pH Dependence of the Binding of Folates to Milk Binder in Radioassay of Folates

We compare the binding of folic acid and N-5-methyltetrahydrofolic acid to milk binder in the pH range 7.4 to 10.1. At pH 7.4 the relative affinities are quite disparate, with folic acid showing the greater affinity for milk binder. As the pH is increased from 7.4 to 9.3, the difference in affinities becomes smaller, and at pH 9.3 the affinities are nearly the same. As the pH is increased from 9.3 to 10.1, the relative affinities again begin to differ, with N-5-methyltetrahydrofolic acid displaying the greater affinity. These results indicate that at a pH of 9.3 the more stable folic acid may be used as the standard in radioassay of endogenous folates.