Chapter 30
Charge Development on Ferritin: An Electrokinetic Study of a Protein Containing a Ferrihydrite Nanoparticle 1
1
2
2
MarkAllen ,TrevorDouglas ,DanielleNest ,Martin Schoonen , and Daniel Strongin Downloaded by UNIV OF GUELPH LIBRARY on May 29, 2012 | http://pubs.acs.org Publication Date: December 14, 2004 | doi: 10.1021/bk-2005-0890.ch030
3
1
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717 Department of Geosciences, Stony Brook University, Stony Brook, NY 11794 Department of Chemistry, Temple University, Philadelphia, PA 19122 2
3
Introduction The iron storage protein ferritin is ubiquitous in biological systems where it functions to sequester iron as a nanoparticle of ferric oxyhydroxide, encapsulated within a protein cage-like structure. Ferritin has several interesting physical characteristics that may lead to new remediation techniques in the future. Both the natural ferritin, synthetic ferritin-like materials, as well as ferritin-derived nano particles have potential use in new remediation techniques. For example, ferritin has already been shown to promote the reduction of hexavalent chromium dissolved in aqueous solutions (1). In addition, by changing the functionality of the surface groups, the surface charge and hydrophobicity of cage can be controlled (2). Hence, it may be possible to tailor the properties of the protein cage for a particular remediation application. Finally, apo-ferritin (i.e., ferritin without a mineral nano particle) can be used for the synthesis of nanometer-sized metal oxyhydroxide or metal sulfide particles (3-6). The protein cage can be removed after synthesizing the nm-size particle within the cage. In this contribution, we report our first results of a collaborative study of the surface charge development of ferritin as a function of solution conditions. This represents a small, but important component of our research program. Similar to contaminant-colloid interactions (7), interaction of dissolved ionic 226
© 2005 American Chemical Society
In Nanotechnology and the Environment; Karn, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
227
Downloaded by UNIV OF GUELPH LIBRARY on May 29, 2012 | http://pubs.acs.org Publication Date: December 14, 2004 | doi: 10.1021/bk-2005-0890.ch030
contaminants with ferritin may be influenced or dictated by the net surface charge of the protein. In addition, binding of ionic species, for example metal ions, can change the surface charge of the protein. Hence, in designing remediation strategies based on ferritin or ferritin-like materials, it is important to understand their surface charge development as function of solution composition,
Ferritin The ferritin protein cage is comprised of 24-subunits, which assemble into a cage-like architecture roughly 12 nm in diameter (8,9). In the assembled structure, the protein subunits of ferritin present charged amino acid side chains on the outer surface. These acidic (glutamic acid, aspartic acid) and basic residues (arginine, lysine, histidine) dominate the charge characteristics of the protein. Calculations of the electrostatic potential of the assembled protein reveal delocalized charge over the surface of the protein with some localized regions of high charge density (10). The overall surface charge of the protein is highly pH dependent due to acid-base ionization of the surface exposed residues. The pH at which the horse-spleen ferritin protein carries no net charge, the isoelectric point (pi), has been reported as 4.5*4.7, when measured by isoelectricfocusing(11). Here, we report the measurement of the isoelectric point of native ferritin (containing Fe 0 ) by measuring the zeta potential as a function of solution pH. 2
3
Light scattering-based Zetapotential Measurements The effective surface charge, at the shear plane of a colloidal particle, can be measured directly as the zeta potential. This is accomplished by measuring the electrophoretic mobility of the particle (μ), during application of an electric field. Given the size of the ferritin, 12 nm diameter, dynamic light scattering techniques must be used to determine the electrophoretic mobility (12,13). There are two light scattering-based techniques to measure electrophoretic mobilities. These are Laser Doppler Velocimetry (LDV) and Phase Analysis Light Scattering (PALS). LDV is routinely used and electrophoretic mobility is related to changes in the correlation function as a result of an applied electrical field. The method requires the use of a DC field on the other of several volts per cm and is limited to solutions with ion strengths below about 10 mmol/L. By contrast, PALS*based zetapotential measurements can be obtained on suspensions with ion strengths as high as 3 M KC1. In this study we report zetapotential data obtained using the PALS method.
In Nanotechnology and the Environment; Karn, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
228
Experimental Native horse spleen ferritin (Calzyme, SanLuis Obispo CA) containing a ferrihydrite (Fe 03»«H 0) mineral core was purified by size exclusion chromatography (Superose 6, Pharmacia-Amersham) and monitored both at 280 nm (protein) and 415 nm (Fe-oxide). Prior to investigation of the zeta potential, the purified protein was dialyzed into an appropriate buffer (50 mM acetate: pH 4.0, 4.5, 5.0, MES: pH 5.5, 6.0, 6.5) or into a salt background (1 mM KC1). The pH of the low salt sample was adjusted by first concentrating the sample in a Microcon ultrafiltration unit and diluting back to the original volume with the appropriate buffer (1 mM) and checking the pH. The electrophoresis measurements were conducted in two different laboratories (Montana State University and Stony Brook University) on the exactly the same ferritin with identical equipment. The equipment in both laboratories is a Birookhaven Instruments Corporation ZetaPlus equipped with a red diode laser (671 nm) and PALS option.
Downloaded by UNIV OF GUELPH LIBRARY on May 29, 2012 | http://pubs.acs.org Publication Date: December 14, 2004 | doi: 10.1021/bk-2005-0890.ch030
2
2
Results The AC zeta potential measurements were performed on mineralized HS ferritin samples at both low (ImM) and high (50 mM) ionic strength. Prior to zeta potential measurements, the protein was first purified by size exclusion chromatography and analysed by dynamic light scattering and size exclusion chromatography to confirm that the sample was monodisperse, with a diameter of 12 nm. As shown in Figure 1, the change in the measured zeta potential as a function of pH follows the typical sigmoidal shape of a titration curve in the pH range from 3 to 7. The measured data could be fit to a sigmoidal curve. There was no significant difference between the curves measured by investigators at the two institutions. In addition, there was no significant difference between measurements made at low and high ionic strength. At a pH of 4.3 to 4.4, the ferritin samples were found to have no net charge, which corresponds to the isoelectric point (pi) of these protein assemblies. This was independent of the ionic strength as shown in Figure 1 measured, at 1 mM and 50 mM respectively, using the AC measurement. This is only slightly lower than the pi value reported from isoelectric focusing methods (11). The results reported here are only the first step toward understanding the interaction of dissolved metal ions and ferritin. Using the results presented here as a reference, we are now conducting electrokinetic experiments to evaluate the interaction of dissolved metal ions, including chromate, with ferritin. The notion is that if a metal ion forms a chemical bond with the sorbent (here ferritin), it will change the sorbent's surface charge. We have used this research strategy before to study the interaction of aqueous metal ions and aqueous organic ions and molecules with iron disulfide (14,15). In Nanotechnology and the Environment; Karn, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
229
Downloaded by UNIV OF GUELPH LIBRARY on May 29, 2012 | http://pubs.acs.org Publication Date: December 14, 2004 | doi: 10.1021/bk-2005-0890.ch030
Φ 50 mM buffer
Ί 3.5
1 4.0
« 4.5
. 5.0
1 55
τ* 50
1 6.5
f7Ό
ΡΗ
Figure 1: Zeta potential of mineralizedferritin measured by AC phase analysis light scattering under low and high ionic strength conditions.
References 1. Kim, I., Hosein, H.-A., Strongin, D. R., Douglas, T. Chemistry of Materials 2002, 14, 4874. 2. Wong, Κ. K . W., Colfen, H., Whilton, N . T., Douglas, T., Mann, S. J. Inorganic Biochemistry 1999, 76, 187. 3. Meldrum, F. C., Wade, V. J., Nimmo, D. L., Heywood, B. R., Mann, S. Nature 1991, 349, 684. 4. Meldrum, F. C., Heywood, B. R., Mann, S. Science 1992, 257, 522. 5. Meldrum, F. C., Douglas, T., Levi, S., Arosio, P., Mann, S. J. Inorg. Biochem. 1995, 58, 59. 6. Douglas, T., Stark, V. T. Inorg. Chem. 2000, 39, 1828. 7. Morel, F. M . M . , Gschwend, P. M., "The role of colloids in the partitioning of solutes in natural waters," 1987. 8. Harrison, P. M., Arosio, P. Biochimica et Biophysica Acta 1996, 1275, 161. 9. Chasteen, N . D., Harrison, P. M . J. Struct. Biol. 1999, 126, 182. 10 Douglas, T., Ripoll, D. Protein Science 1998, 7, 1083. 11.Otsuka, S., Listowsky, I., Niitsu, Y . , Urushizaki, I. J. Biol. Chem. 1980, 255, 6234. 12. Finsy, R. Advances in Colloid and Interface Science 1994, 52, 79. 13. Tscharnuter, W. W. Applied Optics 2001, 40, 3995. 14. J. Bebie and M.A.A. Schoonen, Geochemical Transactions 2000, 47. 15. J. Bebié, M.A.A. Schoonen, D.R. Strongin and M. Fuhrmann, Geochimica Cosmochimica Acta 624. , 633-642, 1998. In Nanotechnology and the Environment; Karn, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
