Intramolecular motility in anti-Dnp antibodies and in their Fab fragments. EPR spectra of the complexes with a spin-labelled hapten in H2O-D2O mixtures at various temperatures.
To study intramolecular motility, the binding sites of the antibodies or Fab fragments were occupied by the spin-labelled hapten Dnp-NO. EPR spectra of the complexes were recorded under various conditions, in particular variable viscosity, and the correlation times of the antibody or its fragment were calculated. Intramolecular motility decreases, i.e. correlation time increases, with temperature in the range from 5 degree C to 30 degrees C. This anomalous dependence points to the important role of hydrophobic bonds in the interactions between domains of the antibody molecule. Substitution of a fraction of H2O by D2O (1 to 13%) is manifested by the increase in correlation time only at 5 degrees C, a maximal effect is already obtained at about 3% D2O. At 20 degrees C or in the presence of perturbants of the hydration shell such as 0.5 M sodium chloride, 1 M urea or 10% butanol, D2O has no appreciable effect. EPR spectra made it possible to study the gradual immobilization of the bound spin-labelled hapten at temperatures lower than 0 degrees C. Water in the vicinity of the bound spin-labelled hapten freezes in three discrete steps, -8 to -13 degrees C, -20 to -40 degrees C, and -40 to -80 degrees C. D2O influences the first and second step so that it shifts the transition to higher temperatures. The results contribute to our understanding of the fine structure of the hydration shell and water in the interdomain space of the antibody molecule.
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001666:Binding Sites, Antibody; D003903:Deuterium / Q000378:metabolism; D017666:Deuterium Oxide; D004136:Dinitrobenzenes / Q000276:immunology; D004578:Electron Spin Resonance Spectroscopy; D006241:Haptens / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D011487:Protein Conformation; D013113:Spin Labels; D013552:Swine; D013696:Temperature; D014867:Water / Q000378:metabolism
D000906:Antibodies; D004136:Dinitrobenzenes; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D013113:Spin Labels; D014867:Water; D003903:Deuterium; D017666:Deuterium Oxide