Biochem. J. (1970) 116, 161-169 Printed in Great Britain Isolation and Spectral Characterization of Phycobiliproteins By F. W. J. TEALE AND R. E. DALE* Department of Biochemistry, University of Birmingham, Edgbaston, Birmingham 15, U.K. (Received 10 June 1969) Several phycobiliproteins were prepared chromatographically pure and their absorp- tion, fluorescence-emission, fluorescence-excitation and fluorescence-excitation polarization spectra determined. Changes in these spectra with ionic strength of the aqueous medium and chromoprotein concentration were interpreted in terms of inter- chromophore energy transfer and protein subunit equilibria. The complexity of the polarization spectra confirms the presence of different types of chromophore, desig- nated sensitizing ('s') and fluorescing ('f'), in a single protein. The phycobiliproteins (6 hEocha, 1965a,b) are high-molecular-weight globular proteins found in three groups of algae, the red (Rhodophyta), blue- green (Schizophyta = Cyanophyta) and cryptomonad (Cryptomonadophyta). They are localized in the stroma of the photosynthetic tissue and not in the lamellae (Giraud, 1966 Fuhs, 1964). Their function in the process of photosynthesis appears to be that of accessory light-absorbers, trapped light-energy being handed on to the primary photochemical agent, chlorophyll (Duysens, 1952). Accordingly they have a very low fluorescence yield in vivo, which increases enormouslyonextractionwhentransferto chlorophyll is prevented. The actual absorbing species are linear tetrapyrrole derivatives, the phycobilins. These are attached to the apoprotein covalently, PUBt by a thioether link (6 hEocha, 1965a), PEB and PCB by an ester link involving a f-hydroxy amino acid (6 hEocha, 1965b 6 Carra, 6 hEocha & Carroll, 1964) and by a second linkage, probably to one of the pyrrole nitrogen atoms (Riudiger & 6 Carra, 1969). The structures of PEB and PCB are now known (Cole, Chapman & Siegelman, 1967 Chapman, Cole & Siegelman, 1967 Riudiger, 6 Carra & 6 hEocha, 1967 Rudiger & 6 Carra, 1969). They are isomeric, the difference being in the extent of the conjugated double-bond systems (Fig. 1). The number and type of chromophores found in different phycobiliproteins varies, and it appears that the same chemical species may exist in forms with different absorption spectra in one protein, probably reflecting different environments (6 hEocha & 6 Carra, 1961 Dale & Teale, 1966). The approximate absorption maxima of the proteins studied here are * Present address: Katedra Fizyki Do6wiadczalnej, Uniwersytetu Mikolaja Kopernika, Torun 1, ul. Grud- ziqdzka 5, Poland. t Abbreviations: PUB, phycourobilin PEB, phyco- erythrobilin PCB, phycocyanobilin. 6 shown in Table 1. The longest-wavelength-absorbing form fluoresces, the others passing on their excitation energy to it. When this sensitization process is (a) CO2H CO2H CH3 CH2 ClH2 CH2 11 CH3 ICH ClH3 CH2 CH2 CH3 CH3 CH o N ---N- H H H (b) C02H CO2H CH3 CH2 ClH2 CH3 CH3 tCH CH3 CH2 CH2 CH3 CH3 CH2 I- N N N N 0 H H H Fig. 1. Proposed chemical structures of (a) PEB and (b) PCB. Table 1. Absorption of phycobiliproteins Phycobiliprotein R-phycoerythrin B-phycoerythrin C-phycoerythrin Schizothrix C-phycocyanin Anacystis C-phycocyanin Approximate absorption maxima (nm) 495 (PUB) 535 (PEBs) 557 (PEBf) 500 (PUB) 540 (PEBs) 565 (PEBf) shoulder , 530 (PEBs) 562 (PEBf) shoulder 610 (PCBs) 630 (PCBf) slight shoulder 580 (PCBs) 620 (PCBf) slight shoulder Bioch. 1970, 116 161

F. W. J. TEALE AND R. E. DALE efficient, little direct fluorescence is seen from the shorter-wavelength-absorbing species. These shorter- and longer-wavelength-absorbing species of the same chemical structure are designated 's' (sensitizing) and 'f'(fluorescing) respectively. Certain chemical reagents differentiate the 'f' and 's'types of protein-bound chromophore. Thus PEBf is the more readily bleached form in 1% hydrogen peroxide, whereas PEBs and PCBs absorptions disappear faster on treatment with dithionite (Jones & Fujimori, 1961 Fujimori & Quinlan, 1963). Ferricyanide in alkaline solution rapidly bleaches PEBs and both forms of PCB, but has hardly any effect on PEBf (Dale, 1967). Mercurials effect a decrease in PEBf absorption with concurrent loss of fluorescence, both being partially recovered on addition of thiols (Fujimori, 1964 Fujimori & Pecci, 1967 Pecci & Fujimori, 1967,1968). These spectral changesmayreflectdissociationofthechromoproteins that also occurs on addition ofmercurials, so that the suggested influence of thiol groups on chromophore spectra and fluorescence properties may not be direct. The differential effects of urea, guanidine or acid pH suggestedthatPEBfwashydrogen-bonded(6hEocha & Carra, 1961), but it has since been argued that unspecified environmental influences other than hydrogen-bondingaccount for the absorption spectral difference between the PEB forms (6 hEocha, 1965b). Thefluorescence spectra ofmanyphycobiliprotei have been reported (see 6 hEocha, 1965b). Those of phycoerythrins have maxima at about 570nm, those of phycocyanins at 630-660nm. The shape of the spectrum is asymmetric with a subsidiary peak or marked shoulder on the long-wavelength side of the ma:ximum. Fully corrected fluorescence-excitation spectra have also been obtained for a number of phycobiliproteins. Their correspondence to absorp- tion spectra is variable and, in particular, widely different efficiencies of transfer from protein aromatic groups to phycobilin chromophores have been repor- ted (Bannister, 1954 Eriksson &Halldal, 1965 Dale, 1967 Macdowall, Bednar & Rosenberg, 1968). R-phycoerythrin from many different sources has a molecular weight of about 290000 and does not readily dissociate (6 hEocha, 1965a Nolan & hEocha, 1967), and B-phycoerythrin has been shown to dissociate in the pH range 6-6.2 into a colourless fragment of molecular weight 159000 and a smaller coloured one of molecular weight 113000 (Brody & Brody, 1961). C-phycocyanins exist as monomer or polymers having three, six or 12 sub- units, the dissociation constants varyingwith pH and ionic strength of the solvent, the molecular weight of the hexamer being approx. 275000 (Berns, Scott & O'Reilly, 1964 Hattori, Crespi & Katz, 1965 Scott & Berns, 1967). C-phycoerythrins probably behave similarly (Pecci & Fujimori, 1968), but are of lower molecular weight, about 226000 for the hexamer (O hEocha, 1965a). Electron microscopy has shown that the hexamer of C-phycocyanin is hexagonally arrangedwithacentralhole (Berns & Edwards, 1965). More extensive dissociation takes place at extremes of pH with all phycobiliproteins (6 hEocha, 1965a,b). The dissociation of C-phycocyanins is attended by changes in chromophore environment, and the consequent absorption-spectral differences have been used to observe the subunit equilibrium quantitative- ly (Hattori et al. 1965). Dissociation also produces changes in fluorescence spectra and fluorescence polarization, which have served as a qualitative index of the equilibrium (Goedheer & Birnie, 1965). EXPERIMENTAL Materials The sources of phycobiliprotein were as follows: R-phycoerythrin from the large rhodophyte Rhodymenia sp. (supplied by the Marine Biological Station, Plymouth, U.K.) C-phycoerythrin and C-phycocyanin from the cyanophyte Schizothrix calcicola (Ag.) Gom. (received as a gift from Dr H. Hoogenhout, Biophysical Laboratory of the State University, Leiden, The Netherlands) C- phycocyanin from the cyanophyte Anacysti8 nidulans (supplied from the Culture Collection of Algae and Protozoa, University of Cambridge, Cambridge, U.K.) B-phycoerythrin from the unicellular rhodophyte Porphyridi,um cruentum NLag. (supplied from the Culture Collection ofAlgae and Protozoa, University ofCambridge, Cambridge, U.K.). Chemicals and solvents used were of analytical grade, or that of highest purity available, from British Drug Houses Ltd., Poole, Dorset, U.K., or Fisons Scientific Apparatus Ltd., Loughborough, Leics., U.K. Whatman DEll DEAE-cellulose for column chromatography was obtained from British Drug Houses Ltd., and Sephadex G-25 (medium grade) from AB. Pharmacia, Uppsala, Sweden. Rhodymenia fronds were stored at -15��C until needed for extraction. The microscopic algae were cultured in 800ml batches with magnetic stirring and a good flow of air (not enriched with C02). After initial inoculation from slopes, the liquid cultures were propagated in a semi- continuous manner by subculturing with 10ml of the previous thick suspension produced. The media and culture conditions employed are shown in Table 2. After being harvested by centrifugation (12 OOOg for 20min) and redispersed in a small volume of distilled water, the algae were frozen for storage until enough had been collected for extraction. F. W. J. Teale (unpublished work) had shown that crystallizable R-phycoerythrin from a Rhodymenia could be purified by chromatography on DEAE-cellulose. The possibility of modifying this rapid and promising method to give not only a highly pure R-phycoerythrin from Rhodymenia but also R-phycocyanin from this and B- and C-phycobiliproteins from other sources was further investigated (Dale, 1967). Flow sheets for the preparation of Rhodymenia R-phycoerythrin, Porphyridium B-phyco- erythrin, Schizothrix C-phycoerythrin and C-phycocyanin and Anacysti8 C-phycocyanin are given in Schemes 1-3 1970 162