Ionic charge, hydrophobicity and tryptophan fluorescence of the folate binding protein isolated from cow's milk.
A high-affinity folate binding protein was isolated and purified from cow's milk by a combination of cation exchange chromatography and methotrexate affinity chromatography. Chromatofocusing studies revealed that the protein possessed isoelectric points in the pH-interval 8-7. Polymers of the protein prevailing at pH values close to the isoelectric points seemed to be more hydrophobic than monomers present at pH 5.0 as evidenced by hydrophobic interaction chromatography and turbidity (absorbance at 340 nm) in aqueous buffer solutions (pH 5-8). Ligand binding seemed to induce a conformation change that decreased the hydrophobicity of the protein. In addition, Ligand binding quenched the tryptophan fluorescence of folate binding protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. There was a noticeable discordance between the ability of individual folate analogues to compete with folate for binding and the quenching effect.
Holm
J
J
Medicinsk Center, Centralsygehuset Roenne, Bornholm, Denmark. HECJH@ringamt.dk
Hansen
S I
SI
Høier-Madsen
M
M
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002417:Cattle; D002845:Chromatography; D005260:Female; D005453:Fluorescence; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions / Q000737:chemistry*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008024:Ligands; D008892:Milk / Q000737:chemistry*; D009391:Nephelometry and Turbidimetry; D011485:Protein Binding / Q000502:physiology; D011869:Radioligand Assay; D011956:Receptors, Cell Surface; D014364:Tryptophan / Q000737:chemistry*
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D007477:Ions; D008024:Ligands; D011956:Receptors, Cell Surface; D014364:Tryptophan; D005492:Folic Acid