Forward-Light-Scattering Characterization of Pre-Crystalline Aggregates in Crystallizing Lysozyme Solutions


We present a method to characterize lysozyme pre-crystalline aggregates using a forward-light-scattering technique, which is highly sensitive to protein aggregates. The static light scattering properties at small angles of crystallizing lysozyme aggregates are discussed, and related to the crystallization conditions based on the concentration of added precipitant NaCl. Lysozyme solutions that started to crystallize because of precipitant exhibited profiles of forward light scattering that could be fitted by non-integer power laws, which indicated fractal aggregations of lysozyme had formed. Pre-crystalline solutions, in which lysozyme crystals later grew, had dense structural fractal clusters with fractal dimensions of D > 1.5. In contrast, solutions with aggregates in which crystals did not grow, had forward-light-scattering profiles that deviated from a power law or had lower power values.

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Wakamatsu, T. (2014) Forward-Light-Scattering Characterization of Pre-Crystalline Aggregates in Crystallizing Lysozyme Solutions. American Journal of Analytical Chemistry, 5, 581-588. doi: 10.4236/ajac.2014.59065.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] McPherson, A. (1982) Preparation and Analysis of Protein Crystals. Wiley, New York.
[2] Chayen, N.E. and Saridakis, E. (2008) Protein Crystallization: From Purified Protein to Diffraction-Quality Crystal. Nature Methods, 5, 147-153.
[3] Muschol, M. and Rosenberger, F. (1997) Liquid-Liquid Phase Separation in Supersaturated Lysozyme Solutions and Associated Precipitate Formation/Crystallization. The Journal of Chemical Physics, 107, 1953-1962.
[4] Tanaka, S., Yamamoto, M., Ito, K., Hayakawa, R. and Ataka, M. (1997) Relation between the Phase Separation and the Crystallization in Protein Solutions. Physical Review E, 56, R67-R69.
[5] Tanaka, S., Ataka, M. and Ito, K. (2002) Pattern Formation and Coarsening during Metastable Phase Separation in Lysozyme Solutions. Physical Review E, 65, 51804-1-6.
[6] Price, W.S., Tsuchiya, F. and Arata, Y. (1999) Lysozyme Aggregation and Solution Properties Studied Using PGSE NMR Diffusion Measuerments. Journal of the American Chemical Society. 121, 11503-11512.
[7] Tanaka, S., Ito, K., Hayakawa, R. and Ataka, M. (1999) Size and Number Density of Precrystalline Aggregates in Lysozyme Crystallization Process. The Journal of Chemical Physics, 111, 10330-10337.
[8] Skouri, M., Lorber, B., Giegé, R., Munch, J. -P. and Candau, J. S. (1995) Effect of Macromolecular Impurities on Lysozyme Solubility and Crystallizability: Dynamic Light Scattering, Phase Diagram, and Crystal Growth Studies. Journal of Crystal Growth, 152, 209-220.
[9] Muschol, M. and Rosenberger, F. (1995) Interactions in Undersaturated and Supersaturated Lysozyme Solutions: Static and Dynamic Light Scattering Results. The Journal of Chemical Physics, 103, 10424-10432.
[10] Sazaki, G., Ooshima, H., Kato, J. Harano, Y. and Hirokawa, N. (1993) Mechanism of Crystallization of Enzyme Protein Thermolysin. Journal of Crystal Growth, 130, 357-367.
[11] Eberstein, W., Georgalis, Y. and Saenger, W. (1994) Molecular Interactions in Crystallizing Lysozyme Solutions Studied by Photon Correlation Spectroscopy. Journal of Crystal Growth, 143, 71-78.
[12] Tanaka, S. and Ataka, M. (2002) Protein Crystallization Induced by Polyethylene Glycol: A Model Study Using Apoferritin. The Journal of Chemical Physics, 117, 3504-3510.
[13] Georgalis, Y., Zouni, A., Eberstein, W. and Saenger, W. (1993) Formation Dynamics of Protein Precrystallization Fractal Clusters. Journal of Crystal Growth, 126, 245-260.
[14] Georgalis, Y., Umbach, P., Saenger, W., Ihmels, B. and Soumpasis, D.M. (1999) Ordering of Fractal Clusters in Crystallizing Lysozyme Solutions. Journal of the American Chemical Society, 121, 1627-1635.
[15] Tanaka, S., Yamamoto, M., Kawashima, K., Ito, K., Hayakawa, R. and Ataka, M. (1996) Kinetic Study on the Early Stage of the Crystallization Process of Two Forms of Lysozyme Crystals by Photon Correlation Spectroscopy. Journal of Crystal Growth, 168, 44-49.
[16] Witten, T.A. and Pincus, P.A. (2004) Structured Fluids: Polymers, Colloids, Surfactants. Oxford, New York.
[17] Schaefer, D.W., Martin, J. E., Wiltzius, P. and Cannell, D.S. (1984) Fractal Geometry of Colloidal Aggregates. Physical Review Letters, 52, 2371-2374.
[18] Weitz, D. A., Huang, J. S., Lin, M. Y. and Sung, J. (1985) Limits of the Fractal Dimension for Irreversible Kinetic Aggregation of Gold Colloids. Physical Review Letters, 54, 1416-1419.
[19] Lin, M. Y., Lindsay, H.M., Weitz, D.A., Ball, R.C., Klein, R. and Meakin, P. (1989) Universality in Colloid Aggregation. Nature, 339, 360-362.
[20] Wakamatsu, T. (2011) Forward Light Scattering for Highly Sensitive Detection of Aggregation in Crystallizing Protein Solutions. Applied Physics Letters, 98, 263701-1-3.
[21] Wakamatsu,T., Toyoshima, S. and Shimizu, H. (2011) Observation of Electric-Field Induced Aggregation in Crystallizing Protein Solutions by Forward Light Scattering. Applied Physics Letters, 99, 153701-1-3.

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