Enzyme mediated synthesis of water-dispersible, naturally protein capped, monodispersed gold nanoparticles; their characterization and mechanistic aspects

Abstract

Inorganic nanomaterials are conventionally synthesized under harsh environments like extremes of temperature, pressure and pH. These methods are eco-unfriendly, expensive, toxic, cumbersome, yield bigger particles which agglomerate due to not being capped by capping agents. In contrast, biological synthesis of inorganic nanomaterials occurs under ambient conditions viz. room temperature, atmospheric pressure, physiological pH and is reliable, eco-friendly and cheap. We have already reported the extracellular biosynthesis of monodispersed gold nanoparticles from the whole cells of novel extremophilic actinomycete Thermomonospora sp. In order to know the exact mechanism of synthesis, we decided to investigating it further. Here we describe the simple protocol for purification of the temperature and SDS resistant sulfite reductase enzyme and organic capping molecule, which are required for the synthesis and stabilization of gold nanoparticles respectively. This purified enzyme was then employed for the synthesis of gold nanoparticles along with the capping molecule, which render gold nanoparticles monodispersed in solution.

---

Introduction

Numerous chemical and physical methods are being employed to fabricate inorganic nanomaterials of different compositions, shapes and sizes. These chemical methods are toxic while the physical ones are very expensive. The growing need of the hour is to develop an alternate route for nanomaterial synthesis which is non-toxic, ecofriendly and cheap. This has led researchers to further investigate the biological green-synthesis routes^1,2 which can produce water-soluble, protein-capped, bio-compatible nanoparticles with control over particle size by preventing agglomeration; at the same time posing absolutely no hazard to the environment. Use of biological organisms such as microorganisms, plant extracts or plant biomass^3,4 and biological molecules such as glucose,^5,6 amino acids,^7–9 enzymes^10–12 or polypeptides^13,14 could be used as an alternative to chemical and physical methods for the production of metal nanoparticles in an eco-friendly manner. Although a very large number of biological systems in general and microbial species in particular are capable of producing metal nanoparticles, the mechanism of nanoparticle bio-synthesis still remains a mystery. The metabolic complexity of viable microorganisms often complicates the analysis and identification of active species in the nucleation and growth of metal nanoparticles. Strategies such as enzymatic oxidation or reduction, absorption on the cell wall and, in some cases, subsequent chelation with extracellular peptides or polysaccharides have been developed and used by microorganisms.¹⁵ Many things about the biochemical and molecular mechanism of enzyme mediated reactions such as reductions and oxidations need a complete study since most often these processes remain unknown and should be revealed. In fact, the biochemical mechanisms refer to finding materials like enzymes, which may mediate the biosynthesis mechanism. The studies of the enzyme structure and the genes which code these enzymes may help improve our understanding as to how metal nanoparticle synthesis is performed. Improvements in chemical composition, size, shape and dispersity of generated nanoparticles could allow the use of nanobiotechnology in a variety of other applications.¹⁶ In vitro biomolecule assisted synthesis could also overcome the problems which arise when particular size, shape, stability, etc. are desired. There have been a few reports where NADPH dependent reductase enzymes have had a peculiar role in the synthesis of nanoparticles.^17,18 Unfortunately, this enzyme mediated synthesis of nanoparticles requires a chemical organic stabilizing molecule to maintain the size of nanoparticles in the nano regime. However, for the biomedical applications of these nanoparticles, it could be of immense importance if one could get a biologically derived stabilizing molecule which could render the obtained nanoparticles water dispersible.

In the current study, we purified an extremely stable sulfite reductase enzyme which could act as a reducing agent for the synthesis of gold nanoparticles and to the best of our knowledge, we for the first time have successfully identified and purified a biological capping molecule (capping protein) which not only maintains the stability of obtained gold nanoparticles, but could also render them monodispersed in solution. This report is novel in the way that both the reducing agent and capping molecule have been isolated and purified from the same source i.e. Thermomonospora sp.

Results and discussion

Purification of sulfite reductase enzyme

Initial fractionation of extracellular broth of Thermomonospora sp. for the purification of sulfite reductase enzyme was carried out using a Fast protein liquid chromatography (FPLC) system. The protein sample (with predetermined concentration) was randomly loaded onto an anionic exchanger (Mono Q). Bound and unbound profiles of the protein were obtained with 2(N-Morpholino)ethanesulfonic acid (MES) buffer at pH 6. From Fig. 1a, it can be seen that the bound protein has been eluted in the linear gradient at five different concentrations of NaCl, whereas the unbound protein emerged as one single peak. These bound and unbound protein fractions were electrophoresed and checked for gold nanoparticle synthesis. Out of these six protein fractions, only one fraction (peak 2) which eluted at 200 mM NaCl showed the capability of forming gold nanoparticles and showed 5–6 closely spaced protein bands on 15% SDS-PAGE (sodium dodecyl polyacryl amide gel electrophoresis) (Fig. 1b). Previous evidence on the role of sulfite reductase enzyme for the synthesis of gold nanoparticles¹⁷ have led us to assay this protein fraction against sulfite reductase activity. Enzyme assay confirmed the presence of sulfite reductase enzyme activity in the fraction (Table S1†). The protein bands were separately eluted^19–21 and individually assayed for enzyme activity²² (Table S1†) as well as gold formation ability.¹⁷ The need for directly eluting the enzyme from the gel arose due to the failure in resolving closely spaced protein bands by gel filtration chromatography (data not shown due to brevity). Here it is to be noted that the enzyme assay was performed directly after eluting the enzyme from the gel without any re-naturation experiment. The extreme stable nature of the enzyme can also be confirmed by the fact that prior to electrophoresis, the enzyme sample was boiled along with reducing agent such as β-mercaptoethanol for a few minutes at 100 °C. It is likely that such harsh treatment did not fully affect the tertiary structure of the enzyme and some part of it remained active which can be justified by the fact that it's a secretional product of an extremophilic microorganism (Thermomonospora sp.). Another proof of the extremely stable nature of this enzyme can be obtained by amino acid analysis which shows the presence of 14 tryptophan and 15 cysteine residues even after the protein sample has been acid hydrolyzed. These amino acids may have been buried deep inside the tertiary structure of the enzyme and thus were not completely destroyed even after treatment with 6 N HCl at boiling temperature (Table S2†).

Fig. 1 Fast protein liquid chromatography and preparative sodium dodecyl sulphate – poly acryl amide gel electrophoresis (SDS-PAGE) (15%) profile of extracellular broth of Thermomonospora sp. (a) Unbound fraction collected in isocratic flow and bound proteins (peaks 1–5) were eluted in linear gradient of 0–0.8 M NaCl. (b) Different fractions obtained from FPLC, lane (2) active fraction for sulfite reductase activity, lane (5) fraction containing capping protein, lane (1), (3) and (4) different fractions eluted from FPLC with no activity.

Identification and purification of capping molecule (protein)

SDS-PAGE analysis of all the protein fractions (peaks) in the above step has shown that the peak 5 fraction contains two closely spaced very low molecular weight proteins. Again, previous published literature on the nature of the size of capping molecules¹⁷ has compelled us to check this fraction for the presence of any low molecular weight capping entity that could be involved in the stabilization of gold nanoparticles. These two closely spaced bands were separately eluted and checked for capping and gold nanoparticle synthesis along with sulfite reductase enzyme obtained in the above step.

Molecular weight determination of sulfite reductase enzyme and capping molecule (protein)

Molecular weight of purified sulfite reductase enzyme through 15% SDS-PAGE was found to be 43 kDa (Fig. 2a, lane 2) and that of capping molecule (protein) was 13 kDa (Fig. 2b, lane 1). Only the first band (upper band) of the fraction 5 was eluted and checked for its role in capping of gold nanoparticles, however any role of the second band (protein) cannot be overruled for the same function or other.

Fig. 2 Molecular weight determination of the purified sulfite reductase enzyme and capping protein by SDS-PAGE. (a) Lane (1) molecular mass markers ranging from 14.4–97 kDa, lane (2) purified sulfite reductase enzyme of 43 kDa. (b) Lane (1) purified capping protein of 13 kDa, lane (2) molecular mass markers ranging from 10–230 kDa.

In vitro synthesis of gold nanoparticles using sulfite reductase enzyme and capping protein

Freshly prepared chloroauric acid (HAuCl₄) when incubated with sodium sulfite (Na₂SO₃), capping protein, nicotinamide adenine dinucleotide phosphate (NADPH) and sulfite reductase enzyme at 50 °C resulted in the formation of highly monodispersed gold nanoparticles just after 4 h, as evidenced by the change in the color of the reaction mixture from colorless (0 h) to ruby-red (4 h) (Fig. 3, tube no. 1 & 2 respectively). The color change indicates the reduction of gold metal ions from Au³⁺ to Au⁰ and arises due to the excitation of SPR (Surface Plasmon Resonance) in the metal nanoparticles.²³ The solution was extremely stable, with no evidence of flocculation of the particles even a month after reaction. From this experiment it is very clear that sulfite reductase enzyme plays a very important role in the reduction of gold ions to metallic gold accompanying the reduction of Na₂SO₃ to sulphide. Sulfite reductase enzyme carried out the 6-electrons reduction with the help of NADPH and hence it's called an NADPH-dependent sufite reductase. NADPH donates electrons to sulfite reductase to reduce sulfite to hydrogen sulfide and water. In doing so, the enzyme also catalyses the reduction of Au³⁺ to Au⁰. After donating the electrons, NADPH oxidizes to NADP⁺ while sulfite reductase enzyme in its oxidized state waits for further electrons to come from NADPH.²⁴ The synthesis of gold nanoparticles by sulfite reductase enzyme can be explained in the following reaction:

Fig. 3 Sulfite reductase enzyme mediated synthesis and UV-vis spectra of gold nanoparticles with and without capping protein. (a) Formation of gold nanoparticles at 0 hour (tube no. 1), formation of gold nanoparticles after 4 hours (tube no. 2) and formation of gold nanoparticles without capping protein after 4 hours (tube no. 3). (b) UV-vis spectra of sulfite reductase enzyme and capping protein mediated synthesized gold nanoparticles (as a function of time) from 0 to 4 h. Curve ‘A’ represents UV-vis spectrum of gold nanoparticles without capping protein after 4 h.

When the same reaction mixture without the capping protein was incubated at 50 °C for 4 h, a less intense color of gold nanoparticles was developed (Fig. 3a, tube no. 3). This less intense color indicates that reduction of Au(III) to Au(0) might have taken place by sulfite reductase enzyme but as there was no capping protein involved in the synthesis, the size of the gold nanoparticles would not have remained in nanodimensions. It is to be noted that the color of gold nanoparticles is a direct function of SPR, which in turn depends on the size of the nanoparticles.²⁵

Characterization of in vitro synthesized gold nanoparticles

UV-vis spectroscopy. The UV-vis spectra for the reaction mixture of gold nanoparticles, as a function of time, (Fig. 3b) showed a well defined Surface Plasmon at 533 nm, which is in very good agreement with the reported SPR band of gold nanoparticles.²⁵ The intensity of sharp SPR band centered at 533 nm increases with increasing time and gets stabilized after 4 h of reaction. Curve ‘A’ represents the UV-vis spectrum of gold nanoparticles synthesized without capping protein which shows a less sharp SPR band centered at 575 nm. This red shift may have arisen due to agglomeration of gold nanoparticles in the reaction mixture, since the exact position of SPR band depends on the size of the particle too.

Transmission electron microscopy (TEM). Transmission electron microscopy (TEM) analysis of the gold nanoparticles showed that they are monodispersed and are essentially spherical (Fig. 4a). The particles are well dispersed and show no sign of aggregation; this separation is mainly because of capping protein which is involved in capping of gold nanoparticles and prevents their aggregation. The dispersity shown in TEM image exactly correlates with the color and UV-vis spectroscopy analysis of the gold nanoparticles which showed the nanoparticles to be very stable and monodispersed. The nanoparticles were crystalline in nature as depicted by Selected Area Electron Diffraction (SAED) pattern (Fig. 4b). Particle size distribution of gold nanoparticles revealed that the particles have diameters in the range of 2–6 nm with an average of 3 nm (Fig. 4c). Fig. 4d shows the TEM image of gold nanoparticles synthesized without capping protein, which shows agglomerated gold nanoparticles forming a clump, each of size 40–50 nm. This is the strongest evidence favouring the role of the capping protein in stabilizing and capping of the gold nanoparticles and also is in good agreement with the UV-vis spectroscopy measurements indicating agglomerated nanoparticles.

Fig. 4 Transmission electron microscopy, selected area electron diffraction and particle size distribution of sulfite reductase enzyme mediated monodispersed gold nanoparticles synthesis. (a) Transmission electron microscope image of sulfite reductase enzyme mediated biosynthesized monodispersed gold nanoparticles. (b) Selected area electron diffraction pattern recorded from one of the enzyme mediated biosynthesized monodispersed gold nanoparticles. (c) Histogram of size distribution of the enzyme mediated biosynthesized monodispersed gold nanoparticles. (d) Transmission electron microscope image of enzyme mediated biosynthesized monodispersed gold nanoparticles synthesized without capping protein.

X-ray diffraction (XRD) analysis. Fig. 5 represents the X-ray diffraction (XRD) pattern of the sulfite reductase enzyme mediated synthesized gold nanoparticles. The X-ray diffraction pattern of gold nanoparticles shows intense peaks at {111}, {200}, {220} and {311} with Bragg's reflection at 2θ = 38.09°, 45°, 65° and 78° respectively, and agrees with those reported for the gold nanocrystals.²⁶ No spurious diffractions due to crystallographic impurities were found. An overwhelmingly strong diffraction peak at 38.09° is assigned to the {111} facets of a face-centered cubic (fcc) metal gold structure.²⁶

Fig. 5 X-ray diffraction pattern of sulfite reductase enzyme mediated biosynthesized monodispersed gold nanoparticles deposited on Si (111) glass plate.

Effect of pH and temperature on sulfite reductase activity. Fig. 6 gives a clear account of the extreme nature of sulfite reductase enzyme where the enzyme was found to be most active at pH 8 and at 50 °C. However, at very high pH values the enzyme is still 22–25% active and at temperatures 80–90 °C the enzyme is capable of retaining more than 20% of its activity. This extreme nature of this enzyme may be attributed to its source which is an extremophilic microorganism that grows strictly at pH 9 and at 50 °C.

Fig. 6 Effect of pH and temperature on the activity of sulfite reductase enzyme. (a) Optimum pH of sulfite reductase enzyme showing maximum activity at pH 8. (b) Optimum temperature of sulfite reductase enzyme showing maximum activity at 50 °C.

Experimental

Materials

The experiments employed malt extract, yeast extract, glucose, bacteriological peptone (Hi Media), Mono Q FPLC column (Biorad), molecular mass markers (G. E. Healthcare), sodium carbonate, dialysis tubing, acrylamide, TEMED, SDS, 2(N-Morpholino)ethanesulfonic acid (MES), nicotinamide adenine dinucleotide phosphate (NADPH), Coomassie Brilliant Blue G-250, chloro auric acid (HAuCl₄), and sodium sulfite (Na₂SO₃) (Sigma Chemicals Co., St Louis, MO, U.S.A). All other chemicals used were of analytical grade.

Methods

For the purification of sulfite reductase enzyme and capping protein, the actinomycete Thermomonospora sp. was grown in 250 ml Erlenmeyer flasks containing 100 ml MGYP medium which is composed of malt extract (0.3%), glucose (1.0%), yeast extract (0.3%) and peptone (0.5%). Sterile 10% sodium carbonate was used to adjust the pH of the medium to 9. The culture was grown with continuous shaking on a rotary shaker (200 rpm) at 50 °C for 72 h. The mycelia (cells) were then separated from the culture broth by centrifugation (5000 rpm) at 10 °C for 20 minutes and were washed thrice with sterile distilled water under sterile conditions and then suspended in 100 ml sterile distilled water (sterile 10% sodium carbonate was used to adjust the pH to 9) in 250 ml Erlenmeyer flask for 72 h under shaking (200 rpm) conditions. The supernatant was collected by centrifugation, concentrated by lyophilization, dialyzed extensively against Milli Q water followed by dialysis (3 kDa, cut off membrane) against 20 mM MES buffer at pH 6.0 and used as a source of sulfite reductase enzyme.

Detection of gold nanoparticles synthesis using crude broth

This was carried out by mixing 100 μl of the extracellular broth of Thermomonospora sp. with 1 ml each of 1 mM HAuCl₄ and NADPH following the development of color. Formation of ruby-red color indicates the formation of gold nanoparticles and presence of reductases in the extra cellular broth.

Purification of sulfite reductase enzyme using fast protein liquid chromatography (FPLC)

Initial fractionation of concentrated and dialyzed extracellular broth of Thermomonospora sp. was performed at ambient temperature using FPLC system (Biorad Biologic Duo flow system) using an anionic exchange resin (source Q packed in 10 ml Triton column, G.E. Healthcare) pre-equilibrated with 20 mM MES buffer, pH 6. The bound fractions were eluted in a linear gradient of 0–0.8 M NaCl in the same buffer at a flow rate of 25 ml h⁻¹.

Determination of sulfite reductase enzyme activity

This was carried out according to Yoshimoto and Sato.²² The total reaction mixture of 1.5 ml contained 1.0 mM of freshly prepared sodium sulfite in 100 mM MES buffer, pH 6.0, containing 1.0 mM EDTA, 0.15 mM NADPH and appropriately diluted enzyme. The reaction was initiated by the addition of NADPH followed by incubation at 50 °C. The oxidation of NADPH was monitored spectrophotometrically at 340 nm. Samples containing sulfite reductase enzyme incubated in the absence of sodium sulfite served as a blank. One unit of sulfite reductase enzyme activity is defined as the amount of enzyme required to oxidize 1 mole of NADPH per min under the assay conditions.

In vitro synthesis of gold nanoparticles using sulfite reductase enzyme

The total reaction mixture of 2 ml containing 1.0 mM each of freshly prepared HAuCl₄ and Na₂SO₃, 100 μg of capping protein, 0.15 mM NADPH and 100 μg of sulfite reductase enzyme was incubated at 50 °C for 4 h. Reaction mixture in the absence of capping protein was also incubated at 50 °C to confirm its role in the stabilization of gold nanoparticles. Samples were removed at regular intervals and subjected to UV-vis spectroscopy to check for the formation of nanoparticles.

Characterization of gold nanoparticles

UV-vis spectrophotometric measurements were carried out on a Perkin Elmer dual-beam spectrophotometer (Model lambda 750) operated at a resolution of 1 nm. The size and shape analysis of gold nanoparticles was done on a TECHNAI G2 F20 S-TWIN instrument operated at voltage of 200 KV. For this purpose, the samples were prepared by drop-coating the particles suspended in aqueous medium on carbon coated copper grids. Selected area electron diffraction (SAED) analysis was carried-out on the same grids. XRD patterns were recorded using a PHILIPS X'PERT PRO instrument equipped X'celerator, a fast solid-state detector on drop-coated sample on glass substrate. The sample was scanned using X'celerator with a total number of 121 active channels. Iron-filtered Cu Kα radiation (λ = 1.5406 Å) was used. XRD patterns were recorded in the 2θ range of 30–80° with a step size of 0.02° and a time of 5 seconds per step at 40 kV voltage and a current of 30 mA.

Effect of pH and temperature on sulfite reductase activity

pH optimum. Into 1.4 ml reaction mixture with various pH (pH 3.0–5.0: 0.2 M citrate buffer; pH 6.0–8.0: 0.2 M phosphate buffer; pH 9.0–10.0: 0.2 M bicarbonate buffer; pH 11.0–12.0: 0.2 M trimethylamine buffer; all buffers containing 1.0 mM EDTA, 0.15 mM NADPH, 1.0 mM of freshly prepared sodium sulfite), 0.1 ml of purified sulfite reductase enzyme was added and incubated at 50 °C for 5 min. The activity was determined from the initial velocity and corrected for the slow oxidation of NADPH in the absence of sulfite.²²

Temperature optimum. Into 1.4 ml reaction mixture [0.2 M MES buffer (pH 6) containing 1.0 mM EDTA, 0.15 mM NADPH, 1.0 mM of freshly prepared sodium sulfite], 0.1 ml of purified sulfite reductase enzyme was added and incubated at various temperatures (25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 °C) for 5 min. The activity was determined from initial velocity and corrected for the slow oxidation of NADPH in the absence of sulfite.²²

Conclusions

We have shown a simple and fast method for obtaining and purifying sulfite reductase enzyme which is responsible for the synthesis of gold nanoparticles. For the very first time, a biological capping molecule (protein) has also been identified and purified from the same microorganism. A prominent role of this capping molecule in the stabilization of gold nanoparticles has been established. These nanoparticles synthesized through an enzyme mediated pathway are monodispersed and of few nanometres in size which is very crucial for their biomedical applications. However, the purified enzyme and capping protein are not restricted for the synthesis of gold nanoparticles but can be exploited for the in vitro tailor made synthesis of several other nanomaterials such as sulfides and quantum dots of immense biological importance. The proof of concept demonstrated in this work that involves the reduction of metal ions by reductase enzyme, a prerequisite for the formation of metal nanoparticles can serve as a template for the production of aforementioned nanomaterials.

Acknowledgements

S.A.K thanks the Council of Scientific and Industrial Research (CSIR), New Delhi for Senior Research Fellowship. A.A thanks the Department of Biotechnology, Govt. of India (New Delhi) for the Tata Innovation Fellowship award and financial support through BSC0112 CSIR, New Delhi. The authors thank the Center for Materials Characterization (CMC, CSIR-NCL), Pune for assistance regarding TEM measurements.

Notes and references

1. P. T. Anastas and J. C. Warner, Green Chemistry: Theory and Practice, Oxford University Press, New York, 1998.
2. M. Sastry, A. Ahmad, M. I. Khan and R. Kumar, Nanobiotechnology, Wiley-VCH, New York, 2004.
3. A. Ahmad, S. Senapati, M. I. Khan, R. Kumar and M. Sastry, Langmuir, 2003, 19, 3550.
4. S. Shiv Shankar, A. Rai, B. Ankamwar, A. Singh, A. Ahmad and M. Sastry, Nat. Mater., 2004, 3, 482.
5. P. Raveendran, J. Fua and S. L. Wallen, Green Chem., 2006, 8, 34.
6. P. Raveendran, J. Fua and S. L. Wallen, J. Am. Chem. Soc., 2003, 125, 13940.
7. Y. Shao, Y. Jin and S. Dong, Chem. Commun., 2004, 1104.
8. S. K. Bhargava, J. M. Booth, S. Agrawal, P. Coloe and G. Kar, Langmuir, 2005, 21, 5949.
9. P. R. Selvakannan, S. Mandal, S. Phadtare, A. Gole, R. Pasricha, S. D. Adyanthaya and M. Sastry, J. Colloid Interface Sci., 2004, 269, 97.
10. A. Bharde, A. Kulkarni, M. Rao, A. Prabhune and M. Sastry, J. Nanosci. Nanotechnol., 2007, 7, 4369.
11. A. Rangnekar, T. K. Sarma, A. K. Singh, J. Deka, A. Ramesh and A. Chattopadhyay, Langmuir, 2007, 23, 5700.
12. T. Yang, Z. Li, L. Wang, C. Guo and Y. Sun, Langmuir, 2007, 23, 10533.
13. Z. Wang, J. Chen, P. Yang and W. Yang, Appl. Organomet. Chem., 2007, 21, 645.
14. R. Djalali, Y. Chen and H. Matsui, J. Am. Chem. Soc., 2003, 125, 5873.
15. N. Krumov, I. Perner-Nochta, S. Oder, V. Gotcheva, A. Angelov and C. Posten, Chem. Eng. Technol., 2009, 32, 1026.
16. A. Bharde, D. Rautaray, V. Bansal, A. Ahmad, I. Sarkar, S. M. Yusuf, M. Sanyal and M. Sastry, Small, 2006, 2, 135.
17. S. A. Kumar, M. K. Abyaneh, S. W. Gosavi, S. K. Kulkarni, A. Ahmad and M. I. Khan, Biotechnol. Appl. Biochem., 2007, 47, 191.
18. S. A. Kumar, M. K. Abyaneh, S. W. Gosavi, S. K. Kulkarni, R. Pasricha, A. Ahmad and M. I. Khan, Biotechnol. Lett., 2007, 29, 439.
19. M. P. Deutscher, Guide to Protein Purification, Academic Press, San Diego, CA, 1990.
20. P. Jeno and M. Horst, Electroelution of Proteins From Polyacrylamide Gels, Humana Press, Totowa, New Jersey, 1996.
21. J. H. Waterborg and H. R. Matthews, The Electrophoretic Elution of Proteins From Polyacrylamide Gels, Humana Press, Totowa, New Jersey, 1994.
22. A. Yoshimoto and R. Sato, Biochim. Biophys. Acta, Bioenerg., 1968, 153, 555.
23. P. Mulvaney, Langmuir, 1996, 12, 788.
24. S. Senapati, A. Ahmad, M. I. Khan, M. Sastry and R. Kumar, Small, 2005, 1, 517.
25. M. B. Mohamed, V. Volkov, S. Link and M. A. El-Sayed, Chem. Phys. Lett., 2000, 317, 517.
26. D. V. Leff, L. Brandt and J. R. Heath, Langmuir, 1996, 12, 4723.

---

Footnote

† Electronic supplementary information (ESI) available: Two dimensional electrophoresis (2-DE), determination of sulfite reductase activity and amino acid composition of sulfite reductase enzyme. See DOI: 10.1039/c3ra43888k

---