Techniques to Quantify the Size of Protein Colloids in Amyloid Fiber Formation

Abstract

A new method for the analysis of protein colloidal diameter has been developed using three existing protein concentration quantification techniques, absorption at 280 nm, colloidal gold assay, and DC protein assay. Protein colloids are formed in the process of aggregation and are thought to be intermediates in protein self-assembly and formation of amyloid fiber. Deposition of the protein fibers in tissues leads to numerous human diseases including Alzheimer’s. Lysozyme was incubated at pH 2.0, 55°C, an environment conducive to amyloid fiber formation. The protein colloids present in the supernatant of the samples after centrifugation were studied over a time course of 30 days. The OD 280 assay detects total protein concentration based on absorption of radiation in the near UV. The colloidal gold assay and DC protein assay only measure colloidal sphere surface protein concentration. Due to the surface plasmon resonance, the light absorption spectrum changes when proteins bind to colloidal gold particles. Using the measured protein concentration on the surface of protein colloids along with the total measured protein concentration in the entire protein colloidal spheres, an interior protein concentration for all colloids is obtained. The protein colloidal sphere size can be calculated by using the ratio between the interior protein concentration and total protein concentration. Results indicate that the colloidal gold assay, DC protein assay, and OD 280 assay can be used to quantify the size of the protein colloids. The colloidal gold assay and DC protein assay are both independently effective in analysis of surface protein concentration in protein colloids. The DC protein assay was found to be much quicker in data production as it only requires 15 minutes of incubation time. The DC protein assay was also more reliable than the colloidal gold assay in accuracy and precision of results.

Share and Cite:

J. Anson, C. Lu, L. Cui, X. Yang and S. Xu, "Techniques to Quantify the Size of Protein Colloids in Amyloid Fiber Formation," Open Journal of Biophysics, Vol. 3 No. 1, 2013, pp. 22-32. doi: 10.4236/ojbiphy.2013.31003.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] S. Xu, “Aggregation Drives ‘Misfolding’ in Protein Amyloid Fiber Formation,” Amyloid, Vol. 14, No. 2, 2007, pp. 119-131. doi:10.1080/13506120701260059
[2] T. H. L. Nghiem, T. H. La, X. H. Vu, V. H. Chu, T. H. Nguyen, Q. H. Le, E. Fort, Q. H. Do and H. N. Tran, “Synthesis, Capping and Binding of Colloidal Gold Nanoparticles to Proteins,” Advances in Natural Sciences: Nanoscience and Nanotechnology, Vol. 1, No. 2, 2010, Article ID: 025009.
[3] J. Cardinal, J. R. Klune, E. Chory, G. Jeyabalan, J. S. Kanzius, M. Nalesnik and D. A. Geller, “Noninvasive Radiofrequency Ablation of Cancer Targeted by Gold Nanoparticles,” Surgery, Vol. 144, No. 2, 2008, pp. 125-132. doi:10.1016/j.surg.2008.03.036
[4] T. Lai, Q. Hou, H. Yang, X. Luo and M. Xi, “Clinical Application of a Novel Silver Nanoparticles Biosensor Based on Localized Surface Plasmon Resonance for Dentecting the Microalbuminuria,” Acta Biochimica et Biophysica Sinica, Vol. 42, No. 11, 2010, pp. 787-792. doi:10.1093/abbs/gmq085
[5] J. Satija, R. Bharadwaj, V. V. R. Sai, et al., “Emerging Use of Nanostructure Films Containing Capped Gold Nano particles in Biosensors,” Nanotechnology, Science and Applications, Vol. 2010, No. 3, 2010, pp. 171-188.
[6] O. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, “Protein Measurement with the Folin Phenol Reagent,” Journal of Biological Chemistry, Vol. 193, No. 1, 1951 pp. 265-275.
[7] J. Bradley, “Amyloid Fiber Formation in Hen Lysozyme.” Dissertation, University College Dublin, Dublin, 2007.
[8] S. Eustis and M. A. El-Say, “Why Gold Nanoparticles Are More Precious than Pretty Gold: Noble Metal Surface Plasmon Resonance and Its Enhancement of the Radiative and Nonradiative Properties of Nanocrystals of Different Shapes,” Chemical Society Reviews, Vol. 35, No. 3, 2006, pp. 209-217. doi:10.1039/b514191e
[9] L. H. Haber, S. J. J. Kwok, M. Semeraro and K. B. Eisenthal, “Probing the Colloidal Gold Nanoparticle/Aqueous Interface with Second Harmonic Generation,” Chemical Physical Letters, Vol. 506, No. 1-3, 2011, pp. 11-14. doi:10.1016/j.cplett.2011.03.042
[10] Bio-Rad, “DC Protein Assay Instruction Manual”. http://www.biorad.com/LifeScience/pdf/Bulletin_9005.pdf
[11] R. Cegielska-Radziejewska, G. Lesnierowski and J. Kijowski, “Properties and Application of Egg White Lysozyme and Its Modified Preparations—A Review,” Polish Journal of Food and Nutrition Sciences, Vol. 58, No. 1, 2008, pp. 5-10.
[12] X. Huang, S. Neretina and M. A. El-Sayed, “Gold Nanorods: From Synthesis and Properties to Biological and Biomedical Applications,” Advanced Materials, Vol. 21, No. 48, 2009, pp. 4880-4910. doi:10.1002/adma.200802789
[13] E. Hutter and J. H. Fendler, “Exploitation of Localized Surface Plasmon Resonance,” Advanced Materials, Vol. 16, No. 19, 2004, pp. 1605-1706. doi:10.1002/adma.200400271
[14] T. Chung, S. Lee, E. Y. Song, H. Chun and B. Lee, “Plasmonic Nanostructures for Nano-Scale Bio-Sensing,” Sensors, Vol. 11, No. 11, 2011, pp. 10907-10929. doi:10.3390/s111110907
[15] J. Mitchell, “Small Molecule Immunosensing Using Surface Plasmon Resonance,” Sensors, Vol. 10, No. 8, 2010, 7323-7346. doi:10.3390/s100807323
[16] S. Ghosh and T. Pal, “Interparticle Coupling Effect on the Surface Plasmon Resonance of Gold Nanoparticles: From Theory to Applications,” Chemical Reviews, Vol. 107, No. 11, 2007, pp. 4797-4862. doi:10.1021/cr0680282
[17] M. Nakkach, P. Lecaruyer, F. Bardin, J. Sakly, Z. B. Lakhdar and M. Canva, “Absorption and Related Optical Dispersion Effects on the Spectral Response of a Surface Plasmon Resonance Sensor,” Applied Optics, Vol. 47, No. 33, 2008, pp. 6177-6182. doi:10.1364/AO.47.006177
[18] Q. Li, Z. Zhang, S. Haque, M. Zhang and L. Xia, “Localized Surface Plasmon Resonance Effects by Naturally Occurring Chinese Yam Particles,” Journal of Applied Physics, Vol. 108, No. 12, 2010. doi:10.1063/1.3520667
[19] S. Yakota, “Effect of Particle Size on Labeling Density for Catalase in Protein A-Gold Immunocytochemistry,” Journal of Histochemistry and Cytochemistry, Vol. 36. No. 1, 1988, pp. 107-109. doi:10.1177/36.1.3335766
[20] S. Xu and M. F. Arnsdorf, “Calibration of the Scanning Atomic Force Microscope with Gold Particles,” Journal of Microscopy, Vol. 173, No. 3, 1994, pp. 199-210.
[21] W. Wang and C. J. Roberts, “Aggregation of Therapeutic Proteins,” John Wiley & Sons, Inc., Hoboken, 2010. doi:10.1002/9780470769829
[22] B. Brian, B. Sepulveda, Y. Alaverdyan, L. M. Lechuga and M. Kall, “Sensitivity Enhancement of Nanoplasmonic Sensors in Low Refractive Index Substrates,” Optics Express, Vol. 17, No. 3, 2015, pp. 2015-2023.
[23] F. M. Huan, D. Wilding, J. D. Speed, A. E. Russell, P. N. Bartlett and J. J. Baumbe, “Dressing Plasmons in Particle-in-Cavity Architechtures,” Nano Letters, Vol. 11, No. 3, 2010, pp. 1221-1226.

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.