Biochem. J. (1980) 187, 303-309 303Printed in Great Britain

The Native Forms of the Phycobilin Chromophores of Algal Biliproteins

A CLARIFICATION

Padraig O'CARRA,* Richard F. MURPHYt and S. Derek KILLILEAtDepartment ofBiochemistry, University College, Galway, Ireland

(Received 3 September 1979)

Pigments released from phycoerythrins and phycocyanins by treatment with hotmethanol are currently regarded as equivalent to the native chromophores phyco-erythrobilin and phycocyanobilin. However, evidence presented here confirms theoriginal views of O'Carra & O'hEocha [(1966) Phytochemistry 5, 993-9971 that thesemethanol-released pigments are artefacts differing in their chromophoric conjugatedsystems from the native protein-bound prosthetic groups. By contrast, the nativespectral properties are retained in pigments released by careful acid treatment of thebiliproteins and these acid-released phycobilins, rather than the methanol-releasedpigments, are therefore regarded as the protein-free forms of the native chromophores.The conclusion reached by Chapman, Cole & Siegelman [(1968) J. Am. Chem. Soc. 89,3643-36451, that all the algal biliproteins contain only phycoerythrobilin andphycocyanobilin, is shown to be incorrect. The identification of a urobilinoidchromophore, phycourobilin, accompanying phycoerythrobilin in B- and R-phycoerythrins is confirmed and supported by more extensive evidence. Thecryptomonad phycocyanins are shown to contain a phycobilin chromophore accom-panying phycocyanobilin. This further phycobilin has the spectral properties of the classof bilins known as violins and the provisional name 'cryptoviolin' is proposed pendingelucidation of its structure.

The algal biliproteins are a group of brilliantlycoloured fluorescent proteins that function as photo-synthetic pigments in red, blue-green and crypto-monad algae (O'hEocha, 1965, 1966). They aresubdivided into two major categories on the basis ofvisual appearance: the phycoerythrins are red with agolden fluorescence; the phycocyanins are blue witha red fluorescence (O'hEocha, 1965, 1966). Severalsubtypes of these chromoproteins are distinguishedon the basis of differences in absorption spectra. R-,B- and C-phycoerythrins and R-, C- and allo-phycocyanins are found in various members of theRhodophyta (red algae) and Cyanophyta (blue-green algae). Cryptomonad algae contain a complexgroup of further variants [see O'hEocha et al. (1964)for a review of these].

The brilliant coloration of the phycoerythrins and* To whom reprint requests should be addressed.t Present address: Department of Biochemistry,

Medical Biology Centre, Queen's University, Belfast,Northern Ireland, U.K.

t Present address: Department of Biochemistry, NorthDakota State University, Fargo, ND 58102, U.S.A.

the phycocyanins is imparted by open-chain tetra-pyrrole chromophores that are covalently attachedto the apoproteins (O'hEocha, 1965, 1966). Thesechromophores are termed phycobilins. They havelabile structures and are difficult to release from theapoproteins without alteration. This has greatlyhindered the complete structural characterization ofthe pigments. We believe that the structures cur-rently accepted for the major phycobilins, phyco-erythrobilin and phycocyanobilin, apply to artefactderivatives of the native chromophores. Since theconfusion of native and artefact forms has beenextensive (Beuhler et al., 1976; Chapman et al.,1967, 1968; Cole et al., 1967; Crespi & Katz, 1969;Crespi et al., 1968; Glazer & Fang, 1973; Glazer &Hixson, 1975; Troxler et al., 1978), we decided thatit was appropriate to clarify this issue beforeproceeding to a consideration of the structures of thenative chromophores [see the following paper(Killilea et al., 1980)].A further issue needing clarification is the

question as to whether phycoerythrobilin and phy-cocyanobilin are the only phycobilins occurring in

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P. O'CARRA, R. F. MURPHY AND S. D. KILLILEA

algal biliproteins, as claimed by Chapman et al.(1968). O'hEocha & O'Carra (1961) and O'Carra etal. (1964) suggested the occurrence of a third typeof phycobilin with urobilinoid spectral propertiesin R-phycoerythrin, and evidence presented herestrongly supports this. In addition, spectral evi-dence suggest that, as well as containing phycocy-anobilin, cryptomonad phycocyanins have a furthertype of phycobilin with violinoid spectral properties(O'hEocha et al., 1964). We propose that thesefurther phycobilins be termed respectively phyco-urobilin and cryptoviolin.

Experimental

Biliprotein preparationsR-phycoerythrin from the red alga Rhodymenia

palmata, C-phycoerythrin from the red algaPhormidium persicinum, C-phycocyanin from theblue-green alga Nostoc punctiforme and R-phyco-cyanin from the red alga Ceramium rubrum wereisolated and purified as by O'Carra (1965). Crypto-monad phycocyanin was isolated from Hemiselmisvirescens as by O'hEocha et al. (1964).Bilin preparations

Methanol-released 'purple pigment' from R-phycoerythrin and 'blue pigment' from C-phyco-cyanin were prepared as by O'Carra & O'hEocha(1966). Preparations of phycoerythrobilin andphycocyanobilin from R-phycoerythrin and C-phycocyanin, respectively, were obtained by incu-bating the biliproteins for successive periods ofl5min at 180C in 11.5-12M-HCl (O'Carra et al.,1964; O'hEocha, 1963). Phycocyanobilin wassimilarly released from R-phycocyanin when most ofthe phycoerythrobilin had been first released fromthe protein by two successive incubations with HCl(O'Carra, 1962; Murphy, 1968). T.l.c. on silica-gelG was used for analysis of the free diacid forms ofthe phycobilin preparations and for purification ofindividual bilin constituents (Murphy, 1968). Thesolvent systems used were: carbon tetra-chloride/acetic acid (2: 1, v/v); carbon tetra-chloride/acetone/acetic acid (4:8:1, by vol.); hep-tane/acetone/acetic acid (16:16:3, by vol.). Bilinswere esterified (Murray, 1966) in a solution (1 ml) ofBF3 in methanol (14%, v/v; Sigma, Poole, Dorset,U.K.) for 25min at 40C. Ice-cold water (4ml) wasthen added and the esterified pigment was extractedwith three 2ml portions of chloroform.Tryptic digestion ofbiliproteins

Previous studies have shown that, under someconditions used to release phycoerythrobilin fromphycoerythrins, random artefact covalent linkage ofthe bilin to the polypeptide chains of the apoproteincan take place and the chromophore is alsoconverted into a urobilinoid artefact derivative

(O'Carra et al., 1964; Riidiger & O'Carra, 1969).Since the artefact attachment takes place by con-densation of the vinyl-side-chain group of ring D ofphycoerythrobilin with thiol groups on the poly-peptide chains (O'Carra et al., 1964; Riidiger &O'Carra, 1969), blocking of the thiol groups byreduction and aminoethylation (Raftery & Cole,1966) before tryptic digestion was carried out toeliminate this complication. To further protect thechromophores, all procedures used were carried outin the dark and under nitrogen. Tryptic digests werecarried out as follows. Reduced and aminoethylatedbiliprotein (50mg) was suspended in 10ml of0.2M-sodium phosphate buffer, pH7.0. A solution(0.1 ml) of diphenylcarbamoyl chloride-treated tryp-sin (2.5 mg; Sigma) in 1 mM-HCl was added. Themixture was incubated with stirring for 6h at 250Cunder nitrogen, and any insoluble material wasremoved by centrifugation. After the addition of afurther portion (0.1 ml) of trypsin solution to thesupematant, digestion was continued for 16 h.Digests were stored at -200C under nitrogen.

Starch-gel electrophoresis of tryptic digests ofR-phycoerythrin

Electrophoresis of tryptic digests of R-phyco-erythrin was carried out on horizontal starch gels byusing 'Starch Hydrolysed' (Connaught MedicalResearch Laboratories, Toronto, Ont., Canada) andadopting the procedure of Smithies (1955). Thebuffer systems used were: glycine/HCl, pH 2.2;sodium acetate, pH4.0, 4.5, 5.0 and 5.5; Tris/mal-ate, pH 7.4 and 8.3. For the glycine/HCl and sodiumacetate buffers, the gel buffer concentration was0.025M and the bridge buffer concentration was0.05M. For the Tris/malate buffers, the gel bufferconcentration was 0.005M and the bridge bufferconcentration was 0.1 M. Electrophoresis was carriedout at 40C in the dark for 16h at 7.5 V/cm. Afterelectrophoresis the starch gels were soaked inethanol/pyridine (1: 1, v/v) for 10min. Thebathing fluid was replaced for each of two subse-quent washings. The gels were then placed in asaturated solution of zinc acetate in ethanol for10min to convert the bilin chromophores into theirzinc complexes. Although the peptide bands thatcontained bilin chromophores could be detectedvisually after electrophoresis, much greater sen-sitivity was achieved by viewing the fluorescent zinccomplexes under u.v. light, when phycoerythrobilinand phycourobilin exhibit orange and green fluores-cence respectively. At each pH value used forelectrophoresis of the tryptic digest, only one peptidecontaining phycoerythrobilin and one containingphycourobilin could be detected. Similar resultswere obtained when the digests were subjectedto electrophoresis on polyacrylamide disc gels bythe method of Dietz & Lubrano (1967).

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THE NATIVE PHYCOBILIN CHROMOPHORES OF ALGAL BILIPROTEINS

Results and Discussion

To simplify discussion, the phycobilins are con-sidered individually.Phycoerythrobilin

This red phycobilin imparts the characteristic redcolour to the phycoerythrins. In 1958, O'hEochareported that careful treatment of phycoerythrinswith cold concentrated HCI releases a chloro-form-soluble pigment from the proteins. The freebilin was considered to be closely related to thenative protein-bound chromophore, and this wasconfirmed later by O'Carra et al. (1964). The acidtreatment, however, resulted in many side reactionsproducing artefacts, so that yields of the acid-released pigment were low.

O'Carra & O'hEocha (1966) showed that treat-ment of phycoerythrins with hot methanol slowlyreleased a more stable derivative of the chromo-phore. This methanol-released pigment was shownto be identical with a bilin extracted from algae withhot methanol and previously considered by Fujita &Hattori (1962, 1963) to be a biosynthetic precursorof phycoerythrobilin. The results of O'Carra &O'hEocha (1966) showed that the extracted pig-ment was a derivative rather than a precursor ofphycoerythrobilin. O'Carra & O'hEocha (1966)considered the acid-released pigment to be muchcloser in properties to the native protein-boundpigment than the methanol-released 'purple pig-ment', which was regarded as a spectrally alteredartefact. However, in much subsequent work byother authors this distinction has been disregarded.Since the methanol-releasing procedure offers amore convenient method of obtaining protein-freepigment, much of the recent structural work(Frackowiak & Skowron, 1978; Gossauer & Weller,1978; Fu et al., 1979) has concentrated on themethanol-released derivative (cf. e.g. Chapman etal., 1967; Crespi et al., 1968).The spectral properties, summarized in Table 1,

clearly demonstrate a distinction between the acid-

released and methanol-released pigment prepara-tions. The two preparations retain their spectraldistinctiveness through a variety of derivatizationprocedures, e.g. formation of the hydrochloride andzinc complex (Table 1), and also when convertedinto the dimethyl esters. The pigments as dimethylesters travel with similar RF values when subjectedto t.l.c. on silica-gel G in some solvent systems, andthis may have helped to create the impression thatthey were identical. In other solvent systems,however [benzene/light petroleum (b.p. 100-1200C)/methanol (9:5:1, by vol.); carbon tetra-chloride/acetic acid (1: 1, v/v) carbon tetrachloride/ethyl acetate (1:1, v/v)], the two pigmentpreparations are readily separated.

In Table 1 and Fig. 1 the spectral properties of thereleased pigments are compared with those ofdenatured C-phycoerythrin. Protein-denaturingconditions were used to ensure unfolding of thepolypeptide chains of the phycoerythrin, thus expos-ing the covalent bound chromophore to the samesolvent conditions as for the protein-free-pigment

1.

0O

0..)

0.0 0O

0

.0

i.8-

~.6 %

i.4 -...

i.2-

C. p~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

350 450 550 650Wavelength (nm)

Fig. 1. Absorption spectra in 0.1M HCl of ( )denatured C-phycoerythrin, (----) acid-released phyco-erythrobilin and (....) methanol-released 'purple pig-

ment' derivative ofphycoerythrobilin

Table 1. Spectral absorption maxima ofdenaturedphycoerythrins, phycoerythrin-derived bilins and i-urobilinThe major maxima are italicized and minor maxima or absorption shoulders are in parentheses. The absorptionmaximum at 278 nm is attributable to aromatic amino acids.

Bilin or denatured phycoerythrin

Denatured C-phycoerythrinPhycoerythrobilin (acid-released)'Purple pigment' derivative of phycoerythrobilinDenatured R-phycoerythrini-Urobilin

Chromophore as hydrochloride Chromophore as zinc complexA- A in 8M urea, pH 7.0,

In acidified containing 1% (w/v)In 0.1 M-HCI chloroform zinc acetate

I, A_

287, 307, 556 Insoluble 278, 321, (542), 586307, 556 312, 576 321, (540), 586326, 590 329, 602 340, (564), 609

287, 307, 498, 556 Insoluble 278, 321, 510, (545), 586495 499 510

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P. O'CARRA, R. F. MURPHY AND S. D. KILLILEA

preparations. In the native proteins the chromo-phores are buried in the interior by the folding of thepolypeptide chains and lie in environment(s) dif-ferent from the external medium (O'hEocha &O'Carra, 1961; Lavorel & Moniot, 1962; O'Carra& O'hEocha, 1965; Murphy & O'Carra, 1970).That the denaturing conditions cause no covalentalteration of the bound chromophore is shown bythe fact that C-phycoerythrin readily renatures whenthe denaturing conditions, low pH or 8M urea, areremoved. The spectral properties of the nativeprotein then return in a rapid spontaneous process.

It can be seen that the protein-bound chromo-phore is almost identical in spectral properties withthe acid-released phycoerythrobilin preparations anddiffers considerably from the methanol-releasedpigment. Muckle & Riidiger (1977) also emphasizedthat the methanol-released pigment exhibits spectralproperties, including absorption coefficients, dif-ferent from those of protein-bound chromophore.

PhycocyanobilinThe situation regarding phycocyanobilin and the

blue pigment released from C-phycocyanin by hotmethanol is analogous to that outlined above forphycoerythrobilin and its methanol-released 'purplepigment' derivative. The 'blue pigment' released fromblue-green algae by methanol treatment wasoriginally considered by Fujita & Hattori (1962,1963) to be a biosynthetic precursor of phyco-cyanobilin, but was shown by O'Carra & O'hEocha(1966) to be an artefact derived from protein-boundphycocyanobilin. As can be seen from the spectralproperties recorded in Table 2, this 'blue pigment'differs spectrally from protein-bound phycocyano-bilin. When C-phycocyanin was treated by themethod of O'hEocha (1963) with 12 M-HCI, aphycobilin preparation that absorbed maximally at630-645nm in acidified chloroform was obtained.By t.l.c. analysis this preparation was shown tocontain a mixture of pigments. The predominantcomponent was a blue-green bilin with an absorp-

tion maximum at 630nm, and this is referred to as'phycobilin-630'. It could be further resolved onchromatograms into two closely spaced compo-nents that were identical by other criteria, includingabsorption spectra, acid/base properties, isomeriza-tions in acids and alkali, chemical reactions anddegradation products (Rudiger & O'Carra, 1969).The two forms of phycobilin-630 may reflect spatialisomerism, which does not involve any rearrange-ment of the double-bond system. The other bilincomponents were a violinoid pigment, which may bea hydrated derivative of phycobilin-630 (Murphy,1968), and a bilin, which was chromatographicallyand spectrally identical with the methanol-releasedblue pigment. The proportion of the componentsextracted into chloroform varied with the con-centration of HCl used to treat the C-phycocyanin.Time-course studies with C-phycocyanin and withchromatographically purified phycobilin-630 in HClat various concentrations between 10M and morethan 13M, however, showed that the blue-greenphycobilin-630 was the bilin initially released. In10-12M-HCI it was converted into the blue pigmenttogether with some violin, but, as the concentrationof HCl was increased to over 13M, the conversioninto violin became the major transformation. Theseobservations were confirmed with acid-releasedphycobilin-630 from R-phycocyanin.

Spectral comparison of denatured C-phyco-cyanin with the chromatographically purified phyco-bilin-630 shows that this pigment closely resemblesthe native protein-bound chromophore (Fig. 2, Table2).As for phycoerythrobilin, the structure and

properties currently accepted (Brown & Troxler,1977; Troxler et al., 1978; Frackowiak & Skowron,1978; Gossauer & Hinze, 1978) for phycocyano-bilin (Cole et al., 1967; Crespi et al., 1968) relate tothe methanol-released 'blue pigment'. This is nowseen to be identical with a spectrally altered artefactderived from the native phycocyanobilin chromo-phore. Because of the differences between the

Table 2. Spectral absorption maxima ofdenatured C-phycocyanin and its released bilinsThe major maxima are italicized. To form the zinc complex of the chromophore in C-phycocyanin, the biliproteinwas dissolved in 8 M-urea containing 1% (w/v) zinc acetate. The zinc complex of protein-free bilins was formedin chloroform, which contained a drop of ethanolic zinc acetate and a trace of pyridine to neutralize protons. Theabsorption maximum at 278nm is attributable to aromatic amino acid residues.

Chromophore as hydrochloride

Bilin or biliprotein

Denatured C-phycocyaninPhycocyanobilin (acid-released phycobilin-630)'Blue pigment' derivative of phycocyanobilin

In aq. methanolic In acidified0.1 M-HCI chloroform

11 A-- - -1.

278, -350, 655-660 Insoluble-360, 655 357, 630374, 684 680

Chromophore as zinccomplex in 8 M-urea

or chloroform278,-375 63278, 375, 630

375, 630378, 665

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THE NATIVE PHYCOBILIN CHROMOPHORES OF ALGAL BILIPROTEINS

1.0r

0.8p-

-., I

.. . ..,I'

00

0.Dci

'.4

).2 -/

0-~~~~~~~~

350 450 550Wavelength (nm)

650

Fig. 3. Absorption spectra in 0.1 M HCI of (- )denatured R-phycoerythrin, (----) acid-released phyco-

erythrobilin and (... . ) i-urobilin

300 400 500 600

Wavelength (nm)700 800

Fig. 2. Absorption spectra in 0.1 M HCI of ( )

denatured cryptomonad phycocyanin, (----) denaturedC-phycocyanin and (....) acid-released phycocyanobilin

(phycobilin-630)

spectral properties of the methanol-released pigmentand those of native phycocyanobilin bound to theprotein, Muckle & Rudiger (1977) state that the useof the absorption coefficient of the methanol-releasedpigment to calculate the chromophore content ofphycocyanin can give erroneous results. The phyco-bilin-630 released by brief acid treatment consistspredominantly of a pigment very close in propertiesto the native chromophore.

PhycourobilinThe grounds on which Chapman et al. (1968)

have disputed the suggestions that R-phycoerythrincontains a urobilinoid chromophore (phycourobilin)accompanying phycoerythrobilin may be sum-marized as follows. (1) The spectral properties ofphycoerythrobilin are distorted by the interaction ofthe chromophore with the native protein environmentand thus the peak at 495-598nm might be attribut-able to such non-covalent modulation of the proper-ties of phycoerythrobilin in the native protein. (2)Free phycoerythrobilin readily undergoes covalentconversion into urobilinoid artefacts both by iso-merization and by oxidation reactions, and theappearance of urobilinoid material in denaturedR-phycoerythrin might be attributable to suchartefact material formed while the chromophore isstill attached. (3) Refluxing R-phycoerythrin inmethanol releases only the 'purple pigment' equated

with phycoerythrobilin by Chapman et al. (1967,1968), and no urobilin is released.

In order to check the first two objections, thespectral data shown in Table 1 and Fig. 3 wereobtained with fully denatured R-phycoerythrin inwhich the effects of the native protein environmentare eliminated by unfolding of the polypeptidechains. The possibility that the procedures used forobtaining these spectra might cause covalent modifi-cation of phycoerythrobilin was investigated bysubjecting purified acid-released phycoerythrobilinand denatured C-phycoerythrin to exactly the sameprocedures. Their spectral properties are comparedin Table 1 and Fig. 1. No significant amount ofurobilinoid material was formed in either case.

As Table 1 and Fig. 3 show, a distinctive peak,characteristic of urobilins, persists in the spectrum ofdenatured R-phycoerythrin. Quantitative sub-traction of the absorption spectrum of phyco-erythrobilin, by using an adjusted spectrum ofdenatured C-phycoerythrin, leaves a spectrum al-most identical with that of i-urobilin. Addition ofZn2+ causes the characteristic shift of the uro-bilinoid absorption peak (Table 1) associated withthe formation of the zinc complex. The fluorescencespectrum of the zinc-complexed denatured R-phyco-erythrin shows two fluorescence bands, one (1max520nm) characteristic of urobilin-zinc and the other(Amax. 600nm) attributable to phycoerythrobilin-zinc.To further eliminate any influence of the protein

environment, denatured R-phycoerythrin wasdigested with trypsin. The digestions were carriedout under carefully controlled conditions (neutralpH, exclusion of oxygen) designed to minimize anyisomerization or oxidative conversion of phyco-erythrobilin units into artefact urobilinoid material.Again, the procedures used were fully checked by

Vol. 187

0.8r

0.7 .

0.6 F

0.5~

D 0.40.0

0.3

0.2 F

0.1~

307

P. O'CARRA, R. F. MURPHY AND S. D. KILLILEA

'-20

0 55 60 65 70 75 80 85Fraction no.

Fig. 4. Elution profiles of(@) the phycourobilin- and (0)the phycoerythrobilin-containing chromopeptides in atryptic digest of R-phycoerythrin from a Sephadex G-50

columnA sample (3 ml) of tryptic digest of R-phyco-erythrin was applied to the column (67.5 cm x2.1cm) and equilibrated with phenol/acetic acid/water (1:1:1, by vol.) that had been flushed withnitrogen. Owing to the highly aromatic natureof the bilin chromophores, the tryptic chromo-peptides were absorbed on to Sephadex whensubjected to gel filtration in conventional aqueousbuffers and were eluted much later than the internalvolume of the column. Such adsorption of aromaticcompounds had been reported previously andeliminated with the above solvent systems (Car-negie, 1965; Janson, 1967). Gel filtration wascarried out in the dark and -the flow rate was12ml/h. Fractions (2ml) were collected and made0.1M with respect to HCI. When the absorptionspectrum was determined, the A495 was corrected bysubtracting any absorbance in this region due tophycoerythrobilin, calculated as A,55 x 0.244. Thisfactor was determined from the absorption spectrumof phycoerythrobilin in acid-denatured C-phyco-erythrin. Since phycourobilin does not absorb at555 nm, no reciprocal correction was necessary.

control experiments in which R-phycoerythrin wasreplaced with C-phycoerythrin, and negligible con-version of phycoerythrobilin, the only chromo-phore of C-phycoerythrin, into a urobilin wasobserved. The tryptic digests of R-phycoerythrinretained the same spectral properties as thedenatured biliprotein, and when the peptide mixturewas fractionated on the basis of a number ofindependent properties (e.g. size or charge charac-teristics), a clear-cut separation of the phyco-erythrobilin-containing and phycourobilin-con-taining peptides was observed (Figs. 4 and 5). Theresults show that the two chromophores reside inquite distinct sections of the polypeptide sequence,

o 10

8

61-

E 40

.o 20o

0

4

.4 4

10

Fig. 5. Effect ofpH on the electrophoretic mobility of(-)the phycourobilin- and (0) the phycoerythrobilin-con-taining chromopeptides in tryptic digests of R-

phycoerythrinTryptic digests of R-phycoerythrin were subjected tostarch-gel electrophoresis at several pH values asdescribed in the Experimental section.

further eliminating the possibility that the urobilinmight be an artefact form of phycoerythrobilin.

Refluxing the phycourobilin-containing peptidefraction, isolated by gel-filtration, with methanolicKOH for 5 min released the chromophore andcoverted it rapidly into a green product that wasidentified as the bilin mesobiliverdin. This resultconfirms that phycourobilin is an open-chain lineartetrapyrrole.

Treatment of the phycourobilin-containing pep-tide with hot methanol did not release the pigment.Structural studies show that the attachment ofphycourobilin to the apoprotein is much strongerthan that of phycoerythrobilin (P. O'Carra & S. D.Killilea, unpublished work). Thus the failure ofChapman et al. (1968) to release urobilinoidmaterial by hot methanol is readily explained.

Although the experiments described here providestrong evidence that phycourobilin is a discrete andnatural chromophore of R-phycoerythrin, this con-cept now also receives acceptance by some othergroups (Frackowiak & Skowron, 1978; Glazer &Hixson, 1977).

CryptoviolinAll the cryptomonad phycocyanins in the native

state exhibited an absorption peak at about 588nmin addition to varied spectral characteristics atlonger wavelengths, usually manifested by a peak ateither 615, 630 or 645nm (O'hEocha et al., 1964).

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THE NATIVE PHYCOBILIN CHROMOPHORES OF ALGAL BILIPROTEINS 309

When these phycocyanins are unfolded by de-naturation, the longer-wavelength absorption in allcases collapses to a spectral peak characteristic ofphycocyanobilin. The shorter-wavelength peak alsoshifts, but remains clearly distinct from the phyco-cyanobilin spectrum. Fig. 2 shows the spectrum of atypical denatured cryptomonad phycocyanin indilute acid; all cryptomonad phycocyanins givealmost identical spectra under such denaturingconditions. The short-wavelength absorption peak isat 6lOnm under these conditions, but if the spectrumis analysed by subtracting out the phycocyanobilinabsorption spectrum with an adjusted spectrum ofacid-denatured C-phycocyanin, the true absorptionmaximum of the peak is found to be about 590nm.This maximum and the shape of the analysed peakcoincides exactly with the absorption characteristicsof the class of bilins known as violins; the absorp-tion maximum of mesobiliviolin in dilute acid is at590-600nm.The bilin nature of the extra chromophore in

cryptomonad phycocyanin was indicated by theobservation that both it and the phycocyanobilinchromophore were converted into mesobiliverdinwhen samples of the phycocyanin were refluxed inmethanolic 1 M-KOH for 30min.The extra chromophore seems clearly to have a

violinoid conjugated system and it is tempting tosuggest the term phycoviolin, which would be in linewith the terminology of the other phycobilins. Asimilar term, phycobiliviolin, has been used byRiudiger (1971) for the 'purple pigment' derivative ofphycoerythrobilin, so, to avoid confusion, it issuggested that the violinoid chromophore of crypto-monad phycocyanin be termed cryptoviolin.

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Brown, A. S. & Troxler, R. F. (1977) FEBS Lett. 82,206-210

Carnegie, P. R. (1965) Biochem. J. 95, 9pChapman, D. J., Cole, W. T. & Siegelman, H. W. (1967)

J. Am. Chem. Soc. 89, 5976-5977Chapman, D. J., Cole, W. T. & Siegelman, H. W. (1968)

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