Cooperative binding of folate to a protein isolated from cow's whey

Abstract

Cooperative binding of [ 3H]folate to a protein ( M r = 35 000) in cow's whey was demonstrated in equilibrium dialysis experiments in Tris buffer (pH 7.4) at 37°C. The folate binding protein was isolated from whey by ion-exchange chromatography (DEAE-Sephadex A-50) in a linear NaCl gradient (0.03–0.3 M). The folate binding protein appeared in the effluent before start of the salt gradient. Fractions eluted after start of the salt gradient contained the major whey proteins β-lactoglobulin and α-lactalbumin but were unable to bind [ 3H]folate. Binding characteristics depended on concentration of folate binding protein. The association constant for folate binding ( K ass) was thus inversely proportional to the folate binding protein concentration. That is binding affinity decreased with increasing concentrations of binding protein. A phenomenon of this type may suggest involvement of a polymerising protein system in binding. Changes in the H + concentration within the interval pH 6–9 did neither decrease maximum bound folate nor the binding affinity. However, lowering pH to 5 produced a 10-fold reduction in K ass. Cooperativity as well as dependence of affinity on the folate binding protein concentration did moreover disappear. Folate binding was virtually insensitive to changes in ionic strength and temperature. Within the interval pH 7.4–5.2 dissociation of [ 3H]folate from binding protein was slow and incomplete. However, dissociation became rapid and complete at pH 3.5. The physiological significance of the folate binding system in milk is obscure but it may play a role in folate homeostasis.