On the mechanism of horse spleen apoferritin assembly: a sedimentation velocity and circular dichroism study.
The apoferritin shell is known to assemble spontaneously from its subunits obtained at acid pH upon neutralization. The reassembly of apoferritin from horse spleen has been followed by means of sedimentation velocity and circular dichroism experiments as a function of the pH and the nature of the assembly buffer in order to obtain information on the assembly pathway. In all the buffer systems tested the subunits sediment as a single peak of varying sedimentation and diffusion coefficients, and shell assembly starts at pH values around 3.5. In dilute glycine-acetate buffers the subunits are essentially dimeric up to this pH value. Therefore, the dimeric building blocks of the apoferritin shell that are apparent in the X-ray structure represent the first assembly intermediates. When the pH is increased to 4.0-4.3, the weight-average sedimentation velocity of the subunits increases to 3.6-4.7 S, respectively, and the subunit population becomes heterogeneous. Concomitantly, significant changes in the circular dichroism properties of the aromatic residues take place. On the basis of the X-ray structure, where aromatic residues appear to be located at or near the fourfold symmetry axes, these data suggest that assembly proceeds from dimers through tetramers and octamers. In the pH range 4.5-6.5 the reassembly process cannot be followed due to reversible precipitation of the subunits near their isoelectric point; at neutral pH values essentially quantitative reassembly is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001052:Apoferritins / Q000378:metabolism*; D002942:Circular Dichroism; D005293:Ferritins / Q000031:analogs & derivatives*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D013154:Spleen / Q000378:metabolism*
D046911:Macromolecular Substances; D005293:Ferritins; D001052:Apoferritins; D007501:Iron