A novel specific heparin-binding activity of bovine folate-binding protein characterized by capillary electrophoresis.
Folate-binding proteins (FBPs) are ubiquitous, soluble and membrane-bound high-affinity receptors for folate, an essential nutrient involved in nucleic and amino acid metabolism. In the course of optimizing CE separation conditions for FBP purified from cow's milk we discovered a novel specific heparin-binding activity of FBP by affinity CE. Heparin is a highly sulfated glycosaminoglycan and thus prone to induce anodic migration shifts of complexing analytes. Prior complexation of FBP with folate abolished heparin binding, and thus folate competes with heparin for binding to FBP. It was estimated that heparin bound several orders of magnitude less strongly than folate with an average dissociation constant in the 1-10 microM range. In contrast to the mobility shifts induced by heparin, free and folate-bound FBP were not separated by CE. However, binding of folate induced a distinct increase in FBP-peak symmetry, and using heparin as an affinity displacer, the free FBP in equilibrium with folate-FBP complexes could readily be separated from the complexes. While the folate-FBP interaction was too strong to be characterized quantitatively because of inadequate detection limits of a UV-based detection system, it was possible to estimate the folate-FBP binding stoichiometry using this approach. The heparin interaction fractionated FBP into distinct subfractions, and the CE approach thus promises to be useful for unraveling the complex oligomerization behavior of FBP isoforms as well as for evaluating the FBP affinity for various species and analogs of glycosaminoglycans and folate.
Heegaard
Niels H H
NH
Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark. nhe@ssi.dk
Hansen
Steen I
SI
Holm
Jan
J
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D066298:In Vitro Techniques; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism*
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D006493:Heparin; D005492:Folic Acid