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

K. Asish Xavier, Kari A. Shick, Sandra J. Smith-Gill, Richard C. Willson

Research output: Contribution to journalArticle

44 Scopus citations

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 ~6-12 water molecules upon formation of the HyHEL-5/BWQL complex.

Original languageEnglish (US)
Pages (from-to)2116-2125
Number of pages10
JournalBiophysical Journal
Volume73
Issue number4
DOIs
StatePublished - Oct 1997

ASJC Scopus subject areas

  • Biophysics

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