Electrofocusing of heparin: Fractionation of heparin into 21 components distinguishable from other acidic mucopolysaccharides

Twenty‐one fractions have been demonstrated in each of 15 different commercially available heparins subjected to electrofocusing. These fractions show a molecular‐weight range from 3000 to 37,500 with a constant interval between molecular weights. Degradation of each fraction by purified enzymes of Flavobacterium heparinum yielded identical end products, suggesting chemical identity. Only fractions with a molecular weight of 7000 and up had significant anticoagulant activities. The phenomenon of electrofocusing of mucopolysaccharides is dependent upon pH, molecular weight, and ampholyte availability. Chemical composition of the mucopolysaccharide is also an essential factor since N‐ and O‐desulfation of heparin markedly changed the focalization pattern. The pattern produced when heparin is subjected to electrofocusing is not duplicated by any other naturally occurring acidic mucopolysaccharide tested. Heparitin sulfate D shows some similarities to heparin and it is probable that heparitin sulfate D is a normal contaminant of heparin preparations (this assumption is supported by molecular‐weight and anticoagulant activity determinations). The technique is specific and reproducible and unequivocally distinguishes heparin from other acid mucopolysaccharides.