A ¹³C-NMR Study on the Interaction of Riboflavin with Egg White Riboflavin Binding Protein

Abstract

The interaction between riboflavin and riboflavin binding protein (RBP) was studied by ¹³C-NMR spectroscopy. The ¹³C-NMR spectra of riboflavin selectively enriched at the 2-, 4-, 4 a-, and lO a-positions and of (3-[¹³C] methyl) riboflavin were measured both in the free and RBP-bound forms. The ¹³C signals of ¹³C-enriched riboflavin or 3-methylriboflavin bound to RBP are broader than those of the free form, reflecting the restriction of flavin mobility. The 2-, 4-, and 10 a-¹³C signals of riboflavin show no pH-dependent shift in the neutral to acidic pH region either in the bound or free form but the 4 a-¹³C signal of bound riboflavin shifts to lower field in the acidic pH region while that of the free form remains unshifted. The 2-, 4-, 4 a-, and 10 a-¹³C signals of free riboflavin exhibited pH-dependent change in the alkaline pH region with a pK value of about 10, in association with the N (3)-H deprotonation. The pH titration profile of the 2-, 4-, and 4 a-¹³C signals of bound riboflavin indicates that the pK of N (3)-H is shifted substantially to the alkaline side when riboflavin is bound to RBP. The 3-methyl-¹³C signal of 3-methylriboflavin shows no pH-dependent shift whether the compound is free or bound to RBP. The binding of riboflavin and 3-methylriboflavin was also studied spectrofluorometrically. The analysis of the pH dependence of the association constant revealed that one ionizable group in RBP with pK of about 5 and N (3)-H of riboflavin play important roles in the binding. We conclude that RBP preferentially binds the neutral, i.e., N (3)-protonated, form of riboflavin and that the neutral form in turn is stabilized by the hydrophobic environment of RBP surrounding the N (3) region of the bound riboflavin molecule.