36] Isolation of avian riboflavin-binding protein

Abstract

Publisher Summary This chapter describes the isolation of avian riboflavin-binding protein (RBP). As avian riboflavin binding protein (RBP) is highly anionic, therefore purification by conventional ion-exchange chromatography is quite effective. The chapter considers the isolation of RBP from egg white, yolk, and hen plasma. The purification of egg white riboflavin-binding protein relies on the affinity of RBP for diethylaminoethyl (DEAE)-cellulose under conditions in which most other proteins do not bind. Egg white RBP can be purified to apparent homogeneity in three steps—namely, batch adsorption to DEAE-cellulose, salt precipitation, and chromatography on CM- or DEAE-cellulose. The strong absorbance of protein-bound riboflavin at 455 nm may be used as a visual indicator for the holoprotein, throughout the purification procedures. As the properties of yolk RBP are similar to those of its egg white counterpart, much of the isolation procedure is identical. The major difference is in the initial preparation of the yolk prior to DEAE batch treatment. The purified preparations of RBP should be examined for homogeneity and assayed for riboflavin-binding capacity. Pure egg white RBP migrates as a single band during electrophoresis on sodium dodecyl sulfate (SDS)–polyacrylamide gels with an apparent molecular weight of 34,000–36,000.