Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

Abstract

The role that heparanase plays during metastasis and angiogenesis in tumours 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 upon labelling variable preparations of the natural substrate heparan sulfate which makes 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 catalysed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogenous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterised giving a Km of 46 mM 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 the Ki of 7.9 nM determined. The simplicity and robustness of this method should, not only, greatly assist routine assay of heparanase activity, but could also be adapted for high-throughput screening of compound libraries with the data generated being directly
comparable across studies.