Further studies of the riboflavin-binding immunoglobulin IgGGar. Resolution into fractions of different riboflavin content and aspects of reassembly.
A previously described human immunoglobulin with unusual flavin-binding activity, IgGGar [Farhangi, M., & Osserman, E. F. (1976) N. Engl. J. Med. 294, 177], is further characterized. The protein can be fractionated into two subpopulations, one of which is nearly completely saturated with riboflavin and one in which the binding sites are largely vacant. Possible differences between these fractions and/or their binding sites are explored. While electrophoretically distinct, the IgGGar-riboflavin complexes possess a basic similarity in the binding sites of both fractions as evidenced by spectroscopic examination. However, an important difference exists in that added riboflavin equilibrates reversibly with the vacant sites of native IgGGar, while the riboflavin in the occupied sites is essentially irreversibly bound. The tight association may be due to an in vivo combination of riboflavin with protein of different conformation than occurs in vitro, such as an incompletely assembled or folded tetramer. Accordingly, in vitro renaturation was examined. Studies of renaturation revealed that the reduced interchain disulfides within a tetramer reoxidize smoothly, although inter-heavy-chain bonds form less readily than inter-heavy-light-chain disulfides. Renaturation of IgGGar, unlike previously studied IgG molecules, does not proceed under conditions in which the protein structure had previously been significantly disrupted. The assembly defect is localized in the inability of the denatured heavy chain to refold into a stable species capable of combining with the light chain.
D001665:Binding Sites; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D012256:Riboflavin / Q000032:analysis*; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis
D007074:Immunoglobulin G; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol; D012256:Riboflavin