Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening
Hammond, Edward, Li, Cai Ping, & Ferro, Vito (2010) Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening. Analytical Biochemistry, 396(1), pp. 112-116.
The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a Km of 46 μM and a kcat of 3.5 s−1 with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a Ki of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.
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|Item Type:||Journal Article|
|Keywords:||heparanase, fondaparinux, colorimetric assay, heparan sulfate, enzyme kinetics|
|Subjects:||Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > BIOCHEMISTRY AND CELL BIOLOGY (060100) > Analytical Biochemistry (060101)|
|Divisions:||Past > QUT Faculties & Divisions > Faculty of Science and Technology|
Past > Schools > School of Physical & Chemical Sciences
|Copyright Owner:||Copyright 2009 Elsevier|
|Copyright Statement:||This is the author’s version of a work that was accepted for publication in Analytical Biochemistry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Analytical Biochemistry, [VOL 396, ISSUE 1, (2010)] DOI: 10.1016/j.ab.2009.09.007|
|Deposited On:||12 Oct 2009 13:08|
|Last Modified:||06 Nov 2013 22:03|
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