Further studies on the riboflavin-binding immunoglobulin IgGGar. Equilibrium and kinetic aspects of the interaction.
The subpopulation of flavin-binding protein IgGGar which differ with respect to the degree of saturation with riboflavin [Chang, M. Y., Friedman, F. K., Beychok, S., Shyong, J. S., & Osserman, E. F. (1981) Biochemistry (preceding paper in this issue) are characterized in terms of hapten binding activity. Riboflavin fluorescence is used as a sensitive probe of the rate and extent of complex formation. The vacant sites on native IgGGar reversibly bind up to a total of 2 equiv of riboflavin/mol of protein. Kinetic and equilibrium measurements yield an affinity constant of (1.7 +/- 0.1) x 10(9) M-1. This value is the highest found to date for a hapten-monoclonal antibody system and is a consequence of an unusually low dissociation rate for the complex. The system exhibits maximal binding efficiency between pH 6 and 8, van't Hoff analysis of binding yields delta H = -15 kcal mol-1 and delta S = -9.5 cal deg-1 mol-1. The occupied sites on native IgGGar irreversibly bind riboflavin under normal reaction conditions. Irreversible denaturation of protein with urea affects the release of riboflavin. The dissociation mechanism is complex and is partially characterized.
D001665:Binding Sites; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011489:Protein Denaturation; D012256:Riboflavin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014508:Urea
D007074:Immunoglobulin G; D014508:Urea; D012256:Riboflavin