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Sonication of proteins causes formation of aggregates that resemble amyloid

¹ Guelph-Waterloo Centre for Graduate Studies in Chemistry and Biochemistry and ² Department of Physics, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
³ Department of Medical Biophysics and Ontario Cancer Institute, University of Toronto, Toronto, Ontario M5G 2M9, Canada

(RECEIVED April 22, 2004; FINAL REVISION July 28, 2004; ACCEPTED August 5, 2004)

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green-gold birefringence upon binding the dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication-induced aggregates have high -content, and proteins with significant native -helical structure show increased -structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with diameters of 5–20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye-binding and structural characteristics, as well as the ability of the sonication-induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.

Keywords: sonication; ultrasound radiation; amyloid; fibrils; protein aggregation; -structure; protein conformational disorder; protein misfolding; Abbreviations: ThT, thioflavin T; CR, Congo red; CD, circular dichroism; TEM, transmission electron microscopy; DLS, dynamic light scattering; PMCA, protein misfolding cyclic amplification; SDS-PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis; BSA, bovine serum albumin; SOD, human cytosolic Cu/Zn superoxide dismutase; GSH, reduced glutathione; K_d, apparent dissociation constant

Reprint requests to: Elizabeth M. Meiering, Guelph-Waterloo Centre for Graduate Studies in Chemistry and Biochemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada; e-mail: meiering{at}uwaterloo.ca; fax: (519) 746-0435.

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