title: Development of new methods for determining the heparanase enzymatic activity.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Biochemistry/Molecular Biology Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil. Electronic address: maspinhal@yahoo.com.br.
authors.forename: Carina Mucciolo
authors.initials: CM
authors.lastname: Melo
authors.affiliation: Biochemistry/Molecular Biology Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil; Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi das Cruzes, Brazil.
authors.forename: Ivarne Luis Santos
authors.initials: IL
authors.lastname: Tersariol
authors.affiliation: Biochemistry/Molecular Biology Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil.
authors.forename: Helena Bonciani
authors.initials: HB
authors.lastname: Nader
authors.affiliation: Biochemistry/Molecular Biology Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil; Biochemistry Department, Faculdade de Medicina do ABC (FMABC), Santo André, Brazil.
authors.forename: Maria Aparecida Silva
authors.initials: MA
authors.lastname: Pinhal
authors.affiliation: Biochemistry/Molecular Biology Department, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil.
authors.forename: Marcelo Andrade
authors.initials: MA
authors.lastname: Lima
mesh_terms: D002457:Cell Extracts / Q000737:chemistry; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli; D000077425:Fondaparinux; D005966:Glucuronidase / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry; D006801:Humans; D061986:MCF-7 Cells; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D000099175:Heparanase
chemical_list: D002457:Cell Extracts; D000077425:Fondaparinux; D005966:Glucuronidase; D006497:Heparan Sulfate; D011134:Polysaccharides; D011994:Recombinant Proteins; D000099175:Heparanase
keywords: Chimeric heparanase; Enzymatic assay; Heparanase; Heparanase activity; Recombinant heparanase; endo-Beta-glucuronidase