35] Competitive binding assays for riboflavin and riboflavin-binding protein

Abstract

This chapter focuses on competitive binding assays for riboflavin and riboflavin-binding protein. Riboflavin-binding protein from chicken egg tightly and specifically binds riboflavin in preference to flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD). In contrast to protein-bound riboflavin, free riboflavin is not retained by anion-exchange resins such as DEAE-cellulose; thus, the two riboflavin pools can be separated and analyzed. The equilibrium distribution of radiolabeled riboflavin in the free and bound pools is determined by the total amounts of riboflavin and its binding protein, and by the final specific radioactivity of the riboflavin. By monitoring the amount of bound, radiolabeled riboflavin as a function of unlabeled riboflavin or the amount of riboflavin-binding protein, respectively, the vitamin or the protein can be assayed. Riboflavin-binding protein is negatively charged and binds tightly to diethylaminoethyl (DEAE)-cellulose while riboflavin is uncharged and will not bind. The content of each reaction mixture is poured directly into a corresponding DE-52 minicolumn and the eluate is discarded. In radioligand binding assay for riboflavin-binding protein, the amount of bound riboflavin varies from near 100% saturation in chicken egg yolk to near 0% in the egg whites of some birds. The indeterminate amount of endogenous riboflavin will dilute added radiolabeled riboflavin such that the binding of the radioligand is not a simple linear function of the concentration of the binding protein.