Peridinin-Chlorophyll a Proteins of the DinoflagellateAmphidinium carterae (Plymouth 450)1
Received for publication September 19, 1975 and in revised form October 24, 1975
FRANCIs T. HAXO,2 J. HELEN KYcIA, G. FRED SOMERS,3 ALLEN BENNEIT,4 AND HAROLD W.SIEGELMANDepartment of Biology, Brookhaven National Laboratory, Upton, New York 11973
ABSTRACT
The marine dinoflageDlate Amphidinium carterae (Plymouth 450) re-leases several water-soluble peridinin-chlorophyDl a proteins after freeze-thawing. These chromoproteins have a molecular weight of 39.2 x 103and are comprised of noncovalently bound peridinin and chlorophyD aand a nonoligomeric protein. They have distinct isoelectric points andmay be resolved into six components by either isoelectric focusing onpolyacrylamide gel or ion exchange chromatography. The predominantchromoprotein, which has a pl of 7.5, constitutes about 90% of theextractable peridinin-chlorophyll a protein. It consists of an alanine-richapoprotein of molecular weight 31.8 x 103 stoichiometricaly associatedwith 9 peridinin and 2 chlorophyUl a molecules. Additionally, the peridi-nin-chlorophyl a proteins with pI values of 7.6 and 6.4 were purified andfound to have amino acid and chromophore composition essentialy identi-cal with the pl 7.5 protein. Peridinin-chlorophyl a protein, pI 7.5, aftertreatment at aLkaline pH was transformed into several more add pI formsof the protein, strongly suggesting that the naturally occurring proteinsare deamidation products of a single protein. Fluorescence excitation andemission spectra demonstrate that light energy absorbed by peridinininduces chlorophyl a fluorescence presumably by intramolecular energytransfer. The peridinin-chlorophyll a proteins presumably function in vivoas photosynthetic light-harvesting pigments.
Several photosynthetic light-harvesting pigments, includingchlorophyll a and the algal bile pigments, are conjugated in vivowith specific proteins (13, 39, 41). Data reported in pioneeringstudies by Schutt (32) suggested that at least a portion of thecharacteristic dinoflagellate carotenoid, peridinin, is protein-bound. His examination of dilute alcoholic extracts of mixeddinoflagellates, which were collected from a natural bloom, ledto the discovery of peridinin, whose structure has only recentlybeen elucidated (38). Aqueous extracts of such collectionsyielded an orange, water-soluble fraction, phycopyrrin, whichcould be precipitated by heat. Schutt emphasized the presenceof Chl in phycopyrrin. Re-examination of his spectral data showa broadened absorption in the blue-green region of the spectrumimplying the occurrence of substantial amounts of peridinin.
1 Research was carried out at Brookhaven National Laboratory un-der the auspices of the United States Energy Research and Develop-ment Administration. A. Bennett was supported by a postdoctoralfellowship from the National Institutes of Health.
2 Permanent address: Scripps Institution of Oceanography, Univer-sity of California, San Diego, Calif. 92093.
3Permanent address: University of Delaware, Newark, Del. 19711.4Present address: University of Miami School of Medicine, Biscayne
Annex, Miami, Fla. 33152.
Bode (3) and Bode and Hastings (4) subsequently found thatclarified aqueous extracts of Gonyaulax polyedra were red.They partially purified the chromoprotein and established thatthe red color was due to peridinin bound to protein. Haidak etal. (9) further demonstrated that the G. polyedra chromoproteinhad a molecular weight of about 38,000, and that it containedboth peridinin and Chl a. Peridinin-Chl a proteins have alsobeen obtained from other unialgal dinoflagellate cultures andfrom the endosymbiotic dinoflagellates ofthe giant clam. Prelim-inary studies (14) showed that these chromoproteins containedperidinin, Chl a, and protein in stoichiometric ratios and ex-hibited efficient sensitization of Chl a fluorescence by peridinin-absorbed light. Each of these chromoproteins chromatographedas reasonably symmetrically eluting species on Sephadex, butcould be electrophoretically resolved into a number of compo-nents, all containing peridinin and Chl a.The marine dinoflagellate Amphidinium carterae (Plymouth
450) was selected for detailed studies. A. carterae is easilycultured on a large scale and provides high yields of the peridi-nin-Chl a proteins predominantly as a single component. Ityields a set of six PCPs5 of about 39,000 mol wt, and this reportis concerned with the detailed properties of three of the PCPs.
MATERIALS AND METHODS
Reagents. Diethylaminoethyl cellulose (DE-52, Whatman)was obtained from Reeve Angel, Sephadex gels from PharmaciaFine Chemicals, and all antibiotics were from Sigma ChemicalCo. Ampholines were purchased from LKB Produkter. Acryl-amide and methylene bisacrylamide were recrystallized fromchloroform and acetone, respectively (23). All other chemicalswere reagent grade and are commonly available.
Culture. Amphidinium carterae (Plymouth 450) was origi-nally isolated by Dr. Mary Parke and was obtained from Dr. L.Provasoli, Haskins Laboratory, Yale University. It was origi-nally identified as A. hoefleri by Dr. D. L. Taylor and subse-quently designated A. carterae (Plymouth 450) (personal com-munication; ref. 40). The alga was grown in ASP7 medium ofProvasoli (29) supplemented with 10 ml of soil extract and45 mg of KNOJA and adjusted to pH 7.9 with I N HCI prior toautoclaving. The soil extract was prepared by autoclaving I kgof forest soil with I liter of H20 for 45 min at 15 p.s.i. Themixture was allowed to stand overnight and centrifuged at2000g. The liquid phase was filtered through Whatman No. Ifilter paper, autoclaved as before, and stored at 4 C. Theaddition of a soil extract to the medium improved cell yields,and the cells did not clump, an advantage here in making cellcounts. Small cultures were grown at 20 C on 14-hr light and 10-hr dark cycles with an illuminance of about 250 ft-c (about 1200.tw cm-2) of cool white fluorescent light. Large scale cultures
5 Abbreviation: PCP: peridinin-chlorophyll a protein.
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were grown at 20 C and were continuously illuminated utilizingprocedures described by Siegelman and Guillard (34). The largescale culture medium was either autoclaved in 18-liter lots or
pasteurized by heating to 80 C twice in the culture container.It proved possible to clone A. carterae and to free it from
contaminating microbes by the addition of antibiotics to ASP7medium in conjunction with plating. Agar plates (0.5%, w/v)were prepared according to the procedure of Allen (1). Concen-trated antibiotic solutions were dissolved in a small volume ofASP7 medium at room temperature, sterilized by passage
through a 0.22-,um Swinnex Millipore filter, and then added towarm (40 C) culture medium immediately prior to pouring theplates. Antibiotics were routinely used at the following concen-
trations per ml of medium: K penicillin G, 50 units: chloram-phenicol, 0.5 ,ug; neomycin, 2.5 jag; polymixin B, 1.5 units;dihydrostreptomycin, 50 ,ug: and tetracycline, 2.5 ,ug/ml ofmedium (30). The substantial tolerance of A. carterae to growthin the presence of antibiotics permitted the concentrations ofantibiotics to be increased 5- to 10-fold if desired.
Harvesting. The algal cells were collected during late logphase growth. Cell proliferation was measured by countingformalin-fixed culture aliquots in a hemacytometer. Prior toharvesting, the cultures were adjusted to pH 8.4 by addition of IM tris base and 0.2M KAI(SO4)2 (alum) added to give a finalconcentration ofI mm KAI (SO4)2. The addition of KAI(SO4)2caused the cells to aggregate and settle rapidly to the bottom ofthe culture vessel. The addition of the tris base compensated forthe acidity of the KAI(SO4)2, maintaining a final pH of 7.9. TheKAI(SO0)2 aggregation procedure does not alter the extractabil-ity or multiplicity of the PCPs in comparison with untreatedcells. The use of KAI(SO)2 increased the apparent fresh weightof the cells by a factor of about 2, presumably by the coprecipi-tation of phosphate as an aluminum phosphate. Following alumprecipitation of the cells, the bulk of the culture medium was
removed with the aid of a mechanical pump. The aggregatedcells were then allowed to settle in a series of progressivelysmaller glass cylinders at room temperature, and the culturemedium removed by decantation after each settling step. About3 hr were required for these operations. Finally, the thick slurryof cells was centrifuged at15OOg for 10 min at 4 C.
Purification. Alum-harvested cells from about 300 liters ofculture having cell densities of about 2.7 x105 cells ml-' weregently dispersed in 140 ml of cold 0.1M tris-HCI buffer, pH 8.4.The slurry was centrifuged at15OOg for 10 min at 5 C, and thesupernatant was discarded. The sedimented cells were resus-pended in 225 ml of 0.1M tris-HCI buffer, pH 8.4. One-third ofthe cell suspension was added dropwise to a large porcelainmortar (Coors, size 6) containing liquid nitrogen. The frozenbeads were ground to a very fine powder in the mortar. Thepowder was thawed at room temperature, and the resultingextract was centrifuged at10,800g for 20 min at 5 C. Thesedimented cells were extracted a second time as describedabove. All purification procedures following the second extrac-tion were carried out at 5 C. The combined extracts wereconcentrated by ultrafiltration (5) to about 50 ml. The darkorange-colored concentrate was subsequently applied to a Seph-adexG-100 (coarse) column (9 x 90cm) equilibrated with1 mMtris-HCl buffer, pH 8.4 (Fig. 1). The column was eluted withbuffer at a pressure head of 45 cm and a flow rate of 1.3 mlmin-'. The principal PCP fraction which eluted off the Sepha-dexG-100 column was then applied to a DEAE-cellulose col-umn (4.25 x 40 cm) equilibrated withI mm tris-HCI buffer, pH8.4. The column was eluted with a linear gradient mixer contain-ing a starting buffer of 2 liters ofI mm tris-HCI, pH 8.4 and a
limit buffer of 2 liters ofI mm tris-HCI, pH 8.4, containing 0.05NaCI followed by a second linear gradient comprised of a
starting buffer of 250 ml ofI mm tris-HCI, pH 8.4, containing0.03M NaCI and a limit buffer of 250 ml ofI mm tris-HCI,pH
8.4, containing 0.25 M NaCl to complete the separation andelution of the chromoproteins. Flow rates were maintained at150 ml hr-' with a peristaltic pump. Selected fractions of theeluate were combined for further purification.The PCPs which eluted ahead of the principal PCP band (Fig.
2) from three large DEAE-cellulose columns were combined.The sample was dialyzed against 1 mm tris-HCI buffer, pH 8.6,and applied to a column (1.25 x 30 cm) of DEAE-celluloseequilibrated with 1 mm tris-HCI buffer, pH 8.6. The column waseluted using a linear gradient mixer containing a starting bufferof 600 ml of 1 mm tris-HCI, pH 8.6, and limit buffer of 600 ml of1 mm tris-HCI, pH 8.6, containing 0.05 M NaCl. Flow rateswere maintained at 48 ml hr-' with a peristaltic pump. Follow-ing ultrafiltration and dialysis of selected fractions, further puri-fication was achieved by a second pH 8.6 DEAE-cellulosecolumn operated exactly as described above. Similarly, thePCP band which emerged as a sharp peak at the end of thegradient (Fig. 2) from three large DEAE-cellulose columns wascombined. It was chromatographed as described above for theearly eluting PCPs, except that the column was eluted with alinear gradient mixer containing a starting buffer of 1 liter of 1
mM tris-HCI, pH 8.4, containing 0.02M NaCI and limit buffer of1 liter of1 mm tris-HCI, pH 8.4, containing 0.20M NaCl.
Analytical Procedures. Analytical gel filtration followed theprocedure of Andrews (2), using SephadexG-100 (superfine) ina column (2 x 53 cm) equilibrated and eluted with 1 mm tris-HCI buffer, pH 8.4, with 0.2M NaCI at a pressure head of 20cmand a flow rate of 6 ml hr-'. Isoelectric focusing on polyacryl-amide gels employed the procedure of Rhigetti and Drysdale(31). SDS polyacrylamide gel electrophoresis was performed bythe method of Weber et al. (42). Gels were scanned with a
Gilford densitometer. Absorption spectra were measured with aCary 14 spectrophotometer and fluorescence spectra in a Hita-chi-Perkin Elmer MPF-4 spectrophotofluorometer. Amino acidanalyses were performed on hydrolysates either prepared withconstant boiling HCI or with 4M methane sulfonic acid by themethod of Liu and Chang (22) in evacuated tubes held forvarying times at 115 C. Performic acid oxidation was by themethod of Hirs (15). Following methane sulfonic acid hydroly-sis, the sample was adjusted to pH 2.1 by addition of 2 N NaOHwithout concentration. The hydrolysates were analyzed by thegeneral method of Spackman et al. (36) on amino acid analyzersequipped with automatic sample injection and computerizeddata processing.
Analyses for content of peridinin and Chi a content were
performed on protein samples of known amino acid composi-tion. Samples containing 5 nm of protein were freeze-dried,extracted four times with either methyl alcohol or 90% acetone,and brought to a final volume of 3 ml. These operations werecarried out rapidly under low illumination. The absorption spec-tra of the individual extracts were measured immediately usingstoppered 1-cm quartz cuvettes. In the case of Chi a, concentra-tions were calculated directly from the absorbance of the redabsorption peak, where peridinin absorption is negligible. Theextinction values used for Chl a were: E%m = 876.7 at 664 nm in90% acetone (18) andElm = 798.1 at 668 nm in methyl alcohol(S. W. Jeffrey, personal communication). The extinction valuesused for peridinin were:El-cm = 1330 at 469 nm in 90% acetoneandEl m = 1360 at 469 nm in methyl alcohol (17). Correctionsfor Chi a Soret absorbance in the determination of peridininwere made by use of the following equations derived by theprocedure outlined by French (6) for two pigment mixtures:peridinin in 90% acetone (Ag ml-') = 7.53E469nm - 0.124E664 nm; peridinin in methyl alcohol(j,g ml-1') = 7.36 E469 nm -
0.134 E668nm. The mol wt of peridinin used in calculations ofmolar concentrations was 630.35 (38).
Chemical deamidation of PCPs was by the method of Funa-koshi and Deutsch (8) using "universal" buffer (0.029M in
boric, citric, and 5,5'-diethyl barbituric acids and KH2PO4)adjusted to pH 11.5 at 4 C with 2 M NaOH or with I mM tris-HCIbuffer, pH 8.4. Alkaline-treated PCPs were held at 4 C for I to 8days and then passed through a Sephadex G-100 column (2.5 x90 cm) equilibrated with I mm tris-HCI, pH 8.4, for bufferexchange and mol wt determination. The treated PCPs werethen examined by isoelectric focusing and SDS-polyacrylamidegel electrophoresis, amino acid analysis, and absorption spec-troscopy.
RESULTS
Large scale culture of Amphidinium carterae (Plymouth 450)in polyethylene drums (150 liters) yielded about 100 g freshweight of algae after 10 days growth. Under the growth condi-tions employed, the cell doubling time was 3 days. Late logphase cell densities in large cultures were about 2.7 x 105 cellsml-'. The extraction of soluble proteins from the alga was mostconveniently achieved by repeated grinding of the cells withbuffer at liquid N2 temperature. Following grinding, thawing,and centrifugation, a clarified dark orange crude extract wasobtained. During subsequent chromatographic procedures, thePCPs were detected by their absorbance at 476 nm and byisoelectric focusing on polyacrylamide gels.Three PCP components from A. carterae were readily ob-
tained. A summary of the purification of the PCP-pI 7.5 compo-nent is shown in Table I. Crude extracts were concentrated andsubstantially freed from contaminants having 260 nm absorb-ance by ultrafiltration. Gel filtration of the concentrated crudeextract on Sephadex G-100 resolved three distinct peaks (Fig.1). The first peak which eluted from the column was paleorange, and it has not been characterized in any detail. Thesecond peak which eluted was dark orange and consisted of thePCPs. The third peak was orange-pink and was identified as Cytf (25). The PCPs from the preparative Sephadex G-100 columnwere examined by analytical gel filtration and were found tohave a mol wt of about 39,000 in comparison with mol wtmarker proteins.A densitometer scan of a pH 9 to 5 isoelectric focusing gel
separation of the PCPs from the preparative Sephadex G-100column is shown in Figure 3 and consists of six components.The gel was scanned at 476 and 669 nm, and all colored compo-nents present had both peridinin and Chl absorbance. The mainPCP component had a pl of 7.5, and it accounted for about 90%of the total PCPs. The PCPs from the preparative Sephadex G-100 column were applied to a column of DEAE-cellulose at pH8.4 and resolved by elution with a shallow linear NaCl gradient(Fig. 2). They were separated into six component PCPs. AfterDEAE-cellulose chromatography, the main PCP component (pl7.5) comprised a single band on isoelectric focusing polyacryl-amide gels as determined by a densitometric scan for eitherperidinin or Coomassie blue stain absorbance (Fig. 4). Follow-ing repeated DEAE-cellulose column chromatography at pH8.6 of the minor PCP fractions, a single PCP was obtained witha pI of 7.6. Similarly, repeated DEAE-cellulose column chroma-tography at pH 8.4 of the PCP which eluted as a sharp band at
Table I. Purification of Peridinin-Chlorophyll a Protein-pl 7.5 fromabout 8 x 10'° Cells of Amphidinium carterae (Plymouth 450)
FIG. 1. Gel filtration of an extract of Amphidinium carterae on acolumn (9 x 90 cm) of Sephadex G-100 with I mM tris-HCI buffer, pH8.4. Absorbance at 476 nm (0), 280 nm (0), and 415 nm (A).
,
4.0-
3.0-9000 1800 2700 30 0.1>-
4 4r2.0 o
In
1.0
9000 1800 2700 3600ml
FIG. 2. Chromatography of the pefidinin-Chl a proteins on a column(4.25 x 40 cm) of DEAE-cellulose equilibrated with 1 mm tfis-HCIbuffer, pH 8.4, eluted with a linear gradient of NaCI. Absorbance at 280nm (0), NaCl molarity (0).
the end of the gradient of the large DEAE-cellulose column(Fig. 2) provided a single PCP with a pI of 6.4. The purifiedPCP-pI 7.6 and pl 6.4 components were judged to be homogene-ous following isoelectric focusing on polyacrylamide gels andCoomassie blue staining. The three remaining PCPs were notavailable in sufficient amounts for further analyses.The purified PCP-pI 7.6, pI 7.5, and pI 6.4 PCP components
were examined on SDS polyacrylamide gels. They all yielded asingle band with a mol wt of about 32,000 for PCP-pI 7.5.Treatment with SDS released the noncovalently linked peridi-nin and Chl a chromophores from the proteins, and Coomassieblue staining was used to visualize the polypeptides.An absorption spectrum of the PCP-pI 7.5, is shown in Fig. 5.
The absorption spectra of the PCP-pI 7.6 and pI 6.4 wereidentical. Absorption maxima occurred at 293, 435, 476, and 669nm. The purified chromoproteins had a 476/283 absorbanceratio of 4.5 in I mm tris-HCl buffer, pH 8.4. The absorptionmaxima at 435 and 669 nm were attributed to the Soret and the abands of Chl a, respectively. The broad absorption band with amaximum at 476 nm is due to peridinin. The extinction coeffi-
FIG. 3. Electrophoresis of the peridinin-Chl a proteins by isoelectricfocusing on polyacrylamide gel. The pI values for each peak are indi-cated. Absorbance at 476 nm ( ), 669 nm (-- -), and pH (@ - ).
1.60
1.201
Lii
z4
r 0.800en
0.400O
2 4 8
detail (19, 20). Spectral identity of the other purified PCPs withPCP-pI 7.5 clearly indicated that peridinin and Chi a were theonly chromophores of the purified PCPs. Quantitive analyses ofthe chromophore composition of PCP-pI 7.5 extracted with 90%acetone or methyl alcohol gave the following results expressedas molar ratios of protein-Chi a-peridinin: 90% acetone (averageof three determinations), 1:2.08:9.25: methyl alcohol (averageof two determinations), 1: 1.96:8.67: mean values, 1:2.02:8.96:integer values, 1:2:9.The fluorescence excitation and emission spectra of the PCP-
pl 7.5 in I mm tris-HCI buffer, pH 8.4, are shown in Figure 6.The concentration of the chromoprotein used for measurementof fluorescence spectra was I ±g ml-'. Energy transfer to Chi ais evident from the excitation spectrum between 240 and600 nm with excitation maxima at 438 and 478 nm. The fluores-cence emission maximum at 672 nm is due to Chl a. Excitationin the 260 to 340 nm region is noteworthy.The amino acid compositions of the three purified PCPs are
shown in Table II. They are rich in alanine and lack cysteine.The effect of alkaline treatments on the PCPs was monitored
by several analytical procedures. Following a 24-hr exposure ofPCP-pI 7.5 at 4 C to "universal buffer," pH 11.5, PCPs with plvalues of 6.9 and 5.4 were observed, in addition to the originalPCP-pI 7.5. After 8 days of exposure, a marked change in thedistribution of the pl values of the PCPs was observed (Fig. 7).The new and more acidic pI forms of the PCPs had the sameamino acid composition, absorption spectra, and mol wt, asdetermined by SDS polyacrylamide gel electrophoresis andSephadex G-100 gel filtration, as the original PCP-pI 7.5 fromwhich they were derived. Incubation of the PCPs for 14 days at4 C with the universal buffer resulted in considerable loss ofperidinin and Chl a chromophores and formation of a proteinaggregate of about 2.5 x 105 daltons. Incubation of PCP-pI 7.5
6
<D
mtr-0
azpH
4
2
cm
FIG. 4. Electrophoresis of peridinin-Chl a protein-pI 7.5 by isoelec-tric focusing on polyacrylamide gel. Absorbance at 476 nm ( ) andpH (-4*).
cients of the PCP-pI 7.5 in 1 mm tris-HCI buffer, pH 8.4 are:
E°9'% = 4.67 at 283 nm, E° '7 = 21.7 at 476 nm and E-19C = 5.3at 669 nm. Thin layer chromatographic and spectral examina-tion (16) of the pigments from the PCP-pI 7.5 component andfrom the PCPs from the preparative Sephadex G-100 columnrevealed only two components, peridinin and Chl a. Identifica-tions were based on spectral properties, RF values and cochro-matography with the full complement of A. carterae pigmentextracts, the composition of which has recently been studied in
FIG. 5. Absorption spectrum of peridinin-Chl a
20 C in 1 mm tris-HCI buffer, pH 8.4.
9
z
bi. 4-
IJ3Lii
260 300 340 380
protein-pI 7.5 at
420 460 500 540 580 660 700WAVELENGTH (nm)
FIG. 6. Fluorescence excitation and emission spectra of peridinin-Chl a protein-pI 7.5. Quantum corrected excitation spectrum is shownfrom 260 to 600 nm. Emission spectra are uncorrected and are shownfor 479 nm excitation (upper) and 330 nm excitation (lower).
Table II. Amino Acid Composition of Peridinin-Chl a Proteins of Amphidinium carterae (Plymouth 450)Protein hydrolysates were prepared with either constant boiling HCI or 4.0 M methane sulfonic acid (23). The average values of the hydrolysates
were determined from 6 analyses of PCP-pI 7.6, 15 of PCP-pI 7.5, and 20 of PCP-pI 6.4. Linear extrapolations to zero time of hydrolysis werecalculated from the 22, 48, and 72 hr values for serine and threonine to correct for decomposition. The values for valine and isoleucine were from %hr hydrolyses. Tryptophan was determined from methane sulfonic acid hydrolysis (23), and cysteic acid and methionine were determined followingperformic acid oxidation (16).
PCP-pI 7.6 PCP-pI 7.5 PCP-pI 6.4
Average Average Average
Hydrolysate integral Hydrolysate integral Hydrolysate integral
Amino acid moles/mole protein ratios moles/mole protein ratios moles/mole protein ratios
Molecular weight without chromophores 31.8 x 103 daltons
Molecularweightwithchromophores39.2x103daltonsMolecular weight with chromophores 39.2 x 10 daltons
in I mM tris-HCI buffer, pH 8.4, at 4 C for 8 days resulted in theformation of PCPs with lower pl values.
DISCUSSION
Several groups of algae, including dinoflagellates, diatoms,and brown algae, utilize carotenoids as light-harvesting pig-ments for photosynthesis. They apparently can make efficientuse of light energy absorbed by two carotenoids, peridinin andfucoxanthin (10-12). Successful isolation of soluble caroteno-proteins from algae has thus far been achieved only from dino-flagellate algae (9, 14, 26, 27, 28). The PCPs of Amphidiniumcarterae (Plymouth 450) comprise a characteristic set of pro-teins having essentially identical molecular weights, chromo-phore, and amino acid composition but differing isoelectricpoints. They can be separated either by isoelectric focusing onpolyacrylamide gels or by ion exchange chromatography. ThePCPs consist of six chromoproteins with pI values ranging from7.9 to 6.4, PCP-pI 7.5 accounting for greater than 90% of thetotal when the alga is cultured under the conditions described.There are several examples of multiple forms of monomeric
proteins (7, 8, 21, 35). The protein heterogeneity observed isassociated with progressive deamidation which results in moreacidic forms of the protein. The conversion of PCPs to more
acidic forms by alkaline treatment clearly implies that the multi-plicity observed in the PCPs of A. carterae is due to partialdeamidation. The constancy of the isoelectric focusing behaviorof the PCPs on polyacrylamide gel electrophoresis suggests thatthe observed multiplicity is associated with an enzymic deami-dation. It was observed that if A. carterae cells are stored at-15 C for 7 days after harvest, the PCPs on extraction had a
markedly changed distribution, on isoelectric focusing on poly-acrylamide gels, to more acidic pl values.
Several other lines of evidence indicate that the multiplicityof PCPs from Amphidinium carterae is not artifactual. Precau-tions were taken to utilize only bacteria-free cultures of the algain good physiological condition. Freshly harvested cells wereextracted as rapidly as possible and all subsequent fractiona-tions were carried out in the cold. To inhibit proteolytic en-zymes and deamidation, cell extracts were made in the pres-ence of 2-mercaptoethanol, 6-aminohexanoic acid, asparagine,glutamine, disopropylphosphofluoridate, phenylmethylsulfon-ylchloride, and HgCI2 in 0.1 M tris-HCI buffer, pH 8.4, invarious combinations. All such treatments failed to alter theisoelectric focusing patterns observed on polyacrylamide gels.Extracts of 40 different A. carterae clones showed qualitativelyidentical PCP banding patterns upon isoelectric focusing on
polyacrylamide gels. Furthermore, the separation of the PCPs
FIG. 7. Electrophoresis of perdinin-Chl a protein-pI 7.5 held for 8days in universal buffer, pH 11.5 at 4 C by isoelectric focusing onpolyacrylamide gel. Absorbance at 476 nm ( ) and pH( -4).
by two different procedures, namely isoelectric focusing on
polyacrylamide gels and ion exchange chromatography onDEAE-cellulose, gave similar separations. The A. c arteraePCPs following storage for 2 yr at -15 C in 5% sucrose-l mMtris-HCI buffer, pH 8.4, were identical in isoelectric focusingpatterns with freshly extracted PCPs. In view of all theseconsiderations, we believe that the intrinsic and constant multi-plicity of the PCPs represents the native state of these proteinsin the dinoflagellate cell.The availability of large amounts of PCP-pI 7.5 permitted a
detailed examination of the composition and properties of theprotein. The molecular weight of PCP-pI 7.5 is about 39.2 x 103(Table II) and it consists of a single polypeptide chain of about31.8 x 103 daltons to which nine peridinin and two Chi a
chromophores are noncovalently bound. The chromophores areeasily removed by lipophilic solvents such as hexane, acetone,or methyl alcohol, or on treatment of the PCP with SDS. ThePCPs contain about 19%, by weight of chromophores. The watersolubility of the protein and the lipophilic character of thechromophores suggest that the chromophores may be internallycomplexed.
Additionally, PCPs-pI 7.6 and 6.4 were purified and com-
pared with PCP-pI 7.5. They all had identical absorption spectraindicating that each PCP contained the same numbers and kindsof chromophores. Detailed amino acid analyses (Table II) of thethree PCPs revealed no significant differences in composition.The remaining three PCPs were present only in very smallamounts but had closely similar mol wt as evidenced by gelfiltration and SDS polyacrylamide gel electrophoresis. Carbohy-drates were not detected in PCPs by the phenol-sulfuric acidtest and no amino sugars were detected by amino acid analysis.The absorption spectra of the PCPs have distinctive proper-
ties associated with the peridinin and Chl a chromophores.Following the first gel filtration purification step, the absorptionspectra of the unresolved PCPs in the visible region of thespectrum was identical with that of purified PCP-pI 7.6, PCP-pI7.5 and PCP-pI 6.4. The general absorbance properties of theChi a of the PCPs are similar to those of other Chl a holo-chromes (39, 41). The stability of the Chl a and perdinin in PCP
is remarkable. No degradation products of the PCP pigmentswere observed by TLC analyses of freeze-dried samples storedin the dark at -15 C over a 2-year period (S. W. Jeffrey and F.T. Haxo, unpublished data).The corrected fluorescence excitation spectra of PCP-pI 7.5
shown in Figure 5 indicates that energy absorbed by bothperidinin and Chl a can be effectively transferred to and excitethe a band fluorescence of Chl a. There is apparently nofluorescence of peridinin in the PCPs. In endosymbiotic dinofla-gellates, Shibata and Haxo (33) demonstrated that the in vivoexcitation spectrum of Chl a a band fluorescence has a maxi-mum in the region of 450 to 540 nm, in correspondence with theaction spectra of photosynthetic 02 evolution in this spectralregion and peridinin absorption (10).The exact cellular location of the PCPs is not yet defined, but
localization within the chloroplast can be strongly inferred fromthe high photosynthetic effectiveness of peridinin-absorbedlight in dinoflagellates known to contain appreciable amounts ofPCP, e.g., Gonyaulax polyedra (12, 26) and in Glenodium sp.(26).
Each dinoflagellate species examined appears to have aunique and distinctive set of PCPs as shown, e.g., isoelectricfocusing on polyacrylamide gels. The PCPs isolated from dino-flagellates likely represent an important class of light-harvestingpigments for photosynthesis (cf. 41). They contribute signifi-cantly to cellular absorption and photosynthetic activity in theportion of the solar spectrum which has maximum penetrationin the water column, e.g. in G. polyedra (12, 26) and Glenodi-nium sp. (26). This is further evidenced by the recent study ofPrezlin et al. (28) which demonstrated, in the case of Glenodi-nium sp., that the fractional absorption by peridinin, in the formof photosynthetically active PCP, increases markedly undergrowth conditions of low light. Thus, the dinoflagellates cancontinue photosynthetic processes through possession of spe-cial light-harvesting pigments which are suitable for effectivelight absorption in diminished and selective light fields, as atdepths in the oceanic environment. This capability may be offurther importance to dinoflagellates during vertical migrations,such as those to depths at which other types of algae maybecome light-limited for photosynthesis, or in survival in deepgrowing corals which contain endosymbiotic dinoflagellates.
Acknowledgments- We thank N. Alonzo for performing the amino acid analyses anddeterminations of chromophore content. We are indebted to L. Provasoli for originally suggest-ing the use of Amphidinium carterae (Plymouth 450) and for advice on its culture.
LITERATURE CITED
I. ALLEN, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates.J. Phycol. 4: 1-4.
2. ANDREWS. P. 1965. The gel filtration behavior of proteins related to their molecular weightsover a wide range. Biochem. J. 96: 595-605.
3. BODE. V. C. 1961. The bioluminescent reaction in Gonvyaulax polvedra: a system forstudying the biochemical aspects of diurnal rhythms. Ph.D. thesis. University of Illinois.Urbana.
4. BODE, V. C.. ANDJ. W. HASTINGS. 1963. The purification and properties of the biolumines-cent system in Gonvuulax polvedra. Arch. Biochem. Biophys. 103: 488-499.
5. CRAIG. L. C. 1967. Techniques for the study of peptides and proteins by dialysis anddiffusion. Methods Enzymol. II: 87()-905.
6. FRENCH. C. S. 1960. the chlorophyl;s it iiro and in *itro. In: W. Ruhland, ed., Encyclope-dia of Plant Physiology. Vol. 1. Springer-Verlag, Berlin. pp. 252-297.
7. FLATMARK, T. 1966. The heterogeneity of beef heart cytochrome c. 111. A kinetic study ofthe nonenzymic deamidation of the main subfractions (Cy I-Cy 111). Acta Chem. Scand.20: 1487-1496.
8. FUNAKOSHI, S. AND H. F. DEUTSCH. 1969. Human carbonic anhydrases. 11. Some proper-ties of native isozymes generated in *itro. J. Biol. Chem. 244: 3438-3446.
9. HAIDAK, D. J., C. K. MATHEWS. AND B. M. SWEENEY. 1966. Pigment protein complexfrom Gon!(aulax. Science 152: 212-213.
10. HALLDAL. P. 1968. Photosynthetic capacities and photosynthetic action spectra of endo-zoic algae of the massive coral Fai-ia. Biol. Bull. 134: 411-418.
11. HALLDAL, P. 1970. The photosynthetic apparatus of microalgae and its adaptation toenvironmental factors: In. D. Halidal, ed., Photobiology of Microorganisms. Wiley-Inter-science, London. pp. 17-55.
12. HAXO, F. T. 1960. The wavelength dependence of photosynthesis and the role of accessory
pigments. In: M. B. Allen. ed.. Comparative Biochemistry of Photoreactive Systems.Academic Press, New York. pp. 339-360.
13. HAXO. F. T. AND OHEOCHA. 1960. Chromoproteins of algae. In: W. Ruhland. ed.,Encyclopedia of Plant Physiology. Vol. 1. Springer-Verlag. Berlin. pp. 497-510.
14. HAXO. F. T., J. H. KYCIA. H. W. SIEGELMAN. AND G. F. SOMERS. 1972. Carotenochloro-phyll proteins from dinoflagellates. Third Int. Symp. Carotenoids (Cluj) (abstr). p. 73.
15. HIRS. C. H. W. 1967. Performic acid oxidation. Methods Enzymol. II: 197-199.16. JEFFREY. S. W. 1968. Quantitative thin layer chromatography of chlorophylls and carote-
noids from marine algae. Biochim. Biophys. Acta 162: 271-285.17. JEFFREY. S. W. AND F. T. HAXO. 1968. Photosynthetic pigments of symbiotic dinoflagel-
lates (zooxanthellae) from corals and clams. Biol. Bull. 135: 149-165.18. JEFFREY, S. W. AND G. F. HUMPHREY. 1975. New spectrophotometric equations for
determining chlorophylls a, b, c, and c2 in algae, phytoplankton, and higher plants.Biochem. Physiol. Pflanzen 167: 191-194.
19. JEFFREY, S. W., M. SIELICKI, AND F. T. HAXO. 1975. Chloroplast pigment patterns indinoflagellates. J. Phycol. 11: 374-384.
20. JOHANSEN, J. E., W. A. SVEC, S. LIAAEN-JENSEN, AND F. T. HAxo. 1974. Carotenoids ofthe dinophyceae. Phytochemistry 13: 2261-2271.
21. LEER. J. C. AND K. HAMMER-JESPERSEN. 1975. Multiple forms of phosphodeoxyribomu-tase. Biochemistry 14: 599-607.
22. Ltu, T. Y. AND Y. H. CHANG. 1971. Hydrolysis of proteins with p-toluene-sulfonic acid.determination of tryptophan. J. Biol. Chem. 246: 2842-2848.
23. LOENING. U. E. 1967. The fractionation of high-molecular-weight ribonucleic acid bypolyacrylamide-gel electrophoresis. Biochem. J. 102: 251-257.
24. MANN. J. E. AND J. MYERS. 1968. On pigments. growth. and photosynthesis of Phueodac-trlum tricornutum. J. Phycol. 4: 349-355.
25. MEHARD. C. W., B. B. PREZELIN. AND F. T. HAXO. 1975. Isolation and characterization ofdinoflagellate and chrysophyte cytochrome f (553-4). Phytochemistry 14: 2379-2382.
26. PREZELIN. B. B. 1975. Characterization of peridinin-chlorophyll a proteins, and their role inphotosynthetic light adaptation. Ph.D. thesis. University of California, San Diego.
27. PREZELIN, B. B. AND F. T. HAXO. 1974. Peridinin-chlorophyll a proteins from the marinedinoflagellate Glenodinium sp. J. Phycol. (Suppl.) 10: II.
303
28. PREZELIN, B. B. AND F. T. HAXO. 1976. Purification and characterization of peridinin-chlorophyll a proteins from the marine dinoflagellates Glenodinium sp. and Gonvcauluxpolvedra. Planta. In press.
29. PROVASOLI, L. 1963. Growing marine seaweeds. In: D. DeVirville and J. Feldmann,ed., Proc. Int. Seaweed Symp. Vol 4. pp. 9-17.
30. PROVASOLI, L. AND K. GOLD. 1962. Nutrition of the American strain of Gyrodiniumcohnii. Arch. Mikrobiol. 42: 196-203.
31. RHIGHEt-ri, P. AND J. W. DRYSDALE. 1971. Isoelectric focusing in polyacrylamide gels.Biochim. Biophys. Acta 236: 17-28.
32. SHUTTT. F. 1890. Uber Peridineenfarbstoffe. Ber. Deut. Bot. Ges. 8: 9-32.33. SHIBATA. K. AND F. T. HAXO. 1969. Light transmission and spectral distribution through
epi- and endozoic algal layers in the brain coral Favia. Biol. Bull. 136: 461-468.34. SIEGELMAN. H. W. AND R. R. L. GuILLARD. 1971. Large scale culture of algae. Methods
Enzymol. 23: 110-115.35. SODETZ. J. M., W. J. BROCKWAY. AND F. J. CASTELLINO. 1972. Multiplicity of rabbit
plasminogen. Physical characterization. Biochemistry II: 4451 -4458.36. SPACKMAN. D. H.. W. H. STEIN. AND S. MOORE. 1958. Automatic recording apparatus for
use in the chromatography of amino acids. Anal. Chem. 30: 1190-1206.37. STRAIN. H. H., W. M. MANNING. AND G. HARDIN. 1944. Xanthophylls and carotenes of
diatoms, brown algae, dinoflagellates and sea anemones. Biol. Bull. 86: 169-191.38. STRAIN. H. H.. W. A. SVEC, K. AITZETMULLER. M. C. GRANDOLFO. J. J. KATZ. H.
KJOSEN. S. NORGARD. S. LIAAEN-JENSEN. F. T. HAXO. P. WEGFAHRT. AND H. RAPO-PORT. 1971. The structure of peridinin. the characteristic dinoflagellate carotenoid. J.Am. Chem. Soc. 93: 1823-1825.
39. TAKAMIYA. A. 1971. Chlorophyll protein complexes. Methods Enzymol. 23: 603-613.40. TAYLOR. D. L. 1971. Taxonomy of some common Amphidinium species. Br. Phycol. J. 6:
129-133.41. THORNBER, J. P. 1975. Chlorophyll proteins: light harvesting and reaction center compo-
nents of plants. Annu. Rev. Plant Physiol. 26: 127-158.42. WEBER. K.. J. R. PRINGLE. AND M. OSBORN. 1972. Measurement of molecular weights by
electrophoresis on SDS-acrylamide gels. Methods Enzymol. 26: 3-27.
