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

Biophys J. 1997 Oct;73(4):2116-25. doi: 10.1016/S0006-3495(97)78242-0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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

Substances

  • Antibodies, Monoclonal
  • Antigen-Antibody Complex
  • Water
  • Muramidase