Photon correlation spectroscopy of human IgG

Abstract

The translational diffusion coefficient D020,w, of monomeric human immunoglobulin G (IgG) has been studied by photon-correlation spectroscopy as a function of pH and protein concentration. At pH 7.6, we find D020,w = 3.89 x 10(-7) cm2/sec, in good agreement with the value determined by classic methods. This value corresponds to an effective hydrodynamic radius R, of 55.1 +/- 0.3 A. As pH is increased to 8.9; with the same ionic strength, the molecule appears to expand slightly (3.5% increase in hydrodynamic radius). The concentration dependence of the IgG diffusion constant is interpreted in terms of solution electrostatic effects and shows that long-range repulsive interactions are negligible in the buffer used. The diffusion coefficient for dimeric IgG has also been determined to be D20,w = 2.81 x 10(-7) +/- 0.04 cm2/sec at 1.6 mg/ml, which corresponds to a hydrodynamic radius of 75 A. For light-scattering studies of protein molecules in the dimension range of 5-10 nm (Mr approximately 10(5)-10(7] we find monomeric horse spleen ferritin well suited as a reference standard. Ferritin is a spherical molecule with a hydrodynamic radius R of 6.9 +/- 0.1 nm and is stable for years in our standard Tris-HCl-NaCl buffer even at room temperature.