Mechanism of the self-assembly of apoferritin from horse spleen |
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Authors: | M Gerl R Jaenicke |
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Institution: | (1) Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, D-8400 Regensburg, Federal Republic of Germany |
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Abstract: | Apoferritin from horse spleen can be reversibly dissociated at pH 2 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.Abbreviations CD
circular dichroism
- DTE
dithioerythritol
- G-HCl
guanidinium chloride
- SDS
sodium dodecylsulfate
- TEA
triethanolamine
- Tris
tris hydroxymethylamino methane
Dedicated to Professor Harold A. Scheraga on the occasion of his 65th birthday |
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Keywords: | Circular dichroism cross-linking ferritin fluorescence self-assembly |
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