Preparation of biologically active fluorescent heparin composed of fluorescein-labeled species and its behavior to antithrombin III.

A hog-mucosal heparin purified gel-chromatographically (total sulfate, 2.20; N-sulfate, 0.82; N-acetyl, 0.15 (mol/mol HexN); activity, 184 USP units/mg) was partially N-desulfated to give a product (total sulfate, 2.12; N-sulfate, 0.71 (mol/mol HexN); activity, 159 USP units/mg). The free amino groups in the product was extensively labeled with fluoresceinylthiocarbamoyl (FTC) groups, and the labeled product was chromatographed on Octyl-Sepharose CL-4B to give a heparin preparation of fluorescein-labeled species (total sulfate, 2.04; N-sulfate, 0.72; degree of substitution, 0.059 (mol/mol HexN); activity, 155 USP units/mg). The fluorescent heparin thus obtained was indicated from its analytical data to have one FTC group pr heparin molecule irrespective of the molecular weight. The fluorescent heparin was separated into the low-affinity (56.6%) and high-affinity (43.4%) fractions for antithrombin III, and the anticoagulant activities of the two types of fluorescent heparin were compared with those of the starting heparin and the partially N-desulfated heparin, suggesting that FTC-substitution in heparin molecules had no effect on the known biological interaction with mobilized or immobilized antithrombin III.