a Spin-Label EPR Study of Immunoglobulin - Lipid Interaction.

The interactions of immunoglobulin-G (IgG) with phospholipid in solution and in model membrane systems (liposomes) were investigated by X-band spin-label EPR. Experimental spectra were analyzed using simulated spectra that reflect the ordering and dynamics characteristic of the liquid crystal state of an aggregation of lipid molecules. The simulation is based on a model in which the spin label undergoes random rotation and tumbling while restricted to a cone. The motional contribution to the linewidth is included by a reformulation of Blume's stochastic theory of lineshape to account for the two independent, simultaneous, and stochastic processes. In order to calculate the fraction of lipid bound to IgG in several titration experiments a data reduction method based on computer simulations was developed because more conventional methods of quantitative spectral analysis were not possible in this study. The simulation approach depends on only the relative amplitudes of the two components of the composite spectrum and the amplitudes of the population normalized simulated spectra of the components. The primary results of experiment and analysis are (1) IgG binds weakly to lipid in solution, and the bond occurs along the stem of the antibody molecule, (2) aggregation of IgG increases binding affinity to lipid significantly but is still much smaller than the affinity of integral membrane proteins for lipid, (3) the binding data of a homogeneous sample of IgG aggregate is accounted for by a calculation based on the law of mass action and that of a heterogeneous mixture of aggregates is explained by treating the binding of each group independently, and (4) no interaction is observed between lipid bilayers and either native or aggregated IgG. Other significant results are the following: (1) the bilayer environments of multilamellar (MLV) and large unilamellar (LUV) vesicles are the same; (2) unperturbed lipid bilayers (MLV and LUV) support a significant amount of internal molecular motion; simulation studies reveal that the spin probe in these model membranes undergoes rotation about its long molecular axis and tumbles within a cone of apex angle 65(DEGREES) with a correlation time of 1.0 nsec at 22(DEGREES)C and within a cone of apex angle 80(DEGREES) with a correlation time of 0.4 nsec at 37(DEGREES)C; (3) the cholestane spin probes 3-doxyl-cholestane (3DC) and 3-doxyl-androstanol (ASL) exhibit identical spectra in liposomes even though their spin label groups are positioned at different depths in the membrane.