The preparation and properties of folate-binding protein from cow's milk.
An improved affinity-chromatographic method for the preparation of folate-binding protein from cow's milk is described. Under dissociating conditions the protein appeared homogeneous in the ultracentrifuge, with a molecular weight of 35 000 +/- 1500, but it was heterogeneous on electrophoresis and ion-exchange chromatography and evidently consisted of several glycoproteins with similar molecular weights that all bound folic acid. Overall, the protein contained a high proportion of half-cystine (18 residues/molecule) and 10.3% of carbohydrate. At saturation it bound approx. 1 mol of folate/mol of protein at pH 7.2. Equilibrium-dialysis measurements of the binding of folic acid and 5-methyltetrahydrofolate to the purified protein gave non-linear Scatchard plots, the shapes of which depended on pH. The results were interpreted in terms of ligand binding to a polymerizing system in which the affinity of ligand for monomer was greater than its affinity for polymer. When the protein concentration was similar to that in cow's milk, dissociation constants (Kd) for folate and 5-methyltetrahydrofolate were 3 nM and 5 nM respectively at pH 7.2 and 37 degrees C, whereas Kd for the binding of folate to monomer was about 50 pM. The properties of the binding protein are discussed in relation to its possible role in folate absorption in the gut.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D004355:Drug Stability; D005260:Female; D058973:Folate Receptors, GPI-Anchored; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands / Q000378:metabolism; D008892:Milk / Q000032:analysis*; D011956:Receptors, Cell Surface; D013696:Temperature; D014461:Ultracentrifugation
D000596:Amino Acids; D002241:Carbohydrates; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D011956:Receptors, Cell Surface