Affinity capillary electrophoresis in binding study of antithrombin to heparin from different sources

Heparin, a highly sulfated polydispersed glycosaminoglycan (GAG), is the most widespread clinical anticoagulant; it binds antithrombin III (AT), a member of serine proteinases superfamily, accelerating its antagonist effect on blood coagulation. The binding interaction with AT is an important aspect in characterization of physicochemical properties of GAGs. With the aim at profiling several clinical and experimental heparin batches from different sources (porcine, bovine and ovine mucosa), a quantitative investigation of the binding heparin-AT, was undertaken by means of Affinity Capillary Electrophoresis (ACE). In dynamic-equilibrium ACE, the electrophoretic mobility of the receptor (AT), analyzed in a BGE containing the ligand (the considered GAG), is correlated to ligand concentration and binding constant. In particular, a 20mM sodium phosphate, pH 7.4 buffer (the BGE) was chosen as the neat medium and the experiments were carried out in a highly hydrophilic poly(vinyl alcohol) coated capillary (effective length 8.5 cm). The applied sample, consisting in the receptor AT (0.30 μM) and phenylacetic acid (PAA; 10.0 μM) used as a reference compound, was electrophoresed at each of the studied concentration levels of the ligand (heparin samples, 0.30-10.0 × 10(-7)M; heparan sulfate, 0.35-8.0 × 10(-5)M) supplemented to the BGE. The migration time ratio of PAA to AT was assumed as the chemical response to be correlated to the ligand concentration and the binding constant estimation was based on the application of a nonlinear regression method (rectangular hyperbola). Under these conditions, a number of heparin samples were analyzed and their binding constants (Kd) were found within 14.2 and 56.1 nM (SD ≤ ± 2.0; n=3; coefficient of determination r(2) ≥ 0.96). The good correlation of Kd values to the in-vitro activity (anti-factor Xa and anti-factor IIa), confirmed that the affinity for the target AT is an important feature of heparin samples and could be included among their physico-chemical characteristics.