TY - JOUR
T1 - Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme
AU - Xavier, K. Asish
AU - Shick, Kari A.
AU - Smith-Gill, Sandra J.
AU - Willson, Richard C.
N1 - Funding Information:
We thank Drs. Susan Chacko, David R. Davies, Shankar Subramaniam, J. Andrew McCammon, Shawn M. McDonald, Jefferson Foote, James N. Herron, Ai-Ping Wei, M. E. Jolley, V. Adrian Parsegian, Donald C. Rau, Clifford R. Robinson, H. R. Bosshard, Catherine Royer, Jannette Carey, Arnold Eskin, David J. Roush, and Davinder S. Gill for helpful discus- sions; Drs. Ellen M. Prager for advice on BWQL purification; and Dr. Mark Hirschel for help with cell culture. We gratefully acknowledge Sam Williston of MicroCal for advice on calorimetric experiments and the help of Karen Rubelowsky and Dr. Jim R. Mattheis of Instruments SA with fluorescence experiments. We thank Dr. Donald O'Connor for performing the fluorescence lifetime measurements, and acknowledge the skilled tech-nical assistance of Rene Ochoa. This work was supported in part by the Office of Naval Research under the accelerated initiative in molecular recognition (ONR N00014-89-J-3052), the National Institute of General Medical Sciences (National Institutes of Health GM44344), and the Welch Foundation (E-1264).
PY - 1997/10
Y1 - 1997/10
N2 - 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.
AB - 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.
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U2 - 10.1016/S0006-3495(97)78242-0
DO - 10.1016/S0006-3495(97)78242-0
M3 - Article
C2 - 9336207
AN - SCOPUS:0030770571
SN - 0006-3495
VL - 73
SP - 2116
EP - 2125
JO - Biophysical Journal
JF - Biophysical Journal
IS - 4
ER -