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Short communication

Extracting and purifying R-phycoerythrin from Mediterranean

red algae Corallina elongata Ellis & Solander

R. Rossano a, N. Ungaro b, A. DAmbrosio a, G.M. Liuzzi c, P. Riccio a,*

a Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, University of Basilicata, Campus Macchia Romana, I-85000 Potenza,

Italyb Laboratorio Provinciale di Biologia Marina, Molo Pizzoli (Porto), Bari, Italyc Dipartimento di Biochimica e Biologia Molecolare, University of Bari, Bari, Italy

Received 8 March 2002; received in revised form 12 December 2002; accepted 19 December 2002

Abstract

R-Phycoerythrin (R-PE) is a protein acting as a photosynthetic accessory pigment in red algae (Rodophyta). This

protein has gained importance in many biotechnological applications in food science, immunodiagnostic, therapy,

cosmetics, protein and cell labelling, and analytical processes. In this paper we report on a new, one step procedure for

the extraction and purification of R-PE from a new source: the Mediterranean red algae Corallina elongata Ellis &Solander. This red algae contains mainly R-PE and is suitable for the production in culture. No other contaminating

phycobiliproteins could be detected in the extracts. The method we propose for the purification is based on the use of

hydroxyapatite, a chromatographic resin that can be produced in the laboratory at very low cost and can be used batch-

wise with large amounts of extracts, alternative to chromatography, and therefore can be scaled up. Both the yield and

the purity of R-PE are very good.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Algae; Corallina elongata ; Phycoerythrin; Purification; Hydroxyapatite

1. Introduction

Phycoerythrin is a major light-harvesting pig-

ment of red algae. R-Phycoerythrin (R-PE) is an

oligomeric protein of 240 kDa, with 6 a (about 20

kDa), 6b (about 20 kDa), and 1g (about 30 kDa)

subunits. R-PE is commonly used as a fluorescent

label in immunology and cell biology (Oi et al.,

1982; Kronik, 1986) and in flow cytometry

(Hardy, 1983; Wilson et al., 1991), but is alsoused as a natural food dye (Mille-Claire et al.,

1993; Qian et al., 1991; DAgnolo et al., 1994) and

as a marker in gel electrophoresis and isoelectro-

focusing (Araoz et al., 1998). It has also been

shown that it could be used as a measure of peroxy

radical damage (DeLange and Glazer, 1989).

R-PE can be purified either by precipitation

with ammonium sulphate, by ion-exchange chro-

matography and gel filtration (Hilditch et al.,

1991; Thammapalerd et al., 1996) or by prepara-

* Corresponding author. Tel.: /39-0971-20-5563; fax: /39-

0971-20-5687.

E-mail address: riccio@unibas.it(P. Riccio).

Journal of Biotechnology 101 (2003) 289/293

www.elsevier.com/locate/jbiotec

0168-1656/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0168-1656(03)00002-6

mailto:riccio@unibas.itmailto:riccio@unibas.it

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tive polyacrylamide gel electrophoresis (Gallard-Irmouli et al., 2000). The use of hydroxyapatite in

association with gel filtration and precipitation

with ammonium sulfate has also been reported

(MacColl et al., 1996).

In this paper we have set up an alternative

procedure for the purification of R-PE extracted

from the Mediterranean red algae Corallina elon-

gata , a new source of R-PE. This procedure is

based on adsorption chromatography on hydro-

xyapatite (HA), a form of calcium phosphate

Ca10(PO4)6(OH)2 (Tiselius et al., 1956; Riccio,1989), and gel filtration. C. elongata was collected

in the southern Adriatic Sea near the town of Bari,

Italy. Algae samples were washed with deionized

water, lyophilised in small aliquots and stored at

/70 8C until use.

The purification procedure is shown in Fig. 1.

Twenty-five grams of lyophilised algae were re-

suspended in three volumes of a buffer containing

10 mM sodium phosphate (NaPi)/100 mM NaCl/

pH 7.0, blended in a cold Waring Blendor for 3

Fig. 1. Flow sheet of R-phycoerythrin purification from Corallina elongata. Protein content was determined using the Bio-Rad

Bradford reagent with BSA as a standard. * S.A. (specific activity)/A566 nm/mg prot. ** P.I. (purity index)/A566 nm/A280 nm.

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min and centrifuged for 10 min at 3000/g at

4 8C. The supernatant, red coloured, was passedthrough a 1.0mm cellulose nitrate membrane filter

(Whatman), adsorbed on a hydroxyapatite column

(16/80 mm) connected to a FPLC system (Phar-

macia Biotech) and eluted with 50 mM NaPi/0.1

M NaCl/pH 7.0. After adsorption the upper

portion of the HA column became red and the

non-adsorbed, pass-through, fraction appeared

yellow. The red coloured fractions were collected,

concentrated by ultrafiltration on YM10 Amicon

membranes, and applied to a Superdex 75 gel

filtration FPLC column. The eluting buffer was 10mM NaPi, 100 mM NaCl, pH 7.0 at a flow rate of

1.0 ml min1. The eluate showed two peaks.

Fractions in the first peak, with purity index

A566/A280 greater than 5.0, were collected. The

absorption spectrum of purified R-PE showed the

characteristic peaks at 495 and 566 nm (Fig. 2).

Purity of R-PE was estimated by means of three

indexes: A566/A280/5.3; A566/A495B/1.5; A620/

A566B/0.005. The 1st, A566/A280, is indicative of

the purity of the preparation with respect to many

contaminating proteins. This index should be

higher than 5.3. Our R-PE showed a purity indexas high as 6.67, corresponding to a 61-fold

enrichment of the specific activity (A566/mg prot.).

The 2nd, A566/A495, indicative of the identity of

the purified pigment, was 1.44. R-PE has a strong

secondary absorbance peak at 495 nm, where B-

phycoerythrin (B-PE) exhibits only a slight

shoulder. When A566/A495B/1.5, the pigment is

not significantly contaminated with B-PE. Finally,

the index A620/A566 of our R-PE preparation was

0.001.

About 15 mg of pure phycoerythrin wereobtained from 25 g of lyophilised algae. Analysis

of purified protein by SDS gel electrophoresis (Fig.

3) gave three bands. The upper band should

correspond to a 55 kDa heterodimer of the a/b

and g subunits, while the second and the third

bands correspond to the g (30 kDa) and a/b

subunits (20 kDa).

The stability of R-PE (1 mg prot. ml1; 10 mM

NaPi; 100 mM NaCl; pH 7.0) was monitored over

time in different conditions (/20 8C/4/8 8C/

Fig. 2. Absorption spectrum of purified R-phycoerythrin. Both the absorption spectrum and the purity indexes of purified R-

phycoerythrin were determined using an UV-Vis spectrophotometer Ultrospec 2000 (Pharmacia Biotech).

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room temperature; in the dark and in the light, and

at pHs between 3.0 and 10.0) measuring the

absorption spectra and the absorbance at 566 nm

after 1:10 dilution. A566 clearly decreased with the

breakdown of the chromophore or with the

aggregation and precipitation of R-PE. After 35

days the sample stored in the dark at 4 8C showed

the greatest stability, and that at /20 8C theworst. Freezing and thawing clearly induced the

formation of red aggregates. When exposed to the

light, R-PE showed a higher trend in losing its

chromophore, and the effect of light was even

higher after 15 days. R-PE was very stable at pHs

in the range 6.0/8.5, but at low pHs showed a

marked decrease of A566. Alternatively, a batch-

wise procedure was used in the first step by adding

HA to the deep red supernatant at the ratio of 0.1

g HA ml1 extract.

2. Conclusions

The aims of our study were to find both a local

natural source of R-PE and a new, simple proce-dure for its purification.

R-PE is commonly extracted from algae differ-

ent from C. elongata . Some examples are: Cer-

amium isogonum (Qian et al., 1991); Corallina

officinalis (Hilditch et al., 1991); Gracilaria longa

(DAgnolo et al., 1994); Gracilaria fisheri (Tham-

mapalerd et al., 1996);Palmaria palmata (Gallard-

Irmouli et al., 2000).

Our results indicate that C. elongata could be a

good alternative source of R-PE. In addition to its

good phycoerythrin content, the choice of C.elongata appears to be very convenient because

no other contaminating phycobiliproteins could be

detected in the extracts. This makes the R-PE

purification procedure presented in this paper very

simple and easily reproducible. When compared

with other purification procedures, our method

which is useful for scale up gives a product with a

high enrichment of specific activity (A566/mg

prot.), one of the highest purity indexes, a good

yield and satisfying stability at 4 8C in the dark

and at pH 6.0/

8.5.The purity index obtained in this study is much

better than those reported earlier for Phyllophora

antarctica (MacColl et al., 1996) or for Palmaria

palmate (Gallard-Irmouli et al., 2000) who ob-

tained purity indexes of 4.4 and 3.2, respectively.

Furthermore, our procedure is less laborious than

those reported by other authors (Hilditch et al.,

1991; MacColl et al., 1996; Thammapalerd et al.,

1996), requiring two or three purification steps.

The possibility of growing C. elongata under

laboratory conditions (Vergara and Niell, 1993)

and applying our one step procedure is anattractive proposition for the production of R-

PE and its purification.

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Fig. 3. SDS gel electrophoresis. Lanes: 1, standard proteins; 2,

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