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Detection of Protease Activity by Fluorescent Peptide Zymography
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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
PubMed

We labeled hyaluronan (HA) with two fluorophores, fluorescein amine and rhodamine B amine. These two fluorophores are suitable for a fluorescence (Foerster) resonance energy transfer (FRET) which results in a fluorescein quenching and an enhanced rhodamine emission. Such labeled HA (HA-FRET) is a potential sensor for HA degradation. We studied fluorescence properties of HA-FRET in the absence and presence of hyaluronidase enzyme (HA-ase). The time-resolved fluorescence measurements indicate more than 50% of FRET in the absence of HA-ase. In the presence of HA-ase FRET decreases with time, and relative fluorescence intensities of fluorescein and rhodamine shifts to fluorescein indicating a release of FRET. The kinetics of the digestion process of HA by HA-ase depends on the concentration of the enzyme. We demonstrate that simultaneous measurements of green and red emission of HA-FRET can be used in ratio metric detection of the HA-ase presence and activity. This in turn, can be utilized for the construction of a robust but reliable HA-ase sensing device.

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