Nature of the riboflavin interaction with the immunoglobulin IgGGAR: analogue binding studies

The human immunoglobulin IgGGAR has been shown to bind riboflavin both in vivo and in vitro with a high binding constant [Farhangi & Osserman (1976), New Engl. J. Med. 294, 177-183; Chang et al. (1981b), Biochemistry 20, 2916-2921; Chang et al. (1981a), Biochemistry 20, 2922-2926] and, therefore, represents a natural human antibody hapten complex. In this study, the parts of the riboflavin molecule which are in intimate contact with the binding pocket have been identified, using a variety of riboflavin analogues. The binding constants of IgGGAR for these flavins were measured by fluorescence spectroscopy. The relative affinities indicate that the pyrimidine edge of the isoalloxazine interacts intimately with the combining site, particularly around N-3. The ribityl side chain and the dimethylbenzene edge of the flavin ring are not critical to the binding interaction and are probably exposed to solvent. Analogues of riboflavin with altered electronic structures have somewhat lowered affinities for IgGGAR, suggesting that the isoalloxazine ring forms stacking interactions. Comparison with other known flavin-binding proteins shows that IgGGAR binds riboflavin in a new way that is different from the sites in other proteins. The isoalloxazine ring binds similarly to the FMN-binding proteins; however, unlike virtually all the other proteins, the ribityl side chain is not essential to binding.