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A simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence
Koji Enomoto1, Hiroyuki Okamoto, Yoshito Numata
1Discovery Research Laboratories, Shionogi & Co., Ltd., 5-12-4, Sagisu, Fukushima-ku, Osaka 553-0002, Japan. koji.enomoto@shionogi.co.jp
Journal of Pharmaceutical and Biomedical Analysis
|February 24, 2006
Summary
A new, rapid assay simplifies heparanase activity measurement. This homogeneous time-resolved fluorescence assay aids research into heparanase (HPSE) in cancer and inflammation.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Heparanase enzyme activity is crucial for tumor and inflammatory cell invasion.
- Existing heparanase assays are complex and time-consuming.
Purpose of the Study:
- To develop a simple, rapid, and efficient assay for measuring heparanase activity.
- To overcome limitations of current heparanase detection methods.
Main Methods:
- Utilized homogeneous time-resolved fluorescence (HTRF) technology.
- Employed biotin and europium cryptate-labeled heparan sulfate proteoglycan as substrate.
- Detected degradation using XL665-labeled streptavidin and fluorescence measurements.
Main Results:
- The developed assay is simple, rapid, and suitable for high-throughput screening.
- Successfully measured heparanase activity in murine melanoma cell extracts.
- Demonstrated efficient separation of degradation products from uncleaved substrates.
Conclusions:
- This novel HTRF assay provides a significant advancement for studying heparanase.
- The assay facilitates research into the role of heparanase in disease pathogenesis.
- Enables rapid processing of large sample numbers for heparanase activity determination.
