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1 Division of Physical Chemistry, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2 Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QT, UK.
Reprint requests to: Daizo Hamada, Department of Developmental Infectious Diseases, Research Institute and Osaka Medical Center for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594–1011, Japan; e-mail: daizo{at}lab.mch.pref.osaka.jp; fax: +81-(0)725-57-3021.
The formation of fibrillar aggregates by ß-lactoglobulin in the presence of urea has been monitored by using thioflavin T fluorescence and transmission electron microscopy (TEM). Large quantities of aggregated protein were formed by incubating ß-lactoglobulin in 3–5 M urea at 37°C and pH 7.0 for 10–30 days. The TEM images of the aggregates in 3–5 M urea show the presence of fibrils with diameters of 8–10 nm, and increases in thioflavin T fluorescence are indicative of the formation of amyloid structures. The kinetics of spontaneous fibrillogenesis detected by thioflavin T fluorescence show sigmoidal behavior involving a clear lag phase. Moreover, addition of preformed fibrils into protein solutions containing urea shows that fibril formation can be accelerated by seeding processes that remove the lag phase. Both of these findings are indicative of nucleation-dependent fibril formation. The urea concentration where fibril formation is most rapid, both for seeded and unseeded solutions, is 
5.0 M, close to the concentration of urea corresponding to the midpoint of unfolding (5.3 M). This result indicates that efficient fibril formation involves a balance between the requirement of a significant population of unfolded or partially unfolded molecules and the need to avoid conditions that strongly destabilize intermolecular interactions.
Keywords: Amyloid fibril; kinetics; ß-lactoglobulin; thioflavin T; electron microscope; MALDI-TOF mass
Abbreviations: NMR, nuclear magnetic resonances • TEM, transmission electron microscopy • ANS, 1-anilino-8-naphthalene sulfonic acid • ß-LG, ß-lactoglobulin • thioT, thioflavin T • MALDI, matrix associate laser disorption • TOF, time of flight • d-1, day -1
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