Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium
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...
Guardado en:
| Publicado: |
2001
|
|---|---|
| Materias: | |
| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01730835_v22_n1_p3_Rozenberg http://hdl.handle.net/20.500.12110/paper_01730835_v22_n1_p3_Rozenberg |
| Aporte de: |
| id |
paper:paper_01730835_v22_n1_p3_Rozenberg |
|---|---|
| record_format |
dspace |
| spelling |
paper:paper_01730835_v22_n1_p3_Rozenberg2023-06-08T15:18:48Z Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium Heparan sutfate Heparin Heparinase Tris (2,2′bipyridine) ruthenium (II) EL 4139 2,2' bipyridine fluorochrome heparan sulfate heparin heparin lyase ruthenium complex tris(2,2' bipyridine)ruthenium unclassified drug adenocarcinoma analytic method animal cell article binding site carcinoma cell densitometry electrophoretic mobility enzyme activity enzyme specificity fluorescence nonhuman polyacrylamide gel electrophoresis 2,2'-Dipyridyl Adenocarcinoma Animals Electrophoresis, Polyacrylamide Gel Female Fluorescent Dyes Heparin Heparin Lyase Heparitin Sulfate Mammary Neoplasms, Animal Mice Molecular Structure Polysaccharide-Lyases Tumor Cells, Cultured 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 μg/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) × 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.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan 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. 2001 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01730835_v22_n1_p3_Rozenberg http://hdl.handle.net/20.500.12110/paper_01730835_v22_n1_p3_Rozenberg |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Heparan sutfate Heparin Heparinase Tris (2,2′bipyridine) ruthenium (II) EL 4139 2,2' bipyridine fluorochrome heparan sulfate heparin heparin lyase ruthenium complex tris(2,2' bipyridine)ruthenium unclassified drug adenocarcinoma analytic method animal cell article binding site carcinoma cell densitometry electrophoretic mobility enzyme activity enzyme specificity fluorescence nonhuman polyacrylamide gel electrophoresis 2,2'-Dipyridyl Adenocarcinoma Animals Electrophoresis, Polyacrylamide Gel Female Fluorescent Dyes Heparin Heparin Lyase Heparitin Sulfate Mammary Neoplasms, Animal Mice Molecular Structure Polysaccharide-Lyases Tumor Cells, Cultured |
| spellingShingle |
Heparan sutfate Heparin Heparinase Tris (2,2′bipyridine) ruthenium (II) EL 4139 2,2' bipyridine fluorochrome heparan sulfate heparin heparin lyase ruthenium complex tris(2,2' bipyridine)ruthenium unclassified drug adenocarcinoma analytic method animal cell article binding site carcinoma cell densitometry electrophoretic mobility enzyme activity enzyme specificity fluorescence nonhuman polyacrylamide gel electrophoresis 2,2'-Dipyridyl Adenocarcinoma Animals Electrophoresis, Polyacrylamide Gel Female Fluorescent Dyes Heparin Heparin Lyase Heparitin Sulfate Mammary Neoplasms, Animal Mice Molecular Structure Polysaccharide-Lyases Tumor Cells, Cultured Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium |
| topic_facet |
Heparan sutfate Heparin Heparinase Tris (2,2′bipyridine) ruthenium (II) EL 4139 2,2' bipyridine fluorochrome heparan sulfate heparin heparin lyase ruthenium complex tris(2,2' bipyridine)ruthenium unclassified drug adenocarcinoma analytic method animal cell article binding site carcinoma cell densitometry electrophoretic mobility enzyme activity enzyme specificity fluorescence nonhuman polyacrylamide gel electrophoresis 2,2'-Dipyridyl Adenocarcinoma Animals Electrophoresis, Polyacrylamide Gel Female Fluorescent Dyes Heparin Heparin Lyase Heparitin Sulfate Mammary Neoplasms, Animal Mice Molecular Structure Polysaccharide-Lyases Tumor Cells, Cultured |
| description |
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 μg/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) × 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.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan 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. |
| title |
Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium |
| title_short |
Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium |
| title_full |
Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium |
| title_fullStr |
Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium |
| title_full_unstemmed |
Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium |
| title_sort |
heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium |
| publishDate |
2001 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01730835_v22_n1_p3_Rozenberg http://hdl.handle.net/20.500.12110/paper_01730835_v22_n1_p3_Rozenberg |
| _version_ |
1768542834378407936 |