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°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°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 μM) 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.
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December 01 2001
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
Jan Holm;
Jan Holm
1Department of Clinical Chemistry, Central Hospital, Herning, DK–7400, Denmark
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Steen Ingemann Hansen
Steen Ingemann Hansen
2Department of Clinical Chemistry, Central Hospital, Hillerød, DK-3400, Denmark
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Publisher: Portland Press Ltd
Online ISSN: 1573-4935
Print ISSN: 0144-8463
© 2001 Plenum Publishing Corporation
2001
Biosci Rep (2001) 21 (6): 733–743.
Citation
Jan Holm, Steen Ingemann Hansen; 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. Biosci Rep 1 December 2001; 21 (6): 733–743. doi: https://doi.org/10.1023/A:1015576522416
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