High and low affinity binding of folate to proteins in serum of pregnancy women.

Binding of [3H]folate to proteins in serum of pregnant women was studied in equilibrium dialysis experiments (pH 7.4, 37 degrees C). A Scatchard analysis revealed the presence of high-affinity (Kass = 10(10) M(-1), N = 0.4 nM folate) and low-affinity sites. The high-affinity folate binding protein (Mr approximately 30 000--35 000) appeared in front effluent after application of serum to a DEAE-Sepharose CL-6B column equilibrated with 0.05 M imidazole buffer (pH 6.3)/30 mM NaCl. Low-affinity binding protein eluted from the column after a rise in NaCl concentration to 1 M was mainly similar to albumin. A minor part was, however, associated with a large molecular size (Mr > 200 000) protein, probably alpha 2-macroglobulin. High-affinity binding which displayed positive cooperativity was saturated at folate concentrations above 10(-10) M. Folate dissociation was a complex process consisting of an initial rapid phase (terminated within 48 h) followed by a slow release. At pH 3.5 dissociation became rapid and complete. Purified methotrexate had no effect on high-affinity binding, whereas N10-methylfolate (an impurity in the methotrexate preparation) acted as a potent inhibitor. Low-affinity binding was proportional to the folate concentration within the range 10(-10)--10(-7) M. Dissociation of folate was rapid.