Isoelectric focusing of heparin. Evidence for complexing with carrier ampholytes

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.