Binding of modified heparins to proteins by affinity coelectrophoresis (ACE).

¹²⁵I-labeled GAGs (HEP, unmodified heparin; De-S HEP, completely desulfated heparin; N-S HEP, N-sulfated heparin; O-S HEP, O-sulfated heparin) were electrophoresed through the proteins (cytokines and matrix components) cast in agarose gels. Results are shown for binding of the GAGs to 100 nM TSP, 25 nM bFGF, and 500 nM each of VEGF, FN, IL-3, and MIP-1α. Ovalbumin (500 nM) was used as a negative control protein. The NaCl electrophoresis buffer was used for TSP, bFGF, and IL-3, and the sodium acetate buffer was used for VEGF, FN, and MIP-1α. Migration of unbound GAGs that are not bound to proteins is dependent on both size and charge. The average size of all 4 GAGs was comparable (8 to 12 kd). Because the negative charge on De-S HEP is the least, its migration was slowest. N-S HEP has an intermediate charge and migrated faster than De-S HEP. O-S HEP and HEP, which are the most highly charged, migrated at comparable rates. Binding to proteins was seen as retardation of the relative rate of migration of the GAGs through the protein lanes, compared to its migration through ovalbumin.