Photon correlation spectroscopy of human IgG

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Abstract
The translational diffusion coefficient D 020,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 D 020,w =3.89×10−7±0.02 cm2/sec, in good agreement with the value determined by classic mehods. This value corresponds to an effective hydrodynamic radius R, of 55.1±0.3 Å. 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=2.81×10−7±0.04 cm2/sec at 1.6 mg/ml, which corresponds to a hydrodynamic radius of 75 Å. For light-scattering studies of protein molecules in the dimension range of 5–10 nm (Mr=105−107) 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.