Effect of Hg(II) and Pb(II) Ions on C-Phycocyanin (Spirulina platensis)

Eteri Gelagutashvili, Ketevan Tsakadze

Andronikashvili Institute of Physics, Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia.

DOI: 10.4236/opj.2013.31020                

Abstract

Influence of Hg(II) and Pb(II) ions on C-Phycocyanin (C-PC) from cyanobacteria Spirulina platensis was investigated using Fluorescence spectroscopy. Fluorescence measurements demonstrate quenching of C-PC emission by Hg(II) and Pb(II), and blue shifts in the fluorescence spectra. The effect of DNA on the fluorescence of Hg(II)-and Pb(II)-C-PC (from Spirulina platensis) complexes was also studied. It was shown that the fluorescence intensity of Hg-C-PC after addition of DNA gave rise to the fluorescence buildup. At the same time, addition of DNA to the Pb(II)-C-PC complexes showed no such effect. In the case of Hg(II)-C-PC, fluorescence intensity significantly decreases in time, while for Pb(II)-C-PC, decrease of the fluorescence intensity is not significant, but blue shift of the peak takes place.

Keywords

C-Phycocyanin; Hg(II); Pb(II) Ions; DNA; Fluorescence

1. Introduction

Blue-green algae Spirulina platensis, like other cyanobacterias, uses proteins called phycobiliproteins to harvest light for photosynthesis. Phycobiliproteins, can be used in photodynamic therapy [1]. One of the basic proteins of Spirulina platensis is C-phycocyanin (C-PC), which is used as fluorescent protein probe in living cells [2]. Phycocyanin, a natural blue pigment that is the major light-harvesting biliprotein in the blue-green alga Spirulina platensis, reduces normal tissue photosensitivity due to its fast metabolism in vivo [3]. Phycocyanins extracted from Spirulina are used as industrial and food coloring agents. The blue color gives the phycocyanins fluorescence properties and the intense fluorescence is exploited in immunoassay tests [4]. In [5], protective role of dietary Spirulina at lead poisoning was studied in Swiss albino mice. Significant increase of the survival time was observed in preand post-treating by Spirulina compared with the control group without Spirulina treatment. Rats with high mercury dosage showed increased levels of blood urea nitrogen (BUN) and serum creatinine, both indicators of acute nephritis. Addition of 30% Spirulina to the diet resulted in significant decrease of (BUN) and serum creatinine levels. Rats showed the similar kidney improvement after Spirulina diet [6]. A tetrapyrrole-based chromophore was obtained through the methanolysis of C-phycocyanin extracted from Spirulina platensis, and was found to act as a selective receptor for Hg(II) at physiological pH conditions [7]. Spirulina contains several active ingredients, notably phycocyanin and β-carotene that have potent antioxidant and antiinflammatory activities. C-PC can be successfully used as an anti-inflammatory drug and liposomal encapsulation is effective in improving its anti-inflammatory activity [8]. Phycocyanin has the ability to scavenge free radicals, including alkoxyl, hydroxyl and peroxyl radicals. It should be noted that “Brilliant Violet” reporters are dramatically brighter than other UV-violet excitable dyes, and are of similar utility to phycoerythrin (PE) and allophycocyanin (APC) [9].

To use algal biomass, it is important to understand the nature of the metal binding process with its components.

In this work, effects of Hg(II) and Pb(II) ions on Spirulina platensis C-PC are presented using Fluorescence spectroscopy. Also influence of DNA on Hg(II)- and Pb(II)-C-PC complexes is explored.

2. Materials and Methods

C-PC was isolated from culture of Spirulina platensis (strain IPPAS B-256) in our laboratory according to the Teale and Dale method [10] with some modifications. Extraction of C-PC was carried out in 0.1 M Na, K phosphate buffer at pH 6.0. Initially, the preparation was purified by deposition in saturated (NH₄)₂SO₄ solution. The final stage of purification was performed on chromatography column filled with DEAE-cellulose, previously equilibrated in 50 mM acetate buffer, pH 5.2. To determine the degree of purity of the samples, spectrophotometric (wavelengths 250 - 750 nm) and electrophoretic methods were used. The concentration of C-PC was determined by ultraviolet-visible spectroscopy using a value of ε_{λ = 615 nm} =279,000 M⁻¹∙cm⁻¹ for the absorption coefficient. The purity of the protein was assessed from the ratio of absorbances at λ = 615 nm and λ = 280 nm (A₆₁₅/A₂₈₀ ≥ 4). Fluorescence spectra were measured in 1 cm³ quartz cells using a laser fluorescence spectroscopy. Fluorescence titration was performed in the range 400 - 700 nm, by adding a metal solution to C-PC and recording the spectrum after each addition. The solutions were excited at 488 nm and the fluorescence intensity was monitored at 635 nm.

3. Results and Discussions

The fluorescence spectra of C-PC in the presence of Hg(II) and Pb(II) ions are shown in Figure 1. The intensity of C-PC fluorescence, observed in the range of 580 - 690 nm, was measured as function of added metal concentration. Metal free C-PC had fluorescence maximum at 635 nm (curve 1). The increase of the metal concentration caused decrease of the peak height accompanied by blue shift in both cases (8 - 15 nm). It is in good agreement with literature data [11], where SPDPC-PC, from Spirulina platensis using one-step anion-exchange chromatography was studied. The obtained C-PC had its absorption maximum at 620 nm and the fluorescence emission maximum at 640 nm. SPDP is an excellent heterobifunctional crosslinker for thiolating amines. Different molar ratios have significant influence on the absorption and fluorescence spectra of C-phycocyanin. The absorption maximum and fluorescence emission maximum both decreased and blue-shifted from 640 nm to 630 nm as the molar ratio of SPDP increased. Similar results were obtained for other metal ion-C-PC interactions [12,13]. The interaction of metal and Phycocyanin was investigated by ultraviolet-visible, fluorescence and infrared spectra. The study showed that the characteristic absorption of PC at 615 nm decreased gradually with increase of metal concentration and interaction time. Accordingly, the fluorescence emission peak and two fluorescence excitation peaks also decreased for Ag(I)-C-PC complexes [13]. Dynamic formation of nano-Ag(0) was also observed by synchronous fluorescence spectra, showing that Ag(0) was formed by the PC in situ reduction.

Figure 1 insert, where normalized fluorescence spectra of C-PC in the absence and in the presence of Hg(II) and Pb(II) ions are shown, also confirms this. It is clear from Figure 1 that quenching has not been saturated even at high concentrations of metal ions. Addition of Hg(II) ions above 7 µM and of Pb(II) ions above 20 µM, has no further effect on the fluorescence intensity, which indicates that their binding to C-PC is saturated. The absence of saturation in the quenching plot even when the binding is saturated indicates that only a fraction of the binding sites quenches the fluorescence. This is clear also from Figure 2 and from Figure 2 inserts. In Figure 2 inserts, there are presented fluorescence spectra of C-PC in the presence Hg(II) and Pb(II) ions at amplification 3.5, C_Hg: 7 µM and C_Pb: 21 µM (curve 1), similar to Figure 1, and the same spectra, when amplification is 8.5 (curve 2). In Figure 2, there are shown fluorescence spectra of C-PC with and without Hg(II) and Pb(II) ions: 1) C-PC spectrum (curve 1); 2) residual fluorescence of C-PC after addition of Hg(II) ions (curve 2); 3) differential spectrum obtained as the difference of (1) and (2) and corresponding to “quenched” residuals of C-PC (curve 3).

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	S.-P. Zhang, J. Xie, J.-P. Zhang, J.-Q. Zhao and L.-J. Jiang, “Electron Spin Resonance Studies on Photosensized Formation of Hydroxyl Radical by C-Phycocyanin from S. platensis,” Biochimica et Biophysica Acta, Vol. 1426, No. 1, 1999, pp. 205-211. doi:10.1016/S0304-4165(98)00153-6
[2]	A. J. Tooley, Y. P. A. Cai and A. N. Gleser, “Biosynthesis of a Fluorescent Cyanobacterial C-Phycocyanin Holo-α Subunit in a Heterologous Host,” Proceedings of National Academy of Sciences of USA, Vol. 98, No. 19, 2001, pp. 10560-10565. doi:10.1073/pnas.181340998
[3]	R. Henrikson, “Earth Food Spirulina. How This Remarkable Blue-Green Algae Can Transform Your Health and Our Planet,” RoNo. re Enterprises Inc., Laguna Beach, 1989, p. 180.
[4]	M. N. Kronick, “The Use of Phycobiliproteins as Fluorescent Labels in Immunoassays,” Journal of Immunological Methods, Vol. 92, No. 1, 1986, pp. 1-13. doi:10.1016/0022-1759(86)90496-5
[5]	D. Shastri, M. Kumar and A. Kumar, “Modulation of Lead Toxicity by Spirulina fusiformis,” Phytotherapy Research, Vol. 13, No. 3, 1999, pp. 258-260. doi:10.1002/(SICI)1099-1573(199905)13:3<258::AID-PTR438>3.0.CO;2-2
[6]	Y. Yamane, H. Fukino, T. Icho and H. Shimamatsu, “Effect of Spirulina (Spirulina platensis) on the Renal Toxicity Induced by Inorganic Mercury and Para-Aminophenol,” Summary of Abstracts, 108th Conference of the Pharmaceutical Society of Japan, Hiroshima, 4-6 April 1998, p. 58.
[7]	M. Suresh, S. K. Mishra, S. Mishra and A. Das, “The Detection of Hg2+ by Cyanobacteria in Aqueous Media,” Chemical Communications, Vol. 14, No. 18, 2009, pp. 2496-2498. doi:10.1039/b821687h
[8]	M. Manconia, J. Pendás, N. Ledón, T. Moreira, C. Sinico, L. Saso and A. M. Fadda, “Phycocyanin Liposomes for Topical Anti-Inflammatory Activity: in-Vitro in-Vivo Studies, ” Journal of Pharmacy and Pharmacology, Vol. 61, No. 4, 2009, pp. 423-430. doi:10.1211/jpp.61.04.0002
[9]	P. K. Chattopadhyay, B. Gaylord, A. Palmer, N. Jiang, M. A. Raven, G. Lewis, M. A. Reuter, A. K. Nur-ur Rahman, D. A. Price, M. R. Betts and M. Roederer, “Brilliant Violet Fluorophores: A New Class of Ultrabright Fluorescent Compounds for Immunofluorescence Experiments,” Cytometry A, Vol. 81, No. 6, 2012, pp. 456-466. doi:10.1002/cyto.a.22043
[10]	F. W. J Teale and R. E. Dale, “Isolation and Spectral Characterization of Phycobiliproterins,” Biochemical Journal, Vol. 116, 1976, pp. 161-165.
[11]	S. G. Yan, X. L. Chen, X. Y. Zhang, B. C. Zhou, Y. Z. Zhang, “Spectral Changes of C-Phycocyanin with Different Molar Ratios of SPDP,” Guang Pu Xue Yu Guang Pu Fen Xi, Vol. 28, No. 5, 2008, pp. 1115-1117 [Article in Chinese].
[12]	E. S. Gelagutashvili, “Comparative analysis of the Titration of Heavy Metals by C-Phycoctanin,” Proceedings of Georgian Academy of Sciences, Vol. 37, No. 1-2, 2011, pp. 112-114.
[13]	F. Yang, Z. J. Guo, Y. Bai and W. J. Zheng, “Spectra of in Situ Reduction of Ag(I) by Phycocyanin and Dynamic Formation of Ag(0),” Guang Pu Xue Yu Guang Pu Fen Xi, Vol. 27,No. 1.2007, pp. 23-27.
[14]	E. S. Gelagutashvili., E. N. Ginturi, N. Kuchava and N. Bagdavadze, “Influene of DNA on Ag(I) C-Phycocyanin Complexes,” Physics, Chemical Physics, 2008. arXiv:0806.3378v1
[15]	L. David, A. Marx and N. Adir, “High-Resolution Crystal Structures of Trimeric and Rod Phycocyanin,” Journal of Molecular Biology, Vol. 405, No. 1, 2011, pp. 201-213. doi:10.1016/j.jmb.2010.10.036
[16]	N. Glazer, “Light Harvesting by Phycobilisomes,” Annual Review of Biophysics and Biophysical Chemistry, Vol. 14, 1985, pp. 44-77. doi:10.1146/annurev.bb.14.060185.000403
[17]	N. Glazer and A. Melis, “Photochemical Reaction Centers; Structure, Organization and Function,” Annual Review of Plant Physiology, Vol. 38, 1987, pp. 11-45. doi:10.1146/annurev.pp.38.060187.000303
[18]	G. Tapia, A. Galetovic, E. Lemp, E. PiNo and E. Lissi, “Singlet Oxygen-Mediated Photobleaching of the Prosthetic Group in Hemoglobin and C-Phycocyanin,” Photochemistry and Photobiology, Vol. 70, No. 4, 1999, pp. 499-504.
[19]	.T. Schirmer, R. Huber, M. Schneider, W. Bode, M. Miller and M. L. Hackert, “Crystal Structure Analysis and Refinement at 2.5 ? of Hexameric C-Phycocyanin from the Cyanobacterium Agmenellum Quadruplicatum. The Molecular Model and Its Implications for Light-Harvest ing,” Journal of Molecular Biology, Vol. 188, No. 4, 1986, pp. 651-676. doi:10.1016/S0022-2836(86)80013-4
[20]	J. M. Womick and A. M. Moran, “Nature of Excited States and Relaxation Mechanisms in C-Phycocyanin,” Journal of Physical Chemistry B, Vol. 113, No. 48, 2009, pp. 15771-15782. doi:10.1021/jp908093x
[21]	S. A. Pizarro, “Spectroscopic Study of the Light-Har vesting Protein C-Phycocyanin Associated with Colorless Linker Peptides,” Photochemistry and Photobiology, Vol. 73, No. 5, 2001, pp. 556-563. doi:10.1562/0031-8655(2001)073<0556:SSOTLH>2.0.CO;2
[22]	W. F. Vermaas, J. A. Timlin, H. D. Jones, M. B. Sinclair, L. T. Nieman, S. W. Hamad, D. K. Melgaard and D. M. Haaland, “In Vivo Hyperspectral Confocal Fluorescence Imaging to Determine Pigment Localization and Distribution in Cyanobacterial Cells,” Proceedings of National Academy of Sciences of USA, Vol. 105, No. 10, 2008, pp. 4050-4055. doi:10.1073/pnas.0708090105
[23]	T. F?rster, “Mechanisms of Energy Transfer,” In: M. Florkin E. H. Stotz, Eds., Comprehensive Biochemistry, Elsevier, Amsterdam, 1967, pp. 22, 61-80.
[24]	K. Sauer and H. Scheer, “Excitation Transfer in C-Phycocyanin. F?rster Transfer Rate and Excitation Calculations Based on a New Crystal Structure Data for C-Phycocyanin from Agmenellum Quadruplicatum and Mastigocladus Laminosus,” Biochiimica et Biophysica Acta, Vol. 936, No. 2, 1988, pp. 157-170. doi:10.1016/0005-2728(88)90232-0
[25]	Y. Tian and D. Pappas, “Energy Transfer and Light Tolerance Studies in a Fluorescent Tandem Phycobiliprotein Conjugate,” Applied Spectroscopy, Vol. 65, No. 9, 2011, pp. 991-995. doi:10.1366/11-06313