorescein quenching and enhanced rhodamine emission.
- The kinetics of HA digestion by HA-ase were dependent on enzyme concentration.
- Simultaneous measurement of green (fluorescein) and red (rhodamine) emissions allowed for ratiometric detection of HA-ase.
Conclusions:
- The developed HA-FRET molecule serves as an effective FRET-based sensor for HA degradation.
- Ratiometric detection using HA-FRET enables sensitive and reliable quantification of HA-ase presence and activity.
- This approach can be utilized to construct a robust HA-ase sensing device.
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