J. Phycol. 46, 285289 (2010) 2010 Phycological Society of America DOI: 10.1111/j.1529-8817.2009.00798.

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ALLOPHYCOCYANIN FROM A LOCAL ISOLATE GEITLERINEMA SP. A28DM (CYANOBACTERIA): A SIMPLE AND EFFICIENT PURIFICATION PROCESS 1
Asha Parmar, Niraj Kumar Singh, and Datta Madamwar2
BRD School of Biosciences, Sardar Patel Maidan, Vadtal Road, Satellite Campus, Post Box No. 39, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India

Allophycocyanin (APC) is the least-studied cyanobacterial bile-pigment invariably present within the phycobilisome core of cyanobacteria. In the present study, we describe a simple, cost-effective, and reproducible method for the purication of APC from a local isolate, Geitlerinema sp. A28DM. The pigment was extracted from the algal biomass and precipitated with 0.25% aqueous solution of the highly aromatic cationic dye ethodin. The precipitated APC was then subjected to a single size-exclusion chromatographic step using Sephadex G-100. Pure cyanobacterial APC (C-APC) (A652 A280 of 3.2) was obtained and characterized by its absorption spectrum with maximum at 652 nm and a shoulder at 620 nm, and by SDS-PAGE, showing two bands with molecular masses of 15 and 17.5 kDa, corresponding to a and b subunits of the biliprotein. The nal yield of C-APC was 66% from its content in the crude extract. The procedure appears to be promising for wider applications and larger production of APC. Key index words: allophycocyanin; ethodin; Geitlerinema; gel permeation chromatography; purication Abbreviations: APC, allophycocyanin; ASN, articial salt nutrient; C-APC, cyanobacterial allophycocyanin; C-PC, cyanobacterial phycocyanin; IEF, isoelectric focusing; IPG, immobilized pH gradient; PC, phycocyanin; PE, phycoerythrin

Phycobiliproteins are homologous chromoproteins that constitute the phycobilisomes, the light-harvesting complexes of the photosynthetic apparatus in the red (Rhodophyta), cyanobacterial (Cyanophyta), and cryptomonad (Cryptophyta) algae (Bogorad 1975, Brown et al. 1975, Simo et al. 2005). Puried phycobiliproteins have a wide range of applications, such as colorants in food and cosmetics, and as labels in different uorescence techniques (Roman et al. 2002). Phycobiliproteins also
1 2

Received 3 February 2009. Accepted 12 October 2009. Address for correspondence: e-mail datta_madamwar@yahoo.com.

have important medical and pharmacological properties (Romay et al. 1998). Recent studies have shown that they also have hepatoprotective (Vadiraja et al. 1998), anti-inammatory (Romay et al. 1998, Gonza lez et al. 1999, Reddy et al. 2000), and antioxidant properties (Bhat and Madyastha 2000, Zhang et al. 2000, Soni et al. 2008). Phycobiliproteins are assignable to three special subclasses: allophycocyanin (APC; kmax: 650655 nm), phycocyanin (PC; kmax: 610620 nm), and phycoerythrin (PE; kmax: 540570 nm), of which APC is invariably present within the core (Gantt et al. 1976, 1977, Koller et al. 1978, Bryant et al. 1979). APC is the most efcient biliprotein for energy transfer (Lemasson et al. 1973, Gantt et al. 1976, Gysi and Zuber 1978) and is the key pigment in funneling excitation energy from the other biliproteins into the chl of the PSII. Despite its obvious importance, APC still remains the least-studied phycobiliprotein. This may be largely because of its miniscule quantity, as APC accounts on a weight basis for 10% or less of the total cellular phycobiliproteins. Hence, absorption maxima in whole cells or crude extract is largely masked by the much greater absorbancy of PC and of chl holochromes in this region (Gysi and Zuber 1978). Various methods have been reported for the purication of C-APC, but all of these involve a large number of chromatographic steps (Troxler et al. 1980, Zilinskas 1982, Gombos et al. 1983) usually in combination with ammonium sulfate precipitation (Brown et al. 1975, Gysi and Zuber 1976). Apart from being time consuming, ammonium sulfate precipitation may hinder the future crystallization process because of the difculty of complete removal of salt by dialysis or even by desalting column (Singh et al. 2009). As general information, even a single increase in chromatography step can reduce 20% of the protein of interest (Soni et al. 2008). We, therefore, looked to develop a method that would be simple, efcient, and easy to perform without compromising the purity and yield. Here, we used ethodin (6,9-diamino-2-ethoxyacridine lactate monohydrate) as an attractive alternative for the precipitation of APC. Ethodin is a strongly yellow-colored, highly aromatic cationic dye that complexes with negatively charged
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proteins. Ethodin has been shown to have certain pharmacological applications and is therefore unlikely to be toxic (Rudolph et al. 1997). There are several reports showing the utility of ethodin as a precipitant for protein purication (Miller 1959, Berns 1967, Neurath and Brunner 1969, Minkova et al. 2003, 2006, Persson and Lester 2004). Gel permeation chromatography has been widely successful in protein purication and has the advantage of not being dependent on the net charge of the protein concerned. To the best of our knowledge, the present study describes for the rst time the purication of APC from Geitlerinema sp. A28DM along with a simple, inexpensive, and efcient procedure based on a size-exclusion chromatographic step after treatment with ethodin. Chemicals. Sephadex G-100 powdered matrix (bead diameter 20300 lm and fractionation range 4150 kDa) was purchased from GE Healthcare UK Limited (Little Chalfont, Buckinghamshire, UK); ethodin, from Fluka Chemei GmbH (Industriestrasse 25, Buchs SG, Switzerland); protein molecular mass standard, from Bangalore Genei (Bangalore, Karnataka, India); bis-acrylamide, from Himedia (Mumbai, Maharashtra, India); sodium dodecyl sulfate and electrophoresis-grade acrylamide, from Merck (Darmstadt, Hesse, Germany); and IPG strip, from BioRad (Hercules, CA, USA). All other chemicals were ultrapure or molecular biology grade and were used without further purication. Organism. Isolation and growth conditions: The enrichment, isolation, and purication of the culture were carried out according to Shah et al. (2001). Twenty-ve cyanobacterial cultures were isolated from the sandy shores parallel to the coast indented by the estuarine mouth of the river Tapi, Gujarat, India. They were cultivated in ASN III medium (Waterbury and Stanier 1981) with 12:12 light:dark (L:D) cycles at 27 2C and illumination of 36W white uorescent lamps at a ux density of 130 lmol photons m)2 s)1 measured at the surface of the ask. Screening of the cultures was done, and the cyanobacterium having maximum CAPC-producing ability was selected. Identication by 16S-rRNA gene sequencing showed that the isolated strain was a Geitlerinema sp.; hence, in the present study, it is referred as Geitlerinema sp. A28DM (accession number FJ410907). Extraction and purication of APC. One gram of 20 d grown wet algal biomass was washed with 10 mM potassium phosphate buffer (pH 6.7) containing 150 mM NaCl and 3 mM sodium azide. It was resuspended in 55 mL of the phosphate buffer. The suspension was frozen at )30C overnight, thawed at 4C for 2 h, incubated at 32C for 90 min under continuous shaking conditions, left at 4C overnight, and then centrifuged (Sigma 3K 18; Sigma Laborzentrifugen GmBH, Osterode am Harz, Germany) at 17,000g for 40 min. The supernatant

was termed as crude extract. All proceeding centrifugations were carried out at 10,000g for 40 min. (a) Treatment with ethodin: Aqueous ethodin solution (0.25%) was added to the crude extract in a ratio of 1:10. The resultant solution after mixing was left at 4C overnight and then centrifuged. The pellet was resuspended in the phosphate buffer and allowed to stand overnight at 4C without disturbance, followed by centrifugation. The pellet was discarded, and the supernatant termed as ethodintreated extract was subjected to size-exclusion chromatography column. (b) Size-exclusion chromatography: Ethodin-treated extract after ltration (Whatman No. 1, Whatman International Ltd., Maidstone, UK) was applied on a Sephadex G-100 column (150 cm 1.5 cm, bed height 105 cm), preequilibrated, and eluted with the phosphate buffer. The ow rate was maintained at 60 mL h)1 using a peristaltic pump (Model P1; Pharmacia, Uppsala, Sweden). Aqua-blue-colored eluate was collected as 1 mL fractions. Experimental estimations and analyses. The cell dry weight was determined according to Boussiba and Richmond (1980). The protein contents were determined by the method of Lowry et al. (1951), with BSA as the standard. Phycobiliprotein content was estimated using the equations of Siegelman and Kycia (1978). The absorption overlay spectra of C-APC from Geitlerinema sp. A28DM at each step of purication was recorded on a UVvisible spectrophotometer (Specord 210, Analytik Jena AG, Jena, Germany). Nondenaturing and SDS-PAGE was carried out as described before (Singh et al. 2009) using 10% and 17% polyacrylamide slab gels, and the same were visualized by silver staining according to Garn (1990). For calibration, marker proteins from Bangalore Genei were used. Zinc-acetate staining to detect the bilin chromophore was done according to Brekelman and Lagarias (1986), and the bilin uorescence was observed under UV light using AlphaEase FC Imaging System (Alpha Innotech Corp., San Leandro, CA, USA). Results reported are the average of three independent experiments, and standard deviation was estimated to be < 5%. Results of extraction and purication of APC. The absorption spectrum of the crude extract of Geitlerinema sp. A28DM (Fig. 1) showed the presence of a cyanobacterial PC (C-PC) peak and a shoulder peak at 652 nm indicating the presence of C-APC (MacColl 2004). The data of C-APC purication from Geitlerinema sp. A28DM are summarized in Table 1. The simplied procedure resulted in pure C-APC (A652 A280 of 3.2), and the product content reached up to 96% from the total proteins. The nal yield of C-APC was 66%, which is signicantly higher than the other reported values (Minkova et al. 2006). The total protein content of the crude extract was 28.2 mg, including that of C-APC, 2.3 mg.

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Fig. 1. UVvisible absorption overlay spectra of C-APC from Geitlerinema sp. A28DM at each step of purication: crude extract, ethodin-treated extract, and gel permeation chromatography eluate. C-APC, cyanobacterial allophycocyanin.

Phycobiliprotein content (C-APC) at each step is shown in Table 1. Characterization of APC. The success of C-APC purication protocol was checked at each step by UVvisible spectroscopy and gel electrophoresis. The purity of C-APC at each step of purication was recorded in terms of purity ratio (A652 A280). The increase in the purity ratio at each step was veried by the overlay spectra (Fig. 1). Nondenaturing gel electrophoresis of puried C-APC showed only one band (Fig. 2a), suggesting the homogeneity of the puried protein. The purity of the isolated C-APC was also veried by SDS-PAGE where only its a and b subunits (Fig. 2b), having molecular masses of 15 and 17.5 kDa, respectively, are visible. The zincassisted uorescence enhancement of the SDS-PAGE conrmed that both the subunits were bilin-linked polypeptides. There were no bilin-linked peptides present in standard molecular mass marker, suggesting the specicity of bili-protein detection by this method. At the nal stage of purication, only one uorescence band was observed in the native-PAGE (Fig. 3a), proving the integrity of the puried C-APC, and two uorescence bands were seen in SDS-PAGE, corresponding to its a and b subunits (Fig. 3b). The isoelectric point (pI) of the puried protein in the nondenaturing conditions was determined according to Soni et al. (2006) using a precasted 310 pH linear gradient, 17 cm ready-made

immobilized pH gradient (IPG) strip in protean isoelectric-focusing (IEF) cell (BioRad). The pI was detected to be 4.7, which is in the range of other phycobiliproteins reported (Glazer 1993). Discussion. Generally described methods for the purication of APC are the combination of chromatographic steps along with ammonium sulfate precipitation. The major limitations of these methods are the nonscalability and the length of time required to complete the process. Hence, we used ethodin as an alternative precipitating agent. The ethodin molecule is positively charged over a wider pH range (Persson and Lester 2004). However, changing the pH will change the charge on the proteins. Hence, it is likely that the pH of the buffer could have a major effect on the purication of CAPC upon precipitation with ethodin. Our strategy for purication herein was based on the fact that there is correlation between the pI of the protein and the pH range at which the protein precipitates. The pI of C-APC was found to be 4.7, and the pH of the buffer used was 6.7. Since the protein becomes negatively charged at pH above pI, C-APC was precipitated after treatment with positively charged ethodin. As ethodin precipitates partly due to the charged characteristics of the molecule, it seems probable that the conductivity of the sample might have an effect on the purity. Hence, addition of a low concentration of NaCl (150 mM) in the buffer considerably improved the effective interaction of ethodin (Persson and Lester 2004) with the crude extract components, leading to a signicant purication of C-APC. The concentration of ethodin plays a major role in protein purication (Persson and Lester 2004). We exploited this property in C-APC purication. Every slight addition of ethodin in the crude extract resulted in increased purication. This effect was noted until the concentration was increased up to 0.25%. However, further increase in ethodin concentration did not result in additional purication. Hence, the concentration of ethodin used in the study was kept at 0.25%. Most of the methods for separation and purication of proteins are based on surface features of proteins, net charge, bioproperties (afnity), or the whole structure (shape and size). Since the methods exploiting surface features depend on solubility properties, such methods were not chosen for the separation of C-APC and C-PC, because both pig-

Table 1. Data of purication of cyanobacterial allophycocyanin (C-APC) from Geitlerinema sp. A28DM.
Volume (mL) Total protein content (mg) Total C-APC content (mg) C-APC from total protein (%) Impurities (%) Yield of C-APC (%)

Purication step

A652 A280

Crude extract Ethodin-treated crude Gel permeation chromatography

55 20 30

28.16 4.02 1.59

2.31 1.94 1.53

8.2 48.2 96.2

91.8 51.8 3.8

0.22 0.83 3.2

100 84 66

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Fig. 2. (a) Silver-stained 10% native gel electrophoresis of pure C-APC from Geitlerinema sp. A28DM. Presence of only one band indicated the homogeneity of C-APC. (b) Silver-stained 17% SDS gel electrophoresis at each stage of purication of C-APC from Geitlerinema sp. A28DM. Lanes: 1, protein molecular mass standard; 2, crude extract; 3, ethodin-treated extract; 4, pure allophycocyanin eluate from gel permeation chromatography. Pure allophycocyanin showed two bands of 15 and 17.5 kDa molecular masses corresponding to characteristic a and b subunits, respectively. Protein concentration was estimated by Lowry assay (Lowry et al. 1951) against BSA as standard, and 10 lg of protein was loaded in each lane. C-APC, cyanobacterial allophycocyanin.

puried C-APC in just one step of gel permeation chromatography. It has an added advantage of not damaging the function of the protein, as there is no binding of proteins to the chromatographic support; hence, the loss of protein is minimal. The nal yield of C-APC was 66% from its content in the crude extract with the purity of 3.2. This is comparatively higher than the other reported values (Minkova et al. 2006). The puried C-APC was characterized in terms of homogeneity and molecular masses of the subunits. The values of a and b subunits with molecular masses of 15 and 17.5 kDa, respectively (Fig. 2b), are in agreement with the previously reported values estimated by SDS-PAGE (Brown et al. 1975, Troxler et al. 1980, Zilinskas 1982, MacColl 1998). Compared to other methods, our method offers the advantage of avoiding lengthy and laborious procedures like ammonium sulfate precipitation, dialysis, and other chromatographic processes. In conclusion, this paper presents a signicant, costeffective, and reproducible method for the purication of C-APC from Geitlerinema sp. A28DM with comparatively higher yield. Hence, the protocol could be suitable for wider applicability to other cyanobacteria and for its larger production. Moreover, it allows us to obtain pure pigment for a broad range of applications.
The authors acknowledge the nancial support provided by the Department of Biotechnology, Government of India (India), and thank Dr. Kaledona Minkova (Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, Soa 1113, Bulgaria) for her unconditional help throughout the purication work, as well as Varun Shah, BRD School of Biosciences, Sardar Patel University (India) for discussion.
Berns, D. S. 1967. Immunochemistry of biliproteins. Plant Physiol. 42:10605. Bhat, V. B. & Madyastha, K. M. 2000. C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro. Biochem. Biophys. Res. Commun. 275:205. Bogorad, L. 1975. Phycobiliproteins and complementary chromatic adaptation. Annu. Rev. Plant Physiol. 26:369401. Boussiba, S. & Richmond, A. E. 1980. C-phycocyanin as a storage protein in the blue-green alga Spirulina platensis. Arch. Microbiol. 125:1437. Brekelman, T. R. & Lagarias, J. C. 1986. Visualization of bilin-linked peptides and proteins in polyacrylamide gels. Anal. Biochem. 156:194201. Brown, A. S., Foster, J. A., Voynow, P. V., Franzblau, C. & Troxler, R. F. 1975. Allophycocyanin from the lamentous cyanophyte, Phormidium luridium. Biochemistry 14:35818. Bryant, D. A., Guglielmi, G., Tandeau de Marsac, N., Castets, A. M. & Cohen-Bazire, G. 1979. The structure of cyanobacterial phycobilisomes. A model. Arch. Microbiol. 123:11327. Gantt, E., Lipschultz, C. A. & Zilinskas, B. A. 1976. Further evidence for a phycobilisome model from selective dissociation, uorescence emission, immunoprecipiation and electron microscopy. Biochem. Biophys. Acta 430:37588. Gantt, E., Lipschultz, C. A. & Zilinskas, B. A. 1977. Phycobilisomes in relation to the thylakoid membranes. Brookhaven Symp. Biol. 28:34757. Garn, D. 1990. One dimensional gel electrophoresis. In Deutscher, M. P., Abelson, J. N. & Simon, M. I. [Eds.] Guide to

Fig. 3. (a) Detection of biliprotein of Geitlerinema sp. A28DM by zinc-assisted uorescence enhancement method as observed under UV light. The 10% native-PAGE of puried C-APC showed only one band, suggesting the homogeneity and integrity of subunits. (b) Detection of biliproteins of Geitlerinema sp. A28DM by zinc-assisted uorescence enhancement method at each step of purication as observed under UV light. SDS electrophoresis was run in 17% gel. Lanes: 1, protein molecular mass marker; 2, crude extract; 3, ethodin-treated extract; 4, pure C-APC eluate from gel permeation chromatography. There were no biliproteins present in molecular mass marker, and so no uorescence observed indicated the specicity of the method for biliprotein detection. The protein loaded was 5 lg in each lane. C-APC, cyanobacterial allophycocyanin.

ment proteins are hydrophilic in nature. Methods based on net charge were eliminated, as both proteins are negatively charged. Exploitation of bioproperties can be a powerful method for separating the desired protein from others. But since a particular biological property of C-APC that differs from C-PC has not been studied by us, we exploited the property based on the whole structure. We, thus,

AL L O P H Y C O C Y A N I N FR O M G E I T L ER I N E M A S P . A 2 8 D M Protein Purication. Academic Press, San Diego, California, pp. 42541. Glazer, A. 1993. Phycobiliproteins a family of valuable, widely used uorophores. J. Appl. Phycol. 6:10512. Gombos, Z., Csizmadia, V. & Csatorday, K. 1983. Two simple procedures for isolation of allophycocyanin II from Anacystis nidulans. Anal. Biochem. 136:4912. Gonza lez, R., Rodriguez, S., Romay, C., Ancheta, O., Gonza lez, A., Armesto, J., Remirez, D. & Merino, N. 1999. Anti-inammatory activity of phycocyanin extract in acetic acid-induced colitis in rats. Pharmacol. Res. 39:559. Gysi, J. & Zuber, H. 1976. Allophycocyanin I-a second cyanobacterial allophycocyanin? Isolation, characterization and comparison with allophycocyanin II from the same alga. FEBS Lett. 68:4954. Gysi, J. & Zuber, H. 1978. Properties of allophycocyanin II and its aand b-subunits from the thermophillic blue-green alga Mastigocladus laminosus. J. Biochem. 181:57783. Koller, K. P., Wehrmeyer, W. & Morschel, E. 1978. Biliprotein assembly in the disc-shaped phycobilisomes of Rhodella violacea. On the molecular composition of energy transferring complexes (tripartite units) forming the periphery of phycobilisome. Eur. J. Biochem. 91:5763. Lemasson, C., Tandeau de Marsac, N. & Cohen-Bazire, G. 1973. Role of allophycocyanin as light-harvesting pigment in cyanobacteria. Proc. Natl. Acad. Sci. U. S. A. 70:31303. Lowry, H. O., Rosenbrough, N. J., Farr, A. L. & Randell, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:26575. MacColl, R. 1998. Cyanobacterial phycobilisomes. J. Struct. Biol. 124:31134. MacColl, R. 2004. Allophycocyanin and energy transfer. Biochim. Biophys. Acta 1657:7381. Miller, K. D. 1959. Rivanol, resin and isolation of thrombins. Nature 184:450. Minkova, K. M., Tchernov, A. A., Tchorbadjieva, M. I., Antova, R. E. & Busheva, M. Ch. 2003. Purication of C-phycocyanin from Spirulina (Arthrospira) fusiformis. J. Biotechnol. 102:559. Minkova, K. M., Tchorbadjieva, M., Tchernov, A., Stojanova, M., Gigova, L. & Busheva, M. 2006. Improved procedure for separation and purication of Arthrospira africanum phycobiliproteins. Biotechnol. Lett. 29:64751. Neurath, A. R. & Brunner, R. 1969. Fraction of proteins with different isoelectric point by Rivanol. Experimentia 25:668. Persson, J. & Lester, P. 2004. Purication of antibody and antibodyfragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipitation agent. Biotechnol. Bioeng. 87:42434. Reddy, C. M., Vadiraja, B. B., Kiranmai, G., Reddy, M. N., Reddanna, P. & Madyastha, K. M. 2000. Selective inhibition of cyclo-oxygenase 2 by C-phycocyanin, a biliprotein from Spirulina platensis. Biochem. Biophys. Res. Commun. 277:599603. Roman, R. B., Alvarez-Pez, J. M., Fernandez, F. G. A. & Grima, E. M. 2002. Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum. J. Biotechnol. 93:7385.

289

Romay, C., Armesto, J., Remirez, D., Gonza lez, R., Ledon, N. & Gracia, I. 1998. Antioxidant and anti-inammatory properties of C-phycocyanin from blue-green algae. Inamm. Res. 47:36 41. Rudolph, M. I., Garcia, M. A., Sepulveda, M., Brandan, E., Reinicke, K., Nicovani, S. & Villan, L. 1997. Ethodin: pharmacological evidence of the interaction between smooth muscle and mast cells in the myometrium. J. Pharmacol. Exp. Ther. 282:25661. Shah, V., Garg, N. & Madamwar, D. 2001. Record of the marine cyanobacterium from the rocky shores of Bet-Dwarka and Okha, India. Acta. Bot. Malacit. 26:18893. Siegelman, H. W. & Kycia, J. H. 1978. Algal biliproteins. In Hellebust, J. A. & Craigie, J. S. [Eds.] Handbook of Phycological Methods, Physiological and Biochemical Methods. Cambridge University Press, Cambridge, UK, pp. 719. Simo , C., Herrero, M., Neusu , C., Pelzing, M., Kenndler, E., Barbas, C., Iba n ez, E. & Cifuentes, A. 2005. Characterization of proteins from Spirulina platensis microalga using capillary electrophoresis-ion-trap-mass spectrometry and capillary electrophoresis-time of ight-mass spectrometry. Electrophoresis 26:267483. Singh, N. K., Parmar, A. & Madamwar, D. 2009. Optimization of medium components for increased production of C-phycocyanin from Phormidium ceylanicum and its purication by single step process. Bioresour. Technol. 100:16639. Soni, B., Kalavadia, B., Trivedi, U. & Madamwar, D. 2006. Extraction, purication and characterization of phycocyanin from Oscillatoria quadripunctulataisolated from the rocky shores of Bet-Dwarka, Gujarat, India. Process Biochem. 41:2017 23. Soni, B., Trivedi, U. & Madamwar, D. 2008. A novel method of single step hydrophobic interaction chromatography for the purication of phycocyanin from Phormidium fragile and its characterization for antioxidant property. Bioresour. Technol. 99:18894. Troxler, R. F., Greenwald, L. S. & Zilinskas, B. 1980. Allophycocyanin from Nostoc sp. phycobilisomes: properties and amino acid sequence at the NH2 terminus of the a and b subunits of allophycocyanin I, II, and III. J. Biol. Chem. 225:93807. Vadiraja, B. B., Giakwad, N. W. & Madyastha, K. M. 1998. Hepatoprotective effect of C-phycocyanin: protection for carbon tetrachloride and R-(+)-pulegone-mediated hepatotoxicity in rats. Biochem. Biophys. Res. Commun. 249:42831. Waterbury, J. B. & Stanier, R. Y. 1981. Isolation and growth of cyanobacteria from marine and hypersaline environments. In Starr, M. P., Stolp, H., Tru per, H. G., Balows, A. & Schlegel, H. G. [Eds.] The Prokaryotes. Springer-Verlag, Berlin, pp. 2213. Zhang, S., Yao, S., Wang, W., Xie, J., Zhang, J., Zhao, J. & Jiang, L. 2000. Studies on the kinetics of reactions between phycobiliproteins and hydroxyl radicals by a pulse radiolytic technique. Chin. Sci. Bull. 45:8969. Zilinskas, B. 1982. Isolation and characterization of the central component of the phycobilisome core of Nostoc sp. Plant Physiol. 70:10605.