Binding of radiolabeled folate and 5-methyltetrahydrofolate to cow's milk folate binding protein at pH 7.4 and 5.0. Relationship to concentration and polymerization equilibrium of the purified protein.
Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7 degrees C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37 degrees C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 microM) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.
Holm
J
J
Department of Clinical Chemistry, Central Hospital, Herning, Denmark. BCMM-JH@BORA.dk
Hansen
S I
SI
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008892:Milk / Q000737:chemistry*; D011108:Polymers / Q000378:metabolism; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism*
D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011108:Polymers; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates; D005492:Folic Acid; D001205:Ascorbic Acid; C005984:5-methyltetrahydrofolate