Detection of hyaluronidase activity using fluorescein labeled hyaluronic acid and Fluorescence Correlation Spectroscopy

Ryan M. Rich, Mark Mummert, Zeno Foldes-Papp, Zygmunt Gryczynski, Julian Borejdo, Ignacy Gryczynski, Rafal Fudala

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The over-expression of hyaluronidase has been observed in many types of cancer, suggesting that it may have utility for diagnosis. Here we present a technique for the detection of hyaluronidase using Fluorescence Correlation Spectroscopy (FCS). Hyaluronan macromolecules (HAs) have been heavily labeled with fluorescein amine resulting in strong self-quenching. In the presence of hyaluronidase, HA is cleaved into smaller, fluorescein-labeled fragments and the self-quenching is released. Such cleavage is manifested by the increased average diffusion rate of the HA fragments, increased concentration of individual, fluorescent HA fragments, and increased intensity. All three of these properties are monitored simultaneously throughout FCS measurements, both as a function of time and hyaluronidase concentration. The method we present provides a sensitive measure of hyaluronidase activity and requires extremely small amounts of the HA substrate.

Original languageEnglish
Pages (from-to)7-12
Number of pages6
JournalJournal of Photochemistry and Photobiology B: Biology
StatePublished - 5 Nov 2012



  • Fluorescence
  • Fluorescence Correlation Spectroscopy
  • Hyaluronan
  • Hyaluronidase
  • Self-quenching

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