ANALYTICAL

BIOCHEMISTRY

188,295-2%

(1990)

Separation of Phycobiliprotein Subunits by ReversePhase High-Pressure Liquid Chromatography’ Ronald

V. Swanson2

Division

and Alexander

N. Glazer3

229 Stanley

of Biochemistry and Molecular Biology, Department of Molecular Hall, University of California, Berkeley, California 94720

Received

March

15,199O

Baseline separation of subunits of diverse phycobiliproteins was achieved by a reverse-phase HPLC gradient method with a C4 large-pore column and a solvent system consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 2: 1 (v/v) acetonitrile:isopropanol. The procedure was successfully applied to cyanobacterial allophycocyanin and C-phycocyanins, an unusual phycocyanin from a marine cyanobacterium, red algal B- and R-phycoerythrins, and a cryptomonad phycoerythrin. The subunit sizes in these proteins range from about 7.5 to 30 kDa. Sample recovery was in excess of 85% in all cases. On-line spectroscopic analysis with a multiple diode array detector allowed determination of the type and number of bilins carried by each subunit. 0 1990 Academic Press, Inc.

Phycobiliproteins are components of the light-harvesting antenna complexes of cyanobacteria (blue-green algae) and of two groups of eukaryotic algae, the red algae and the cryptomonads. These proteins carry covalently linked open-chain tetrapyrrole (bilin) prosthetic groups. The spectroscopic properties of individual phycobiliproteins depend in large measure on the chemical nature of the bilins they carry (1,2). In cyanobacteria and red algae, the phycobiliproteins are assembled through specific interactions with polypeptides called “linker polypeptides” (3,4) into macromolecular complexes called phycobilisomes (5,6). The nature of the r This research was supported in part by National Institute of General Medical Sciences Grant GM-28994, by National Science Foundation Grant DMB 8816727, and by the Lucille P. Markey Program in Biomolecular Structure and Design at the University of California, Berkeley. ’ Recipient of a predoctoral fellowship from Department of Health and Human Services Training Grant 5 T32 GM-07232-12. 3 To whom correspondence should be addressed. 0003.2697/90 Copyright All rights

and Cell Biology,

$3.00 0 1990 by Academic Press, of reproduction in any form

Inc. reserved.

higher-order assemblies of the cryptomonad phycobiliproteins has not yet been determined. On breakage of cells, the macromolecular assemblies dissociate and the phycobiliproteins are released as water-soluble complexes varying in composition depending on the particular protein and source organism. The major cyanobacterial and red algal phycobiliproteins-phycoerythrin, phycocyanin, and allophycocyanin-are each composed of two dissimilar polypeptide chains, (Y and p, of approximately 17 and 18 kDa, respectively. Allophycocyanin carries one bilin on each subunit, phycocyanin carries one bilin on the (Ysubunit and two on the fi subunit, and phycoerythrin carries two or in some cases three bilins on the LYsubunit and three on the /3 subunit. Highly purified native phycobiliproteins are complexes of the form (c$?)~, n = l-6. In addition, B- and R-phycoerythrins are isolated as very stable (a&r complexes, where the -30-kDa y subunit carries four bilins (1,7,8). Four chemically distinct bilins are known to occur in cyanobacteria and red algae. The attachment of each of the bilins to the polypeptide involves one of two modes of linkage, either a single thioether bond between ring A of the bilin and a cysteinyl residue or two such thioether bonds between rings A and D of the bilin and the polypeptide. Less extensive information is available on the cryptomonad phycobiliproteins. The quaternary structure of the purified native phycobiliproteins is (Y(Y’&. The two a-type subunits differ from each other significantly in amino acid sequence (9). Their molecular weights are in the range 7-9 kDa and each carries a single bilin. The p subunits, of about 18 kDa, each carry three bilins. The p subunits are highly homologous to the p subunits of red algal phycoerythrins (9,lO). In addition to bilins identical to those of cyanobacterial and algal cryptomonad phycobiliproteins, certain cryptomonad phycobiliproteins carry different bilins of unknown structure (lo12). 295

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Characterization of phycobiliproteins with respect to subunit composition, bilin type and content, and amino acid sequence requires separation of the subunits. Several methods have been employed in the past. Of necessity, all are performed under denaturing conditions. These include chromatography on Bio-Rex 70 in acid urea (13), gel filtration under denaturing conditions (14,X), semipreparative electrophoresis (16), ion-exchange chromatography on Whatman microgranular cellulose CM-52 at pH 5.0 (7) or DEAE-Sephadex A-50 at pH 7-8 (17,18), and sucrose density gradient centrifugation at acid pH (19). These methods all share the following disadvantages. They are not generally applicable to all phycobiliproteins. The separations require long periods, in some instances under conditions where the bilin prosthetic groups undergo some oxidative degradation. The amounts of protein required are in the several milligram range. The procedure described in this report is generally applicable to all types of phycobiliproteins, is rapid, and requires less than a milligram of protein. Information is obtained on the subunit composition and stoichiometry, bilin type, and content. The sample recovery is high and the subunits are isolated in a form suitable for gas-liquid phase sequencing. The separations are achieved by a reverse-phase HPLC gradient method with a Cd4 largepore column and a solvent system consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 2:l (v/v) acetonitrile:isopropanol. The peptide-linked bilins are stable in this low-pH solvent system. Moreover, the solvent does not interfere with on-line spectroscopic identification and quantitation of the bilins. MATERIALS

AND

METHODS

Chemicals. HPLC-grade acetonitrile and isopropano1 were obtained from Fisher Scientific and TFA from Pierce Chemical Company (Milwaukee, WI). All other chemicals were of reagent grade. Organisms and culture conditions. Anabaenu sp. PCC 7120 and Synechococcus sp. PCC 6301 were cultured in liquid medium BG-11 (20) at room temperature under constant warm white illumination in an atmosphere of 5% C02-95% Nz. Cryptomonad strain STAN M2 was obtained from R. Guillard (Woods Hole Oceanographic Institution, Woods Hole, MA) and cultured at 20°C in “K” medium (21) with inorganic phosphate containing thiamine, 10 pg liter-‘; biotin, 50 pg liter-‘; and cyanocobalamin, 50 pg liter-‘. Cultures were gassed with 5% C02--95% Nz and maintained on an illumination cy-

AND

GLAZER

cle of 16 h light: 8 h dark. Synechocystis sp. WH 8501 cells were obtained from J. B. Waterbury (Woods Hole Oceanographic Institution). Proteinpurification. Anabaena sp. PCC 7120 C-phycocyanin and Synechococcus sp. PCC 6301 C-phycocyanin and allophycocyanin were purified by the procedure of Glazer and Fang (13), followed by chromatography on hydroxylapatite (22) and FPLC mono Q. Briefly, the proteins were dialyzed against 20 mM piperazine-HCl, pH 6.0 (buffer A), and applied to an FPLC mono Q HR 5/5 column (Pharmacia) connected to a Perkin-Elmer 410 LC Bio pump. The samples were eluted with 1 M NaCl, 20 mM piperazine-HCl, pH 6.0 (buffer B) at a flow rate of 2 ml min-’ according to the following program: 100% buffer A, 2 min; linear gradient to 50% buffer B, 15 min; 100% buffer B, 2 min. Phycocyanin from Synechocystis sp. WH 8501 was purified by hydroxylapatite (22) and FPLC mono Q chromatography, as above. Bphycoerythrin from Porphyridium cruentum was purified as described in Ref. (7). R-phycoerythrin from Gastroclonium coulteri was purified as described in Ref. (8). Phycoerythrin 545 from cryptomonad strain STAN M2 was isolated by the procedure described by Wilbanks et al. (10) except that 50 mM sodium phosphate, pH 7.0, 1 mM EDTA, 1 mM NaN3 was substituted for the sodium acetate buffers used in their procedure. HPLC chroHigh-performance liquid chromatography. matography was performed using two Waters (Waters Chromatography Division, Milford, MA) Model 510 pumps and a Model 680 automated gradient controller or a Waters 600E multisolvent delivery system. Data were acquired using a Waters 991 photodiode array detector; spectra were collected from 210 to 750 nm at 2-s intervals. Protein, typically 0.05-1.0 mg in 400 ~1, was injected onto a Hipore RP-304 (Bio-Rad, Richmond, CA) column previously equilibrated in 65% TFA (0.1%) in water (buffer A), and 35% 2:l acetonitrile:isopropanol containing 0.1% TFA (buffer B). Proteins were eluted from the column at a flow rate of 1.5 ml mini according to the following program: O-2 min, 65% buffer A-35% buffer B; 2-37 min, linear gradient to 30% buffer A-70% buffer B; 37-42 min, linear gradient to 100% buffer B; 42-47 min, linear gradient to 65% buffer A-35% buffer B. Prior to HPLC chromatography, all proteins were dialyzed against 5 mM NaP04, pH 7.0,l mM Z-mercaptoethanol. Samples were diluted 1:l with 9 M urea, pH 2.0, and spun for 5 min in a microcentrifuge immediately prior to injection. RESULTS

4 Abbreviations used: C, , butyl bonded phase; TFA, trifluoroacetic acid, AUFS, absorbance units full scale; FPLC, fast protein liquid chromatography; PCB, phycocyanobilin; PEB, phycoerythrobilin; PUB, phycourobilin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Separation of subunits of differentphycocyanins. The separation conditions were developed with a well-characterized phycobiliprotein, Synechococcus sp. PCC 6301 C-phycocyanin (13,23). Denaturation of the protein in

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FIG. 1. Cyanobacterial C-phycocyanin subunit separation. (a) Synechococcus sp. PCC 6301 C-phycocyanin, AUFS 0.26. (b) Anabuena sp. PCC 7120 C-phycocyanin, AUFS 0.53. Elution profiles were monitored at 660 nm, the peak of visible absorbance of peptide-linked PCB chromophores in acid solution.

4.5 M urea at pH 2 prior to injection was found to be preferable to simple acidification of the sample with dilute TFA. The latter procedure resulted in partial precipitation of the sample with preferential loss of the 0 subunit. A linear gradient of 2:l (v/v) acetonitrile:isopropanol containing 0.1% TFA on a large-pore C4 column gave a baseline separation of the CYand /3 subunits (Fig. la). Integration of peak areas at 660 nm, a measure of phycocyanobilin (PCB) content, gave results within experimental error of the 1:2 ratio for o$ expected from the known bilin content of the two subunits (13,23). A reproducible sample recovery of 290% was determined by collecting both peaks and comparing the absorbance at 660 nm to that of an identical sample, which had not been injected, adjusted to the same volume. The above procedure was found to be generally applicable. This is illustrated by the subunit separation for a different C-phycocyanin, isolated from the filamentous cyanobacterium Anabaena sp. PCC 7120, shown in Fig. lb, and that for a novel phycourobilin (PUB)-containing phycocyanin (24) from a marine cyanobacterium, Synechocystis sp. WH 8501, shown in Fig. 2. Spectroscopic analysis of the elution profile of the Synechocystis sp. WH 8501 phycocyanin, obtained with a multiple diode array detector, showed that a PUB (X,,, 495 nm) group was attached to the (Ysubunit and that PCB groups (X,,, 660 and 350 nm) were attached to the p subunit. The spectroscopic data in conjunction with the known extinction coefficients for these peptide-linked bilins in acid solution (8,13) gave a PUB:PCB ratio of 1:2. Allophycocyanin. The (Yand @subunits of allophycocyanin are very similar to each other in molecular weight and each carries a single PCB group. The various methods previously applied to the separation of phycobiliproteins did not separate the subunits of allophycocy-

FIG. 2. Synechocystis sp. WH8501 phycocyanin subunit separation. The elution profile was monitored over the range 220-700 nm, 0.45 AUFS. The a subunit, which carries a single PUB (X,.. 495 nm), elutes at 23 min and is followed by the 0 subunit, which carries two PCB chromophores (X,,, 660 nm).

anin. This required the development of ion-exchange procedures optimized for the allophycocyanin subunit preparation (17,lB). It was particularly gratifying to find that the separation method described here gave baseline separation of allophycocyanin subunits. Under the conditions described above for phycocyanins, Synechococcus sp. PCC 6301 allophycocyanin was resolved into two components of equal PCB content (Fig. 3). Again, the sample recovery was 290%. SDS-PAGE showed that for both allophycocyanin and the phycocyanins the first component eluted represented the (Ysubunit and the second, the /3 subunit. R- and B-F’hycoerythrins. R- and B-phycoerythrins offer additional challenges to subunit separation. These proteins have the composition (a&r (7,25). Moreover, SDS-PAGE of Porphyridium cruentum B-phycoerythrin shows the presence of at least three y subunits, differing slightly in molecular weight (26). The elution profile for R-phycoerythrin from the red alga Gastroclonium coulteri is shown in Fig. 4. The -30-kDa y subunit which carries one phycoerythrobilin (PEB) and three PUBS elutes first, followed by the (Y (2 PEB) and @(2 PEB, 1 PUB) subunits. All three subunits are well re-

RFITEN'CION TIME (mh)

FIG. 3. Synechococcus sp. PCC 6301 allophycocyanin ration. The elution profile was monitored at 660 nm. carries one PCB chromophore.

subunit sepaEach subunit

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FIG. 4. Separation of the subunits of G. coulteri R-phycoerythrin. The elution profile was monitored over the range 250-700 nm. The identity of each peak was determined from the visible absorption spectra and the known number and distribution of PEB and PUB on the subunits of this protein (8). The order of subunit elution is y (3 PEB, 1 PUB), 01(2 PEB), /3 (1 PUB, 2 PEB).

solved from each other and analysis of the spectroscopic data (Fig. 4) is consistent with the information on bilin distribution and content based on structural studies of this protein (8). Peak integration showed greater loss of the 0 relative to the (Ysubunit. Overall sample recovery was -85%, slightly less than that for phycocyanins and allophycocyanin, but comparable to that reported by others for reverse-phase HPLC of hemoglobin subunits (27,28) and ribosomal proteins (29). Similar results were obtained with P. cruentum B-phycoerythrin (Fig. 5). In this instance, three distinct components are resolved in the y-subunit region of the chromatogram in keeping with the SDS-PAGE results cited above. With both R- and B-phycoerythrin, blank gradient runs performed following a subunit separation occasionally eluted additional @subunit. This observation is not unprecedented in the literature on reverse-phase HPLC of proteins (30) and the potential problem of cross-contamination of samples was effectively avoided by running a steep blank gradient between injections of different proteins.

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FIG. 6. Separation of the subunits of P. cruentum B-phycoerythrin. The elution profile monitored at 495 nm is shown. The identity of each peak was determined from the visible absorption spectra and the known distribution and content of PEB and PUB on the subunits of this protein (7,25). The order of subunit elution is y, 01,fl. The elution profile shows a partial resolution of at least three y-subunit species.

20 TIME (min)

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t 30

FIG. 6. Separation of the subunits of cryptomonad phycoerythrin 545. The elution profile was monitored at 575 nm. The (Y and (Y’ subunits elute early in the gradient while the more highly retarded fi subunit elutes as a doublet. The (Y subunits carry an unidentified bilin with carries three PEB groups. a km. at about 614 nm, and the fl subunit

Cryptomonad phycoerythrin 545. The same conditions led to an excellent separation of the subunits of cryptomonad phycoerythrin 545 (Fig. 6). The a! and (Y’ subunits of this protein are much smaller than those of the proteins discussed above and eluted early in the gradient. The fl subunit eluted as a partially resolved doublet. Heterogeneity of the /3 subunit of cryptomonad phycobiliproteins has been noted previously (9), but the basis is not known. DISCUSSION

The phycobiliproteins are a complex family of proteins with respect both to polypeptide composition and to the diversity of bilin prosthetic groups. Figures 1, 4, 5, and 6 offer examples of the diversity in polypeptide compositions, while Figs. l-6 illustrate the diversity of bilin types, contents, and distribution between the various subunits. In addition to the obvious interest in these proteins as components of photosynthetic antenna systems, they are also widely used as fluorescent tags for cell sorting (31,32) and in fluorescence-based assays for reactive oxygen species (33,34). Limited screening of marine cyanobacterial strains (24), of higher red algae (35)) and of cryptomonads (2,lO) indicates that a number of phycobiliproteins with novel bilins and bilin contents remain to be fully characterized. The methodology described in this report will facilitate both the screening of phycobiliproteins for interesting structural features and the preparation of subunits in pure form for amino acid sequence determination and isolation of bilin peptides. It will also permit rapid characterization of mutant phycobiliproteins lacking one or more bilins. Such mutant proteins have been generated by uv mutagenesis (36) as well as by insertional inactivation of open reading frames in the C-phycocyanin operon (37). Other applications include analysis of the products of in vitro bilin addition to apophycobiliproteins (38). ACKNOWLEDGMENTS The authors express their appreciation to G. Wedemayer for helpful discussions and preparation of the cryptomonad phycoerythrin sample and to C. Chan for the B-phycoerythrin sample.

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21. Keller, (1987)

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Separation of phycobiliprotein subunits by reverse-phase high-pressure liquid chromatography.

Baseline separation of subunits of diverse phycobiliproteins was achieved by a reverse-phase HPLC gradient method with a C4 large-pore column and a so...
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