Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium (II)
Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2′-bipyridine) ruthenium (II)
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DOI：
10.1002/1522-2683(200101)22:1
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发表时间：
2001-01-01
期刊：
影响因子：
2.9
通讯作者：
Bertolesi, GE
中科院分区：
文献类型：
--
作者：
Rozenberg, GF;Espada, J;Bertolesi, GE
本文展示了荧光染料三（2,2'-联吡啶）钌（II） （Rubipy）在天然聚丙烯酰胺凝胶电泳（PAGE）中检测M3小鼠乳腺腺癌细胞硫酸肝素、肝素和肝素酶活性以及细菌肝素酶I、II和III的能力。该技术是基于高分子量肝素的电泳迁移率和随后的红宝石染色（50马克杯/毫升）。紫外透照器荧光检测肝素的最低含量为25 ~ 50 ng。与肝素结合的红宝石分子数量与双糖单位（DU）的数量的关系表明，每个荧光染料分子结合了2至6个肝素双糖单位。Scatchard图分析显示1个rubipy结合位点(Kd = （8.56 +/- 2.97) × 10(-5) M）。肝素酶活性通过凝胶含肝素带荧光强度的密度分析测定。肝素酶I （EC 4.2.2.7.）对肝素的降解程度较低，对部分n -脱硫n -乙酰化肝素（N-des N-Ac）的降解程度较低，而肝素酶II（无EC编号）对硫酸肝素（HS）和部分N-des N-Ac肝素的降解效果较好。最后，肝素酶III （EC 4.2.2.8.）几乎完全降解HS。M3肿瘤细胞肝素酶的底物只有肝素和N-des N-Ac肝素。我们描述了一种定性、敏感和简单的方法来检测肝素酶活性，并确定其底物特异性使用红宝石荧光与肝素和硫酸肝素在多个生物样品平行测试。
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 mug/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.) 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.