PROTEIN DIFFUSION IN POROUS CHROMATOGRAPHIC MEDIA STUDIED BY PROTON AND FLUORINE PFG-NMR

Diffusivities of several proteins or protein variants in nonadsorbing rigid-polymer and bonded-phase-silica chromatographic media were deter mined using pulsed-field-gradient NMR of both proton and fluorine nucl ei. Solute size, pore size, and concentration all affect the intrapart icle diffusivity. The intraparticle diffusivity of proteins is well re presented by a hindered diffusion of a sphere in a long cylinder and a tortuosity factor of 2.0 for a ratio of solute diameter to pore diame ter ranging from 0 to 0.3. An investigation of the effects of fluorine labeling on protein chemistry and hydrodynamics revealed that the ext ensive fluorine labeling denatured the protein, increasing its effecti ve Stokes radius. The fluorine labeling did not, however, significantl y alter the intraparticle diffusivity variation dependence on protein and pore sizes.