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Effects of PAR and UV Radiation on the Structural and Functional Integrity of Phycocyanin, Phycoerythrin and Allophycocyanin Isolated from the Marine Cyanobacterium Lyngbya sp. A09DM Rajesh Prasad Rastogi#*, Ravi Raghav Sonani# and Datta Madamwar* BRD School of Biosciences, Sardar Patel University, Anand, Gujarat, India Received 10 December 2014, accepted 4 March 2015, DOI: 10.1111/php.12449
ABSTRACT
DNA. However, intense photosynthetically active radiation (PAR) or UV-A radiation can induce DNA damage indirectly by the generation of singlet oxygen (1O2) or reactive oxygen species (ROS) via indirect photosensitizing reactions (11–14). UV-B radiation has direct effects on key cellular machinery such as proteins and DNA having the absorption maxima in the range of short wavelength UV radiation (15). Moreover, increased UV-B flux over the Earth’s atmosphere can induce a number of debilitating effects, including the pigment photo-oxidation and inactivation of photosystem II (PSII) in cyanobacteria (16–22). The phycobiliproteins (PBPs) such as phycocyanin (PC, kmax: 610–620 nm), phycoerythrin (PE, kmax: 540–570 nm) and allophycocyanin (APC, kmax: 650–655 nm) are major photosynthetic accessory pigments of cyanobacteria, which are assembled into supramolecular light-harvesting complexes, phycobilisomes (PBS), on the stromal surfaces of the thylakoid membranes. PBPs play an important role in electron/energy transfer chain in photosynthesis. UV-induced degradation of PBPs followed by a decline of photosynthetic activity can directly influence the primary productivity and community structure of the ecosystems. Furthermore, PBPs are being used as natural dyes in food, cosmetics and pharmaceutical industries as well as in different biomedical research (23,24). Nevertheless, stability of any compounds, is the utmost requirement for their commercial application, and a few studies have been conducted on these relevant biomolecules, PBPs. Past research mainly focused on the effects of UV-B on the photosynthetic apparatus and D1/D2 proteins of photosystem-II (PSII) (16,25,26). Hence, the main objective of the present study was to investigate the effects of intense PAR and UV radiation on the integrity of the ecologically and industrially important biomolecules PC, PE and APC. Furthermore, the information regarding the UV effects, particularly on all PBPs (i.e. PC, PE and APC) isolated from different or the same cyanobacteria are still limited (27–29) and poorly understood. In the present study, we have isolated and purified the PC, PE and APC from a marine cyanobacterium Lyngbya sp. A09DM and an in vitro study was performed to analyze the structural as well as functional stability of all PBPs and associated bilin chromophores under artificial intense PAR and extremely high energy of UV-A and UV-B radiation fluxes.
An in vitro analysis of the effects of photosynthetically active and ultraviolet radiations was executed to assess the photostability of biologically relevant pigments phycocyanin (PC), phycoerythrin (PE) and allophycocyanin (APC) isolated from Lyngbya sp. A09DM. Ultraviolet (UV) irradiances significantly affected the integrity of PC, PE and APC; however, PAR showed least effect. UV radiation affected the bilin chromophores covalently attached to phycobiliproteins (PBPs). Almost complete elimination of the chromophore bands associated with a- and β-subunit of PE and APC occurred after 4 h of UV-B exposure. After 5 h of UV-B exposure, the content of PC, PE and APC decreased by 51.65%, 96.8% and 96.53%, respectively. Contrary to PAR and UV-A radiation, a severe decrease in fluorescence of all PBPs was observed under UV-B irradiation. The fluorescence activity of extracted PBP was gradually inhibited immediately after 15–30 min of UV-B exposure. In comparison to the PC, the fluorescence properties of PE and APC were severely lost under UV-B radiation. Moreover, the present study indicates that UV-B radiation can damage the structural and functional integrity of phycobiliproteins leading to the loss of their ecological and biological functions.
INTRODUCTION Cyanobacteria are most ancient (1,2), ubiquitous (3) and one of the dominant microfloras in terms of total biomass and productivity in aquatic as well as terrestrial ecosystems. They are an immense source of several natural products of ecological and industrial significance (4). Moreover, during the past few decades, the increase in ultraviolet (UV: 280–400 nm) radiation (5– 7) due to anthropogenically released ozone-depleting substances has generated tremendous concern about its negative impact on the biota (8–10). The core requirement of solar energy to perform some essential metabolic functions such as N2-fixation and photosynthesis often exposes cyanobacteria to harmful UV-A (315–400 nm) and UV-B (280–315 nm) radiations in their natural habitats that are exposed to direct solar radiation. In comparison to UV-B radiation, UV-A radiation has a poor efficiency in inducing the cell damage, because it is not absorbed by native
MATERIALS AND METHODS
*Corresponding authors e-mails: [email protected] (Rajesh Prasad Rastogi), [email protected] (Datta Madamwar) #These authors have contributed equally to the paper. © 2015 The American Society of Photobiology
Experimental organism and growth conditions. The filamentous cyanobacterium Lyngbya sp. A09DM was originally isolated from rocky sea shores of Okha, Gujarat, India and identified by 16S rRNA gene
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sequence analysis (accession no. HM446280). The culture was maintained and routinely grown under axenic conditions in an autoclaved ASN III liquid medium (30) in a culture room at 25 2°C under 12/ 12 h dark/light illumination of 6 W m 2 provided by cool white fluorescent lamps. Extraction and purification of PBPs. The PBPs PC, PE and APC were extracted and purified from exponentially growing cells of Lyngbya sp. A09DM as described earlier (31). In brief, the cell-mass of cyanobacterium was lysed in an extraction buffer (20 mM potassium phosphate buffer, pH 7.2) by two successive cycles of freezing ( 25°C) and thawing (4°C). The extraction was followed by 20% ammonium sulfate fractionation to salt-out suspended impurities. The red supernatant obtained after 20% saturation was subjected to 40% ammonium sulfate saturation to precipitate PE. The resultant dark-blue supernatant was treated with 0.1% Triton X-100 (w/v) at room temperature to get a PC from solution as a precipitate. Remaining aqua blue supernatant was further saturated up to 70% ammonium sulfate to obtain APC as a precipitate. The precipitated PC, PE and APC were further purified by passing through manually packed gel permeation matrix (Sephadex G-150, Column dimensions: 500 mm 9 10 mm, bed height: 350 mm) with 20 mM potassium phosphate buffer (pH 7.2) as a mobile phase. Flow rate (40 mL h 1) was maintained using a peristaltic pump (Model P1, Pharmacia, Sweden). Elutes were collected in 1 mL fractions. The fractions of PC, PE and APC with a high purity ratio were desalted and concentrated by 30 kDa MWCO centrifugal devices (Macrosepâ, Pall Corporation). All centrifugations throughout the purification were carried out at 17 000 g and 4°C for 20 min. All buffers and solutions were prepared in autoclaved ultrapure water. Protein estimation and quantification of PBPs. Total protein contents were estimated by following the method of Lowry et al. (1951) (32). Bovine serum albumin (BSA) was used as the standard. PBPs contents were determined from UV-visible absorbance spectra by using Bennet and Bogorad (1973) equations (33). Source and mode of irradiation. The extracted and purified PBPs (2 mg mL 1 of each) were irradiated inside an incubator (Newtronic) manually fitted with cool white fluorescent, UV-A (TL-D, Philips, Holland) and UV-B (G15T8E, SankyoDenki, Japan) tubes as a source of PAR, UV-A and UV-B radiations, respectively, at a constant temperature of about 6 2°C. The irradiance of PAR and UV radiation was measured, using a digital Lux meter and a UVX-digital radiometer (UVP. Inc.) equipped with specific UV sensors, respectively. The irradiances effectively received by the proteins were 15 W m 2, 3.6 W m 2 and 1.5 W m 2 for the PAR, UV-A and UV-B, respectively. Time-course UV photo-stability experiment. The photostability of all PBPs (i.e. PC, PE and APC) and their associated bilin chromophores was investigated individually under different sets of experiments. They were exposed to PAR, UV-A and UV-B radiations continuously for 5 h. Subsequently, equal amounts (100 lL) of individual PBP were removed from each treatment after 15, 30, 60, 90, 120, 180, 240 and 300 min of continuous exposure and analyzed for photo-stability by means of SDS-PAGE, UV-visible (UV-Vis) and fluorescence spectroscopy. UV-Vis and fluorescence spectroscopic analysis. Absorption and fluorescence spectroscopy was performed using an UV-Vis (Analytik JenaAG Specord 210, Germany) and fluorescence (F-7000, Hitachi High Technologies, Japan) spectrophotometer, respectively. The samples were diluted
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uniformly within each experiment for spectral analysis. Dilution factor for single set of experiment was identical; however, it may vary between sets due to the dissimilarity in molar absorption coefficient factor (e) of PBPs. The purity ratio of PBPs at each step of purification was measured using the absorbance ratio at A615/A280, A564/A280, and A653/A280 for PC, PE and APC, respectively. The fluorescence of PC, PE and APC was measured at room temperature with an excitation wavelength of 589, 559 and 645 nm, respectively. The raw data were transferred to a microcomputer and both absorption and emission peaks were analyzed with the respective software provided by the manufacturer. SDS-PAGE analysis of PBPs and associated bilin chromophores. Denaturing gel electrophoresis of control as well as irradiated PBPs was carried out using a vertical slab gel apparatus (Mini Protean III, BioRad) as described earlier (31). Briefly, the concentration of polyacrylamide for the resolving and stacking gel was 15% and 4%, respectively. The PBP (5 lg of each) sample was mixed with gel loading dye (2% sodium dodecyl sulfate, 10% glycerol, 4.5% b-mercaptoethanol, 0.02% bromophenol blue dye and 50 mM Tris–Cl of pH 6.8). The mixture was incubated for 10 min in a boiling water bath. The gels were run using a tank buffer (25 mM Tris, 200 mM glycine, 0.1% SDS) at 25 2°C and 120 volt power supply. The apoproteins and bilin chromophores were visualized by silver (34) and zinc-acetate (35) staining, respectively. The standard protein marker ranging from 6 kDa to 43 kDa (Bangalore Genei, Karnataka, India) was used to determine the molecular mass. The gels were imaged and analyzed using an AlphaEase FC imaging system (Alpha Innotech Corp., San Leandro, CA).
RESULTS AND DISCUSSION Cyanobacteria are an immense source of several natural products, including dyes and pigments such as PC, PE and APC of high economic values. The present investigation shows that the studied cyanobacterium is able to synthesize large amounts of PBP pigments. SDS-PAGE (Fig. 1A) as well as UV-Vis absorption spectra (Fig. 1B) of crude extracts (a) and purified PC (b), PE (c) and APC (d) shows the success of purification process. The occurrence of two bands in each purified PBPs is corresponding to their a and b-subunits (Fig. 1A). Molecular weight analysis on the basis of migration on SDS-PAGE has revealed that the size of a and b-subunits were 17.5 and 19.0 kDa (for PC), 19.0 and 21.5 kDa (for PE), and 15.5 and 17.0 kDa (for APC) (Fig. 1A). UV-Vis spectra of purified PBPs showed a significant decrease in the absorption peak at 280 nm and increase in the peaks of respective PBPs, clearly indicating the removal of other protein impurities during the purification process (Fig. 1B). Purity and contents of PBPs at each step were probed via calculation of purity ratio, PBP content and total protein content as shown in Table 1. PC (kmax: 615 nm), PE (kmax: 563 nm) and APC (kmax: 652 nm) were successfully purified up to equitable purity ratios of 2.37, 6.71,
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Table 1. Summery of purification protocol in terms of protein content, purity ratio and percentage yield. (GPC=gel permeation chromatography).
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Figure 2. UV-visible absorption spectra of the purified PC (A, B, C), PE (D, E, F) and APC (G, H, I) exposed under PAR, UV-A and UV-B radiations. The down arrow (↓) denotes the absorption spectra of control and irradiated samples for 5 h from top to bottom, respectively (Control, 15, 30, 60, 90, 120, 180, 240 and 300 min).
and 2.48, respectively, which are comparable to previously reported values (Table 1) (36–38). PBPs are major light-harvesting pigments in cyanobacteria and other algae, used in maintaining the basic mode of energy metabolism by means of photosynthesis (39). Among cyanobacteria, extensive research shows that UV-B radiation has a number of harmful effects on cell physiology and biochemistry (18,21,40). In the present work, in vitro analysis of the effects of PAR and UV radiation was observed in the cyanobacterium Lyngbya sp. A09DM. The absorbance spectra of PC (Fig. 2A– C), PE (Fig. 2D–F) and APC (Fig. 2G–I) measured under different radiation conditions of PAR, UV-A and UV-B are shown in Fig. 2. The absorption spectra (Fig. 2) clearly show the dose-dependent decrease in absorbance of the PBPs PC (Fig. 2C), PE (Fig. 2F) and APC (Fig. 2I) under UV-B irradiation up to 5 h; however, no significant difference in absorption properties was observed under PAR and UV-A radiations.
Furthermore, in comparison to the PC, the PE and APC were more susceptible to UV-B radiation. Figure 3 shows the percentage of PBPs in the cyanobacterium Lyngbya sp. after 5 h of PAR and UV-A/B irradiation. The percentage decrease in PC content after 5 h of PAR, UV-A and UV-B was 11.89 2.4, 6.88 3.62 and 51.65 2.86, respectively. About 5 2.34, 6.23 1.51 and 96.8 1.1% decreases in PE was observed after 5 h of PAR, UV-A and UV-B irradiation, respectively. Similarly, the content of APC after 5 h of PAR, UV-A and UV-B irradiation was decreased by 14.53 2.89, 21.27 4.86 and 96.53 1.3%, respectively (Fig. 3). The elevated level of PAR has also been reported to reduce the level of PE, APC and PC by 69%, 75% and 93%, respectively, in the cyanobacterium Nostoc spongiaeforme (41). The light-harvesting complexes, including PBPs and chlorophyll are supposed to absorb more than 99% of incoming solar UVB in photosynthetic organisms (42). Moreover, UV-B radiation
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Figure 3. The content of PBPs PC, PE and APC after 5 h of PAR, UVA and UV-B exposure. The error bar represents standard deviation of mean (means SD, n = 3).
is known to cause effective damage to the photosynthetic apparatus and its components (19,27,28,43). The effects of PAR and UV radiation were also studied with great emphasis on the measurement of PBP monomers (PC, PE and APC) and their associated bilin cromophores. Figure 4 shows the electrophoretic pattern of PBPs PC, PE and APC (each of them composed of a and β monomers) after various durations of PAR and UV exposure. In comparison to PAR, UV-A and UV-B radiations exhibited more damaging effects which were dose-dependent. Moreover, UV-B radiation drastically influences the integrity of all PBP monomers. Almost every band of PC (Fig. 4A), PE (Fig. 4B) and APC (Fig. 4C) shows a
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gradual decrease in protein content with increasing UV exposure time. Contrary to PC and PE, APC was more susceptible to UVB exposure; 5 h of exposure resulted in almost complete elimination of the protein bands of a/β monomers (Fig. 4C). A rapid destruction in PBS isolated from the thylakoid membrane of Nostoc sp. was also observed under UV irradiation (27). Moreover, a linear decrease in the number of protein bands has been reported in several cyanobacteria under increased durations of UV-B exposure (16,19,28). PBPs are highly fluorescent by virtue of their covalently bound linear tetrapyrrole bilin chromophores. Analysis of these bilin chromophores can provide insight into the structural stability of PBP exposed to UV stress. The chromophore-containing PBPs PC, PE and APC were observed after increasing the fluorescence of phycobilin groups by soaking the gel in a 20 mM zinc acetate solution (Fig. 5). Similar to apoproteins, the bilin chromophores of each PBP monomer was also affected under UV exposure. Contrary to PAR and UV-A radiation, a dramatic decrease in phycobilin fluorescence of PC (Fig. 5A), PE (Fig. 5B) and APC (Fig. 5C) was observed following the increase in UV-B exposure time. Severely decreased fluorescence of PE (Fig. 5B) and APC (Fig. 5C) phycobilin groups were recorded after 4 h of UV-B exposure owing to either the presence of a very low level or complete degradation of bilin chromophores. Exposure of isolated cyanobacterial PBS to intense UV-B radiation has been shown to induce a decrease in PBP PC and/or APC (42,44,45). Similarly, UV-B irradiation showed photodestruction of both a and β-PC, but not of APC isolated from Synechococcus sp. PCC 7942 (46). Furthermore, our results revealed that when PBPs PC, PE and APC isolated from Lyngbya sp. A09DM were irradiated with UV-B, PE and APC were
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C PAR UV-A UV-B Figure 4. Electrophoretic pattern showing the effects of PAR, UV-A and UV-B radiations on PBPs PC (A), PE (B) and APC (C) with an increase in UV exposure time up to 5 h.
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Figure 5. Effects of PAR, UV-A and UV-B radiations on bilin chromophores of PC (A), PE (B) and APC (C) after 0 (C) trol sample.
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Figure 6. Effects of PAR and UV exposure time (0–5 h) on the fluorescence emission spectra of the PC (A), PE (B) and APC (C) isolated from Lyngbya sp. A09DM. The down arrow (↓) denotes the fluorescence emission spectra of control and irradiated samples for 5 h from top to bottom, respectively (Control, 15, 30, 60, 90, 120, 180, 240 and 300 min).
more affected than PC (Figs. 4 and 5), suggesting more complex mechanisms toward the structural integrity of individual PBPs isolated from different organisms under diverse environmental stress (42,47). Degradation of protein by UV-B exposure may occur either by direct photochemical reaction or indirectly by the UV-mediated ROS-induced oxidative stress. It has been supposed that the bilin chromophore is the main target of UV-B irradiation, causing structural changes of PBPs due to the fact that phycobilins can act as photosensitizers and produce ROS under UV or visible light by the reaction of the chromophore with atmospheric oxygen (11,28,48). In order to investigate further the functional properties of isolated PBPs exposed under PAR and UV radiation, fluorescence activity was monitored. The fluorescence spectra of the isolated PBPs PC, PE and APC after 0 (control) 5 h of PAR and UV exposure is illustrated in Fig. 6. Fluorescence emission was measured using the excitation wavelengths, 589 nm, 559 nm and 645 nm to monitor PC (peak at 641 nm) (Fig. 6A–C), PE (peak at 581 nm) (Fig. 6D–F) and APC (peak at 655 nm) (Fig. 6G–I), respectively. The decrease in the fluorescence measured for PC, PE and APC followed similar kinetics as were observed under UV-Vis (Fig. 2) and electrophoretic (Figs. 4 and 5) analysis. Maximum loss in fluorescence emission of all PBPs was observed under UV-B followed by UV-A and PAR exposure. Contrary to PC (Fig. 6A–C), the fluorescence properties of PE (Fig. 6D–F)/APC (Fig. 6G–I) were decreased significantly after 5 h UV-B irradiation. The decrease in PC, PE and APC fluorescence also indicated the structural changes in the PBPs induced by UV radiation. The effect of light on the fluorescence activity
of R/B-PE was also investigated, and similar patterns of fluorescence decrease were observed after 48 h of exposure (47). Moreover, decreased absorption and fluorescence emission of isolated PBPs under UV exposure might be due to changes in conformation of bilin chromophores within the native polypeptide chain (49). The photodestruction quantum yields for UV-B photons of purified PBPs and of DNA revealed that PBPs will be destroyed about 20 times faster than DNA bases (42). Furthermore, exact mechanisms of the changes in the structural integrity or destruction of isolated PBPs in response to UV exposure have not been fully clarified, and more extensive studies are needed to explore the mystery behind this interesting phenomenon at the molecular level. Overall, the data presented here clearly show that increased UV-B radiation has great efficacy for disturbing the structural as well as the functional integrity of purified PBPs by means of changes in intrinsic property of biliprotein and associated bilin chromophores. Acknowledgements—Rajesh P Rastogi is thankful to the University Grant Commission (UGC), New Delhi, India for Dr. D. S. Kothari Postdoctoral Research Grant. RRS thanks the DST, New Delhi for INSPIRE fellowship. We are also thankful to Dr. Jisha Elias, Home Science Department, Sardar Patel University, Gujarat, India for help during fluorescence analysis.
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Effects of PAR and UV Radiation on the Structural and Functional Integrity of Phycocyanin, Phycoerythrin and Allophycocyanin Isolated from the Marine Cyanobacterium Lyngbya sp. A09DM Rajesh Prasad Rastogi#*, Ravi Raghav Sonani# and Datta Madamwar* BRD School of Biosciences, Sardar Patel University, Anand, Gujarat, India Received 10 December 2014, accepted 4 March 2015, DOI: 10.1111/php.12449
ABSTRACT
DNA. However, intense photosynthetically active radiation (PAR) or UV-A radiation can induce DNA damage indirectly by the generation of singlet oxygen (1O2) or reactive oxygen species (ROS) via indirect photosensitizing reactions (11–14). UV-B radiation has direct effects on key cellular machinery such as proteins and DNA having the absorption maxima in the range of short wavelength UV radiation (15). Moreover, increased UV-B flux over the Earth’s atmosphere can induce a number of debilitating effects, including the pigment photo-oxidation and inactivation of photosystem II (PSII) in cyanobacteria (16–22). The phycobiliproteins (PBPs) such as phycocyanin (PC, kmax: 610–620 nm), phycoerythrin (PE, kmax: 540–570 nm) and allophycocyanin (APC, kmax: 650–655 nm) are major photosynthetic accessory pigments of cyanobacteria, which are assembled into supramolecular light-harvesting complexes, phycobilisomes (PBS), on the stromal surfaces of the thylakoid membranes. PBPs play an important role in electron/energy transfer chain in photosynthesis. UV-induced degradation of PBPs followed by a decline of photosynthetic activity can directly influence the primary productivity and community structure of the ecosystems. Furthermore, PBPs are being used as natural dyes in food, cosmetics and pharmaceutical industries as well as in different biomedical research (23,24). Nevertheless, stability of any compounds, is the utmost requirement for their commercial application, and a few studies have been conducted on these relevant biomolecules, PBPs. Past research mainly focused on the effects of UV-B on the photosynthetic apparatus and D1/D2 proteins of photosystem-II (PSII) (16,25,26). Hence, the main objective of the present study was to investigate the effects of intense PAR and UV radiation on the integrity of the ecologically and industrially important biomolecules PC, PE and APC. Furthermore, the information regarding the UV effects, particularly on all PBPs (i.e. PC, PE and APC) isolated from different or the same cyanobacteria are still limited (27–29) and poorly understood. In the present study, we have isolated and purified the PC, PE and APC from a marine cyanobacterium Lyngbya sp. A09DM and an in vitro study was performed to analyze the structural as well as functional stability of all PBPs and associated bilin chromophores under artificial intense PAR and extremely high energy of UV-A and UV-B radiation fluxes.
An in vitro analysis of the effects of photosynthetically active and ultraviolet radiations was executed to assess the photostability of biologically relevant pigments phycocyanin (PC), phycoerythrin (PE) and allophycocyanin (APC) isolated from Lyngbya sp. A09DM. Ultraviolet (UV) irradiances significantly affected the integrity of PC, PE and APC; however, PAR showed least effect. UV radiation affected the bilin chromophores covalently attached to phycobiliproteins (PBPs). Almost complete elimination of the chromophore bands associated with a- and β-subunit of PE and APC occurred after 4 h of UV-B exposure. After 5 h of UV-B exposure, the content of PC, PE and APC decreased by 51.65%, 96.8% and 96.53%, respectively. Contrary to PAR and UV-A radiation, a severe decrease in fluorescence of all PBPs was observed under UV-B irradiation. The fluorescence activity of extracted PBP was gradually inhibited immediately after 15–30 min of UV-B exposure. In comparison to the PC, the fluorescence properties of PE and APC were severely lost under UV-B radiation. Moreover, the present study indicates that UV-B radiation can damage the structural and functional integrity of phycobiliproteins leading to the loss of their ecological and biological functions.
INTRODUCTION Cyanobacteria are most ancient (1,2), ubiquitous (3) and one of the dominant microfloras in terms of total biomass and productivity in aquatic as well as terrestrial ecosystems. They are an immense source of several natural products of ecological and industrial significance (4). Moreover, during the past few decades, the increase in ultraviolet (UV: 280–400 nm) radiation (5– 7) due to anthropogenically released ozone-depleting substances has generated tremendous concern about its negative impact on the biota (8–10). The core requirement of solar energy to perform some essential metabolic functions such as N2-fixation and photosynthesis often exposes cyanobacteria to harmful UV-A (315–400 nm) and UV-B (280–315 nm) radiations in their natural habitats that are exposed to direct solar radiation. In comparison to UV-B radiation, UV-A radiation has a poor efficiency in inducing the cell damage, because it is not absorbed by native
MATERIALS AND METHODS
*Corresponding authors e-mails: [email protected] (Rajesh Prasad Rastogi), [email protected] (Datta Madamwar) #These authors have contributed equally to the paper. © 2015 The American Society of Photobiology
Experimental organism and growth conditions. The filamentous cyanobacterium Lyngbya sp. A09DM was originally isolated from rocky sea shores of Okha, Gujarat, India and identified by 16S rRNA gene
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sequence analysis (accession no. HM446280). The culture was maintained and routinely grown under axenic conditions in an autoclaved ASN III liquid medium (30) in a culture room at 25 2°C under 12/ 12 h dark/light illumination of 6 W m 2 provided by cool white fluorescent lamps. Extraction and purification of PBPs. The PBPs PC, PE and APC were extracted and purified from exponentially growing cells of Lyngbya sp. A09DM as described earlier (31). In brief, the cell-mass of cyanobacterium was lysed in an extraction buffer (20 mM potassium phosphate buffer, pH 7.2) by two successive cycles of freezing ( 25°C) and thawing (4°C). The extraction was followed by 20% ammonium sulfate fractionation to salt-out suspended impurities. The red supernatant obtained after 20% saturation was subjected to 40% ammonium sulfate saturation to precipitate PE. The resultant dark-blue supernatant was treated with 0.1% Triton X-100 (w/v) at room temperature to get a PC from solution as a precipitate. Remaining aqua blue supernatant was further saturated up to 70% ammonium sulfate to obtain APC as a precipitate. The precipitated PC, PE and APC were further purified by passing through manually packed gel permeation matrix (Sephadex G-150, Column dimensions: 500 mm 9 10 mm, bed height: 350 mm) with 20 mM potassium phosphate buffer (pH 7.2) as a mobile phase. Flow rate (40 mL h 1) was maintained using a peristaltic pump (Model P1, Pharmacia, Sweden). Elutes were collected in 1 mL fractions. The fractions of PC, PE and APC with a high purity ratio were desalted and concentrated by 30 kDa MWCO centrifugal devices (Macrosepâ, Pall Corporation). All centrifugations throughout the purification were carried out at 17 000 g and 4°C for 20 min. All buffers and solutions were prepared in autoclaved ultrapure water. Protein estimation and quantification of PBPs. Total protein contents were estimated by following the method of Lowry et al. (1951) (32). Bovine serum albumin (BSA) was used as the standard. PBPs contents were determined from UV-visible absorbance spectra by using Bennet and Bogorad (1973) equations (33). Source and mode of irradiation. The extracted and purified PBPs (2 mg mL 1 of each) were irradiated inside an incubator (Newtronic) manually fitted with cool white fluorescent, UV-A (TL-D, Philips, Holland) and UV-B (G15T8E, SankyoDenki, Japan) tubes as a source of PAR, UV-A and UV-B radiations, respectively, at a constant temperature of about 6 2°C. The irradiance of PAR and UV radiation was measured, using a digital Lux meter and a UVX-digital radiometer (UVP. Inc.) equipped with specific UV sensors, respectively. The irradiances effectively received by the proteins were 15 W m 2, 3.6 W m 2 and 1.5 W m 2 for the PAR, UV-A and UV-B, respectively. Time-course UV photo-stability experiment. The photostability of all PBPs (i.e. PC, PE and APC) and their associated bilin chromophores was investigated individually under different sets of experiments. They were exposed to PAR, UV-A and UV-B radiations continuously for 5 h. Subsequently, equal amounts (100 lL) of individual PBP were removed from each treatment after 15, 30, 60, 90, 120, 180, 240 and 300 min of continuous exposure and analyzed for photo-stability by means of SDS-PAGE, UV-visible (UV-Vis) and fluorescence spectroscopy. UV-Vis and fluorescence spectroscopic analysis. Absorption and fluorescence spectroscopy was performed using an UV-Vis (Analytik JenaAG Specord 210, Germany) and fluorescence (F-7000, Hitachi High Technologies, Japan) spectrophotometer, respectively. The samples were diluted
kDa A 43.0 S
M
PC
PE
APC
B 0.30
uniformly within each experiment for spectral analysis. Dilution factor for single set of experiment was identical; however, it may vary between sets due to the dissimilarity in molar absorption coefficient factor (e) of PBPs. The purity ratio of PBPs at each step of purification was measured using the absorbance ratio at A615/A280, A564/A280, and A653/A280 for PC, PE and APC, respectively. The fluorescence of PC, PE and APC was measured at room temperature with an excitation wavelength of 589, 559 and 645 nm, respectively. The raw data were transferred to a microcomputer and both absorption and emission peaks were analyzed with the respective software provided by the manufacturer. SDS-PAGE analysis of PBPs and associated bilin chromophores. Denaturing gel electrophoresis of control as well as irradiated PBPs was carried out using a vertical slab gel apparatus (Mini Protean III, BioRad) as described earlier (31). Briefly, the concentration of polyacrylamide for the resolving and stacking gel was 15% and 4%, respectively. The PBP (5 lg of each) sample was mixed with gel loading dye (2% sodium dodecyl sulfate, 10% glycerol, 4.5% b-mercaptoethanol, 0.02% bromophenol blue dye and 50 mM Tris–Cl of pH 6.8). The mixture was incubated for 10 min in a boiling water bath. The gels were run using a tank buffer (25 mM Tris, 200 mM glycine, 0.1% SDS) at 25 2°C and 120 volt power supply. The apoproteins and bilin chromophores were visualized by silver (34) and zinc-acetate (35) staining, respectively. The standard protein marker ranging from 6 kDa to 43 kDa (Bangalore Genei, Karnataka, India) was used to determine the molecular mass. The gels were imaged and analyzed using an AlphaEase FC imaging system (Alpha Innotech Corp., San Leandro, CA).
RESULTS AND DISCUSSION Cyanobacteria are an immense source of several natural products, including dyes and pigments such as PC, PE and APC of high economic values. The present investigation shows that the studied cyanobacterium is able to synthesize large amounts of PBP pigments. SDS-PAGE (Fig. 1A) as well as UV-Vis absorption spectra (Fig. 1B) of crude extracts (a) and purified PC (b), PE (c) and APC (d) shows the success of purification process. The occurrence of two bands in each purified PBPs is corresponding to their a and b-subunits (Fig. 1A). Molecular weight analysis on the basis of migration on SDS-PAGE has revealed that the size of a and b-subunits were 17.5 and 19.0 kDa (for PC), 19.0 and 21.5 kDa (for PE), and 15.5 and 17.0 kDa (for APC) (Fig. 1A). UV-Vis spectra of purified PBPs showed a significant decrease in the absorption peak at 280 nm and increase in the peaks of respective PBPs, clearly indicating the removal of other protein impurities during the purification process (Fig. 1B). Purity and contents of PBPs at each step were probed via calculation of purity ratio, PBP content and total protein content as shown in Table 1. PC (kmax: 615 nm), PE (kmax: 563 nm) and APC (kmax: 652 nm) were successfully purified up to equitable purity ratios of 2.37, 6.71,
PC
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Figure 1. Silver-stained (S) and zinc acetate-stained (Z) 15% SDS-PAGE of purified PC, PE and APC (A), and UV-Vis absorption spectrum of crude extract (a), purified PC (b), PE (c) and APC (d) from Lyngbya sp. A09DM (B).
Photochemistry and Photobiology
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Table 1. Summery of purification protocol in terms of protein content, purity ratio and percentage yield. (GPC=gel permeation chromatography).
A 0.5
Purity ratio Amax/A280
Yield (%)
Crude GPC elutes Crude GPC elutes Crude GPC elutes
52.55 12.5 52.55 7.24 52.55 5.84
16.16 12.27 11.94 6.40 6.55 4.71
0.57 6.74 0.21 2.37 0.09 2.48
100.00 75.93 100.00 53.60 100.00 71.91
B 0.6
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PE/PC/APC content (mg)
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Total protein content (mg)
Absorbance
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Purification
350 450 550 650 Wavelength [nm]
750
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0.08 0.04 0.00
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Figure 2. UV-visible absorption spectra of the purified PC (A, B, C), PE (D, E, F) and APC (G, H, I) exposed under PAR, UV-A and UV-B radiations. The down arrow (↓) denotes the absorption spectra of control and irradiated samples for 5 h from top to bottom, respectively (Control, 15, 30, 60, 90, 120, 180, 240 and 300 min).
and 2.48, respectively, which are comparable to previously reported values (Table 1) (36–38). PBPs are major light-harvesting pigments in cyanobacteria and other algae, used in maintaining the basic mode of energy metabolism by means of photosynthesis (39). Among cyanobacteria, extensive research shows that UV-B radiation has a number of harmful effects on cell physiology and biochemistry (18,21,40). In the present work, in vitro analysis of the effects of PAR and UV radiation was observed in the cyanobacterium Lyngbya sp. A09DM. The absorbance spectra of PC (Fig. 2A– C), PE (Fig. 2D–F) and APC (Fig. 2G–I) measured under different radiation conditions of PAR, UV-A and UV-B are shown in Fig. 2. The absorption spectra (Fig. 2) clearly show the dose-dependent decrease in absorbance of the PBPs PC (Fig. 2C), PE (Fig. 2F) and APC (Fig. 2I) under UV-B irradiation up to 5 h; however, no significant difference in absorption properties was observed under PAR and UV-A radiations.
Furthermore, in comparison to the PC, the PE and APC were more susceptible to UV-B radiation. Figure 3 shows the percentage of PBPs in the cyanobacterium Lyngbya sp. after 5 h of PAR and UV-A/B irradiation. The percentage decrease in PC content after 5 h of PAR, UV-A and UV-B was 11.89 2.4, 6.88 3.62 and 51.65 2.86, respectively. About 5 2.34, 6.23 1.51 and 96.8 1.1% decreases in PE was observed after 5 h of PAR, UV-A and UV-B irradiation, respectively. Similarly, the content of APC after 5 h of PAR, UV-A and UV-B irradiation was decreased by 14.53 2.89, 21.27 4.86 and 96.53 1.3%, respectively (Fig. 3). The elevated level of PAR has also been reported to reduce the level of PE, APC and PC by 69%, 75% and 93%, respectively, in the cyanobacterium Nostoc spongiaeforme (41). The light-harvesting complexes, including PBPs and chlorophyll are supposed to absorb more than 99% of incoming solar UVB in photosynthetic organisms (42). Moreover, UV-B radiation
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C
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20 0
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Figure 3. The content of PBPs PC, PE and APC after 5 h of PAR, UVA and UV-B exposure. The error bar represents standard deviation of mean (means SD, n = 3).
is known to cause effective damage to the photosynthetic apparatus and its components (19,27,28,43). The effects of PAR and UV radiation were also studied with great emphasis on the measurement of PBP monomers (PC, PE and APC) and their associated bilin cromophores. Figure 4 shows the electrophoretic pattern of PBPs PC, PE and APC (each of them composed of a and β monomers) after various durations of PAR and UV exposure. In comparison to PAR, UV-A and UV-B radiations exhibited more damaging effects which were dose-dependent. Moreover, UV-B radiation drastically influences the integrity of all PBP monomers. Almost every band of PC (Fig. 4A), PE (Fig. 4B) and APC (Fig. 4C) shows a
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gradual decrease in protein content with increasing UV exposure time. Contrary to PC and PE, APC was more susceptible to UVB exposure; 5 h of exposure resulted in almost complete elimination of the protein bands of a/β monomers (Fig. 4C). A rapid destruction in PBS isolated from the thylakoid membrane of Nostoc sp. was also observed under UV irradiation (27). Moreover, a linear decrease in the number of protein bands has been reported in several cyanobacteria under increased durations of UV-B exposure (16,19,28). PBPs are highly fluorescent by virtue of their covalently bound linear tetrapyrrole bilin chromophores. Analysis of these bilin chromophores can provide insight into the structural stability of PBP exposed to UV stress. The chromophore-containing PBPs PC, PE and APC were observed after increasing the fluorescence of phycobilin groups by soaking the gel in a 20 mM zinc acetate solution (Fig. 5). Similar to apoproteins, the bilin chromophores of each PBP monomer was also affected under UV exposure. Contrary to PAR and UV-A radiation, a dramatic decrease in phycobilin fluorescence of PC (Fig. 5A), PE (Fig. 5B) and APC (Fig. 5C) was observed following the increase in UV-B exposure time. Severely decreased fluorescence of PE (Fig. 5B) and APC (Fig. 5C) phycobilin groups were recorded after 4 h of UV-B exposure owing to either the presence of a very low level or complete degradation of bilin chromophores. Exposure of isolated cyanobacterial PBS to intense UV-B radiation has been shown to induce a decrease in PBP PC and/or APC (42,44,45). Similarly, UV-B irradiation showed photodestruction of both a and β-PC, but not of APC isolated from Synechococcus sp. PCC 7942 (46). Furthermore, our results revealed that when PBPs PC, PE and APC isolated from Lyngbya sp. A09DM were irradiated with UV-B, PE and APC were
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C PAR UV-A UV-B Figure 4. Electrophoretic pattern showing the effects of PAR, UV-A and UV-B radiations on PBPs PC (A), PE (B) and APC (C) with an increase in UV exposure time up to 5 h.
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Figure 5. Effects of PAR, UV-A and UV-B radiations on bilin chromophores of PC (A), PE (B) and APC (C) after 0 (C) trol sample.
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Rajesh Prasad Rastogi et al.
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Figure 6. Effects of PAR and UV exposure time (0–5 h) on the fluorescence emission spectra of the PC (A), PE (B) and APC (C) isolated from Lyngbya sp. A09DM. The down arrow (↓) denotes the fluorescence emission spectra of control and irradiated samples for 5 h from top to bottom, respectively (Control, 15, 30, 60, 90, 120, 180, 240 and 300 min).
more affected than PC (Figs. 4 and 5), suggesting more complex mechanisms toward the structural integrity of individual PBPs isolated from different organisms under diverse environmental stress (42,47). Degradation of protein by UV-B exposure may occur either by direct photochemical reaction or indirectly by the UV-mediated ROS-induced oxidative stress. It has been supposed that the bilin chromophore is the main target of UV-B irradiation, causing structural changes of PBPs due to the fact that phycobilins can act as photosensitizers and produce ROS under UV or visible light by the reaction of the chromophore with atmospheric oxygen (11,28,48). In order to investigate further the functional properties of isolated PBPs exposed under PAR and UV radiation, fluorescence activity was monitored. The fluorescence spectra of the isolated PBPs PC, PE and APC after 0 (control) 5 h of PAR and UV exposure is illustrated in Fig. 6. Fluorescence emission was measured using the excitation wavelengths, 589 nm, 559 nm and 645 nm to monitor PC (peak at 641 nm) (Fig. 6A–C), PE (peak at 581 nm) (Fig. 6D–F) and APC (peak at 655 nm) (Fig. 6G–I), respectively. The decrease in the fluorescence measured for PC, PE and APC followed similar kinetics as were observed under UV-Vis (Fig. 2) and electrophoretic (Figs. 4 and 5) analysis. Maximum loss in fluorescence emission of all PBPs was observed under UV-B followed by UV-A and PAR exposure. Contrary to PC (Fig. 6A–C), the fluorescence properties of PE (Fig. 6D–F)/APC (Fig. 6G–I) were decreased significantly after 5 h UV-B irradiation. The decrease in PC, PE and APC fluorescence also indicated the structural changes in the PBPs induced by UV radiation. The effect of light on the fluorescence activity
of R/B-PE was also investigated, and similar patterns of fluorescence decrease were observed after 48 h of exposure (47). Moreover, decreased absorption and fluorescence emission of isolated PBPs under UV exposure might be due to changes in conformation of bilin chromophores within the native polypeptide chain (49). The photodestruction quantum yields for UV-B photons of purified PBPs and of DNA revealed that PBPs will be destroyed about 20 times faster than DNA bases (42). Furthermore, exact mechanisms of the changes in the structural integrity or destruction of isolated PBPs in response to UV exposure have not been fully clarified, and more extensive studies are needed to explore the mystery behind this interesting phenomenon at the molecular level. Overall, the data presented here clearly show that increased UV-B radiation has great efficacy for disturbing the structural as well as the functional integrity of purified PBPs by means of changes in intrinsic property of biliprotein and associated bilin chromophores. Acknowledgements—Rajesh P Rastogi is thankful to the University Grant Commission (UGC), New Delhi, India for Dr. D. S. Kothari Postdoctoral Research Grant. RRS thanks the DST, New Delhi for INSPIRE fellowship. We are also thankful to Dr. Jisha Elias, Home Science Department, Sardar Patel University, Gujarat, India for help during fluorescence analysis.
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