The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation |
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| Authors: | Carrie A. May John K. Grady Thomas M. Laue Maura Poli Paolo Arosio N. Dennis Chasteen |
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| Affiliation: | 1. Department of Chemistry, University of New Hampshire, Durham, NH 03824-2544, USA;2. Center to Advance Molecular Interaction Science, University of New Hampshire, Durham, NH 03824-2544, USA;3. Department Materno Infantile e Tecnologie Biomediche, University of Brescia, 25125, Italy |
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| Abstract: | BackgroundFerritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.MethodsAnalytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.ResultsMore uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe2+ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s20,w = 17.0 S and D20,w = 3.21 × 10−7 cm2/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s20,w = 18.3 S and D20,w = 3.18 × 10−7 cm2/s.SignificanceThe methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals. |
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| Keywords: | AUC, analytical ultracentrifugation DTT, dithiothreitol HoSF, horse spleen ferritin HuHF, recombinant human H-chain ferritin MOPS, 3-(N-morpholino)propanesulfonic acid TEM, transmission electron microscopy D, diffusion coefficient (cm2/s) under the specific conditions of protein concentration, buffer and temperature c, concentration (g/cm3) D20,wo, diffusion coefficient of infinite dilute protein in pure water at 20 ° C (cm2/s) D20,w, diffusion coefficient of the protein at the stated concentration in pure water at 20 ° C (cm2/s) d, Stokes diameter (nm) f, frictional coefficient (g/s) g(s*), concentration distribution function (see Fig. 2 legend) η, viscosity (poise) ρ, density (g/mL) nFe, number of iron atoms per protein shell as determined by AUC or ferrozine assays M, molar mass (g/mol) Mr, relative molar mass (unitless) r, Stokes radius (nm) rm, radius of meniscus (cm) s, Svedberg constant for the protein at stated concentration and temperature in buffer (10&minus 13 s) sW, weighted average of s over the g(s*) curve s20,wo, Svedberg constant for infinitely dilute protein in pure water at 20 ° C (10&minus 13 s) s20,w, Svedberg constant of protein at stated concentration in pure water at 20 ° C (10&minus 13 s) ω, angular velocity (rad/s) σ, linewidth of Gaussian function as defined in the text (svedbergs) ν?, partial specific volume (mL/g) |
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