97] Purification of folate binding factors

Abstract

This chapter discusses the purification of folate binding factors. Specific binding proteins for folic acid (pteryolmonoglutamic acid, PteGlu), and some of its derivatives and analogs have been found in a wide variety of tissues. The small amount of binder that is present in most tissues and fluids, coupled with the fact that the binder is usually saturated with endogenous folate, have hampered large-scale purification and analysis of these factors. The binding of folic acid is determined by incubating [ 3 H]PteGlu with the binder preparation, and then separating the [ 3 H]PteGlu-binder complex from free [ 3 H]PteGlu. Several different methods have been used to achieve the separation: (a) precipitation of the complex with 75% ethanol; (b) filtration through sephadex G-25 or G-50; (c) sucrose density gradient centrifugation; and (d) selective adsorption of free PteGlu by activated charcoal that has been “coated” with protein or dextran. The use of coated charcoal has proved to be the most rapid, facile, and economical method when large number of samples are to be processed. Moreover, when proper ratio of dextran or protein to charcoal is used, the results are comparable to those obtained by other methods.