Development of new methods for determining the heparanase enzymatic activity

Heparanase is a mammalian endo-β-glucuronidase. Notwithstanding its importance in various pathological and non-pathological events few straightforward methods for heparanase enzymatic activity has been stated. The aim of this study was to develop two heparanase activity assays to cover a whole range of applications. First, a fast and easy method based on commercial homogenous substrate, fondaparinux, was described. The other method is a quantitative assay based on biotinylated heparan sulfate that uses an easier technique to immobilize the substrate in a 96-well plate.

1): The heparanase recombinant enzyme and fondaparinux were incubated overnight. After incubation, a fluorescent redox marker, resazurin, was added. The reduction of resazurin depends on the amount of glucuronic acid released by heparanase digestion. Fluorescence measurements were done using excitation and emission wavelengths of 560 nm and 590 nm, respectively.

2): The 96-well plate was incubated with protamine sulfate. Subsequently, biotinylated heparan sulfate was immobilized. The enzymatic assay was performed using chimeric recombinant heparanase at different concentrations. In sequence, the immobilized biotinylated heparan sulfate that was not digested by recombinant heparanase was bound to streptavidin conjugated with europium. Fluorescence was measured using a time-resolved fluorometer.

Both methods have high sensitivity and can be used to detect heparanase activity. Fondaparinux assay is a quick and easy method for screening of heparanase inhibitors using recombinant enzyme or bacterial crude extract. Biotinylated heparan sulfate assay can be used for quantitative analysis in biological samples and protamine sulfate showed been capable to immobilized heparan sulfate.