Mechanism of the self-assembly of apoferritin from horse spleen. Cross-linking and spectroscopic analysis.
Apoferritin from horse spleen can be reversibly dissociated at pH2 or in 7.2 M G-HCl (pH 3.5). Reconstitution of the native icositetramer in 0.1 M TEA buffer (pH 7.9) in the presence of 1 mM EDTA and 3 mM dithioerythritol leads to yields higher than 80%. To monitor the kinetic mechanism, intrinsic fluorescence, far-UV circular dichroism, and covalent cross-linking with glutaraldehyde were applied. The overall mechanism of assembly is characterized by a sequence of concentration-dependent association reactions involving "structured monomers" and a dimeric intermediate as the most prominent species, apart from trimers and dodecamers. The parallel decrease in monomers, dimers and trimers indicates that association equilibria precede the formation of the final assembly product. The assembly reaction is accompanied by characteristic changes in fluorescence emission and dichroic absorption. To a first approximation, renaturation and reassociation may be quantitatively described by one single rate-determining second-order process, subsequent to fast folding steps at the monomer level.
Gerl
M
M
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Federal Republic of Germany.
Jaenicke
R
R
D000818:Animals; D001052:Apoferritins / Q000378:metabolism*; D002942:Circular Dichroism; D004226:Dithioerythritol / Q000494:pharmacology; D005293:Ferritins / Q000031:analogs & derivatives*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013154:Spleen / Q000378:metabolism*
D006146:Guanidines; D046911:Macromolecular Substances; D004226:Dithioerythritol; D005293:Ferritins; D001052:Apoferritins; D019791:Guanidine