- Email: [email protected]
Alkaline transition of horse heart cytochrome c in the presence of ZnO nanoparticles
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 410–414
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Alkaline transition of horse heart cytochrome c in the presence of ZnO nanoparticles Michaela Šimšíková a, Marián Antalík a,b,⇑ a b
Department of Biochemistry, Faculty of Science, PJ Šafárik University, Šrobárova 2, 041 54 Košice, Slovakia Department of Biophysics, Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Košice, Slovakia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" The influence of ZnO nanoparticles
to the cytochrome c in alkaline pH have been studied. " The structural stability around the heme of cyt c assembled with ZnO nanoparticles was increased. " The pKa constant of cyt c-ZnO complex was took place to the higher value. " The alkaline transition of cytochrome c in the presence of ZnO nanoparticles was more cooperative.
a r t i c l e
i n f o
Article history: Received 2 May 2012 Received in revised form 4 October 2012 Accepted 8 October 2012 Available online 16 October 2012 Keywords: Cytochrome c Zinc oxide Nanoparticles Alkaline transition
a b s t r a c t The effect of zinc oxide nanoparticles (ZnO NPs) on cytochrome c (cyt c) in alkaline pH was studied with absorption spectroscopy and UV circular dichroism (CD). Spectral data from UV–vis spectroscopy and circular dichroism indicate only small changes in the native structure of the protein at neutral pH after the interaction with ZnO nanoparticles. The stability around the heme crevice of cyt c and therefore the switch of the axial ligand Met80 to Lys which occurs in conditions of higher pH was proven following the interaction of cytochrome c with ZnO nanoparticles. The formation of cyt c-ZnO NPs complex based on electrostatic attraction was accompanied by a significant increase in the apparent pKa constant of the alkaline transition of cyt c. Ó 2012 Elsevier B.V. All rights reserved.
Introduction Research into the hierarchy of interactions which stabilize the native states of proteins is vital in order to understand the factors that govern the formation of their three-dimensional structure. One major focus of this research has been the study of the structure and stabilizing mechanism of protein conformational changes. One of the most extensively studied proteins is cytochrome c which is an essential component of mitochondrial electron transfer ⇑ Corresponding author at: Department of Biochemistry, Faculty of Science, PJ Šafárik University, Šrobárova 2, 041 54 Košice, Slovakia. Tel.: +421 55 7204138; fax: +421 55 7204139. E-mail address: [email protected] (M. Antalík). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.10.007
activity, an important mediator in pro-apoptotic [1] and apoptotic pathways and a regulator of biological activity [2,3]. The multifunctional nature of cytochrome c is thought to be a result of the conformational transitions which the protein undergoes. The alkaline transition, a high pH induced conformational change in which the axial ligand Met80 dissociates and is replaced by a lysine (Lys) [4,5], has been the subject of great interest as a model of the potential regulation of cyt c for use as a biological function [6]. The interconversion from the native to the alkaline form of cytochrome c operates as an efficient binary molecular switch which has a dramatic effect on the electron-transfer activity of the protein [7–9]. Recent years have seen great advances in the study of nanoparticles, both in terms of their unique properties and their potential
M. Šimšíková, M. Antalík / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 410–414
applications, i.e. biolabeling [10], biosensing [11], therapy [12,13], or as scaffolds for tissue engineering [14]. One result of the use of nanoparticles in bio-applications has been the discovery of structural and functional changes of biomolecules caused by the exposure of proteins to inorganic surfaces. Our group has previously published work on the influence of various polyanions with different lengths and structures and negatively charged gold nanoparticles on the structure and stability of horse heart cytochrome c under conditions of varying temperatures and pH factors [15– 19]. In this study, we follow up on this work and examine the effects of ZnO nanoparticles on the conformation of cytochrome c in alkaline pH conditions.
Materials and methods Reagents and chemicals Horse heart cytochrome c (type IV) was purchased from Sigma as salt-free, dry powder and was used without further purification. The concentration of protein solution in the native state was determined with absorption spectroscopy using a Shimadzu UV-3600 UV–vis–NIR photospectrometer, with an extinction coefficient of e408 = 106,100 mol1 cm1 [20]. Zinc chloride (ZnCl2 P 99.995%) and sodium hydroxide (NaOH P 98%) were obtained from Sigma and were of analytical grade. Ultra pure water (Merck Millipore) was used throughout the experiments.
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UV–vis spectroscopy The alkaline transition changes of cytochrome c were performed on a Shimadzu UV-3600 UV–vis–NIR photospectrometer. Difference absorption spectra were recorded in range of 215– 850 nm with a spectral resolution of 2 nm. Alkaline titrations were performed by adding very small amounts of concentrated NaOH to a solution of free cytochrome c or a solution of cytochrome associated with ZnO nanoparticles. Potassium ferricyanide (1 lM ferricyanide for 6 lM cytochrome c solutions or 2 lM for 60 lM cytochrome c solutions) was added to each solution in order to ensure complete oxidation of the proteins. The spectrum at pH 6.00 ± 0.05 was used as a reference; the absorption differences were followed at 400–415 nm in the Soret region and at 695–800 nm in the charge transfer band which is specific for the native Met80 to heme Fe ligation. The pH dependences of all transitions were analyzed using the following formula:
pH ¼ pK app
Aprotonated Aobserved 1 log n Aobserved Adissociated
ð1Þ
where Aobserved is the spectral difference at a given pH, Aprotonated and Adissociated are spectral differences at pH 6.50 and 12.00, respectively, 1/n is the slope, the measurement of the number of protons (n) participating in the transition, and pKapp represents the apparent pK constant of the observed transition. Circular dichroism (CD)
Preparation of zinc oxide nanoparticles The nanoparticles of zinc oxide were prepared using a precipitation method [21], with zinc chloride and sodium hydroxide servings as precursors. The mixture of 0.2 M ZnCl2 (0.06885 g, 2.5 mL) and equimolar aqueous solution of NaOH (0.1 g, 12.5 mL) was stirred for 3 h at room temperature (25 °C ± 1) producting a milky white solution. After continuous stirring, the precipitate was collected through centrifugation (Eppendorf 5804R centrifuge; 6 min, 25 °C) at 100 rpm and subsequently at 15,000 rpm, which removed the eventual aggregates from the sample. The resulting fraction was washed in ultrapure Millipore water several times and allowed to evaporate at room temperature in order to obtain ZnO nanoparticles in white powder form.
The structural change of ferricytochrome c induced by the addition of ZnO NPs was evaluated with CD spectra measured with a JASCO J-815 spectropolarimeter. The cyt c was converted to a completely oxidized form by the addition of small amounts of potassium ferricyanide. The CD measurements of 10 lM free cytochrome c and cyt c complexed with zinc oxide nanoparticles (0.137; 0.416; 1.0; 2.0, and 3.0 mg mL1) were recorded in far UV region of 180–260 nm and 0.2 M cyt c (free or as a complex with ZnO-NPs at concentrations of 2.74 and 8.32 mg mL1) was used in Soret region of 350–450 nm. Alkaline titrations were performed by adding very small amounts of concentrated NaOH to the solution of free cytochrome c (10 lM) or solution of cytochrome c conjugated with zinc oxide nanoparticles (0.137 mg mL1). All measurements were performed at room temperature using a rectangular cuvette with an optical path length of 1 mm, a scan rate of 100 nm min1 with an average of five consecutive scans.
Atomic Force Microscopy (AFM) Results and discussion AFM images of ZnO NPs were collected using an Innova AFM system (Veeco Instruments) in tapping mode following 5 min of absorption on a freshly cleaved mica surface. The AFM tips used for the measurement were commercial antimony-doped silicon cantilevers (NCHV, Veeco Instruments) with a nominal tip radius of approximately 10 nm and a resonance frequency 301–331 kHz. The scanning rate of the measurements was 1 Hz and the scan size was in the range of 500 500 points. The data was analyzed with NanoScope Analysis (1.20) to obtain particle sizes and morphology.
Size characterization of ZnO nanoparticles The shape and diameter of zinc oxide nanoparticles were analyzed with Atomic Force Microscopy (AFM). Fig. 1 shows that the nanoparticles are spherical with a diameter of 50–80 nm. The size of ZnO nanoparticles was confirmed by Malvern Zetasizer Nano ZS. The particle size distribution demonstrates a relatively narrow range (Fig. 1, detail) which suggests the complete absence of aggregated particles.
Particle size distribution of ZnO nanoparticles
UV–vis spectroscopy
The average particle size of ZnO nanoparticles was determined by Malvern Zetasizer Nano ZS (4 mW, 633 nm laser) at 25 °C and pH 6.98. During the experiments, 0.1 mg mL1 of ZnO solution which had been sonicated for approximately 1 min was used.
It is possible to demonstrate the native conformation of cytochrome c from optical spectra results, and Fig. 2 shows the UV– vis absorption spectrum of native ferricytochrome c. It can be seen that cyt c has characteristic Soret and Q transitions bands at
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Fig. 1. Atomic force micrograph of the ZnO nanoparticles (bar 500 nm). The detail shows the size of nanoparticles determined by small angle scattering which remained in a narrow range.
Fig. 3. (A) The pH dependencies of absorbance changes measured in a Soret band region for free ferricytochrome c (black squares) and ferricytochrome c associated with ZnO at concentrations of 0.l mg mL1 (red left triangles). The concentration of ferricytochrome c was 6 lM. (B) The pH dependencies of absorbance changes measured in the 695 nm charge transfer band region for free ferricytochrome c (black squares) and ferricytochrome c associated with ZnO NPs at concentrations of l mg mL1 (red left triangles). The concentration of ferricytochrome c was 60 lM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Absorption spectra of 6 lM ferricytochrome c (purple dash line) and 6 lM ferricytochrome c adsorbed on ZnO nanoparticles (0.1 mg mL1; olive straight line). The detail shows the absorption spectra of 60 lM ferricytochrome c (purple dash line) and 60 lM ferricytochrome c adsorbed on ZnO nanoparticles (0.1 mg mL1; olive straight line) observed at the range of 600–850 nm. All measurements were performed in ultrapure water, pH 7 at 25 °C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
409 nm and near 528 nm, both of which originate in the heme chromophore. The spectrum of cytochrome c complexed with ZnO NPs (Fig. 2) reveal that the native heme structure of cytochrome c was preserved, indicating that the conjugation of cyt c with nanoparticles does not affect the spectra of the heme microenvironment at pH 7. The detail in Fig. 2 displays a weak band at 695 nm that is highly specific for the correctly folded and functional form of the protein [22]. This band is a diagnostic for intact Met80 ligation of the heme iron and is used to define native state III spectroscopically [6,23]. The inset in Fig. 2 shows the presence of a 695 nm absorption band for complexed cytochrome c at pH 7 which also confirms the correctly folded local protein conformation. Under these conditions, no denaturation of cyt c was induced upon exposure to the zinc oxide nanoparticles Fig. 2.
The conformational changes of oxidized cytochrome c at increasing levels of pH, i.e., the alkaline isomerisation, can be monitored easily using absorption spectroscopy due to the marked shift in most of the major absorption bands which occurs during the process. We monitored the changes in the Soret region because of its higher sensitivity and the absorption band at 695 nm, which has been the main focus of study because of its proven links with the Met80-iron bond in the native protein [24,25]. By fitting the experimental data obtained for free cytochrome c into Eq. (1), it was established that the alkaline transition resulted in the apparent release of 0.54 proton (Fig. 3A, black squares). The effect of zinc oxide nanoparticles on cytochrome c in alkaline pH conditions was studied for various concentrations of ZnO NPs (0.045 mg mL1, 0.l mg mL1, 0.2 mg mL1 and 0.5 mg mL1). The set of results indicate that the optimal concentration for the stabilization of the heme crevice is approximately 0.1 mg mL1. The alkaline transition of cyt c in the presence of 0.1 mg mL1 ZnO NPs (Fig. 3A, red left triangles) was more cooperative and resulted in the apparent release of 0.69 proton. This shift to a higher value of n was coupled with steeper titration curves for cytochrome c complexed with nanoparticles. The results indicate that pH-induced conformational changes of ionic complex of cytochrome c and ZnO nanoparticles occurred in higher pH conditions. The up-shift of the pKapp values was from 8.44 (in accordance with other studies [19,26,27]) to 9.38 (at 0.1 mg mL1). Further increases in the concentration of ZnO-NPs led to a slight decrease in the value of pKapp and n. The pKapp was approximately
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cles, while studies in the Soret region (350–450 nm) (Fig. 4B) can offer data on protein structural integrity in the vicinity of heme region. Fig. 4A shows that the addition of ZnO nanoparticles (0.137 mg mL1) did not cause any significant changes in the position or intensity of the negative dichroic band at 222 nm and 208 nm at neutral pH conditions which is characteristic for a-helix structure [24]. The CD measurements were also performed with 0.416 mg mL1, 1 mg mL1, 2 mg mL1, and 3 mg mL1 of ZnO nanoparticles; the results were identical to those of previous experiments. The negligible changes in structure were also confirmed by observations in the Soret region (Fig. 4B). Circular dichroism was also used to monitor of the behavior of free ferricytochrome c and cyt c-ZnO nanoparticle complexes in alkaline pH conditions (Fig. 4A). The observations indicate an increase in pKa constant of cyt c after association with zinc oxide nanoparticles from 8.44 to 9.40 which is consistent with the results obtained through UV–vis measurements. The spectrum of cyt c-ZnO NPs is similar to that of free ferricytochrome at a final condition of pH 11 which confirms the unchanged secondary structure of cytochrome c after conjugation with ZnO nanoparticles. Conclusions
Fig. 4. (A) Far-UV circular dichroism (CD) spectra of 10 lM free oxidized cytochrome c at pH 7.01 (pink right triangles), 8.50 (purple down triangles), 10,98 (olive left triangles) and 10 lM oxidized cytochrome c in the complex with 0.137 mg mL1 ZnO at pH 7.01 (dark cyan squares), 8.52 (blue stares), 9.40 (red circles), and 11.00 (black diamonds). (B) Soret CD spectra of 0.2 mM free (pink right triangles) and adsorbed cyt c on ZnO-NPs (2.74 mg mL1; dark cyan squares) at pH 7.00. All measurements were performed in ultrapure water, at 25 °C, and in a 1 mm path length cuvette. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
9.11 at a concentration of 0.2 mg mL1 (supplement1, blue circles) and 9.03 at 0.5 mg mL1 (supplement1, pink diamonds), respectively. The value of n was approximately 0.67 at a concentration of 0.2 mg mL1, and 0.66 at a concentration of 0.5 mg mL1. These changes are very similar at the Soret and Q transition bands of pure and associated cyt c despite the fact that each probe recorded changes at substantially different protein concentrations. The increasing part of the profile indicates a significant increase in pKapp values, as is suggested by the loss of absorption at 695 nm (Fig. 3B) and by monitoring the Soret band changes (Fig. 3A); this may be a consequence of progressive binding of nanoparticles to the cytochrome c. These results may also be complicated by the pH-dependent chemistry of ZnO in aqueous solutions [28]. However, this increase in pKapp of alkaline isomerisation of cytochrome c has been reported in several studies in which negatively charged hydrophilic surfaces, such as heparin, polyglutamate, polygalacturonate, polyadenylate, and glutathione-covered gold nanoparticles [15,18,19,29] were used. The value of apparent pKa of alkaline transition of cyt c is affected by the ionic strength of the medium and also by the type of ion used. Anions which conjugate with high affinity to a site not far from the top heme crevice bond have been shown to increase pKa at relatively low concentrations, while other anions which do not interact with this site are effective only at much higher ionic strengths [30]. Circular dichroism (CD) Far UV CD analysis (Fig. 4A) can provide information regarding the secondary structures of cyt c associated with ZnO nanoparti-
The structure and stability of cytochrome c was studied following association with ZnO nanoparticles. The results from UV–vis spectroscopy and circular dichroism confirm that cytochrome c retained its native secondary structure. It was also demonstrated that structural stability around the heme of complexed cyt c was increased and that the zinc oxide nanoparticles caused a significant increase in the apparent pK values (from 8.44 to 9.40) of the cyt c alkaline transition. Acknowledgments This work was financially supported by the projects funded from the European Union Structural Funds, namely from the Operational Programme, Research and Development‘‘, identification ITMS codes 26220120021, 26220220061, and project CEX NANOFLUIDS SAS. The authors thank the Slovak Grant Agency for support through VEGA Grant No. 2/0025/12. Support from the Slovak Research and Development Agency under the Contract Nos. APVV-0171-10 and APVV-0280-11 is gratefully acknowledged too. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2012.10.007. References [1] V.E. Kagan, V.A. Tyurin, J. Jiang, Y.Y. Tyurina, V.B. Ritov, A.A. Amoscato, A.N. Osipov, N.A. Belikova, A.A. Kapralov, V. Kini, Vlasova II, Q. Zhao, M. Zou, P. Di, D.A. Svistunenko, I.V. Kurnikov, G.G. Borisenko, Nat. Chem. Biol. 1 (2005) 223– 232. [2] E. Margoliash, A. Schejter, Adv. Protein Chem. 21 (1966) 113–286. [3] B. Mignotte, J.L. Vayssiere, Eur. J. Biochem. 252 (1998) 1–15. [4] F.I. Rosell, J.C. Ferrer, A.G. Mauk, J. Am. Chem. Soc. 120 (1998) 11234–11245. [5] M. Assfalg, I. Bertini, A. Dolfi, P. Turano, A.G. Mauk, F.I. Rosell, H.B. Gray, J. Am. Chem. Soc. 125 (2003) 2913–2922. [6] G.R. Moore, G.W. Pettigrew, Cytochromes c: Evolutionary, Structural and Physiochemical Aspects, Springer-Verlag, Berlin, 1990. [7] H.B. Gray, J.R. Winkler, Annu. Rev. Biochem. 65 (1996) 537–561. [8] S. Döpner, P. Hildebrandt, F.I. Rosell, A.G. Mauk, J. Am. Chem. Soc. 120 (1998) 11246–11255. [9] P.M. Gadsby, J. Peterson, N. Foote, C. Greenwood, A.J. Thomson, Biochem. J. 246 (1987) 43–54.
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[10] C.A.J. Lin, T. Liedl, R.A. Sperling, M.T. Fernandez-Arguelles, J.M. CostaFernandez, R. Pereiro, A. Sanz-Medel, W.H. Chang, W.J. Parak, J. Mater. Chem. 17 (2007) 1343–1346. [11] S. Sudhagar, S. Sathya, K. Pandian, B.S. Lakshmi, Biotechnol. Lett. 33 (2011) 1891–1896. [12] I. Brigger, C. Dubernet, P. Couvreur, Adv. Drug Deliv. Rev. 54 (2002) 631– 651. [13] L. Brannon-Peppas, J.O. Blanchette, Adv. Drug Deliv. Rev. 56 (2004) 1649– 1659. [14] M. Goldberg, R. Langer, X. Jia, J. Biomater. Sci. Polym. Ed. 18 (2007) 241–268. [15] M. Antalik, M. Bona, J. Bagelova, Biochem. Inter. 28 (1992) 675–682. [16] E. Sedlak, M. Antalik, Biopolymers 46 (1998) 145–154. [17] E. Sedlak, M. Antalik, Biochim. Biophys. Acta 1434 (1999) 347–355. [18] M. Antalik, J. Bagel’ova, Z. Gazova, A. Musatov, D. Fedunova, Biochim. Biophys. Acta 1646 (2003) 11–20. [19] E. Valusova, P. Svec, M. Antalik, J. Biol. Inorg. Chem. 14 (2009) 621–630. [20] W.D. Butt, D. Keilin, R. Soc, Lond. B Biol. Sci. 156 (1962) 429–458.
[21] S.S. Ashtaputre, A. Deshpande, S. Marathe, M.E. Wankhede, J. Chimanpure, R. Pasricha, J. Urban, S.K. Haram, S.W. Gosavi, S.K. Kulkarni, PRAMANA -J. Phys. 65 (2005) 615–620. [22] T.D. Perroud, M.P. Bokoch, R.N. Zare, Proc. Natl. Acad. Sci. USA 102 (2005) 17570–17575. [23] H. Theorell, Å. Åkesson, J. Am. Chem. Soc. 63 (1941) 1812–1818. [24] E. Schechter, P. Saludjian, Biopolymers 5 (1967) 788–790. [25] W.A. Eaton, R.M. Hochstrasser, J. Chem. Phys. 46 (1967) 2533–2539. [26] P. Weinkam, J. Zimmermann, L.B. Sagle, S. Matsuda, P.E. Dawson, P.G. Wolynes, F.E. Romesberg, Biochemistry 47 (2008) 13470–13480. [27] C.J. Wallace, Biochem. J. 217 (1984) 601–604. [28] S.W. Bian, I.A. Mudunkotuwa, T. Rupasinghe, V.H. Grassian, Langmuir 27 (2011) 6059–6068. [29] J. Bagel’ova, Z. Gazova, E. Valusova, M. Antalik, Carbohyd. Polym. 45 (2001) 227–232. [30] N. Osheroff, D. Borden, W.H. Koppenol, E. Margoliash, J. Biol. Chem. 255 (1980) 1689–1697.
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Alkaline transition of horse heart cytochrome c in the presence of ZnO nanoparticles Michaela Šimšíková a, Marián Antalík a,b,⇑ a b
Department of Biochemistry, Faculty of Science, PJ Šafárik University, Šrobárova 2, 041 54 Košice, Slovakia Department of Biophysics, Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Košice, Slovakia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" The influence of ZnO nanoparticles
to the cytochrome c in alkaline pH have been studied. " The structural stability around the heme of cyt c assembled with ZnO nanoparticles was increased. " The pKa constant of cyt c-ZnO complex was took place to the higher value. " The alkaline transition of cytochrome c in the presence of ZnO nanoparticles was more cooperative.
a r t i c l e
i n f o
Article history: Received 2 May 2012 Received in revised form 4 October 2012 Accepted 8 October 2012 Available online 16 October 2012 Keywords: Cytochrome c Zinc oxide Nanoparticles Alkaline transition
a b s t r a c t The effect of zinc oxide nanoparticles (ZnO NPs) on cytochrome c (cyt c) in alkaline pH was studied with absorption spectroscopy and UV circular dichroism (CD). Spectral data from UV–vis spectroscopy and circular dichroism indicate only small changes in the native structure of the protein at neutral pH after the interaction with ZnO nanoparticles. The stability around the heme crevice of cyt c and therefore the switch of the axial ligand Met80 to Lys which occurs in conditions of higher pH was proven following the interaction of cytochrome c with ZnO nanoparticles. The formation of cyt c-ZnO NPs complex based on electrostatic attraction was accompanied by a significant increase in the apparent pKa constant of the alkaline transition of cyt c. Ó 2012 Elsevier B.V. All rights reserved.
Introduction Research into the hierarchy of interactions which stabilize the native states of proteins is vital in order to understand the factors that govern the formation of their three-dimensional structure. One major focus of this research has been the study of the structure and stabilizing mechanism of protein conformational changes. One of the most extensively studied proteins is cytochrome c which is an essential component of mitochondrial electron transfer ⇑ Corresponding author at: Department of Biochemistry, Faculty of Science, PJ Šafárik University, Šrobárova 2, 041 54 Košice, Slovakia. Tel.: +421 55 7204138; fax: +421 55 7204139. E-mail address: [email protected] (M. Antalík). 1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2012.10.007
activity, an important mediator in pro-apoptotic [1] and apoptotic pathways and a regulator of biological activity [2,3]. The multifunctional nature of cytochrome c is thought to be a result of the conformational transitions which the protein undergoes. The alkaline transition, a high pH induced conformational change in which the axial ligand Met80 dissociates and is replaced by a lysine (Lys) [4,5], has been the subject of great interest as a model of the potential regulation of cyt c for use as a biological function [6]. The interconversion from the native to the alkaline form of cytochrome c operates as an efficient binary molecular switch which has a dramatic effect on the electron-transfer activity of the protein [7–9]. Recent years have seen great advances in the study of nanoparticles, both in terms of their unique properties and their potential
M. Šimšíková, M. Antalík / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 410–414
applications, i.e. biolabeling [10], biosensing [11], therapy [12,13], or as scaffolds for tissue engineering [14]. One result of the use of nanoparticles in bio-applications has been the discovery of structural and functional changes of biomolecules caused by the exposure of proteins to inorganic surfaces. Our group has previously published work on the influence of various polyanions with different lengths and structures and negatively charged gold nanoparticles on the structure and stability of horse heart cytochrome c under conditions of varying temperatures and pH factors [15– 19]. In this study, we follow up on this work and examine the effects of ZnO nanoparticles on the conformation of cytochrome c in alkaline pH conditions.
Materials and methods Reagents and chemicals Horse heart cytochrome c (type IV) was purchased from Sigma as salt-free, dry powder and was used without further purification. The concentration of protein solution in the native state was determined with absorption spectroscopy using a Shimadzu UV-3600 UV–vis–NIR photospectrometer, with an extinction coefficient of e408 = 106,100 mol1 cm1 [20]. Zinc chloride (ZnCl2 P 99.995%) and sodium hydroxide (NaOH P 98%) were obtained from Sigma and were of analytical grade. Ultra pure water (Merck Millipore) was used throughout the experiments.
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UV–vis spectroscopy The alkaline transition changes of cytochrome c were performed on a Shimadzu UV-3600 UV–vis–NIR photospectrometer. Difference absorption spectra were recorded in range of 215– 850 nm with a spectral resolution of 2 nm. Alkaline titrations were performed by adding very small amounts of concentrated NaOH to a solution of free cytochrome c or a solution of cytochrome associated with ZnO nanoparticles. Potassium ferricyanide (1 lM ferricyanide for 6 lM cytochrome c solutions or 2 lM for 60 lM cytochrome c solutions) was added to each solution in order to ensure complete oxidation of the proteins. The spectrum at pH 6.00 ± 0.05 was used as a reference; the absorption differences were followed at 400–415 nm in the Soret region and at 695–800 nm in the charge transfer band which is specific for the native Met80 to heme Fe ligation. The pH dependences of all transitions were analyzed using the following formula:
pH ¼ pK app
Aprotonated Aobserved 1 log n Aobserved Adissociated
ð1Þ
where Aobserved is the spectral difference at a given pH, Aprotonated and Adissociated are spectral differences at pH 6.50 and 12.00, respectively, 1/n is the slope, the measurement of the number of protons (n) participating in the transition, and pKapp represents the apparent pK constant of the observed transition. Circular dichroism (CD)
Preparation of zinc oxide nanoparticles The nanoparticles of zinc oxide were prepared using a precipitation method [21], with zinc chloride and sodium hydroxide servings as precursors. The mixture of 0.2 M ZnCl2 (0.06885 g, 2.5 mL) and equimolar aqueous solution of NaOH (0.1 g, 12.5 mL) was stirred for 3 h at room temperature (25 °C ± 1) producting a milky white solution. After continuous stirring, the precipitate was collected through centrifugation (Eppendorf 5804R centrifuge; 6 min, 25 °C) at 100 rpm and subsequently at 15,000 rpm, which removed the eventual aggregates from the sample. The resulting fraction was washed in ultrapure Millipore water several times and allowed to evaporate at room temperature in order to obtain ZnO nanoparticles in white powder form.
The structural change of ferricytochrome c induced by the addition of ZnO NPs was evaluated with CD spectra measured with a JASCO J-815 spectropolarimeter. The cyt c was converted to a completely oxidized form by the addition of small amounts of potassium ferricyanide. The CD measurements of 10 lM free cytochrome c and cyt c complexed with zinc oxide nanoparticles (0.137; 0.416; 1.0; 2.0, and 3.0 mg mL1) were recorded in far UV region of 180–260 nm and 0.2 M cyt c (free or as a complex with ZnO-NPs at concentrations of 2.74 and 8.32 mg mL1) was used in Soret region of 350–450 nm. Alkaline titrations were performed by adding very small amounts of concentrated NaOH to the solution of free cytochrome c (10 lM) or solution of cytochrome c conjugated with zinc oxide nanoparticles (0.137 mg mL1). All measurements were performed at room temperature using a rectangular cuvette with an optical path length of 1 mm, a scan rate of 100 nm min1 with an average of five consecutive scans.
Atomic Force Microscopy (AFM) Results and discussion AFM images of ZnO NPs were collected using an Innova AFM system (Veeco Instruments) in tapping mode following 5 min of absorption on a freshly cleaved mica surface. The AFM tips used for the measurement were commercial antimony-doped silicon cantilevers (NCHV, Veeco Instruments) with a nominal tip radius of approximately 10 nm and a resonance frequency 301–331 kHz. The scanning rate of the measurements was 1 Hz and the scan size was in the range of 500 500 points. The data was analyzed with NanoScope Analysis (1.20) to obtain particle sizes and morphology.
Size characterization of ZnO nanoparticles The shape and diameter of zinc oxide nanoparticles were analyzed with Atomic Force Microscopy (AFM). Fig. 1 shows that the nanoparticles are spherical with a diameter of 50–80 nm. The size of ZnO nanoparticles was confirmed by Malvern Zetasizer Nano ZS. The particle size distribution demonstrates a relatively narrow range (Fig. 1, detail) which suggests the complete absence of aggregated particles.
Particle size distribution of ZnO nanoparticles
UV–vis spectroscopy
The average particle size of ZnO nanoparticles was determined by Malvern Zetasizer Nano ZS (4 mW, 633 nm laser) at 25 °C and pH 6.98. During the experiments, 0.1 mg mL1 of ZnO solution which had been sonicated for approximately 1 min was used.
It is possible to demonstrate the native conformation of cytochrome c from optical spectra results, and Fig. 2 shows the UV– vis absorption spectrum of native ferricytochrome c. It can be seen that cyt c has characteristic Soret and Q transitions bands at
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Fig. 1. Atomic force micrograph of the ZnO nanoparticles (bar 500 nm). The detail shows the size of nanoparticles determined by small angle scattering which remained in a narrow range.
Fig. 3. (A) The pH dependencies of absorbance changes measured in a Soret band region for free ferricytochrome c (black squares) and ferricytochrome c associated with ZnO at concentrations of 0.l mg mL1 (red left triangles). The concentration of ferricytochrome c was 6 lM. (B) The pH dependencies of absorbance changes measured in the 695 nm charge transfer band region for free ferricytochrome c (black squares) and ferricytochrome c associated with ZnO NPs at concentrations of l mg mL1 (red left triangles). The concentration of ferricytochrome c was 60 lM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Absorption spectra of 6 lM ferricytochrome c (purple dash line) and 6 lM ferricytochrome c adsorbed on ZnO nanoparticles (0.1 mg mL1; olive straight line). The detail shows the absorption spectra of 60 lM ferricytochrome c (purple dash line) and 60 lM ferricytochrome c adsorbed on ZnO nanoparticles (0.1 mg mL1; olive straight line) observed at the range of 600–850 nm. All measurements were performed in ultrapure water, pH 7 at 25 °C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
409 nm and near 528 nm, both of which originate in the heme chromophore. The spectrum of cytochrome c complexed with ZnO NPs (Fig. 2) reveal that the native heme structure of cytochrome c was preserved, indicating that the conjugation of cyt c with nanoparticles does not affect the spectra of the heme microenvironment at pH 7. The detail in Fig. 2 displays a weak band at 695 nm that is highly specific for the correctly folded and functional form of the protein [22]. This band is a diagnostic for intact Met80 ligation of the heme iron and is used to define native state III spectroscopically [6,23]. The inset in Fig. 2 shows the presence of a 695 nm absorption band for complexed cytochrome c at pH 7 which also confirms the correctly folded local protein conformation. Under these conditions, no denaturation of cyt c was induced upon exposure to the zinc oxide nanoparticles Fig. 2.
The conformational changes of oxidized cytochrome c at increasing levels of pH, i.e., the alkaline isomerisation, can be monitored easily using absorption spectroscopy due to the marked shift in most of the major absorption bands which occurs during the process. We monitored the changes in the Soret region because of its higher sensitivity and the absorption band at 695 nm, which has been the main focus of study because of its proven links with the Met80-iron bond in the native protein [24,25]. By fitting the experimental data obtained for free cytochrome c into Eq. (1), it was established that the alkaline transition resulted in the apparent release of 0.54 proton (Fig. 3A, black squares). The effect of zinc oxide nanoparticles on cytochrome c in alkaline pH conditions was studied for various concentrations of ZnO NPs (0.045 mg mL1, 0.l mg mL1, 0.2 mg mL1 and 0.5 mg mL1). The set of results indicate that the optimal concentration for the stabilization of the heme crevice is approximately 0.1 mg mL1. The alkaline transition of cyt c in the presence of 0.1 mg mL1 ZnO NPs (Fig. 3A, red left triangles) was more cooperative and resulted in the apparent release of 0.69 proton. This shift to a higher value of n was coupled with steeper titration curves for cytochrome c complexed with nanoparticles. The results indicate that pH-induced conformational changes of ionic complex of cytochrome c and ZnO nanoparticles occurred in higher pH conditions. The up-shift of the pKapp values was from 8.44 (in accordance with other studies [19,26,27]) to 9.38 (at 0.1 mg mL1). Further increases in the concentration of ZnO-NPs led to a slight decrease in the value of pKapp and n. The pKapp was approximately
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cles, while studies in the Soret region (350–450 nm) (Fig. 4B) can offer data on protein structural integrity in the vicinity of heme region. Fig. 4A shows that the addition of ZnO nanoparticles (0.137 mg mL1) did not cause any significant changes in the position or intensity of the negative dichroic band at 222 nm and 208 nm at neutral pH conditions which is characteristic for a-helix structure [24]. The CD measurements were also performed with 0.416 mg mL1, 1 mg mL1, 2 mg mL1, and 3 mg mL1 of ZnO nanoparticles; the results were identical to those of previous experiments. The negligible changes in structure were also confirmed by observations in the Soret region (Fig. 4B). Circular dichroism was also used to monitor of the behavior of free ferricytochrome c and cyt c-ZnO nanoparticle complexes in alkaline pH conditions (Fig. 4A). The observations indicate an increase in pKa constant of cyt c after association with zinc oxide nanoparticles from 8.44 to 9.40 which is consistent with the results obtained through UV–vis measurements. The spectrum of cyt c-ZnO NPs is similar to that of free ferricytochrome at a final condition of pH 11 which confirms the unchanged secondary structure of cytochrome c after conjugation with ZnO nanoparticles. Conclusions
Fig. 4. (A) Far-UV circular dichroism (CD) spectra of 10 lM free oxidized cytochrome c at pH 7.01 (pink right triangles), 8.50 (purple down triangles), 10,98 (olive left triangles) and 10 lM oxidized cytochrome c in the complex with 0.137 mg mL1 ZnO at pH 7.01 (dark cyan squares), 8.52 (blue stares), 9.40 (red circles), and 11.00 (black diamonds). (B) Soret CD spectra of 0.2 mM free (pink right triangles) and adsorbed cyt c on ZnO-NPs (2.74 mg mL1; dark cyan squares) at pH 7.00. All measurements were performed in ultrapure water, at 25 °C, and in a 1 mm path length cuvette. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
9.11 at a concentration of 0.2 mg mL1 (supplement1, blue circles) and 9.03 at 0.5 mg mL1 (supplement1, pink diamonds), respectively. The value of n was approximately 0.67 at a concentration of 0.2 mg mL1, and 0.66 at a concentration of 0.5 mg mL1. These changes are very similar at the Soret and Q transition bands of pure and associated cyt c despite the fact that each probe recorded changes at substantially different protein concentrations. The increasing part of the profile indicates a significant increase in pKapp values, as is suggested by the loss of absorption at 695 nm (Fig. 3B) and by monitoring the Soret band changes (Fig. 3A); this may be a consequence of progressive binding of nanoparticles to the cytochrome c. These results may also be complicated by the pH-dependent chemistry of ZnO in aqueous solutions [28]. However, this increase in pKapp of alkaline isomerisation of cytochrome c has been reported in several studies in which negatively charged hydrophilic surfaces, such as heparin, polyglutamate, polygalacturonate, polyadenylate, and glutathione-covered gold nanoparticles [15,18,19,29] were used. The value of apparent pKa of alkaline transition of cyt c is affected by the ionic strength of the medium and also by the type of ion used. Anions which conjugate with high affinity to a site not far from the top heme crevice bond have been shown to increase pKa at relatively low concentrations, while other anions which do not interact with this site are effective only at much higher ionic strengths [30]. Circular dichroism (CD) Far UV CD analysis (Fig. 4A) can provide information regarding the secondary structures of cyt c associated with ZnO nanoparti-
The structure and stability of cytochrome c was studied following association with ZnO nanoparticles. The results from UV–vis spectroscopy and circular dichroism confirm that cytochrome c retained its native secondary structure. It was also demonstrated that structural stability around the heme of complexed cyt c was increased and that the zinc oxide nanoparticles caused a significant increase in the apparent pK values (from 8.44 to 9.40) of the cyt c alkaline transition. Acknowledgments This work was financially supported by the projects funded from the European Union Structural Funds, namely from the Operational Programme, Research and Development‘‘, identification ITMS codes 26220120021, 26220220061, and project CEX NANOFLUIDS SAS. The authors thank the Slovak Grant Agency for support through VEGA Grant No. 2/0025/12. Support from the Slovak Research and Development Agency under the Contract Nos. APVV-0171-10 and APVV-0280-11 is gratefully acknowledged too. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2012.10.007. References [1] V.E. Kagan, V.A. Tyurin, J. Jiang, Y.Y. Tyurina, V.B. Ritov, A.A. Amoscato, A.N. Osipov, N.A. Belikova, A.A. Kapralov, V. Kini, Vlasova II, Q. Zhao, M. Zou, P. Di, D.A. Svistunenko, I.V. Kurnikov, G.G. Borisenko, Nat. Chem. Biol. 1 (2005) 223– 232. [2] E. Margoliash, A. Schejter, Adv. Protein Chem. 21 (1966) 113–286. [3] B. Mignotte, J.L. Vayssiere, Eur. J. Biochem. 252 (1998) 1–15. [4] F.I. Rosell, J.C. Ferrer, A.G. Mauk, J. Am. Chem. Soc. 120 (1998) 11234–11245. [5] M. Assfalg, I. Bertini, A. Dolfi, P. Turano, A.G. Mauk, F.I. Rosell, H.B. Gray, J. Am. Chem. Soc. 125 (2003) 2913–2922. [6] G.R. Moore, G.W. Pettigrew, Cytochromes c: Evolutionary, Structural and Physiochemical Aspects, Springer-Verlag, Berlin, 1990. [7] H.B. Gray, J.R. Winkler, Annu. Rev. Biochem. 65 (1996) 537–561. [8] S. Döpner, P. Hildebrandt, F.I. Rosell, A.G. Mauk, J. Am. Chem. Soc. 120 (1998) 11246–11255. [9] P.M. Gadsby, J. Peterson, N. Foote, C. Greenwood, A.J. Thomson, Biochem. J. 246 (1987) 43–54.
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