Amino acid end-group in commercial heparin. III. Determination of the number of amino groups of amino acid and hexosamine residues per heparin molecule

The reactions of heparin with 2,4,6-trinitrobenzenesulfonic acid (TNBS) were studied spectrometrically. Seven different commercial heparins were used in this study. The amino groups react with TNBS to form equimolar amounts of trinitrophenylated (TNP) amino groups and bisulfite ions. The TNP-amino groups further react with bisulfite ions to form the monosubstituted anionic sigma complex. The absorption spectrum with two maxima at approximately 350 nm and approximately 420 nm, characteristic of either the TNP-amino groups or the complex, was analyzed for the reaction of TNBS with heparin. It was shown that the reactivities of TNBS with amino groups from α-amino acid and hexosamine residues are greatly different. By combining the results of the reaction kinetics and the reaction of heparin with Sanger's reagent, the number of the α-amino groups and the free amino groups in hexosamine residues were determined. These data have been performed with a range of heparins from different commercial sources, of different activities and physical characteristics. No correlation was found between the free amino contents of these heparins and biological potency. © 1993 John Wiley & Sons, Inc.