Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme.
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.
Xavier
K A
KA
Department of Chemical Engineering, University of Houston, Texas 77204-4792, USA.
Shick
K A
KA
Smith-Gil
S J
SJ
Willson
R C
RC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000936:Antigen-Antibody Complex / Q000737:chemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D002151:Calorimetry; D002645:Chickens; D005454:Fluorescence Polarization; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D009997:Osmotic Pressure; D017354:Point Mutation; D011784:Quail; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D014867:Water; D009113:Muramidase