Righetti P G, Gianazza E
Biochim Biophys Acta. 1978 Jan 25;532(1):137-46. doi: 10.1016/0005-2795(78)90456-7.
The basis for heparin fractionation into 21 components by isoelectric focusing has been shown to be a strong interaction between the polysaccharide and different amphoteric species in the Ampholine mixture. This was demonstrated by altering the heparin/Ampholine ratio, by loading the sample either before focusing or to a prefocused gel slab and by re-running single heparin bands. The complexes exhibiting apparent pI values in the pH range 3.2--4.5 appear to be particularly stable, probably because an optimal amount of amino groups (4 to 5) in the Ampholine molecules are protonated. When stained with Toluidine blue, the heparin/Ampholine complexes precipitated in the gel exhibited different degrees of metachromasia, reflecting competition of the dye and individual components of Ampholine with respect to binding sites of heparin: at least three colours, violet, blue and indigo, are distinguishable. Ampholine, when added to a heparin . Toluidine blue complex in solution displaces the dye from the polysaccharide.
通过等电聚焦将肝素分离成21种成分的基础已被证明是多糖与两性电解质混合物中不同两性物质之间的强相互作用。通过改变肝素/两性电解质的比例、在聚焦前或预聚焦的凝胶板上加载样品以及重新运行单个肝素条带，证明了这一点。在pH值3.2 - 4.5范围内表现出表观pI值的复合物似乎特别稳定，这可能是因为两性电解质分子中最佳数量的氨基（4至5个）被质子化。用甲苯胺蓝染色时，凝胶中沉淀的肝素/两性电解质复合物表现出不同程度的异染性，反映了染料和两性电解质的各个成分在肝素结合位点上的竞争：至少可以区分出三种颜色，紫色、蓝色和靛蓝色。当将两性电解质添加到溶液中的肝素-甲苯胺蓝复合物中时，它会将染料从多糖中置换出来。
