title: Detection of hyaluronidase activity using fluorescein labeled hyaluronic acid and Fluorescence Correlation Spectroscopy.
abstract: 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.
authors.affiliation: Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Rich
authors.forename: Mark
authors.initials: M
authors.lastname: Mummert
authors.forename: Zeno
authors.initials: Z
authors.lastname: Foldes-Papp
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: Julian
authors.initials: J
authors.lastname: Borejdo
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
mesh_terms: D000818:Animals; D002417:Cattle; D057075:Enzyme Assays / Q000191:economics / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013997:Time Factors
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase