Development of single and double spin-labeling methods and their applications to the study of antibody active sites

Abstract

Two specific methods have been developed, utilizing the electron spin resonance (ESR) spin-labeling technique, to probe the dimensional and structural nature of the macromolecular active site and to determine the correlation of active site structure with its biological specificities. The hapten combining sites (HCS) of rabbit anti-dinitrophenyl antibodies have been studied using two classes of twenty-two specifically prepared spin-labeled haptens. The single spin-labeling method employs a hapten labeled with only one spin. The hapten may be either a monovalent or bivalent homologous hapten, or a cross reacting hapten. The line-width and maximum splitting of the anisotropic ESR spectrum of the spin-label is a function of its correlation time Tc. The spectral variation is very sensitive to changes in Tc within the range of 1 to 100 nanoseconds. This range is most suitable for studying dimensional variations of antibody HCS in solution since 100 nanoseconds is approximately the average rotational diffusion time of the antibodies. Pertinent results obtained with this method are: (1) the dominant contribution to the correlation time Tc was found to be associated with freedom of rotation about a single bond for the bound spin label; (2) the average depth of HCS was determined to be 10 Å, and heterogeneity of depth was observed; (3) differences in specificity of antibodies isolated with homologous and cross reacting antigens were found due to structural and dimensional differences at the HCS; (4) homologous haptens form a rigid complex and/or have preferred orientation at HCS, however, cross reacting haptens form a less rigid complex; (5) rotational relaxation time (ρ) for antibody monomer and dimer was determined; (6) a sensitive method has been developed in determining antibody hapten affinity, taking advantage of the complete separation of ESR absorption spectrum of the bound and free spin-label; (7) affinity spin-labels have been demonstrated to be able to distinguish specific and non-specific labeling. Double spin-labeled haptens have two unpaired electrons per molecule. The intensities of the isotropic hyperfine and maximum splitting of the anisotropic ESR spectra are very sensitive to electron-electron exchange and dipolar interactions as well as to the relative orientation to the hyperfine tensors of the two electrons. The anisotropic spectra of the bound labels were only slightly perturbed. This may indicate that the HCS is quite flexible. However, the same spin-labeled hapten containing only one electron was rigidly immobilized at the antibody HCS. From the results of this work, the HCS was found to be dimensionally heterogeneous and probably consists of an inner rigid charge transfer complex region of approximately 5~6 Å with a flexible outer region of 4~5 Å which is partially hydrophobic.