y-polypeptides of b-phycoerythrin porphyridium cruentum

7/28/2019 Y-polypeptides of B-Phycoerythrin Porphyridium Cruentum

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El:B IOLOGYE L S E V I E R Journal of Photochemistry and Photobiology B: Biology 39 (1997 ) 19-23

T w o ,y-polypeptides of B -phycoerythr in from P o r p h y r i d i u m c r u e n t u m 1I .N . S t a d n i c h u k a , . , N . V . K a r a p e t y a n a, L . D . K i s lo v b, V . E . S e m e n e n k o b , M . B . V e r y a s o v c

a A.N. Bakh ins t i tute~fBioc hem is try, Russ ian Aca dem y of Sciences, Leninsky prospe kt 33, Moscow 117071, Russ iab K.A. Timiryazev Ins t itute o f Plant Physiology, Russ ian A cadem y of Sciences , Mo scow 127262, Russ ia

M.V. Lomonosov State Universi ty, Mosco w 119899, Russ iaReceived 14 March 1996; accepted 9 Sep tember 1996

AbstractB -P h y c o e ry th r in , t h e m a jo r p h y c o b i l ip ro te in o f Bangiophyceae red a lgae , i s composed o f chromophory la ted a - , /3 - and "y-po lypep t idechains . Tw o " , /-subunitswith d is t inc t molecu la r m asses were chromatograph ica l ly separa ted f rom B-ph ycoery th r in o f the red a lga Porphyridiumcruentum. Both 7 -po lypep t ides were spec tropho tometr ica l ly iden t if ied as hav ing the same comp os i t ion o f f ive chrom ophoric p ros the t ic g roups :

th ree phycouro b i l ins and two ph ycoery th rob i l ins . 1997 Elsev ie r Sc ience S .A.Keywords: B-Phyc6erythrin ; Phycoerythrobil in ; Phycourobil in; Porphyridium cruentum; y-Subunit

1 . I n t r o d u c t i o nRed-co lou red B -phycoery th r in i s the majo r phycob i l ip ro -

tein of Bangiophyceae and som e spe c ies o f h igher red a lgae .I t s abso rp t ion spec t rum resu l t s f rom the p resence o f two typeso f ch romophore cova len t ly l inked to apopro te in : phycouro -b i li n ( s h o u l d e r a t 4 9 8 n m ) a n d p h y c o e r y t h ro b i l in ( m a x i m ain the range 540--565 nm ) . S im i la r to a l l know n phyco-b i l ip ro te in s , B-phycoery th r in cons i s t s o f two d i s s imi la r a -and f l -po lypep t ide cha ins o f abou t 17 -20 kDa in 1 : 1 s to i-ch iome t ry . In B- , R- and some CU -phycoery th r in s , the th i rdtype o f po lypep t ide i s a T-subun i t wi th an apparen t mo lecu la rmass o f 30 --33 kDa [ 1 -3 ]. T-Subun i t s a re ch romo phory la tedin add i tion to a - and g -cha ins and fo rm wi th them very s t ab led i s c - s h a p e d ( a / / ) 6 y - a g g r e ga t e s .T h e T - s u b u n it s o f so m e R - a n d C U - p h y c o e r y t h r in s h a v ebeen charac te r i zed wi th respec t to the i r number , b i l in typeand con ten t ( see Refs . [4 ,5 ] ) . Fo r B-phycoery th r in , the a -and f i -po lypep t ides have been wel l descr ibed . Spec i f i ca l ly ,t h e a - c h a i n b e a r s t w o p h y c o e r y t h r o b i li n c h r om o p h o r e s , a n dthe f i - subun i t i s connec ted wi th th ree such g roups [ 6 ] . Lessex tens ive in fo rmat ion i s ava i l ab le on the T-subun i t . Thusacco rd ing to e lec t rophore t i c da ta [7 ] and the par t i a l ch ro -ma tog raph ic reso lu t ion o f the T-po lypep t ide f rac t ion [ 8 ] , B -phyco ery th r in agg rega tes f rom the u n ice l lu la r a lga Porphy r -id ium cruentum may inc lude th ree d i f fe ren t T-subun i t s [ 8 ] .

* Corresponding author . Tel .: + 7 (095) 954-1473; fax: + 7 (095) 954-2732; e-mail : inbio@ glas .apc .org .t De dicated to Professor H. Se nger on the occasion of h is 65th b ir thday.1011-1344/9 7/$17.00 1997 Elsevier Science S.A. All tights reservedP i l S I O I 1 - 1 3 4 4 ( 9 6 ) 0 7 4 5 3 - 2

On the o ther h and~ the pu r i f ied B-p hycoery th r in f romP. cruentum in ano ther s tudy [6 ] show ed on ly a s ing le po ly -p e p t i d e o f 3 0 k D a o n s o d i u m d o d e c y l s u l p h a t e ( S D S )po lyacry lam ide ge l e lec t rophores is . B-Ph ycoery th r in s f romP. aerugineum and Rhodella violacea probab ly have morethan one T-subun i t [9 ] .The number o f ch romophor ic p ros the t i c g roups bound toT-subun i t ( s ) o f B-phycoery th r in has been repo r ted to beequa l to four : tw o phyc ourob i l in s and tw o phycoery th rob i l in s[10 ] . Th i s eh romophore de te rmina t ion dea l t wi th the to ta ly - f rac t ion , because the nu mbe r o f T -subun i ts i s no t per fec tlyc lear , the mo lar ra t io o f 7 - to a - and / / -po lypep t ides i s lowand the c lo se s imi la r i ty o f the T-po lypep t ides compl ica testhe i r ind iv idua l separa t ion . The ob jec t ive o f th i s s tudy w asto a t t empt to iden t i fy the ind iv idua l T -subun i t s o f B-phyco -ery th r in by means o f ch romatog raph ic separa t ion , and tocoun t the i r ch rom ophore g roups .

2 . E x p e r i m e n t a l d e t a i l s2.1 . Chemicals

B i o - R e x 7 0 ( m i n u s 4 0 0 m e s h ) w a s o b t a i n e d f r o m B i o -R a d ( U S A ) . H i g h p re s s u r e l iq u i d c h r o m a t o g r a p h y ( H P L C )grade ace ton i t r i l e and i sop ropano l were ob ta ined f romCryochrom (Russ ia ) and t r i f luo roace t i c ac id (TFA) f romP i e rc e C h e m i c a l C o m p a n y ( U S A ) . A l l o t h e r c h e m i c a l s w e r eo f reagen t g rade .


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20 I.N. Stadnichuk et al. /Journ al o f Photochemistry and P hotobiology B: B iology 39 (1997) 19-23

2.2. Algal growthThe culture of P. cruentum strain Visher 107 was grownaxenicaUy at 28 C in the m edium descr ibed by Brody and

Emerson [ 11 ] in 220 ml flasks, bub bled w ith air enriched to1.7% in CO2 and exposed to 100 t tE m -~ s- t continuousdaylight . The cells grown to a density of 3.0 g l- t wereharvested by centrifugation.2.3. Isolation o f B-phycoerythrin

The ce l ls ofP . cruentum were suspended to 0.15 mg m l-in 10 mM Na-phosphate, 1 mM ethylenediaminetetraaceticacid (EDTA), I mM phenylmethanesulphonylfluoride and 1mM 2-mercaptoethanol (pH 7 .0) at 4 C. The suspensionwas passed three times through a French press at 18 000 l bfin -2 an d centrifuged at 21 0 0 0 g for 30 min. The superna-tant was used for the isolation of B-phycoerythrin by theprocedure o f Glazer and Hixso n [ 10]. Purification stepsincluded chrom atograph y on hydroxylapatite followed by ionexchange chromatography on Toypearl DEAE -650M and gelfiltration on Sephadex G-200. Final chromatographic frac-tions of B-phycoerythrin with a ratio of A542/A5oo- 2.35 andA542/A2s0 > 6 .0 were taken for subu nit studies.2.4. Separation of polypeptide subunits o f B-phycoerythrin

Three sequential chromatographic steps were carried outin the conditions [12] minimizing the loss of phycobilinabsorbance. The first step was the procedure originally devel-oped for B-phycoerythrin by Glazer and Hixs on [ 10]. Proteinin 10 mM Na-phosphate, pH 7.0, was adjusted to pH 3.0 withglacial acetic acid, and 2-mercaptoethanol was added to 10raM. The solution was applied to a 1.5 cm 14 cm B io-Rex70 column developed stepwise with 2, 4, 6, 7.4, 8 and 9 Murea in 0 . 4 % ( v / v ) acetic acid-10 mM 2-mercaptoethanol,pH 3.0. The last three fractions containing red material werepooled separately. For the next chromatographic step, thefraction eluted with 7.4 M urea was d iluted with one par t ofacetic acid and then secondarily applied to a Bio-Rex 70column ( 1 cm 4 c m ) equilibrated w ith 3.7 M urea, pH 3.0.The coloured m ater ial was eluted stepwise with 6, 7.9 and8.8 M acidic urea solutions. The addition of 0.1% Zwittergen t3-12 (Calbiochem ) to the 6 M urea solution improved thesubsequent poly peptide separation. Th e fractions containing7.9 and 8.8 M urea were used for reverse phase HPLC (RP-HPL C) on an analytical Nucleosil 300 C4 colum n (Eisico,Russia , 4.6 m m 150 mm) with a solvent system consistingof 0 .1% TFA (buf fer A) and 0 .1% TFA in 2 : ] (v /v) ace-tonitr ile- isopropanol (buffer B) . T he samples were appliedto the c olum n in buffer A containing 5 M urea and eluted ata f low rate o f 1 ml m in - ~ according to the following pro-gramme: 1 ~ % buffer A, 5 rain; l inear gradient to 50% bufferB, 25 min. A model 344 gradient liquid chrorr~atograph(Beckman, USA) was used; samples were continuouslyde tec ted a t 495 and 555 nm (PU4021 UV-vis ible lye Uni-

cam detector , UK) with automatic peak integrations by aShimadzu C-R .1A integrator (Japan) . The result ing proteinfractions were dr ied at 277 K on a Speedvac concentrator(Savant Instruments, U SA ).2.5. Polyacrylamide gel electrophoresis

The protein fractions of B-phycoerythr in subunits dr iedaf te r RP-HPLC were dissolved in 12.5 mM Tr is-H Cl buf fe r(pH 8.0) containing 3% SDS and 5% 2-mercaptoethanol,heated to 373 K fo r 10 min and analysed by disc electropho-resis according to the standard procedure o f Laem mli [ 13 ] .The separation gel ( thickn ess, 0.7 mm; length, abou t 14 cm)contained 12.5% polyacrylamide. Polypep tide markers f romPharmacia w ere used for calibration. Ge ls were stained withCoom assie (Bril l iant Blue) R-250. The fractions of B-phy-coerythrin polypeptides, obtained af ter chromatography o n aBio-Rex 70 column and containing concentrated urea (pH3) , were dia lysed aga ins t pH 8.0 , 12.5 mM Tr is-HC l buf ferand precipitated from solution with cold acetone. Th e precip-i tate was dissolved in the same buffer containing 3% SDSand 5% 2 -mercaptoethanol, and subjected to electrophoresisas above.2.6. Bilin analysis

The chromophore content of B-phycoerythr in polypep-tides was calculated from the absorption spectra in 8 M ureaat pH 3 0 using extinction coeff icients at 495 and 555 nm of94 000 and 0 M - ! cm - I for phycourobil in and 18 600 and53 700 M -~ cm - ~ for phycoerythrobil in respectively [ 14] .The ratio of phy courobil in absorbance to phycoerythrobil inabsorbance in the total fraction of ~/-subunits belon ging to B -phycoerythrin does not change significantly during chroma-tography and possible chromophore al ter ing [10] . Theabsorption spectra were recorded at ambient temperature ona Specord M4 00 spectrophotometer (C arl Zeiss, Germ any).The absorption of the p olypeptide fractions of B-phy c3ery-thr in after the chromatographic separation on a Bio-Rex 70column w as recorded imm ediately, whereas the dr ied frac-tions after reverse phase separation were pre-dissolved inacidic 8 M urea.

3 . Resu l t sThe three polypeptide fractions o f 7.4, 8.0 and 9.0 M acidicurea, obtained after the first chrom atograp hic step of B-phy-

coerythrin on a B io-Rex 70 column, w ere used for electro-phoretic analysis. According to the electrophoretic data, the8.0 M and 9.0 M urea eluates contained polypeptides of 19and 20 kD a respectively (Fig . 1 (A )) . They were identifiedas the a- and/3-subun its of B-phycoerythr in by their molec-ular masses and known absorption spectra (not shown) inwhich the 495 nm band be longing to phycourobi l in wasabsent. The polypeptide content of these fractions coincided


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I .N. Stadnichuk et al . / Journal of Photochemis try and Photobiology B: Biology 39 (1997) 19 -23 21

k D a k D a

( a )

- - 4 3 - - 4 3

- - 3 0 - - 3 0

- - 20 .1 - - 20 .1

- - 14 .4 - - 14 .4( b )I 2 1 2 3

Fi g . 1 . SD S p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f B- p h y c o e r y t h r i n p o l y -p e p t i d e s : ( A ) l a n e 1 , a - p o l y p e p t i d e ; l a n e 2 , f l - p o l y p e p t i d e ; ( B) l a n e I , ~ ,t -p o l y p e p t i d e ; l a n e 2 , f r a c t i o n e n r i c h e d i n - / - p o l y p e p ti d e s a f t e r s i n g l e c h r o -m a t o g r a p h y o n a B i o - R e x 7 0 c o l u m n ; l a n e 3 , 7 2 - p o l y p e p ti d e .

B.phycoerythrln

Lt in t s tepwise chromatographyon Bio- rex 7 07 .4 M u r t s f r a ct i o n 8 M u r e a f r s c t i o n(?- and ct-subunits) (ct4u bun i0

~ c h m m a t o ~ p h yon Bio-rtz 7 0

9 M ares f rac t ion(J3-subunit)

6 M urea f rac t ion 7 .9 M urea Frac tion(ct-subunit) (t- and (Hu bun its)

Irever ie phsse HPLCl(t-subunit)F i g . 2 . Ch r o m a t o g r a p h i c s e p a r a t i o n o f 7 - su b u n i t s o f B- p h y c o e r y t h r i n i nd e n a t u r a t i n g c o n d i t i o n s .

8.8 M-"~rel raction(7 :" and a4 ubun i ts)

Ir e v e r i e p b s s e H PL CI(rsubunit)

w i t h t h o s e o r i g i n a l l y o b t a i n e d b y t h i s c h r o m a t o g r a p h i cm e t h o d [ 1 0 ] .

T h e f r a c t i o n o f 7 . 4 M a c i d i c u r e a c o n s i s t e d o f p o l y p e p -t id e ( s ) w i t h a m o l e c u l a r m a s s o f a b o u t 3 0 k D a a n d a n a d m i x -t u r e o f t h e a - s u b u n i t . T h e e l e c t r o p h o r e t i c b a n d b e l o n g i n g t ot h e 30 k D a p o l y p e p t i d e ( s ) w a s n o t s h a rp ( F i g . 1 ( B ) ) . T h e3 0 k D a p o l y p e p t i d e ( s ) , a c c o r d i n g t o t h e m o l e c u la r m a s s a n dt h e p r e s e n c e o f t h e 4 9 5 n m p e a k i n t h e a b s o r p t io n s p e c t r u m( n o t s h o w n ) , s h o u l d b e c o n s i d e r e d a s t h e 3 ' - s u b u n i t ( s) . T h eu s e o f u r e a c o n c e n t r a t i o n s b e l o w 7 . 4 M r e s u l t e d i n a r a p i ddec r ease in the po lypep t ide y ie ld ; the r e f o r e the s ing le u t i l i -z a t io n o f t h e B i o - R e x 7 0 c o l u m n d i d n o t a l lo w t h e r e m o v a lo f a ll t h e a - p o l y p e p t i d e t o g i v e p u r e 3 " - s u b u n it ( s) .

T h e a d d i t i o n a l s e p a r a t io n f r o m t h e w s u b u n i t w a s a c h i e v e db y r e p e a t e d a p p l i c at i on o f B i o - R e x 7 0 c h r o m a t o g r a p h y t o t h e

7 . 4 M a c i d ic u r e a f r a c t i o n e n r i c h e d i n 7 - p o l y p e p t i d e s . S i m u l -t aneous ly , i t was pos s ib le to s epa r a te two f r ac t ions o f 3 ' -s u b u n i t ( s ) . T h e e l u t i o n s e q u e n c e a n d t h e n e c e s s a r y u r e aconcen t r a t ions wer e d i f f e r en t f r om the f i r s t f r ac t iona t ion ,w h e n t h e / 3 - s u b u n i t w a s p r e s e n t i n t h e p r o t e i n p r e p a r a t i o n .F i r st , th e a - s u b u n i t w a s e l u t e d w i t h 6 M u r e a , a n d th e n t h e3 ' - p o l y p e p t i d e ( s ) w e r e e h t e d w i t h 7 . 9 a n d 8 . 8 M u r e a i ns u c c e ss i o n . B o t h f r a c t io n s s t il l c o n t a i n e d s c m e a - p o l y p e p t i d ec o n t a m i n a t i o n ( e l e c t r o p h o r e t i c d a t a o f t h i s i n t e r m e d i a t ec h r o m a t o g r ap h y n o t s h o w n ) .

The f ina l pur i f i ca t ion o f 3 ' - po lypep t ides was ach ieved byt h e a p p l ic a t i o n o f R P - H P L C t o 7 .9 M a n d 8 . 8 M u r e a fr a c -t i o n s , a - P o l y p e p t i d e w a s r e m o v e d d u r i n g t h i s l a s t s t a g e o fc h r o m a t o g r a p h y . T h e 3 ' -p o l y p ep t id e i n t h e 7 . 9 M u r e a f ra c -t i o n h a d a m o l e c u l a r m a s s o f 3 0 k D a ; t h e s e c o n d p o l y p e p t i d ew i t h a m o l e c u l a r m a s s o f 3 1 k D a i s p r e s e n t i n t h e 8 . 8 M a c i d i cu r e a f r ac t i o n ( F i g . 1 ( B ) ) . A c c o r d i n g t o t h e d i f f e r en t m o l e c -u l a r m a s s e s, t h e s e t w o p o l y p e p t i d e s w e r e d e s i g n a t e d a s 3 " rand 3 '2 - subuni ts r e spec t ive ly . I t was im p oss ib le to f r ac t iona tet h e t w o 3 ' -s u b u n it s b y R P - H P L C a l o n e , o m i t t i n g t h e B i o - R e x70 co lu m n , a s bo th 3" -subuni ts had the s am e r e ten t ion t im e int h i s c h r o m a t o g r a p h i c s y s te m . T h e s u c c e s s i o n o f c h r o m a t o -gr aph ic p r o cedur e s , used to ob ta in the 3" -subunit s o f B- phy -coe r y th r in , i s p r e sen te d in F ig . 2 .

I n t h e a b s o r p t i o n s p e c t r a o f d e n a t u r a t e d p u r e 3 ' -p o l y pe p -t i d e s , t h e p h y c o u r o b i l i n m a x i m u m a t 4 9 5 n m d o m i n a t e s i nthe v i s ib le r eg ion . I n con t r a s t , pur i f i ed na t ive B- phycoe r y-th r in shows a cha r ac te r i s t i c absor p t ion spec t r u m w i th a wea ks h o u l d e r a t 4 9 8 - 5 0 0 n m , c o r r e s p o n d i n g t o t h i s m a x i m u m ,a n d t w o i n t e n se p e a k s a t 5 4 2 an d 5 4 6 n m ( F i g . 3 ) . T h e l a r g e

0 . ~

0 . 2

0 . I

PU B!IIIIIII

III

// I/ \4 O O 5 O O 6 0 0

| a v e l e n s t h ( r i m )F i g . 3 . A b so r p t i o n sp e c t r a o f B- p h y c o e r y t h r i n f r o m P. cruentum in 0.01 MN a - p h o s p h a te b u f f er , p H 7 . 0 ( - - - ) a n d o f i t s 7 - p o ly p e p t i de i n 8 M u r e a ,p H 3 . 0 ( - - - ) ; PE B , p h y c o e r y t h r o b i l i n : PU B , p h y c o u r o b i l i n .


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2 2 1 .N . S t adn i chuk e t a l . ~J ourna l o f P ho t oc he mi s t r y and P hot ob io i ogy B : B i o l ogy 39 (1997) 19-23

difference between the two spectra in the 4 95 -500 nm regionis du et o th e fact that all phycourobilin chrom ophore groupsof B-phy coerythrin are connected on ly to 3"-chain(s) [ 10]and t o the low part of 3,-subunits in the (c~/3)6 3'-aggregatesof B-phyco erythr in.The absorp tion spectra of the 3'a- and 3'2-polypeptides in 8M acidic urea are identical; therefore both 3"-subunits haveidentical chrom oph ore compositions. T he ratio of the absorp-t ion peaks a t 495 nm (phycourobil in) and 555 nm (phy-coerythrobilin) is 3.10:t:0.10. The calculation based on theknown ex tinction coeff icients ofpolyp eptide-bound phycou-robilin and phycoerythrobilin in concentrated acidic ureashow s that each 3,-subunit contains these chromopho res in amo lar ratio of 1.55 + 0.05. Ac cording to this ratio an d to theobvious fact that the numbers of phyc obilins should be inte-gers, the 3'1- and 3"2-subunits of B -phyco erythrin h ave fivechromophofic prosthetic groups: three phycourobilins andtwo phycoerythrobilins. Twice these num bers wou ld lead toan uncharacteristically large chromop hore content.

4 . D i s c u s s i o nChromatography under denaturating con ditions using con-centrated acidic u rea is one of the mo st effective methods for

the fractionation of phycobilin polypeptides. It was used toderive the 3,-polypeptides of B-phycoerythrin from P. c ruen-turn [ 10] an d R -phycoerythrin from the red algae Gast ro-c lon ium cou l t e r i [ 14] and Cal l i t hamnion corymbosum [ 15].However, in all of these cases, a single run over a Bio-Rex70 column only allowed to obtain the total fraction of ~/-polypeptides. At the same time, RP-HPLC allowed us toseparate two (R-phycoerythrin from Aglao thamnion neg lec -turn [ 4 ] ) and three (R-phycoerythrin from C. corymbosum[ 16]) V-subunits, omitting the use of the Bio-Rex 70co lumn .However, o nly partial resolution of the 3'-polypeptides wasrealized by the application of the same method to B-phyco-etythrin of P . c r u e n t u m [ 8 ] and to R-phycoerythrin o f A n t i -t h a m n io n s p a r s u m [ 15 ]. The s traight utilization of RP-H PLCwas not successful in the present work . The separation of twoV-polypeptides could b e achieved on ly by the combined useof two types of chromatography and re-chromatography ona Bio-Rex 70 colum n.

The co ntent of 3 '-subunits in phycoerythrins varies in dif-ferent cyanobacteria and red algae. R-Phycoerythrins fromC . c o r y m b o s u m and A. sparsum [ 15 ] h ave three T-subun its,while those fromA , neglec tum [4] , Cal l i t hamnion bysso ides ,CaUithamnion roseum [17] , Audonie l la sav iana [18] andGrac i lar ia l ong a [ 19] co ntain tw o ~/-subunits. Three ~,-poly-peptides were detected in CU-phycoerythrin of the cyano-bacterium S y n e c h o c o c c u s sp. WH 8301 [ 1 , bu t only one 3'-subunit was found in another CU-phycoerythrin from5 y n e c h o c o c c u s sp. WHS02 0 [3 ] . W e have obtained two 3' -subunits of B-phycoerythrin from P . c r u e n t u m . No signs wereobserv ed of a third 3'-subunit [7,8] durin g all the separationpcocedures. Th e con tent of various 3'-polypeptides belon ging

to the (a ~) ~ T-aggregates of one phycoerythr in may dependon the algal species and algal strain, as well as on the lightand culture condit ions [4] .The numbers of phy cobil ins connected with 3' -subunits ofvarious phycoerythr ins have been determined to be one [ 3 ] ,four [4,10,1-~] and five [5 ]. Specifically, the 3"-polypeptideof B-phy ceerythr in from P. c ruen tum has been found to beassociated w ith two phycoerythrobil ins and two ph ycouro-bilins; the chromoph ore content was determ ined spectropho-tometrically for the total fraction of 3'-polypeptides obtainedby single chromatography on a Bio-Rex 70 co lumn [ 10] .T h e As55/A495 ratio in the absorption spectrum of the 3'-fraction was found to be equal to 2.5; for the calculation ofthe chromophore groups, the bilin extinction coefficients of100 300 M - *c m - ! ( i ns te a d o f 94 000 M - ~c m - m f o r phy-courobil in and 43 000 M - l cm - t ( ins tead of 53 700 M -cm - ~) for phycoerythrobil in were used [ 10] .The absorption coefficients of phycou robilin and phycoer-ythrobilin ha ve been corrected several times [ 1,14,2 0]. Inour work, the most recent values of the molar extinctioncoefficients applicable to protein-bound bilins in 8 M urea atpH 3 [ 13] w ere used. Tw o 3'-subunits were sep arated fromeach other and additionally purified from the a-polypeptide.The calculated m inimum num ber of f ive chromoph ore groupsper T-subunit correspon ds to the spectral measu reme nts.

It would be highly preferable to compare th ese results withdata on the chromophore contents obtained by other m ethods.There are two basic po ssibilities of determ ination of the chro-mophore numbers. It can be performed spectrophotometri-caily, as in this w ork, or by the separation and num bering ofdifferent chrom opeptide s obtained after partial proteolysis ofphycobiliproteins. However, the latter method has not yetbeen used to count the chromophore groups of T-subunitsfrom B-phycoerythrin. The m ost exact data can be obtainedby X-ray crystal structure analysis o f phycobilipro teins, per-mitting a significantly improved definition of the chromo-phore locations. All chromophore groups have beenvisualized in the tertiary structure up to 2.2/~ resolution inthe a- and ~-subunits of B-phycoerythr in [21] . Unfortu-nately, the disordering of the 3'-subunit(s) in the crystal, asa consequence of crystal and local symm etry averaging, hasmade it impossible hitherto to use X-ray crystallographicdetermination in this case [ 21 ].

A c k n o w l e d g e m e n t sFinancial support by the International Scien ce Foundation(Grant M O 1000) is acknowledged.

R e f e r e n c e s[ ! ] L.J . Ong , A.N. Glazer, $ .B. Waterbury , An unu sual phyco ery thrin from

a m a r i n e c y a n o b a c te r i u m, Science. 2 2 4 ( 1 9 8 4 ) 8 0 - 8 3 .


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LN. Stadnichuk e t al. I Journal of Photochemistry a nd Photobiology B: Biology 39 (1997) 19 -23 23[2] L. Ho ffmann , L. Talarico, A. W ilmot te, Presence of CU-phycoerythrin

in t he mar ine benth i c b lue-green a lga OsciUatoria toria ct : coralinae,Phycologia, 29 (1990) 19-26 .[31 S.M. W ilbank s, A.N. Glazer, Rod structure of phycoerythrin ll-

containing phy cobi l isome. II . Com plete sequence and bi lin at tachmentsi te o fa phycoery thrin 7-subunit , J. Biol . Chem., 268 (1 993) 1 2 3 6 -1241.

[4] K.E. Apt , N.E. H offman, A.R. Grossman, Th e 7-subunits of R-phycoerythrin and i t s possible mod e of t ransport into the plast ids ofred algae, J. Biol. Chem., 268 (1993) 16 208-16 215 .

[5] I .N. S tadnichuk, Phycobi liproteins: determinat ion of chromo phorecom posi t ion and content, Phytochem. Analysis, 6 (1995) 281-288 .

[6] D.J . Lunde l l , A.N. Glazer, R.J. de Lange, D .M . Brown, Bi l inat tachment si tes in the a- a nd/3-subuni ts of B-phycoery!hrin. Am inoacid seque nce studies, Biol . Chem., 259 (1984) 5472-5480.

[7] T. Red l inger, E. Gant t, Ph ycobi l isome structure o f Porphyridiumcruentum. Polypept ide composit ion, Plan t Physiol., 68 ( 1981 ) 1375 -1379.

[8] R.W. Sw anso n, A.N. Glazer, Separation o f phycobiliprotein subunitsby reverse-phase high-pressure l iquid chromatography, Anal.Biochem., 188 (1990) 295-299 .

[9] E. MSrschel , A ccessory polypept ides in phycob i l isomes of red algaeand cyanobacteria, Pianta, 154 (198 2) 251-258 .

[10] A.N. Glazer, C.S. Hixson, Subun i t structure and chrom ophorecom posi t ion of rhodophytan phyc oerythrins. B-Phycoerythrin and b-phycoerythrin, d. Biol . Chem., 252 (197 7) 32-42 .

[ 11 ] M. B rody, R. Emerso n, The effect of wavelen gth and intensi ty o f l ighton the p roport ion of pigm ents in Porphyridium cruentum, Am. J. Bot.,46 (1959) 433-440 .

[12]

1131

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D. Guard-Friar, R. MacColl, Spectroscopic properties oftetrapyrrolesof denaturated biliprotein s,Arch. Biochem. Bicphys., 23 0 ( 1 9 8 7 ) 3 0 0 -305.U.K. Laemm li , Cleava ge of the st ructural proteins during he assemblyof t he head of bac t e r iophage T4 , Nature (London), 227 (19 70) 6 8 0 -685.A.V. K lotz, A.N. Glaze r, Characterization of the bilin attachnmnt sitesin R-phycoerythrin, J. Biol. Chem ., 260 (1 985) 4856--4863.I.N . Stadnichnk, T.I. Odintsova, A.Ya. Strongin, Molecularorganizat ion and c hrom opho re composi t ion of R -phycoerythrin fromthe red alga Callithamnion, MoL Biol. (Moscow), 18 (1984) 343-350(in Russian).i .N. Stadnichuk, A.V . Khokhlacbev, Ye.V. Tikho nov a, Polypeptide 7-subunits of R -phycoerythrin, Photochem. Pho tobiol. B: Biol., 18(1993) 169-175.M.-H. Yu, A.N. Glazer, K.G. Spencer, J.A. West, Phycoerythrins ofthe red alga Callithamnion. Variation in phycoerythrobilin andphycourobilin content, Plan t Physiol., 68 ( 1981 ) 48 2-488.L. Talarico, R-Phycoe rythrin from Audoniella saviana (Nemaliales,Rhodophyta) nul t ra stm ctura l and bioch emical analysis of aggregatesand subunits, Phycologia, 29 (1990) 292-302 .E. Dagnolo, R. Rizzo, S. Paoletti , E. Murano, R-Phycoerythrin fromthe red alga Gracilaria longa, Phytochemistry, 35 (1994) 693-695 .G. M uckle, W . R i idiger, Ch romoph ore content of C-phycoerythrinfrom various cyanobacteria, Z. Naturforsch., T ell C, 32 (1977) 9 57-962.R. Ficner, K. Lo beck , G. Schmidt, R. Huber, Isolation, crystallization,crystal st ructure analysis and refinement of B- phycoerythrin from thered alga Porphyridium cruentum at 2.2 angstrom resolution, J. Mol.Biol. , 228 ( 1992 ) 935-950 .

y-polypeptides of b-phycoerythrin porphyridium cruentum

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