Abstract
Affinity CE (ACE) was used to study interactions of small, highly sulfated, aromatic molecules with antithrombin (AT). The high charge density of the small molecules induces differential migration of the complex resulting in a versatile method of assessing binding affinities, nature of interactions and site of binding on the inhibitor. Scatchard analysis of the interaction of three tetrahydroisoquinoline-based polysulfated molecules with AT results in monophasic profiles with affinities in the range of 40-60 microM in 20 mM sodium phosphate buffer, pH 7.4. For a pentasulfated molecule, a biphasic profile with affinities of 4.7 and 30 microM was observed. Measurement of K(D) as a function of ionic strength of the medium indicated that ionic and non-ionic forces contribute 2.4 and 1.9 kcal/mol, respectively, at pH 7.4 and 100 mM NaCl. Competitive binding studies showed that the tetrahydroisoquinoline-based molecules do not compete with a high-affinity heparin pentasaccharide. In contrast, the affinity of these tetrahydroisoquinoline derivatives decreases dramatically in the presence of an extended heparin-binding site ligand. Overall, ACE analysis of small, sulfated aromatic molecules interacting with AT is relatively easy and obviates the need for an external signal, e.g. fluorescence, for monitoring the interaction. In addition to affording biochemical knowledge, the small sample requirement and fast analysis time of ACE could be particularly advantageous for high-throughput screening of potential anticoagulants.