J. Phys. Chem. C 2009, 113, 9993–9997
9993
Relative Functionality of Buffer and Peptide in Gold Nanoparticle Formation Steve Diamanti, Andrea Elsen, Rajesh Naik, and Richard Vaia* Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 45433 ReceiVed: NoVember 20, 2008; ReVised Manuscript ReceiVed: April 23, 2009
The templated growth of nanoparticles via biological agents, such as peptides, provides an exciting complement to abiotic routes, opening facile means to combine the specificity of biomacromolecules with nanoparticle platforms. The specific role of the peptide sequence, and its state relative to the buffer, is still unclear with respect to the processes underlying nanoparticle formation. By investigation of Au mineralization in two commonly used buffers (sodium borate and (2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)), the role of the phage-display-identified A3 peptide, and its residues, is examined. In a nonreducing buffer (e.g., borate), mineralization is very slow, suggesting that the tyrosine residue in the A3 motif has at most a minor role in reduction of Au(III). In a buffer with substantial reducing capability (e.g., HEPES), the peptide retards nucleation relative to synthetic additives such as poly(ethylene glycol). Furthermore, it also functions to regulate the concentration of free Au in the growth medium, resulting in a diffusion limited process that yields larger nanoparticles with increased peptide concentration. These roles are consistent with the phage-display process that identified the A3 peptide sequence with respect to its binding strength to metal surfaces and not with regard to a specific reduction capability. Introduction Numerous microorganisms are capable of synthesizing inorganic structures. These natural processes of biomineralization and assembly into hierarchical structures have led to the development of a variety of synthetic techniques that mimic the recognition, nucleation, and growth capabilities of biological systems.1-4 One of the key components in this process is the organic matrix, which templates the spatial arrangement of the inorganic structure.5,6 Although there has been much attention paid to the identification and demonstration of mineralization of different inorganic salts by peptides, less is known about the specific influence of the peptide and buffer conditions on the particular steps of the process: reduction, nucleation, growth, and stabilization. In general, control of the nanoparticle size, shape, and the associated distributions arise from a delicate balance of reduction, nucleation, and growth rates. This control is achieved through the use of relatively small amounts of additives with preferential affinity for the reactants and products. The A3 peptide (A3-Y) has previously been shown to form both silver and gold nanoparticles under different synthetic conditions.7-9 This mineralization ability is thought to arise from the amino acid sequence, which contains a tyrosine residue and is rich in serine (hydroxyl-containing amino acid) and hydrophobic amino acids (proline and phenylalanine). As with other metal mineralization peptides established by phage display,10 the specific role of the amino acid sequence on the various steps of metal reduction and nanoparticle formation is not well established. Chelating amino acid residues, such as serine, have been speculated to be important in the nucleation and growth process due to their ability to bind metal salts and stabilize gold particles in solution.11,12 Tyrosine is well-known for its ability to reduce gold salts to Au(0).13-16 Hydrophobic amino acids such as proline and phenylalanine have also been shown to be conserved * Corresponding author,
[email protected].
10.1021/jp8102063
in panning for gold binders, although their role in the mineralization process is not as well understood.11 In addition to the peptide structure, the solution pH, temperature, buffer salt, and electrolyte content are major factors determining its functionality. In this contribution, the relative impact of a metal binding peptide to the mineralization steps is established by comparing the Au nanoparticle formation process in different buffers and with different macromolecular additives. Specifically, buffers such as 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) dominate the reduction of Au(III) and, in such cases, the peptide functions as a stabilizing agent that solubilizes the growing nanoparticle. In contrast to synthetic polymer additives with Au binding affinity, such as poly(ethylene glycol) (PEG), the peptide regulates the concentration of free Au, resulting in a diffusion limited growth process. Results and Discussion For commonly used reaction conditions in HEPES and borate buffer (pH ) 7.5), Figures 1 and 2 summarize the relative rate of formation and final morphology of Au nanoparticles for various mineralization additives including A3-Y (amino acid sequence GGAYSSGAFPPMPPFGG), A3-S (amino acid sequence GGASSSGAFPPMPPFGG), and monomethoxy-terminated poly(ethylene glycol) (PEG) (MW ) 5000). A3-S is similar to A3-Y but with the central tyrosine residue replaced by serine. The increase in absorbance of the Au nanoparticle plasmon resonance (550 nm) reflects the mineralization rate.17 The two most striking observations are (1) the Au nanoparticle formation process depends as much on the type of buffer as it does on the macromolecular additive (Figure 1) and (2) within the HEPES series, PEG addition results in nanoparticles with almost the same size and shape dispersity as A3-Y (Figure 2). To begin, pure freshly prepared HEPES buffer (pH 7.5) is observed to rapidly reduce Au(III) leading to the formation of polydisperse and fused Au particles (Figure 2a, 38.4 ( 8.4 nm) as indicated by the very fast increase in the plasmon resonance
This article not subject to U.S. Copyright. Published 2009 by the American Chemical Society Published on Web 05/18/2009
9994
J. Phys. Chem. C, Vol. 113, No. 23, 2009
Figure 1. Representative summary of Au nanoparticle mineralization in (a) HEPES and (b) borate buffer at constant pH of 7.5 containing A3-Y (circle), A3-S (open-circle), PEG (square), and buffer only (triangle). Reaction conditions: HAuCl4:buffer:macromolecular additive (1:100:0.25). 0.5 mM HAuCl4, 50 mM HEPES (pH ) 7.5), 50 mM borate buffer (pH ) 7.5), 0.125 mM A3, A3-S, or PEG in Milli-Q water. Absorbance at 520 nm for 1 cm cuvette where error bars represent data range from three independent reactions. Note time is presented logarithmically.
absorbance peak (Figure 1a). Due to the absence of stabilizing agents in the aqueous solution, black precipitates comprised of fused Au particles form, decreasing the NP concentration in solution and reducing the absorbance at longer times (Figure 1a). This particle formation process occurs over a wide range of Au(III):HEPES molar ratios (0.005-0.05). These observations are consistent with triethylamine acting as a reducing agent based on its redox properties relative to Au(III) and Au(I) species.18 Previous reports of Good’s buffers, such as HEPES, 1,4-piperazine-diethanesulfonic acid (PIPES), and 4-morpholinopropanesulfonic acid (MOPS), acting as efficient reduction agents may also be found.19,20 As with the triethylamine, the piperzaine ring in HEPES generates a nitrogen-centered, cationic free-radical through the reduction of Au(III). This reduction tendency should be enhanced at higher pH due to the increased concentration of HEPES molecules with unprotonated amine. The high electrolyte concentration necessary for pH adjustment to 7.5 (nearly 1:1 NaOH:HEPES) however destabilizes the colloidal system, resulting in rapid precipitation as observed. In contrast, comparable borate buffer (pH 7.5) solutions containing HAuCl4 shows no appearance of a surface plasmon resonance absorption, or particle formation, after almost 1 month of incubation time (Figure 1b). The inactivity of borate buffer enables the separation of reduction events from nanoparticle nucleation and subsequent growth. For representative borate buffered reactions in the presence of A3-Y, A3-S, and PEG, absorbance increase and particle formation only occur after very long incubation times (>100 h, 4 days). We believe this reflects the impact of unknown contamination in the reaction solution potentially arising from slow degradation of the peptide due to
Diamanti et al.
Figure 2. Transmission electron micrographs of Au nanoparticles formed in (a) HEPES (pH 7.5) (38.4 ( 8.4 nm) and in (b-d) HEPES (pH 7.5) containing PEG (13.6 ( 2.4 nm), A3-Y (11.5 ( 2.1 nm), and A3-S (11.0 ( 3.6 nm), respectively. For comparison, see examples of particle morphology at extremely long incubation times (700 h) for borate buffer (pH 7.5) containing (e) A3-Y (32.4 ( 13.9 nm) and (f) A3-S (10.9 ( 5.3 nm). Note that for borate buffer the reaction has not reached a quasi-static limit. The reaction conditions are the same as shown in Figure 1.
sample handling or adventitious impurities associated with the peptide synthesis. Seed formation due to photon-based reduction of Au(III) is not believed to occur since nanoparticle formation was not observed in the borate buffer alone. The inactivity of A3-Y implies that the tyrosine residue in the amino acid sequence at most plays a minor role in Au(III) reduction and does not appear to be sufficient to initiate substantial Au NP formation in a reasonable time scale under the inert borate buffer conditions. Although differing from initial conclusions,13 this is consistent with the commonly assumed nonradical process where 1.5 tyrosine residues donating two electrons apiece are required to reduce Au(III) to Au(0). Thus, the tyrosine residue in A3-Y could only account for ∼17% of the reduction of all the Au(III) precursor to Au(0) at the normal reaction conditions. As anticipated, a substantial increased NP formation rate occurs in borate if A3-Y is replaced on an equimolar basis with tri- or hexatyrosine, thereby increasing the concentration of reducing agent (results not shown).21 Overall, these observations emphasize that the choice of buffer is critical to provide reduction of the gold precursor salt. For the HEPES buffered reactions, HEPES is present in a 100fold excess relative to Au(III), thus providing sufficient concentration to fully reduce Au(III) as well as maintain its buffering capability, as reflected by the constant solution pH throughout the particle formation process. The macromolecular additive, therefore impacts critical nuclei formation, mediates
Buffer and Peptides in Gold Nanoparticle Formation
Figure 3. Nanoparticle formation for various ratios of Au(III) (0.5 mM) to A3-Y (13-250 µM) in 50 mM HEPES (pH 7.5) (Au:HEPES: A3-Y 1:100:0.026 to 1:100:0.5). Growth rate is reflected by the relative change in the absorption at 520 nm, and incubation time is represented by the intersection of the linear growth region with the abscissa. Error bars are the spread in data from two independent reactions. Note, time is presented logarithmically.
particle growth, and/or serves to solubilize the growing nanoparticles. Relative to pure HEPES (Figure 1a), PEG addition only slightly increases incubation time, thus not altering the supersaturation of the solution. However, the Au nanoparticles are substantially smaller and more uniform (Figure 2b, 13.6 ( 2.4 nm). The absorbed PEG is an efficient solubilizing agent, even at high salt concentrations, therefore increasing the stability of the colloidal dispersion.22 The smaller nanoparticle size indicates that PEG also regulates surface reactivity, most likely due to its ability to chelate cations and modify the electrostatic double layer. Furthermore, initial observations indicate that the impact of PEG on nanoparticle size and dispersity increases as the buffering capability of HEPES is stressed, such as due to extrogenous absorption of CO2/carbonic acid. This reduces the amine concentration, overall reduction capacity, and rate and thereby increases the efficacy of nonspecific additives such as PEG. In comparison to PEG, the peptides have a substantial impact on both the nucleation and growth processes, increasing incubation time and providing further reductions in nanoparticle formation rate. The resultant nanoparticle size is comparable to PEG, however. The peptide sequence though has a secondary impact on the dispersity of nanoparticle size; A3-Y exhibits slightly narrower dispersity than A3-S (Figure 2c, 11.5 ( 2.1 nm; Figure 2d, 11.0 ( 3.6 nm, respectively). The different dispersity between A3-Y and A3-S peptides is consistent with prior conclusions the tyrosine residue has a role in modulating or templating nanoparticle growth through its stronger interaction with gold atoms.13 Recent molecular simulations confirm the selective binding of several short peptide sequences to surfaces of Au and Pd and distinguish the affinity of specific residues relative to their confirmation, affinity for water, and polarization and charge transfer with the metal surface.23 To further understand the relative roles of the peptides in the particle formation process, Figures 3 and 4 compare the impact of A3-Y concentration on the formation rate and particle size, respectively. Overall, the incubation time increases and growth rate decreases with increasing peptide concentration (Au(III):A3-Y from 38 to 2). In parallel, the average hydrodynamic size of the nanoparticle increases, and the relative
J. Phys. Chem. C, Vol. 113, No. 23, 2009 9995
Figure 4. Hydrodynamic diameter (DDLS) of nanoparticles for mineralization reactions in 50 mM HEPES (pH 7.5) for various ratios of Au(III) (0.5 mM) to A3-Y (13-250 µM). The DLS dispersity index linearly decreases from 0.22 to 0.07 at 188 µM. Error bars are the spread in data from two identical reactions.
dispersity (0.22 to 0.08) decreases, with peptide concentration (13 to 188 µM). If the peptide determined the number of critical nuclei, a decrease in particle size would be expected. For a constant concentration of Au(0), this would reflect an increase in the number of critical nuclei with increasing addition of the peptide (recall, HEPES rapidly reduces Au(III)). If, on the other hand, the peptide’s major impact is on the particle growth rate due to passivation of the particle surface, the rate of growth and possibly the dispersity in particle size would decrease with increased peptide concentration. However, for a given interfacial binding strength, the size of the resultant particles would be expected to be approximately constant with peptide concentration, definitely not expected to increase by almost a factor of 4 (13 to 51 nm) for a 5-fold increase in peptide concentration (50-250 µM). For example, the size of hexadecanethiol stabilized Au NPs via a single phase route24 only decreases from ∼7.5 to 5 nm over a 1000-fold range of Au(I):alkanethiol (0.03-300). Size focusing and digestive ripening25,26 processes use additions of excess surfactant to narrow polydispersity, but not increase size. One hypothesis consistent with the HEPES and borate observations is that in addition to solubilizing the nanoparticles free peptide strongly chelates Au(0) in solution prior to and during the growth of Au nanoparticles. Since HEPES acts as a strong reducing agent, rapidly converting Au(III) to Au(0), the concentration of Au(0) in the initial HEPES solution is high. This results in rapid nucleation of Au nanoparticles in the absence of an additive. PEG addition only modestly delays incubation (Figure 1a) but slows down growth rate, effectively acting as a surface passivation agent, but not chelating sufficient Au(0) to alter the supersaturation of the initial solution. In contrast, the A3-Y and A3-S peptides substantially delay nucleation, reflecting the impact of binding Au(0) atoms as well as possibly stabilizing and retarding growth of subcritical nuclei. The positive correlation between A3-Y concentration and hydrodynamic particle size is consistent with the peptide reducing the concentration of free Au(0) in solution. This reduced effective concentration of free Au(0) will reduce the number of critical nuclei leading to larger particles for an initial Au salt concentration. The free Au(0) concentration available to add to the growing nanoparticle is thus controlled by the
9996
J. Phys. Chem. C, Vol. 113, No. 23, 2009
equilibrium concentration of bound peptide-Au(0) complexes. As peptide concentration increases, the further reduced growth rate and decreased polydispersity are consistent with a more controlled, reactant-limited mineralization. Overall, the resultant rapid reduction of Au(III) by HEPES buffer, followed by rapid nucleation and diffusion limited growth is ideal for the formation of highly monodisperse particles. For example, this is the objective of hot-solvent injection methods, widely used to synthesize II-VI quantum dots, including CdS and CdSe.27 In this method the rapid temperature quench resulting from injection of room temperature solvent into the hot precursor results in an almost instantaneous formation of stable nuclei, whose growth is subsequently controlled by slight temperature increases. Conclusion In conclusion, the choice of buffer is critical in the investigation of peptide-mediated mineralization phenomena, particularly the mineralization of gold salts. In a buffer with substantial reducing ability (e.g., HEPES), the peptide only appears to be involved in the growth and stabilization of the nanoparticles, as well as in the regulation of the concentration of free Au(0). The latter role is critical in effectively reducing the available concentration of Au reactant in the growth media after the initial supersaturation driven nucleation. This typifies a common synthetic additive approach to control particle growth. Further investigation to elucidate the ability of these metal binding peptides to stabilize free solvated metal atoms and trap subcritical nuclei would lead to synthetic schemes providing even greater control of nanoparticle size and shape. Determining the possible structure and optical spectrum of these Au(0)peptide complexes provides alternatives to Aun clusters (n ) 5, 8, 13, etc.) stabilized by thiol and phosphine molecules.28,29 Finally, by uncoupling the roles of hydroxyl and aromatic residues, refinement of the peptide design should lead to a better understanding of the role of peptides in nanoparticle growth and stabilization. Methods Mineralizations. All mineralizations were based on an adaptation of a previously reported procedure:13 to a 10 mL reaction vessel was added 3.5 mL of 50 mM freshly prepared HEPES or borate buffer in Milli-Q water (pH ) 7.5), to this solution was added a stock solution (10 mg/mL) of the additive (A3 or PEG) in Milli-Q water (in reactions performed in pristine HEPES and borate buffer this step was skipped), the reaction was mixed by vortex, then a stock solution of HAuCl4 in Milli-Q water was added, the reaction was mixed by vortex, and UV-vis spectra were collected immediately after addition of HAuCl4 and vortex mixing. Final concentration of reagents was 125 µM additive (PEG, A3-Y, or A3-S) and 500 µM HAuCl4. Note that previous studies13 used 100 mM HEPES with A3-Y which resulted in 12.8 ( 2.9 nm diameter (TEM) Au NPs. Also for reference, the molar ratio of metal salt to peptide (1:0.25) is substantially less than those for common synthetic alternatives (Turkevitch Au/citrate, 1:16000; two-phase Brust Au:surfactant, 1:4.4). Characterization. UV-vis analysis was performed on a CARY 5000 UV-vis-NIR spectrophotometer using undiluted reaction mixture in a 1 cm cuvette. No precipitation was observed in reactions with the exception of mineralization in pristine HEPES buffer. Hydrodynamic size, DDLS, was determined with a Zeta PALS dynamic light scattering instrument (Brookhaven Instruments). Consistent with other reports of
Diamanti et al. oligomeric stabilized nanoparticles,30 the hydrodynamic diameter, which includes the effect of the organic corona, is nominally greater than the nanoparticle diameter determined by transmission electron microscopy (TEM), which reflects the inorganic core. Nanoparticle size, shape, and crystallinity were determined with a Phillips CM200 transmission electron microscope with a field emission gun operating at 200 kV. TEM samples were prepared by pipetting one drop (∼10 µL) of reaction solution after 30 min of reaction time onto a 3 mm diameter copper grid coated with carbon film (Ted Pella). Quantitative image analysis used Image J (NIH Open Source Software) to determine mean and average of at least 250 particles per condition. Materials. Poly(ethylene glycol) monomethyl ether, 5000 molecular weight, was used as received from Fluka. A3-Y and A3-S peptides were used as received from New England Peptide (crude, 70%). 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (1 M solution, biotechnology grade) from Amresco was diluted with Milli-Q water as received to a 50 mM solution which was used throughout the course of experiments. Similar results were obtained from a fresh stock of HEPES buffer made from the free-acid salt (Sigma-Aldrich) and titrated to pH 7.5 via dilute sodium hydroxide in Milli-Q water. Borate buffer (50 mM) was purchased from Amresco (50 mM, pH ) 7.5, ACE tetraborate solution) and was used as received. Comparable results were observed from borate buffer of various pH values (100 mM, Amresco) as well as fresh stock buffer using boric acid, sodium tetraborate and adjusting pH with either HCl or NaOH as needed. Note that as with all biological buffers, the stability and capacity of a buffer to mediate pH decreases with exposure to CO2 (formation of carbonic acid). HEPES is a “Good” buffer, containing both positive and negative ionizable groups, where the tertiary amine groups provide the positive charge and the negative charges are offered by the sulfonic acid group. These buffers have shown reactivity toward H2O231 and complexation with adventitious metallic cations dissolved from glassware surfaces, such as Cu(II) and Ni(II).32,33 Acknowledgment. The authors thank R. MacCuspie, J. Slocik, and W. Goodson for insightful discussions. Funding was graciously provided by the Air Force Office of Scientific Research and the Air Force Research Laboratory, Materials and Manufacturing Directorate. References and Notes (1) Lowenstam, H. A. Science 1981, 211, 1126–1130. (2) Mann S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: Oxford, 2001. (3) Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Nature 2000, 403, 289–292. (4) Naik, R. R.; Brott, L. L.; Clarson, S. J.; Stone, M. O. J. Nanosci. Nanotechnol. 2002, 2, 95–100. (5) Klaus, T.; Joerger, R.; Olsson, E.; Granquist, C. G. Proc. Natl. Acad. Sci.U.S.A. 1999, 96, 13611–13614. (6) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129– 1132. (7) Slocik, J. M.; Stone, M. O.; Naik, R. R. Small 2005, 1, 1048– 1052. (8) Naik, R. R.; Jones, S. E.; Murray, C. J.; McAuliffe, J. C.; Vaia, R. A.; Stone, M. O. AdV. Funct. Mater. 2004, 14, 25–30. (9) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nat. Mater. 2004, 1, 169–172. (10) Kehoe, J. W.; Kay, B. K. Chem. ReV. 2001, 105, 4056–4072. (11) Tamerler, C.; Oren, E. E.; Duman, M.; Vekatasubramanian, E.; Sarikaya, M. Langmuir 2006, 22, 7712–7718. (12) Tamerler, C.; Dincer, S.; Heidel, D.; Zareie, M. H.; Sarikaya, M. Prog. Org. Coatings 2003, 47, 267–274. (13) Slocik, J. M.; Naik, R. R.; Stone, M. O.; Wright, D. W. J. Mater. Chem. 2005, 15, 749–753.
Buffer and Peptides in Gold Nanoparticle Formation (14) Zhou, Y.; Chen, W.; Itoh, H.; Naka, K.; Ni, Q.; Yamane, H.; Chujo, Y. Chem. Commun. 2001, 2518–2520. (15) Si, S.; Bhattacharjee, R. R.; Banerjee, A.; Mandal, T. K. Chem.sEur. J. 2006, 12, 1256–1265. (16) Bhattacharjee, R. R.; Das, A. K.; Haldar, D.; Si, S.; Banerjee, A.; Mandal, A. K. J. Nanosci. Nanotechnol. 2005, 5, 1141–1147. (17) Creighton, J. A.; Eadon, D. G. J. Chem. Soc., Faraday Trans. 1991, 87, 3881–3891. (18) Newman, J. D. S.; Blanchard, G. J. Langmuir 2006, 22, 5882– 5887. (19) Habib, A.; Tabata, M.; Wu, Y. G. Bull. Chem. Soc. Jpn. 2005, 78, 262–269. (20) Xie, J.; Lee, J. Y.; Wang, D. I. C. Chem. Mater. 2007, 19, 2823– 2830. (21) Akbarzadeh, A.; Zare, D.; Farhangi, A.; Meharabi, M. R.; Norouzain, D.; Tangestaninejad, S.; Moghadam, M.; Bararpour, N. Am. J. Appl. Sci. 2009, 6, 691. (22) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226–8230. (23) Heinz, H.; Farmer, B. L.; Pandey, R. B.; Slocik, J. M.; Patnaik, S. S.; Pachter, R.; Naik, R. R. J. Am. Chem. Soc., submitted.
J. Phys. Chem. C, Vol. 113, No. 23, 2009 9997 (24) Zheng, N. F.; Fan, J.; Stucky, G. D. J. Am. Chem. Soc. 2006, 128, 6550–6551. (25) Prasad, B. L. V.; Stoeva, S. I.; Sorensen, C. M.; Klabunde, K. J. Langmuir 2002, 18, 7515–7520. (26) Stoeva, S.; Klabunde, K. J.; Sorensen, C. M.; Dragieva, I. J. Am. Chem. Soc. 2002, 124, 2305–2311. (27) de Mello Doneg, C.; Liljeroth, P.; Vanmaekelbergh, D. Small 2005, 1, 1152–1162. (28) Schichibu, Y.; Negishi, Y.; Tsukuda, T.; Teranishi, T. J. Am. Chem. Soc. 2005, 127, 13464–13465. (29) Zheng, J.; Zhang, C.; Dickson, R. M. Phys. ReV. Lett. 2004, 93, 077402–1. (30) Rahme, K.; Oberdisse, J.; Schweins, R.; Marty, J.-D.; Mingotaud, C.; Gauffre, F. ChemPhysChem 2008, 9, 2230–2236. (31) Zhao, G.; Chasteen, N. D. Anal. Biochem. 2006, 349, 262–267. (32) Simpson, J. A.; Cheeseman, K. H.; Smith, S. E.; Dean, R. T. Biochem. J. 1988, 254, 519–523. (33) Sokolowska, M.; Bal, W. J. Inorg. Biochem. 2005, 99, 16531660.
JP8102063
