TY - JOUR
T1 - Detection of hyaluronidase activity using fluorescein labeled hyaluronic acid and Fluorescence Correlation Spectroscopy
AU - Rich, Ryan M.
AU - Mummert, Mark
AU - Foldes-Papp, Zeno
AU - Gryczynski, Zygmunt
AU - Borejdo, Julian
AU - Gryczynski, Ignacy
AU - Fudala, Rafal
N1 - Funding Information:
This work was supported by NIH Grants R01EB12003 (Z.G.) and 1RO1HL090786-01A2 (J.B.). Zeno Foldes-Papp wants to thank the ISS team, in particular Drs. Beniamino Barbieri, Tiefeng You and Shih-Chu Jeff Liao.
PY - 2012/11/5
Y1 - 2012/11/5
N2 - 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.
AB - 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.
KW - Fluorescence
KW - Fluorescence Correlation Spectroscopy
KW - Hyaluronan
KW - Hyaluronidase
KW - Self-quenching
UR - http://www.scopus.com/inward/record.url?scp=84865432940&partnerID=8YFLogxK
U2 - 10.1016/j.jphotobiol.2012.07.007
DO - 10.1016/j.jphotobiol.2012.07.007
M3 - Article
C2 - 23018154
AN - SCOPUS:84865432940
SN - 1011-1344
VL - 116
SP - 7
EP - 12
JO - Journal of Photochemistry and Photobiology, B: Biology
JF - Journal of Photochemistry and Photobiology, B: Biology
ER -