Document Detail

Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening.
MedLine Citation:
PMID:  19748475     Owner:  NLM     Status:  MEDLINE    
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 K(m) of 46 microM and a k(cat) of 3.5 s(-1) with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a K(i) 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.
Edward Hammond; Cai Ping Li; Vito Ferro
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Publication Detail:
Type:  Journal Article     Date:  2009-09-11
Journal Detail:
Title:  Analytical biochemistry     Volume:  396     ISSN:  1096-0309     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2010 Jan 
Date Detail:
Created Date:  2009-11-25     Completed Date:  2010-01-20     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  112-6     Citation Subset:  IM    
Drug Design Group, Progen Pharmaceuticals, Toowong, Qld 4066, Australia.
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MeSH Terms
Colorimetry / methods*
Enzyme Assays / methods*
Enzyme Inhibitors / analysis*,  pharmacology*
Glucuronidase / antagonists & inhibitors*
Oligosaccharides / pharmacology
Polysaccharides / chemistry,  metabolism
Reducing Agents / pharmacology
Time Factors
Reg. No./Substance:
0/Enzyme Inhibitors; 0/Oligosaccharides; 0/Polysaccharides; 0/Reducing Agents; 0/fondaparinux; 0/phosphomannopentaose sulfate; EC 3.2.1.-/heparanase; EC

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