The preparation and properties of folate-binding protein from cow's milk

Abstract

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.

The complete amino acid sequence of the folate-binding protein from cow's milk

“…SALTER el al. (14) have determined the content of the individual carbohydrates in FBP and showed that approximately 40% is contributed by N-acetylglucosamine and N-acetylgalactosamine. These authors found a carbohydrate content of 10%.…”

“…SALTER et al (14) determined the molecular weight to 35.000 from sedimentation equilibrium studies. We are unable to explain the large discrepancy between our and their values but a difference in experimental technique was that they used 6 M-guanidinium chloride as solvent.…”

“…Proteins which specifically bind folate have been isolated from various body fluids and tissues of mammals (1,7,12,14,15,16,18,19,20) and also from bacteria (9), In order to understand the function of these proteins it is important to know their structures, In a previous communication we have shown the partial amino acid sequence of the folate binding protein from cow's milk based on the cyanogen bromide fragments (16). Homology between this protein and the high and low molecular weight folate binding protein from human milk has been demonstrated (17).…”

Red cell or serum folate: what to do in clinical practice?

“…These assays use folate binding protein (FBP) for analyte capture, which is typically a β -lactoglobulin isolated from cow ' s milk. These β -lactoglobulins will capture 5-methylTHF, as well as other forms of folate depending on pH [ 7 ]. These assays can be used to quantify folate both in serum and red blood cells.…”

Bioavailability of folic acid from fortified pasteurised and UHT-treated milk in humans

“…In the fortified pasteurised milk, the amount of folic acid was 7.5 times higher than the amount of FBP (Table 3). According to Salter et al (1981), 1 molecule FBP binds 1 molecule of folate at pH 7.2. Thus, only a small amount of the added folic acid is expected to be bound to FBP in the fortified pasteurised milk.…”