Abstract
We have compared the size and shape of ferritins from human heart, rat heart, and skeletal muscle with those of rat liver and horse spleen ferritins, using sedimentation and gel filtration techniques. The electrophoretically "fast" form of heart ferritin was partially separated from both the "slow" heart form and from rat liver ferritin by gel filtration (Stokes radii 72 A versus 69 and 68.5 A). Sedimentation velocity after iron removal showed an 18.5 S boundary even for mixtures of the two heart species, versus 17.3 S for rat liver and horse spleen apoferritins. Holoferritins gave a broad boundary, with coefficients from 66-80 S depending on iron content. Variable amounts of disaggregated ferritin (2.6 S) were also present in the heart ferritin preparations. Removal of iron significantly increased the electrophoretic migration of the fast but not the slow heart ferritin species. By sedimentation equilibrium, the molecular weights of all apoferritins save the fast heart form were about 490,000; that for the latter was near 750,000. Since electrophoresis revealed no major differences in subunit size, it is concluded that the larger, more asymmetric form of muscle ferritin contains 34 to 38 rather than 24 subunits.
Highlights
The temperature of theheat treatment was not allowed to Ferritins are large molecular weight iron storage proteins found in all animal ceils and in the cells of most other phyla (1).Studies, especially on ferritin from horse spleen, cornbining evidence fromx-ray diffraction (2,3),SDS-gel electrophoexceed 70 “C,and gel filtration was on Ultrogel AcA34 (LKB) rather than Sephadex G-200
In 1969, we demonstrated by disc polyacrylamide gel elec- Gel Filtration-For determination of apparent molecular weight trophoresis the presence of two major forms of ferritin in rat heart (9);other tissues showed only one major form by this technique (10).Later, we found that some human hearts contained two forms,a finding since confied by other workers (12)
Since the properties of rat heart anddiaphragm ferritins were indistinguishable in gel filtration, sedimentation, and electrophoresis, in later studies, ferritins were purified froma pool of both tissues to obtain more material
Summary
Iron thioglycolate was removed by filtration on 50-ml columns of Sephadex G-100 or Ultrogel AcA34, equilibrated with 0.02 M phosphate, pH 7.0 (containing NaN3 and PMSF, as above), or in some cases, with 0.05 M sodium acetate,pH 5.5 (with the same (1, 6). Shape of Heart, Liver, and Spleen Ferritins some cases, buffers were 0.05 M sodium acetate, pH 5.5, or 0.10 M Tris/glycine, pH 8.9 (containing the same additives). Electrophoresis-Electrophoresis was performed on holoferritin and apoferritin samples under nondenaturing conditions, as previously described (9,19),or afterdissociation in 2%SDS, 2% 2-mercaptoethanol(in 0.02 M potassium phosphate, pH 7.0) in SDS slab, gradient polyacrylamide gels (IO to 15% T) (19). Ferrocyanide staining (9)was used to visualize ferritin-iron bands on the gels
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