Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme

• Published in: Biophysical journal 1997-10, Vol.73 (4), p.2116-2125
• Main Authors: Xavier, K.A., Shick, K.A., Smith-Gil, S.J., Willson, R.C.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - chemistry; Antigen-Antibody Complex - chemistry; Biophysical Phenomena; Biophysics; Calorimetry; Chickens; Fluorescence Polarization; Kinetics; Muramidase - chemistry; Muramidase - genetics; Muramidase - immunology; Osmotic Pressure; Point Mutation; Quail; Thermodynamics; Water - chemistry
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(97)78242-0

Fluorescence polarization spectroscopy and isothermal titration calorimetry were used to study the influence of osmolytes on the association of the anti-hen egg lysozyme (HEL) monoclonal antibody HyHEL-5 with bobwhite quail lysozyme (BWQL). BWQL is an avian species variant with an Arg-->Lys mutation in the HyHEL-5 epitope, as well as three other mutations outside the HyHEL-5 structural epitope. This mutation decreases the equilibrium association constant of HyHEL-5 for BWQL by over 1000-fold as compared to HEL. The three-dimensional structure of this complex has been obtained recently. Fluorescein-labeled BWQL, obtained by labeling at pH 7.5 and purified by hydrophobic interaction chromatograpy, bound HyHEL-5 with an equilibrium association constant close to that determined for unlabeled BWQL by isothermal titration calorimetry. Fluorescence titration, stopped-flow kinetics, and isothermal titration calorimetry experiments using various concentrations of the osmolytes glycerol, ethylene glycol, and betaine to perturb binding gave a lower limit of the uptake of approximately 6–12 water molecules upon formation of the HyHEL-5/BWQL complex.