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Irreversible Nature of Mesoscopic Aggregates in Lysozyme Solutions

Abstract

Study of protein stability in solutions is important for better understanding the pathogenesis of diseases caused by abnormal protein folding and aggregation. Lysozyme is known to form mesoscopic aggregates (30–100 nm radius) in concentrated solutions (>30 mg/mL), however the origin and thermodynamic status of these aggregates remain unclear. In this work we have investigated the effects of concentration, filtration, and temperature on the sizes and relative amount of mesoscale aggregates in solutions of lysozyme. We have used dynamic light scattering, small-angle X-ray scattering, and size exclusion chromatography (SEC). Mesoscopic protein aggregates were commonly thought to be in equilibrium with protein monomers in solution, resulting from a reversible self-assembly of the monomers. We instead show that systematic filtration through 20 nm pore size filters completely removes the aggregates from solution. The aggregates do not reemerge. Without filtering, the relative number of monomers decreases with increasing solution temperature, indicating formation of more aggregates. SEC was used to search for the presence of lysozyme dimers, which have been previously hypothesized to be related to the formation of mesoscopic aggregates. SEC did not detect dimers in solutions of filtered or unfiltered lysozyme. Taken together, our results strongly suggest that the mesoscopic aggregates in lysozyme are not caused by reversible self-assembly of lysozyme monomers and are not an intrinsic property of lysozyme monomers in their native state. We hypothesize that the lysozyme aggregation is likely due to some impurities in lysozyme introduced during purification or lyophilization and/or to traces of misfolded lysozyme.

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ACKNOWLEDGMENTS

We thank Prof. Peter Vekilov for consulting us on the issue of mesoscopic aggregation in lysozyme solutions and Prof. Marc Taraban for advice on Size Exclusion Chromatography.

Funding

Acknowledgment is made to the donors of the American Chemical Society Petroleum Research Fund for support of this research (Grant no. 59 434-ND6). T.J.W. acknowledges partial funding from the Maryland Industrial Partnerships program.

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Correspondence to T. J. Woehl or M. A. Anisimov.

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Nikfarjam, S., Ghorbani, M., Adhikari, S. et al. Irreversible Nature of Mesoscopic Aggregates in Lysozyme Solutions. Colloid J 81, 546–554 (2019). https://doi.org/10.1134/S1061933X19050090

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  • DOI: https://doi.org/10.1134/S1061933X19050090