title: Fluorescence detection of hyaluronidase.
abstract: 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.
authors.affiliation: Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX 76107, USA. Rafal.Fudala@unthsc.edu
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
authors.forename: Mark E
authors.initials: ME
authors.lastname: Mummert
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
mesh_terms: D019793:Fluorescein / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000032:analysis* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D012235:Rhodamines / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; C029773:rhodamine B; D019793:Fluorescein