Characterization of a high affinity folate binding protein in porcine serum: ionic charge, concentration-dependent polymerization and ligand binding mechanism.
The folate binding protein in porcine serum, present at concentrations of 50-100 nM, is cationic at near neutral pH as evidenced by ion exchange chromatography. The gel filtration profile of the protein isolated from porcine serum by methotrexate affinity chromatography exhibited one peak at 48 kDa and an additional peak of 91 kDa at higher protein concentrations. This could suggest the involvement of concentration-dependent polymerization phenomena. Binding of [3H] folate was of a high-affinity type with upward convex Scatchard plots and Hill coefficients > 1.0 indicative of apparent positive cooperativity. However, binding to protein isolated from porcine serum after affinity chromatography was biphasic (high/low-affinity) in the absence of Triton X-100, 1 g/l. These findings which are similar to those reported for purified milk folate binding proteins are consistent with a model predicting association between unliganded and liganded monomers to weak-ligand affinity heterodimers. Amphiphatic substances, e.g. Triton X-100, form micelles which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers are hydrophilic in the liganded state) thereby preventing hetecrodimerization. The folate analogue N10 methyl folate was a potent and competitive inhibitor of [3H] folate binding to the folate binding protein, and moreover changed the binding type to apparent negative cooperativity.
Holm
Jan
J
Department of Clinical Chemistry, Hillerod Hospital, DK-3400, Denmark.
Hansen
Steen Ingemann
SI
D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry*; D008024:Ligands; D008970:Molecular Weight; D011108:Polymers / Q000737:chemistry*; D011956:Receptors, Cell Surface; D044967:Serum / Q000737:chemistry*; D013552:Swine
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D007477:Ions; D008024:Ligands; D011108:Polymers; D011956:Receptors, Cell Surface; D005492:Folic Acid