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Journal of Photochemistry and Photobiology B: Biology 39 (1997) 19-23

Two ,y-polypeptides of B-phycoerythrin from P o r p h y r i d i u m

I.N. Stadnichuk
a,.,

cruentum 1

N.V. Karapetyan a, L.D. Kislov b, V.E. Semenenko b, M.B. Veryasov c

a A.N. Bakh institute~fBiochemistry, Russian Academy of Sciences, Leninsky prospekt 33, Moscow 117071, Russia b K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow 127262, Russia M.V. Lomonosov State University, Moscow 119899, Russia Received 14 March 1996; accepted 9 September 1996

Abstract
B-Phycoerythrin, the major phycobiliprotein of Bangiophyceae red algae, is composed of chromophorylated a-,/3- and "y-polypeptide chains. Two ",/-subunitswith distinct molecular masses were chromatographically separated from B-phycoerythrin of the red alga Porphyridium cruentum. Both 7-polypeptides were spectrophotometrically identified as having the same composition of five chromophoric prosthetic groups: three phycourobilins and two phycoerythrobilins. 1997 Elsevier Science S.A.
Keywords: B-Phyc6erythrin; Phycoerythrobilin; Phycourobilin; Porphyridium cruentum; y-Subunit

1. Introduction

Red-coloured B-phycoerythrin is the major phycobiliprotein of Bangiophyceae and some species of higher red algae. Its absorption spectrum results from the presence of two types of chromophore covalently linked to apoprotein: phycourobilin (shoulder at 498 nm) and phycoerythrobilin (maxima in the range 540--565 nm). Similar to all known phycobiliproteins, B-phycoerythrin consists of two dissimilar aand fl-polypeptide chains of about 17-20 kDa in 1 : 1 stoichiometry. In B-, R- and some CU-phycoerythrins, the third type of polypeptide is a T-subunit with an apparent molecular mass of 30--33 kDa [ 1-3 ]. T-Subunits are chromophorylated in addition to a- and g-chains and form with them very stable disc-shaped (a//)6 y-aggregates. The T-subunits of some R- and CU-phycoerythrins have been characterized with respect to their number, bilin type and content (see Refs. [4,5] ). For B-phycoerythrin, the aand fi-polypeptides have been well described. Specifically, the a-chain bears two phycoerythrobilin chromophores, and the fi-subunit is connected with three such groups [ 6]. Less extensive information is available on the T-subunit. Thus according to electrophoretic data [7] and the partial chromatographic resolution of the T-polypeptide fraction [ 8 ], Bphycoerythrin aggregates from the unicellular alga Porphyridium cruentum may include three different T-subunits [ 8 ].
* Corresponding author. Tel.: + 7 (095) 954-1473; fax: + 7 (095) 9542732; e-mail: inbio@glas.apc.org. t Dedicated to Professor H. Senger on the occasion of his 65th birthday. 1011-1344/97/$17.00 1997 Elsevier Science S.A. All tights reserved PilSIOI 1-1344(96)07453-2

On the other hand~ the purified B-phycoerythrin from P. cruentum in another study [6] showed only a single polypeptide of 30 kDa on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. B-Phycoerythrins from P. aerugineum and Rhodella violacea probably have more than one T-subunit [9]. The number of chromophoric prosthetic groups bound to T-subunit(s) of B-phycoerythrin has been reported to be equal to four: two phycourobilins and two phycoerythrobilins [10]. This ehromophore determination dealt with the total y-fraction, because the number of T-subunits is not perfectly clear, the molar ratio of 7- to a- and//-polypeptides is low and the close similarity of the T-polypeptides complicates their individual separation. The objective of this study was to attempt to identify the individual T-subunits of B-phycoerythrin by means of chromatographic separation, and to count their chromophore groups.

2. Experimental details
2.1. Chemicals

Bio-Rex 70 (minus 400 mesh) was obtained from BioRad (USA). High pressure liquid chromatography (HPLC) grade acetonitrile and isopropanol were obtained from Cryochrom (Russia) and trifluoroacetic acid (TFA) from Pierce Chemical Company (USA). All other chemicals were of reagent grade.

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I.N. Stadnichuk et al. /Journal of Photochemistryand PhotobiologyB: Biology 39 (1997) 19-23

2.2. Algal growth

The culture of P. cruentum strain Visher 107 was grown axenicaUy at 28 C in the medium described by Brody and Emerson [ 11 ] in 220 ml flasks, bubbled with air enriched to 1.7% in CO2 and exposed to 100 ttE m-~ s-t continuous daylight. The cells grown to a density of 3.0 g l-t were harvested by centrifugation.

cam detector, UK) with automatic peak integrations by a Shimadzu C-R.1A integrator (Japan). The resulting protein fractions were dried at 277 K on a Speedvac concentrator (Savant Instruments, USA).

2.5. Polyacrylamide gel electrophoresis

The protein fractions of B-phycoerythrin subunits dried after RP-HPLC were dissolved in 12.5 mM Tris-HCl buffer (pH 8.0) containing 3% SDS and 5% 2-mercaptoethanol, heated to 373 K for 10 min and analysed by disc electrophoresis according to the standard procedure of Laemmli [ 13 ]. The separation gel (thickness, 0.7 mm; length, about 14 cm) contained 12.5% polyacrylamide. Polypeptide markers from Pharmacia were used for calibration. Gels were stained with Coomassie (Brilliant Blue) R-250. The fractions of B-phycoerythrin polypeptides, obtained after chromatography on a Bio-Rex 70 column and containing concentrated urea (pH 3), were dialysed against pH 8.0, 12.5 mM Tris-HCl buffer and precipitated from solution with cold acetone. The precipitate was dissolved in the same buffer containing 3% SDS and 5% 2-mercaptoethanol, and subjected to electrophoresis as above.

2.3. Isolation of B-phycoerythrin

The cells ofP. cruentum were suspended to 0.15 mg mlin 10 mM Na-phosphate, 1 mM ethylenediaminetetraacetic acid (EDTA), I mM phenylmethanesulphonylfluoride and 1 mM 2-mercaptoethanol (pH 7.0) at 4 C. The suspension was passed three times through a French press at 18 000 lbf in -2 and centrifuged at 21 000g for 30 min. The supernatant was used for the isolation of B-phycoerythrin by the procedure of Glazer and Hixson [ 10]. Purification steps included chromatography on hydroxylapatite followed by ion exchange chromatography on Toypearl DEAE-650M and gel filtration on Sephadex G-200. Final chromatographic fractions of B-phycoerythrin with a ratio of A542/A5oo- 2.35 and A542/A2s0> 6.0 were taken for subunit studies.

2.6. Bilin analysis 2.4. Separation of polypeptide subunits of B-phycoerythrin

Three sequential chromatographic steps were carried out in the conditions [12] minimizing the loss of phycobilin absorbance. The first step was the procedure originally developed for B-phycoerythrin by Glazer and Hixson [ 10]. Protein in 10 mM Na-phosphate, pH 7.0, was adjusted to pH 3.0 with glacial acetic acid, and 2-mercaptoethanol was added to 10 raM. The solution was applied to a 1.5 cm 14 cm Bio-Rex 70 column developed stepwise with 2, 4, 6, 7.4, 8 and 9 M urea in 0.4% (v/v) acetic acid-10 mM 2-mercaptoethanol, pH 3.0. The last three fractions containing red material were pooled separately. For the next chromatographic step, the fraction eluted with 7.4 M urea was diluted with one part of acetic acid and then secondarily applied to a Bio-Rex 70 column ( 1 cm 4 cm) equilibrated with 3.7 M urea, pH 3.0. The coloured material was eluted stepwise with 6, 7.9 and 8.8 M acidic urea solutions. The addition of 0.1% Zwittergent 3-12 (Calbiochem) to the 6 M urea solution improved the subsequent polypeptide separation. The fractions containing 7.9 and 8.8 M urea were used for reverse phase HPLC (RPHPLC) on an analytical Nucleosil 300 C4 column (Eisico, Russia, 4.6 mm 150 mm) with a solvent system consisting of 0.1% TFA (buffer A) and 0.1% TFA in 2 : ] (v/v) acetonitrile-isopropanol (buffer B). The samples were applied tothe column in buffer A containing 5 M urea and eluted at a flow rate of 1 ml min- ~ according to the following programme: 1~% buffer A, 5 rain; linear gradient to 50% buffer B, 25 min. A model 344 gradient liquid chrorr~atograph (Beckman, USA) was used; samples were continuously detected at 495 and 555 nm (PU4021 UV-visible lye UniThe chromophore content of B-phycoerythrin polypeptides was calculated from the absorption spectra in 8 M urea at pH 30 using extinction coefficients at 495 and 555 nm of 94 000 and 0 M- ! cm- I for phycourobilin and 18 600 and 53 700 M-~ cm- ~ for phycoerythrobilin respectively [ 14]. The ratio of phycourobilin absorbance to phycoerythrobilin absorbance in the total fraction of ~/-subunits belonging to Bphycoerythrin does not change significantly during chromatography and possible chromophore altering [10]. The absorption spectra were recorded at ambient temperature on a Specord M400 spectrophotometer (Carl Zeiss, Germany). The absorption of the polypeptide fractions of B-phyc3erythrin after the chromatographic separation on a Bio-Rex 70 column was recorded immediately, whereas the dried fractions after reverse phase separation were pre-dissolved in acidic 8 M urea.

3. Results
The three polypeptide fractions of 7.4, 8.0 and 9.0 M acidic urea, obtained after the first chromatographic step of B-phycoerythrin on a Bio-Rex 70 column, were used for electrophoretic analysis. According to the electrophoretic data, the 8.0 M and 9.0 M urea eluates contained polypeptides of 19 and 20 kDa respectively (Fig. 1(A)). They were identified as the a- and/3-subunits of B-phycoerythrin by their molecular masses and known absorption spectra (not shown) in which the 495 nm band belonging to phycourobilin was absent. The polypeptide content of these fractions coincided

I.N. Stadnichuk et al. / Journal of Photochemistry and Photobiology B: Biology 39 (1997) 19-23 kDa
-43 --

21

kDa
43

--

30

--

30

-- 20.1

-- 20.1

-- 14.4

-- 14.4

(a)
I 2

(b)
1 2 3

Fig. 1. SDS polyacrylamide gel electrophoresis of B-phycoerythrin polypeptides: (A) lane 1, a-polypeptide; lane 2, fl-polypeptide; (B) lane I, ~,tpolypeptide; lane 2, fraction enriched in -/-polypeptides after single chromatography on a Bio-Rex 70 column; lane 3, 72-polypeptide. B.phycoerythrln

tint stepwise chromatography on Bio-rex 70

7.4

Murts fraction (?- and ct-subunits)

8 M urea frsction (ct4ubuni0

9 M ares fraction (J3-subunit)

~chmmato~phy on Bio-rtz 70

7.4 M acidic urea fraction enriched in 7-polypeptides. Simultaneously, it was possible to separate two fractions of 3'subunit(s). The elution sequence and the necessary urea concentrations were different from the first fractionation, when the/3-subunit was present in the protein preparation. First, the a-subunit was eluted with 6 M urea, and then the 3'-polypeptide(s) were ehted with 7.9 and 8.8 M urea in succession. Both fractions still contained scme a-polypeptide contamination (electrophoretic data of this intermediate chromatography not shown). The final purification of 3'-polypeptides was achieved by the application of RP-HPLC to 7.9 M and 8.8 M urea fractions, a-Polypeptide was removed during this last stage of chromatography. The 3'-polypeptide in the 7.9 M urea fraction had a molecular mass of 30 kDa; the second polypeptide with a molecular mass of 31 kDa is present in the 8.8 M acidic urea fraction (Fig. 1 (B)). According to the different molecular masses, these two polypeptides were designated as 3"r and 3'2-subunits respectively. It was impossible to fractionate the two 3'-subunits by RP-HPLC alone, omitting the Bio-Rex 70 column, as both 3"-subunits had the same retention time in this chromatographic system. The succession of chromatographic procedures, used to obtain the 3"-subunits of B-phycoerythrin, is presented in Fig. 2. In the absorption spectra of denaturated pure 3'-polypeptides, the phycourobilin maximum at 495 nm dominates in the visible region. In contrast, purified native B-phycoerythrin shows a characteristic absorption spectrum with a weak shoulder at 498-500 nm, corresponding to this maximum, and two intense peaks at 542 and 546 nm (Fig. 3). The large

6 M urea fraction (ct-subunit)

7.9 M urea Fraction (t- and (Hubunits)

8.8 M-"~relfraction (7:" and a4ubunits)

reverie phsse HPLC

(rsubunit) (t-subunit) Fig. 2. Chromatographic separation of 7-subunits of B-phycoerythrin in denaturating conditions.

I l

reverie pbsse

I I

PUB 0.~ HPLC !

0.2

with those originally obtained by this chromatographic method [ 10]. The fraction of 7.4 M acidic urea consisted of polypeptide (s) with a molecular mass of about 30 kDa and an admixture of the a-subunit. The electrophoretic band belonging to the 30 kDa polypeptide(s) was not sharp (Fig. 1 (B)). The 30 kDa polypeptide (s), according to the molecular mass and the presence of the 495 nm peak in the absorption spectrum (not shown), should be considered as the 3'-subunit(s). The use of urea concentrations below 7.4 M resulted in a rapid decrease in the polypeptide yield; therefore the single utilization of the Bio-Rex 70 column did not allow the removal of all the a-polypeptide to give pure 3"-subunit(s). The additional separation from the wsubunit was achieved by repeated application of Bio-Rex 70 chromatography to the

I I I I I I I

I I

I
0.I

/
4OO

\
600

5OO

|avelensth (rim) Fig. 3. Absorption spectra of B-phycoerythrin from P. cruentum in 0.01 M Na-phosphate buffer, pH 7.0 ( - - - ) and of its 7-polypeptide in 8 M urea, pH 3.0 (- - -); PEB, phycoerythrobilin: PUB, phycourobilin.

22

1.N. Stadnichuk et al. ~Journal of Photochemistry and Photobioiogy B: Biology 39 (1997) 19-23

difference between the two spectra in the 495-500 nm region is dueto the fact that all phycourobilin chromophore groups of B-phycoerythrin are connected only to 3"-chain(s) [ 10] and to the low part of 3,-subunits in the (c~/3)6 3'-aggregates of B-phycoerythrin. The absorption spectra of the 3'a- and 3'2-polypeptides in 8 M acidic urea are identical; therefore both 3"-subunits have identical chromophore compositions. The ratio of the absorption peaks at 495 nm (phycourobilin) and 555 nm (phycoerythrobilin) is 3.10:t:0.10. The calculation based on the known extinction coefficients ofpolypeptide-bound phycourobilin and phycoerythrobilin in concentrated acidic urea shows that each 3,-subunit contains these chromophores in a molar ratio of 1.55 + 0.05. According to this ratio and to the obvious fact that the numbers of phycobilins should be integers, the 3'1- and 3"2-subunits of B-phycoerythrin have five chromophofic prosthetic groups: three phycourobilins and two phycoerythrobilins. Twice these numbers would lead to an uncharacteristically large chromophore content.

4. Discussion
Chromatography under denaturating conditions using concentrated acidic urea is one of the most effective methods for the fractionation of phycobilin polypeptides. It was used to derive the 3,-polypeptides of B-phycoerythrin from P. cruenturn [ 10] and R-phycoerythrin from the red algae Gastroclonium coulteri [ 14] and Callithamnion corymbosum [ 15]. However, in all of these cases, a single run over a Bio-Rex 70 column only allowed to obtain the total fraction of ~/polypeptides. At the same time, RP-HPLC allowed us to separate two (R-phycoerythrin from Aglaothamnion neglecturn [4] ) and three (R-phycoerythrin from C. corymbosum [ 16]) V-subunits, omitting the use of the Bio-Rex 70column. However, only partial resolution of the 3'-polypeptides was realized by the application of the same method to B-phycoetythrin of P. cruentum [ 8 ] and to R-phycoerythrin of Antithamnion sparsum [ 15 ]. The straight utilization of RP-HPLC was not successful in the present work. The separation of two V-polypeptides could be achieved only by the combined use of two types of chromatography and re-chromatography on a Bio-Rex 70 column. The content of 3'-subunits in phycoerythrins varies in different cyanobacteria and red algae. R-Phycoerythrins from C. corymbosum and A. sparsum [ 15 ] have three T-subunits, while those fromA, neglectum [4], Callithamnion byssoides, CaUithamnion roseum [17], Audoniella saviana [18] and Gracilaria longa [ 19] contain two ~/-subunits. Three ~,-polypeptides were detected in CU-phycoerythrin of the cyanobacterium Synechococcus sp. WH8301 [ 1], but only one 3'subunit was found in another CU-phycoerythrin from 5ynechococcus sp. WHS020 [3 ]. We have obtained two 3'subunits of B-phycoerythrin from P. cruentum. No signs were observed of a third 3'-subunit [7,8] during all the separation pcocedures. The content of various 3'-polypeptides belonging

to the (a~)~ T-aggregates of one phycoerythrin may depend on the algal species and algal strain, as well as on the light and culture conditions [4]. The numbers of phycobilins connected with 3'-subunits of various phycoerythrins have been determined to be one [ 3 ], four [4,10,1-~] and five [5]. Specifically, the 3"-polypeptide of B-phyceerythrin from P. cruentum has been found to be associated with two phycoerythrobilins and two phycourobilins; the chromophore content was determined spectrophotometrically for the total fraction of 3'-polypeptides obtained by single chromatography on a Bio-Rex 70 column [ 10]. The As55/A495ratio in the absorption spectrum of the 3'fraction was found to be equal to 2.5; for the calculation of the chromophore groups, the bilin extinction coefficients of 100 300 M- *cm- ! (instead of 94 000 M - ~cm- m ) for phycourobilin and 43 000 M - l cm- t (instead of 53 700 Mcm- ~) for phycoerythrobilin were used [ 10]. The absorption coefficients of phycourobilin and phycoerythrobilin have been corrected several times [ 1,14,20]. In our work, the most recent values of the molar extinction coefficients applicable to protein-bound bilins in 8 M urea at pH 3 [ 13] were used. Two 3'-subunits were separated from each other and additionally purified from the a-polypeptide. The calculated minimum number of five chromophore groups per T-subunit corresponds to the spectral measurements. It would be highly preferable to compare these results with data on the chromophore contents obtained by other methods. There are two basic possibilities of determination of the chromophore numbers. It can be performed spectrophotometricaily, as in this work, or by the separation and numbering of different chromopeptides obtained after partial proteolysis of phycobiliproteins. However, the latter method has not yet been used to count the chromophore groups of T-subunits from B-phycoerythrin. The most exact data can be obtained by X-ray crystal structure analysis of phycobiliproteins, permitting a significantly improved definition of the chromophore locations. All chromophore groups have been visualized in the tertiary structure up to 2.2/~ resolution in the a- and ~-subunits of B-phycoerythrin [21]. Unfortunately, the disordering of the 3'-subunit(s) in the crystal, as a consequence of crystal and local symmetry averaging, has made it impossible hitherto to use X-ray crystallographic determination in this case [ 21 ].

Acknowledgements
Financial support by the International Science Foundation (Grant MO 1000) is acknowledged.

References
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[12] D. Guard-Friar, R. MacColl, Spectroscopic properties oftetrapyrroles of denaturated biliproteins, Arch. Biochem. Bicphys., 230 (1987) 300305. 1131 U.K. Laemmli, Cleavage of the structural proteins during the assembly of the head of bacteriophage T4, Nature (London), 227 (1970) 680685. [14] A.V. Klotz, A.N. Glazer, Characterization of the bilin attachnmnt sites in R-phycoerythrin, J. Biol. Chem., 260 (1985) 4856--4863. [151 I.N. Stadnichnk, T.I. Odintsova, A.Ya. Strongin, Molecular organization and chromophore composition of R-phycoerythrin from the red alga Callithamnion, MoL Biol. (Moscow), 18 (1984) 343-350 (in Russian). [161 i.N. Stadnichuk, A.V. Khokhlacbev, Ye.V. Tikhonova, Polypeptide 7subunits of R-phycoerythrin, Photochem. Photobiol. B: Biol., 18 (1993) 169-175. [171 M.-H. Yu, A.N. Glazer, K.G. Spencer, J.A. West, Phycoerythrins of the red alga Callithamnion. Variation in phycoerythrobilin and phycourobilin content, Plant Physiol., 68 ( 1981 ) 482-488. [18] L. Talarico, R-Phycoerythrin from Audoniella saviana (Nemaliales, Rhodophyta) nultrastmctural and biochemical analysis of aggregates and subunits, Phycologia, 29 (1990) 292-302. [19] E. Dagnolo, R. Rizzo, S. Paoletti, E. Murano, R-Phycoerythrin from the red alga Gracilaria longa, Phytochemistry, 35 (1994) 693-695. [201 G. Muckle, W. Riidiger, Chromophore content of C-phycoerythrin from various cyanobacteria, Z. Naturforsch., Tell C, 32 (1977) 957962. [211 R. Ficner, K. Lobeck, G. Schmidt, R. Huber, Isolation, crystallization, crystal structure analysis and refinement of B- phycoerythrin from the red alga Porphyridium cruentum at 2.2 angstrom resolution, J. Mol. Biol., 228 (1992) 935-950.