RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Muramidasa/química , Muramidasa/inmunología , Animales , Complejo Antígeno-Anticuerpo/química , Fenómenos Biofísicos , Biofisica , Calorimetría , Pollos , Polarización de Fluorescencia , Cinética , Muramidasa/genética , Presión Osmótica , Mutación Puntual , Codorniz , Termodinámica , Agua/químicaRESUMEN
The energetics of association of the murine anti-hen egg lysozyme antibody HyHEL-5 with bobwhite quail lysozyme, California quail lysozyme, and the Arg45-->Lys mutant of hen egg lysozyme was characterized by isothermal titration calorimetry. The association of each lysozyme with HyHEL-5 is enthalpically driven in the temperature range 10 degrees C to 37 degrees C. The calorimetric results indicate that the salt-links between Arg45 and Arg68 of hen egg lysozyme and GluH50 on the HyHEL-5 paratope are energetically important in HyHEL-5/HEL association. In contrast to previous studies, the results suggest that the three characteristic 'quail' mutations affect the energetics of antibody/antigen association, even though they are buried and not in direct contact with the antibody.
Asunto(s)
Anticuerpos/inmunología , Muramidasa/inmunología , Codorniz/genética , Animales , Anticuerpos/química , Complejo Antígeno-Anticuerpo/química , Ratones , Muramidasa/genética , Mutación , TermodinámicaRESUMEN
The HyHEL-5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg-white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL-5, an antihen egg-white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side-chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL-5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side-chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL-5 is similar to that in previously determined lysozyme-HyHEL-5 complexes.
Asunto(s)
Complejo Antígeno-Anticuerpo/química , Fragmentos Fab de Inmunoglobulinas/química , Muramidasa/química , Animales , Pollos , Cristalización , Cristalografía por Rayos X , Proteínas del Huevo/química , Mapeo Epitopo , Enlace de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Muramidasa/inmunología , Muramidasa/metabolismo , Mutación/genética , Conformación Proteica , Codorniz , Agua/metabolismoRESUMEN
The adsorption of recombinant soluble tryptic fragment of rat cytochrome b5 on the strong anion exchanger Mono Q was studied using isothermal titration calorimetry and differential scanning calorimetry (DSC). Titration calorimetry results obtained at low levels of adsorbed protein show increasingly endothermic (unfavorable) enthalpies of binding with increasing surface coverage, confirming the heterogeneous nature of binding. The enthalpy of adsorption declines toward zero at higher loadings. At low surface coverage, enthalpies increase linearly with temperature, giving rise to a positive value of delta Cp. Enthalpies of adsorption depend strongly on the history of the adsorbent. DSC is used to show that cytochrome b5 is stable in both free and adsorbed states at all temperatures used in the titration calorimetric experiments. Site-directed mutants of recombinant cytochrome b5 carrying single charge-neutralizing substitutions are used to test the contributions of particular residues to the thermodynamics of adsorption. Like those derived from van't Hoff analysis of equilibrium adsorption isotherms and HPLC retention data, calorimetric enthalpies of adsorption are positive, confirming the dominant role of entropic effects in ion-exchange adsorption in this system.