Molecular diffusion into ferritin: pathways, temperature dependence, incubation time, and concentration effects.
The detailed kinetics of permeation and effusion of small nitroxide spin probe radicals with the protein shells of horse spleen ferritin (HoSF) and human H-chain ferritin (HuHF) and a 3-fold channel variant D131H+E134H of HuHF were studied by electron paramagnetic resonance spectroscopy and gel permeation chromatography under a variety of experimental conditions. The results confirm that the permeation of molecular species of 7-9-A diameter into ferritin is a charge selective process and that the threefold channels are the likely pathways for entry into the protein. Studies with holoHoSF show that increased temperature increases the rates of penetration and effusion and also increases the concentration of positively charged spin probe accumulated within the protein in excess of that in the external solution. The interior of HoSF is much more accessible to small molecules at physiological temperature of approximately 40 degrees C than at room temperature. The large activation energy of 63-67 kJ/mol measured for the effusion/penetration and the small diffusion coefficient, D approximately 5 x 10(-22) m(2)/s at 20 degrees C, corresponding to a time of approximately 60 min for traversing the protein shell, is consistent with the kinetics of diffusion being largely controlled by the restrictive porosity of the protein itself. An inverse dependence of the first-order rate constant for effusion out of the protein channel on the incubation time used for radical penetration into the protein is attributed to increased binding of the radical within the funnel-shaped channel.
Yang
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Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, USA.
Arosio
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Chasteen
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D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002850:Chromatography, Gel; D003497:Cyclic N-Oxides; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry; D014644:Genetic Variation; D006736:Horses; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D009589:Nitrogen Oxides / Q000737:chemistry; D011759:Pyrrolidines; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013113:Spin Labels; D013816:Thermodynamics
D003497:Cyclic N-Oxides; D005609:Free Radicals; D009589:Nitrogen Oxides; D011759:Pyrrolidines; D011994:Recombinant Proteins; D013113:Spin Labels; C048737:tempo carboxylic acid; C077429:3-aminomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxyl; D005293:Ferritins; C039900:nitroxyl; C001803:tempol