Molecular diffusion into ferritin: Pathways, temperature dependence, incubation time, and concentration effects

The detailed kinetics of permeation and effusion of small nitroxide spin pr obe radicals with the protein shells of horse spleen ferritin (HoSF) and hu man H-chain ferritin (HuHF) and a 3-fold channel variant D131H+E134H of HuH F were studied by electron paramagnetic resonance spectroscopy and gel perm eation chromatography under a variety of experimental conditions. The resul ts confirm that the permeation of molecular species of 7-9-Angstrom diamete r into ferritin is a charge selective process and that the threefold channe ls are the likely pathways for entry into the protein. Studies with holoHoS F show that increased temperature increases the rates of penetration and ef fusion and also increases the concentration of positively charged spin prob e accumulated within the protein in excess of that in the external solution . The interior of HoSF is much more accessible to small molecules at physio logical temperature of similar to 40 degrees C than at room temperature. Th e large activation energy of 63-67 kJ/mol measured for the effusion/penetra tion and the small diffusion coefficient, D similar to 5 x 10(-22) m(2)/s a t 20 degrees C, corresponding to a time of similar to 60 min for traversing the protein shell, is consistent with the kinetics of diffusion being larg ely controlled by the restrictive porosity of the protein itself. An invers e dependence of the first-order rate constant for effusion out of the prote in channel on the incubation time used for radical penetration into the pro tein is attributed to increased binding of the radical within the funnel-sh aped channel.