Flavin affinity chromatography: General methods for purification of proteins that bind riboflavin

Abstract

Analogs of riboflavin that were altered at positions N(3), 8α, and N(10) of the 7,8-dimethylisoalloxazine ring were immobilized by covalent attachment to aminoalkylated agarose and polyacrylamide beads. These materials were used for affinity chromatographic purification of the riboflavin-carrier protein from egg white, egg yolk, and blood from laying hens, of flavokinase from rat liver, and of partially purified flavodoxin from Azotobacter vinelandii (FMN). The apo-carrier protein, which tightly complexes riboflavin ( K d ≈ 2 n m), was bound by the N(3)-, 8α-, and N(10)-flavinyl beads and was selectively displaced in moderate to high yield by 10 μ m riboflavin or 1 m NaCl at pH 3.5. Flavokinase, which complexes less tightly with riboflavin ( K m ≈ 12 μ m), was bound by the 8α- and N(10)-flavinyl beads. Binding to the latter was sufficiently tight that the addition of riboflavin was needed to displace flavokinase from the beads. The A. vinelandii flavodoxin, which normally complexes riboflavin 5′-phosphate ( K 3 ≈ 5 n m) but less avidly complexes riboflavin ( K d ≈ 0.6 μ m), was bound by the N(10)-flavinyl beads and eluted in low yield upon addition of FMN; most of the apoprotein denatured on the column despite the inclusion of thiol-protecting reagents. These flavin affinity materials may be generally useful for isolating a variety of other proteins that bind riboflavin.