Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris (2,2’-bipyridine) ruthenium (II)

G.ROZENBERG,; J.ESPADA,; L.LAURIA DE CIDRE,; ANA MARÍA EIJÁN,; G.E.BERTOLESI

ELECTROPHORESIS

WILEY-V C H VERLAG GMBH

Año: 2001 vol. 22 p. 3 - 11

0173-0835

13. Electrophoresis. 2001 Jan;22(1):3-11. Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2´-bipyridine) ruthenium (II). Rozenberg GI, Espada J, de Cidre LL, Eiján AM, Calvo JC, Bertolesi GE. Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. garozenb@qb.fcen.uba.ar The paper shows the ability of the fluorochrome tris(2,2´-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent  staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degradedheparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparin sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate  in multiple biological samples tested in parallel.  PMID: 11197174 [PubMed - indexed for MEDLINE]