Plant Physiol. (1969) 44, 1629-1638

Properties and Ultrastructure of PhycoerythrinFrom Porphyridium cruentum"2

E. GanttRadiation Biology Laboratory, Smithsonian Institution, Washington, D. C. 20560

Received June 23, 1969.

Abstract. Phycoerythrin, a photosynthetic accessory pigment, was isolated from Porphyri-dium cruentum and examined by eleotron microscopy and disc gel electrophoresis. The absorp-tion monomer, with maxima at 563, 545, and a shoulder at 500 nm, has a molecular weight ofabout 300,000. With phosphatungstic acid staining it appears as a tightly structured disc-shapedparticle possessing a mean diameter of 101 0.4A and height of 54 + 0.7A. The absorptionmaxima remained the same in glutaraldehyde fixed material, and in dimer and trimer aggregates.Treatment with sodium dodecyl sulfate caused a breakdown into smaller units acoompanied bya loss of the 563 nm peak. It is suggested that this absorption monomer is the in vivo func-tional species and comparable to the phycocyanin hexamer, but structurally distinguishable atthe ultrastructural level. It has been oalculated that about 35 phycobiliprotein molecules canbe contained within each phycobilisome. There are 1.4 X 103 chlorophyll molecules perphyoobilisome, but not contained within it.

Our previous work (10) has shown that thephotosynthetic accessory pigments in Porphyridiumcruentum are located on the lamellae in the form ofphycobhilisomes (in vivo phycobilin aggregates).Absorption spectra of isolated phycobilisomes indi-cate that phycoerythrin as well as phycocyanin arepresen,t. Because only 1 type of phycobilisome ispresent in P. cruentum, and since phycocyanin is anecessary intermediate in the effective energy trans-fer between phycoerythrin and chlorophyll (6, 8) the2 phycobilins must be very closely associated. Bothpigments are believed to be within each individualaggregate.

To understand the in vivo relationship of the 2phycobiliproteins they must be distinguished at theultrastructural level. Therefore, phycoerythrin wasisolated from the red alga P. cruentum to study itsstructure in order to be able to compare it withphycocyanin. The work of Berns and Edwards (2)indicates that the purified C-phycocyanin, isolatedfrom Plectonema calothricoides, in its most stableform exists as a hexameric ring with a central hole,an overall diameter of 130A and a molecular weightof ab-outt 200,000. It remains to be shown whetheror not phycoerythrin has a similar shape and ap-pearance as the phycocyanin hexamer, and in whatway, if any, it differs. To permit a meaningfulcomparison of the 2 phycobilins in the present work,

1 Research was supported by the United States AtomicEnergy Commission Contract No. AT(30-1)3913, andthe Smithsonian Institution.

2 Published with the approval of the Secretary ofthe Smithsonian Institution.

preparation.s of phycoerythrin were monitored byelectron microscopy, absorption spectrophotometry,and the molecular weight of the smallest stableparticle was determined.

Materials and Methods

Phycoerythrin was obtained from Porphyridiumcruentum which had been grown in axenic culturesin an artificial sea water medium (14) at roomtemperature. The liquid cultures, aerated with 1 %carbon dioxide and 99 % air, were continuouslyagitated and illuminated with cool white fluorescentlamps (incident intensity 350 ft-c).

Cells were harvested by centrifugation (10,000g)and rinsed twice in 0.01 M phosphate buffer (sodiumand potassium) pH 6.8. The pellets were frozen inpolyethylene tubes in an acetone-dry ice bath andstored in a freezer until used. For use the pelletswere thawed and the slurry of cells was passedseveral times through a No. 20 syringe needle inorder to break the cell clumps prior to breaking inan An4nco-French pressure cell at 8 to 12,000 psi.

The phycoerythrin isolation method, involvingn-butanol and ammonium sulfate fractionation, de-scribed by Leibo and Jones (15) was followed inmost essentials. Throughout the entire isolation thepreparation was kept at about 4°. For the precipi-tations a solution of supersaturated ammonium sul-fate was used in decreasing concentrations (35-20 %of saturation) and repeated until a 545/275 nmabsorbance ratio of 5 or higher was obtained. Finalcrystallization of phycoerythrin was accomplished byaddition of recrystallized ammonium sulfate powder(15-20 % of saturation).

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PLANT PHYSIOLOGY

The criteria of purity were the following: a) a545/275 nm absorbance ratio of 5 or higher, b)homogeneity on polvacrylamide gel (see resuilts),and c) uniformity of structure when examined byelectron microscopy.

Electron Microscopy. Phycoerythrin crystalswere dissolved in 0.001 m phosphate buffer pH 6.0,6.8, and 7.0 and dialyzed against the same buffer,then centrifuged for 30 min at 100,000g. It wasnecessary to use low ionic strength phosphate bufferin order to prevent obliteration of detail when thematerial was negatively stained. For glutaraldehydefixation the ammonium sulfate was removed bydialysis against 0.01 M phosphate (pH 6.8 generally),then 2 % or 4 % buffered glutaraldehyde was addeddirectly and allowed to react for 1 hr, or it wasallowed to en-ter through the dialysis membraneovernight. The negative stains were always pre-pared shortly before use. The 2 % solutions ofphosphotungstic acid (PTA) were adjusted to theappropriate pH (usually 6.8) with ammonium hy-droxide, while uranyl oxalate was prepared accordingto the procedure of Mellema et al. (17). Othernegative stains such as potassium tungstate, sodiumtungstate, sodium silicotungstate, and uranyl acetatewere also tried. However, because of inconsistentresults or precipitation of the protein (with uranylacetate) they were not normallv used. Phospho-tungstic acid at pH 6.8 was commonly employedbecause it yielded the most consistent results andbecauise it made more valid comparisons with other1l)hcobiliproteins possible (2, 5).

Phycoerythrin was diluted with buffer and furtherby phosphotungstic acid or uranyl oxalate to a finalconcentration of 5 to 25 ,ug/ml and applied to formvarcarbon-coated grids which had been ionized with a5000 volt glow discharge in a vacuum evaporator, toenhance spreading. Carbon-coated "holey" formvarnets were tried and discontinued after observing thatdistortions in shape and size of the phycoerythrinparticles occurred. Control grids were prepared byapplying the buffer plus PTA, but no reproduciblepatterns could be seen which corresponded to thephycoerythrin preparations. The grids were studiedin a Philips EM 200 electron microscope at 60 or 80kv with double condenser illumination and liquidnitrogen specimen cooling. Records were made on35 mm fine grain positive film (Eastman Kodak No.FRP 426) at a magnification of 33,200. Measure-ments of the particles were made from photographi-cally enlarged areas of preparations such as Fig. 1.Only those particles were measured which had thelargest diameters as well as the most distinct bounda-ries. This was justified by assuming that those moreclearly visible were least buried in the stain, andsince we dealt with a homogenous population therewas little possibility of selecting for one particularspecies. This usually was not a problem since withvery thin spreading it was possible to measure almostevery particle.

Disc Gel Electrophoresis. For the molecularweight determination of phycoerythrin the acrylamidegel electrophoresis method of Hedrick and Smith(13) was followed with 1 minor modification. Itwas necessary to reduce the recommended concen-tration of the ammonium persulfate catalyst to pre-vent too rapid gelation and release of 'heat. Theammonium persulfate concentration was 0.047g/ 100ml for 4 % and 5 % gel, and 0.0228g/ml for 6 to8 % gel. The final pH of the -small pore solutionwas pHl 7.9, and the large pore solution pH 5.7,while the reservoir buffer (0.034 M asparagine neu-tralized with Tris) wvas pH 7.3. The commerciallyavailable Buchler Polyanalyst (Buchler Instruments,Incorporated, Fort Lee, New Jersey) apparatus wasused.

Small pore gel concentrations ranged in 1 %increments from 4 to 8 %. The phycoerythrin wasdiluted in a 50 % glycerol solution, and layeredbetween the spacer gel and reservoir buffer. Eachtuibe received about 25 ,ug of protein. Electrophoresiswas at room temperature (2 mA per tube initiallyand 4 mA after the dye front passed into the smallpore gel). After putting the gels into ice water, tofacilitate their intact removal from the tube, the dyefront was marked by insertion of a thin piece ofnichrome wire. All visible bands were measuredbefore and after fixing in trichloroacetic acid andstaining with Coomassie blue or amido sch'warz.

To verify the reliability of the method in ourhands, several proteins of known molecular weightwere used as reference markers: 'bovine serumalbumin (Nutritional Biochemical Corporation,Cleveland, Ohio), catalase (CalBiochem, Los An-geles, California), alpha-amylase (Sigma ChemicalCompany, St. Louis, Missouri), and ferritin (MannResearch Laboratories, New York, New York).

For the recovery of the phycoerythrin bands fromthe gels the same procedure was followed except theprotein concentration was increased to 50 to 75 ,ugper tube. Advantage was taken of the fact thatglutaraldehyde stabilizes the phycobiliprotein aggre-gates, which then allows their recovery in greaterconcentieation than in the unfixed material. Bestresults were obtained with phycoerythrin which hadbeen fixed in glutaraldehyde and then stored in20 % ammonium sulfate (once fixed, phycoerythrindoes not crystallize again in ammonium sulfate).Of the several d'istinct pink bands which formed onthe gel, the 3 which had migrated the farthest, werecut out with a razor blade. The excess gel wasremoved and -the separate pink band's were collectedin 5 ml tubes and macerated with a close fittingstirring rod. Twelve or 24 gel slices were used foreach extraction. To extract the pigment about 2 mlof cold blffer or 2 % PTA (pH 6.8) was added toeach tube. This gel slurry was then transferred toa larger centrifuge tube and agitated on a magneticstirrer for 1 to 2 hr then centrifuged at top speedin an International clinical centrifuge. The clear

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GANTT-PROPERTIES AND ULTRASTRUCTURE OF PHYCOERYTHRIN

pink supernatant solutions were used for spectro-photometric readings (on a Cary 14 spectrophotom-eter) and/or preparation of electron microscopegrids within 30 to 120 min after extraction. It wasnecessary to use the material as soon as possiblebecause some dissociation occurred on s'torage.

Sodium Dodecyl Sulfate Treatment. Phycoery-thrin in 0.01 M sodium phosphate buffer pH 6.8 and1 % sodium dodecyl sulfate (SDS) was incubatedfor 3 hr at 370 and dialyzed overnight against thesame buffer concentration but with a reduction to0.1 % SDS. The control preparations were exposedto the same conditions with the omission of SDS.At the termination of the incubation the SDS-treatedpigment had changed in color from pink to purple.The treated material did not have the usual brightorange fluorescence noticeable in white light.

Results

Structure of Phycoerythrin. Phycoerythrin isone of the most stable chromoproteins and its isola-tion from P. cruentum and purification were readilyaccomplished. It is known to be stable over a widerange of pH (3-10) (19,20) and the presentconditions were well within that limit. A purepreparation characterized by a 545/275 nm absorb-ance ratio of 5 or higher displayed one major pinkband on polyacrylamide gel. Sometimes a faintsecond and suggestion of a third band were present.After staining for protein, the only visible bandswere those corresponding to the original pink phy-coerythrin bands.A preparation of phycoerythrin when negatively

stained with uranyl oxalate, is seen to consist pri-marily of monomers (see Discussion for definition).In Fig. 1 various aspects of the phycoerythrinparticles are evident. Those with a rounded ap-pearance are believed to be views of the flat side ofthe particles which are envisioned as being discshaped with an axial ratio of 2:1. Since there is atendency for the particles to dry on the grid withtheir flat side up, views of their narrow side areless common. Side views are more easily foundwhere there is a build up of stain and pigment, andof course in the case of linear aggregates (Fig. 3).The first 2 enlargements (a and b) in Fig. 2 are ofthe broad face view of phycoerythrin particles, andc and d are side views.

Uranyl oxalate staining resulted in pictures withgreater detail. However, with either stain theparticles appear very tightly structured. Some sub-structure is evident in Figs. 1 and 2, although onecannot detect a definite subunit shape nor anyregular array of the substructure. There were nostructural differences among preparations at pH 6.0,6.8, and 7.0.

Measurements were made of the particle widthand height on uranvl oxalate and PTA stainedpreparations. With uranyl oxalate staining the mean

diameter of the broad face view was 115 ± 0.8A(SE). This also is the approximate diameter of thelong aspects of the side views. The mean particleheight (the short axis) measured 64 ± 0.6A. Withphosphotungstic acid staining the mean diameter ofthe rounded face view is 101 ± O.4A and the heightis 54 + 0.7A. A d'iscrepancy of about 14A isreadily evident in- the values- obtained from the 2differently stained preparations, however, as ispointed out in the- Discussion the values obtainedfrom the PTA preparations are believed to be morereliable.

Properties of Phycoerythrin. A typical absorp-tion spectrum of phycoerythrin from P. cruentutmshown in Fig. 6 characteristically has a shoulder at500 nm and peaks at 545 and 563 nm. Treatmentwith glutaraldehyde stabilizes the phycoerythrinaggregates but causes no noticeable shift in theabsorption peaks. However, the bright orangefluorescence noted under white light is diminishedupon glutaraldehyde fixation.A preparation fixed in glutaraldehyde contains

aggregates of various sizes and shapes (Fig. 3). Amixture of linear and globular aggregates is seen.There appears to be considerable freedom of choicein the manner of aggregation. The linear figuresare formed by association of the particles along theirbroad faces. However, they can also associate alongtheir narrow faces, or along the narrow of one andbroad face of another. This allows formation of3 dimensional configurationis such as the phycobili-somes.

When a glutaraldehyde-fixed phycoerythrin prep-aration (Fig. 3) is subjected to discontinuous elec-trophoresis on polyacrylamide gel several distinctbands result (Inset Fig. 7). As in the untreatedphycoerythrin, aill the material visible on the stainedgel corresponded to the phycoerythrin bands priorto staining. The 3 bands which migrated fastestwere extracted and studied. The first band, nearestthe front, when examined by electron microscopyconsisted entirely of monomers. Dimers (Fig. 4)were the predominant species in the second band,and trimers (Fig. 5) in the third band. Somemonomers were found in the negative stained prep-arations from band IIL while dimers and monomerswere present in preparations from band III. Thiscan either be due to incomplete separation on thegel, or what is more likely, the larger aggregatesare more readily dissociated by handling and dryingon the electron microscope grids.

Of the 3 bands examined, the monomer band wasalways the most dense and had a bright orangefluorescence. As the aggregates increased in size,the intensity of the bands decreased. Monitoringthe extracts from the gel bands revealed (Fig. 7)that the absorption maxima are the same in themonomer, dimer, and trimer preparations. There-fore, aggregation of phycoerythrin particles at thislevel does not cause a shift in the characteristic

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PLANT PHYSIOLOGY

absorption peaks of phycoerythrin (563, 545, and ashoulder at 500 nm).

Molecular Weight Determination. The methodof Hedrick and Smith (13) was used for themolecular weight determination of phycoerythrin.By this method, using disc gel electrophoresis, sizeisomers can be distinguished from charge isomers.The mobility of the 3 fastest moving bands of phy-coerythrin was followed on 4 to 8 % gel. Whenthe log of the phycoerythrin mobility relative to thedye front was plotted against gel concentration, agroup of non-parallel lines intersecting around 0 %gel concentration resulted (Fig. 8). This indicatesthat these are size isomers, i.e. the aggregates sepa-rated according to size.

To determine the reliajbility of this techniqueunder our conditions, proteins of known molecularweight were run on disc gels. Then the slopes fromplots of the log relative mobility versus gel concen-tration were determined. When the negative slopesof the chosen proteins (alpha-amyilase 45,000, bovineserum albumin monomer 65,000, catalase 240,000,and ferritin monomer 450,000) were plotted againstmolecular weight (Fig. 9) there was very goodagreement between our results and those of Hedrickand Smith (13). The plot of Fig. 9 was taken asthe standard. By plotting the negative slope of12.5, the phycoerythrin monomer comes very nearcatalase and has a molecular weight of about 270,000while the dimer (20.5) is close to ferritin and has arel'ative molecukar weight of about 500,000. Themonomer molecullar weight value is considered to bemost reliable because the dimer is near the limit ofresolution. The trimer had to be ignored, since thissystem's accuracy is limited to the molecular weightrange of 50,000 to 500,000.

Exposure of phycoerythrin to 1 % sodium dodecylsulfate for 3 hr at 370 resulted in the loss of fluores-cence and a shift in color from the usual pink topurple. The absorption spectrum of such a prepara-tion showed that the 563 nm peak was absent. Whensubjected to electrophoresis on 5 % polyacrylamidegel a fast moving purple band appeared followed bya more slowly moving pink band, which containedthe monomers. Examination by electron microsrcopyof the extracted purple material revealed that onlyvery small units were present which were difficultto distinguish from the background. It was, there-fore, concluded that sodium dodecyl sulfate causeda breakdown of the phycoerythrin monomer, and thatdue to this breakdown there was a change in thecolor.

Discussion

The smallest stable phycoerythrin particle possess-ing absorption maxima at 563, 545, and a shoulderat 500 nm with an approximate molecular weight of300,000 is considered a monomer. This is the"native" (1,5) B-phycoerythrin molecule (for no-

menclature designation see 20,22), and probably thein vivo functional species. Supporting reasons are:A) Phycoerythrin is a stable chromoprotein whichwhen isolated, normally has a molecular weight ofabout 200,-300,000, and B) has absorption maximaof 563, 545, and 500 nm, corresponding closelv to,n vivo absorption, C) when this particle is .brokendown into smaller units a change in-the absorptionresults, while upon aggregation there is no aibsorp-tion change.

Most reported phycoerythrin molecular weightsare in the 200,-300,000 range (1,3, 7, 19, 20, 22).Therefore, the value of 270,000 obtained here bydisc-gel electrophoresis and the 340,000 calculatedfrom the electron micrograph measurements are inreasonable agreement.

T'his particle is by no mean's the smallest subunitobtainable, as shown for instance by the sodiumdodecyl sulfate treatment, but it is the smallestparticle which has the typical absorption maxima.Since absorption spectra of various smaliler fractionsare often not reported, it is difficult to judge if allmanifest an absorption change. Neufeld (19) iden-tified a purple component, which lacked the 563 nmpeak, as a subunit with a molecular weight of 40,000.Mieras and Wall (18) who recently reported molecu-lar weights of 23,000, 36,000 and 85,000 for B-phy-coervthrin also mentioned a purple component, whichimplies the loss of the 563 nm peak. Fujimori andPecci (9) 'found that one of the subunits created byP-chiloromercuribenzoate treatment lacked the samepeak. They believed that they were dealing with asubunit which had only one of the 2 phycoervthrinchromophore types.

If the various results are considered in terms ofthe existence of one physiologically active phycoery-thrin chromophore (phycoerythrobilin), demonstratedby Chapman et al. (4), one is led to assume that thechanges are due to alterations of the protein environ-ment to which the phycoerythrobilins are attached.A diameter of 101A and particle height of 54A

with phosphotungstic acid are believed to be thetrue values, not those of 115A by 64A derived fromuranyl oxal'ate stained material. The molecularweight calculations, using a radius of 50.5A andheight of '54A with an assumed density of 1.3, givea value of 340,000 which i's close to that derived byother methods (1, 3, 19). Using a radius of 57.5Aand height of 64A, the calculated molecular weightis 530,000 a'bout 2 times *as high. Uranyl oxalategives an excellent image as Mellema et al. (17)claim. However, it appears that a swelling is causedby this stain which could accoun't for the larger sizeas well as for greater detail in the phycoerythrinparticle (compare Figs. 1 and 3). It 'should benoted that very interesting piatterns can be obtainedby the photographic rotation enhancement technique(16) showing 5, 6, or more distinct units per par-ticle. They were not held to be meaningful becauseit was not possible to observe these patterns directly

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GANTT-PROPERTIES AND ULTRASTRUCTURE OF PHYCOERYTHRIN 1633

2a b c dFIG. 1. An electron micrograph of phycoerythrin stained with uranyl oxalate. Most of the particles are ori-

ented with their broad faces in full view. Side views are also present but less common. Substructure can be noted,however, individual subunits cannot be discerned. 302,000 X.

FIG. 2. Enlargements of phycoerythrin particles stained with uranyl oxylate. Two broad face views of thedisc shaped particles are seen in a and b, and side views in c and d. All are at the same magnification. 2,100,000x.

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GANTT-PROPERTIES AND ULTRASTRUCTURE OF PHYCOERYTHRIN

NX,~ ~ K

FIG. 3. Phycoerythrin fixed in glutaraldehyde and stained with phosphotungstic acid consists of a mixture ofparticles in various states of aggregation. Side views of the particles are clearly evident in most of the linear aggre-gates. 266,000X.

FIG. 4. Phosphotungstic acid stained preparation extracted from the second phycoerythrin band obtained on poly-acrylamide gel. Most are dimers. 266,OOOX.

FIG. 5. Preparation from the third phycoerythrin band on polyacrylamide gel. Trimers are the predominantspecies. Stained with phosphotungstic acid. 266,OOOX.

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GANTT-PROPERTIES AND ULTRASTRUCTURE OF PHYCOERYTHRIN

0.9 I

0.8-

0.7

0.6.

wj 0.5-

z

f'i: 04/|m

0.3 4(U)

< 0.2-

0.1

0.0

240 300 400 500 600 700

WAVELENGTH (nm)FIG. 6. Absorption spectra of phycoerythrin in 0.01

M phosphate buffer pH 6.8 (93 jg/ml ( ), anda similar preparation (68 ,ug/ml) which had been treatedin glutaraldehyde With glutaraldehydetreatment no shift in the absorption maxima is apparent.

from a through focus series, and because one caneasily create artifacts with this technique.

Phycocyanin in its most stable form has a molec-ular weight around 200,000 (3,12,19,20,21), veryclose to that of phycoerythrin. Berns and Edwards(2) regard phycocyanin of this size to be the in vivofunctional species. It is described as a ring with 6distinct subunits, a central hole, and overiall diameterof 130A (PTA staining). Phycoerythrin, a com-pact molecule with no definable sulbunits, and diam-eter of 101A, although designated as a monomer isbelieved to be functionally comparable. The ultra-structure of phycoerythrin is also quite distinct fromphytochrome, which seems to exist as a tetramer or

hexamer (5). Certain calculations can be madeusing some of our previous data. The phycobili-somes (10, 11) have a diameter of about 350A andare composed of closely associated particles. It hasbeen calculated for P. cruentum that for every phy-coerythrin modecule there are 40 chlorophyll mole-cules (1.4 X 103 chlorophylls to every phycobili-some) although the chlorophyll is not in the phyco-bilisomes. On a volume basis about 60 phycobili-protein molecules could fit into a phycobilisome.This would be the case if the molecules were solidlypacked. However, the electron micrographs showthat they are not solidly packed because the negativestain can penetrate. It is reasonable to assume thateach phycobilisome contains about 35 phycobilipro-teins (phycoerythrin and phycocyanin) with phy-

w

z

< 0.4Ca~0

oo~~~I

%.

0.0

400 500 600 700WAVELENGTH (nm)

FIG. 7. Results of glutaraldehyde fixed phycoerythrinafter disc electrophoresis on 5 % polyacrylamide gel.The inset is a photograph of a stained gel with themonomer, dimer, and trimer bands marked on the right.The larger phycoerythrin aggregates were unable to pene-trate into the small pore gel and have accumulated inthe large pore gel, as well as at the interface betweenthe 2 gel phases. Sample concentration was about 75 ,ug.The wire marks the distance the dye front migrated.Absorption spectra in 0.001 M phosphate buffer pH 6.8of the monomer (I), dimer (II), and trimer (III) bandsafter extraction from the gel have the same maxima(563, 545, and 500 nm shoulder).

coerythrin predominating. From these results itshould now be possible to distinguish between severalprobable molecular organizations in phycobilisomes:a model in which phycocyanin is present as a coredirectly attached to the photosynthetic lamellae witha phycoerythrin shell, or a spoke model where chainsof phycocyanin and phycoerythrin molecules areinterspersed.

Acknowledgments

The author expresses appreciation to Dr. K. M.Towe, of the Paleobiology Department of the Smith-sonian Institution, for the use of the electron microscopeand associated facilities, and to Miss Claudia A. Lip-schultz for her able technical assistance.

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PLANT PHYSIOLOGY

0202 200

=12.5

100 - DIMER01 ~~~~~~~~~~=20.5

0

0 50 RIMER=25.2

02 3 4 5 6 7 8 9

GEL CONCENTRATION %FIG. 8. The effect of different gel (4-8 %) con-

centrations on the relative mobility (RM) of the 3 fastestmoving phycoerythrin aggregates. The negative slopesof the lines are noted at the right of the figure.

(L0

(I)

zLIz

25 -T-- 1- l lID

020 - ol--"F

15-M0

10 _BC

~05 __-

IN A ^ ;0 10 20 30 40

MOLECULAR WEIGHT x 104

FIG. 9. The slope-molecular weight relationship ofproteins of known molecular weight and the phycoerythrinmonomer (M), and dimer (D). A-alpha amylase, B-bovine serum albumin monomer, C-catalase, F-ferritin.

Literature Cited

1. AIRTH, R. L. AND L. R. BLINKS. 1956. A new

phycoerythrin from Porphyra naiadumt. Biol. Bull.111: 321.

2. BERNS, D. S. AND M. R. EDWARDS. 1965. Electronmicrographic investigations of C-phycocyanin.Arch. Biochem. Biophys. 110: 511-16.

3. BRODY, S. S. AND M. BRODY. 1961. A quatitativeassay for the number of chromophores on a chro-moprotein; its application to phycoerythrin andphycocyanin. Biochim. Biophys. Acta 50: 348-52.

4. CHAPMIAN, D. J., W. J. COLE, AND H. W. SIEGEL-MAN. 1968. A comparative study of the phyco-erythrin chromophore. Phytochemistry 7: 1831-35.

5. CORRELL, D. L., E. STEERS, K. M. TOWE, AND W.SHROPSHIRE, JR. 1968. Phytochrome in etiolated

annual rye. IV. Physical and chemical characteri-zation of phytochrome. Biochim. Biophys. Acta168: 46-57.

6. DUYSENS, L. N. M. 1952. Transfer of ExcitationEnergy in Photosynthesis. Thesis. 'Jtrecht.

7. ERICKSSON-QUENSEL, I. 1938. The molecularweights of phycoerythrin and phycocyanin. Bio-chem. J. 32: 585.

8. FRENCH, C. S. AND V. K. YOtJXG. 1952. Thefluorescence spectra of red algae and the transferof energy from phycoerythrin to phycocyanin andchlorophyll. J. Gen. Phvsiol. 35: 873-90.

9. FujIMORi, E. AND J. PECCI. 1967. Distinct subunitsof phycoerythrin from Porphyridium cruentum andtheir spectral characteristics. Arch. Biochem. Bio-phys. 118: 448-55.

10. GANTT, E. AND S. F. CONTI. 1966. Phycobilipro-tein localization in algae. In: Energy Conversionby the Photosynthetic Apparatus, BrookhavenSymposia in Biology, No. 19: 393405.

11. GANTT, E. AND S. F. CONTI. 1966. Granules as-

sociated with the chloroplast lamellae of Porphy-ridiumn cruentum. J. Cell Biol. 29: 423-34.

12. HATTORT, A. AND Y. FUJITA. 1959. Crystallinephycobilin chromoproteins obtained from blue-green alga, Tolypothrix tenuis. J. Biochem. To-kyo 46: 633.

13. HEDRICK, J. L. AND A. J. SMITH. 1968. Size andcharge isomer separation and estimation of molecu-lar weights of proteins by disc gel electrophoresis.Arch. Biochem. Biophys. 126: 155-64.

14. JONES, R. F., H. L. SPEER, AND W. KURY. 1963.Studies on the growth of the red alga Porphy-ridi1lm cruentium. Physiol. Plantarum 16: 636.

15. LEIBO, S. P. AND R. F. JONES. 1964. Freezing ofthe chromoprotein phycoerythrin from the red algaPorphyridiumii cruentutni. Arch. Biochem. Biophys.106: 78-88.

16. MARKHAM, R., S. FREY, AND G. J. HILLS. 1963.Methods for the enhancement of image detail andaccentuation of structure in electron microscopy.Virology 20: 88-102.

17. MELLEMA, J. E., F. F. J. VAN BRUGGEN, AND M.GRUBER. 1967. Uranyl oxalate as a negative stainfor electron microscopy of proteins. Biochim.Biophys. Acta 140: 180-82.

18. MIERAS, G. A. AND R. A. WALL. 1968. Sub-unitsof the algal biliprotein phycoerythrin. Biochem.J. 107: 127-28.

19. NEUFELD, G. J. 1966. Structural characterizationof phycocyanin and phycoerythrin. Thesis. Uni-versity of Texas.

20. O'HEOCHA, C. 1965. Biliproteins in algae. Ann.Rev. Plant Physiol. 16: 415.

21. SCOTT, E. AND D. S. BERNS. 1965. Protein - pro-

tein interaction. The phycocyanin system. Bio-chemistry 4: 2597-2606.

22. SVEDBERG, T. AND T. KATSURAI. 1929. The mo-

lecular weights of phycocyanin and of phycoery-thrin from Porphyra tenera and of phycocyaninfrom Aphanizornenon flosaquae. J. Am. Chem.Soc. 51: 3573.

1638

Z)50

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