Distinct subunits of phycoerythrin from Porphyridium cruentum and their spectral characteristics

ARCHIVES

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BIOCHEMISTRY

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Subunits

BIOPHYSICS

448-455 (1967)

118,

of Phycoerythrin and EIJI

Their

from

Spectral

FUJIMORI

Porphyridium

cruentum

Characteristics JOSEPH PECCI

AND

Photochemistry Section, Energetics Branch, Space Physics La.boratory, Air Force Cambridge Research Laboratories, Bedford, Afassachusetts 01790 Received

June 23, 1966

Phycoerythrin from Porphyridium cruentum (.s~o,~= 11.58) exhibits three visible absorption bands at 500,545, and 565 rnp and is split by p-chloromercuribenzoate into two types of subunit which are easily separated by either centrifugation or gel filtration. One of the subunits is insoluble and exhibits a major absorption maximum at 590 maximum rnp. The other soluble subunit (s& = 2.35) exhibits a single absorption at 545 rnp. The 565-rnp band disappears as a result of this treatment. Native phycoerythrin is not completely reconstituted from the unseparated subunits upon removal of the p-chloromercuribenaoate through the addition of mercaptoethanol or glutathione. Aggregation among the soluble subunim occurs to some extent producing an associated form exhibiting a better regeneration of the 565-rnr absorption band than does the unassociated form.

Absorption spectra of the photosynthetic chromoprotein, phycoerythrin, obtained from the red alga Porphyridium cruentum exhibit three absorption bands in the visible region: a shoulder at 500 rnp, the principal maximum at 545 rnp, and a subsidiary maximum at 565 mp. The chief effect of the sulfhydryl-blocking reagent p-chloromercuribenzoate (PCMB)’ on phycoerythrin is the elimination of the 565-rnp absorption band (1, 2) resulting in a considerable decrease of fluorescence (2). When cells of P. cruentum are treated with PCMB, it is possible to isolate a modified phycoerythrin characterized by a single absorption band at 545 rnp. A second component having an absorption band at 500 mp remains in the cell. Addition of the sulfhydryl compound glutathione to the isolated modified phycoerythrin brought about a partial regeneration of the 565-rnp absorption band as well as an efficient regeneration of its fluorescence (3). These investigations suggest. that it is possible for phycoerythrin from P. wuenlum 1 The abbreviation PCMB shall be used designate p-chloromercuribenzoat.e, Na salt’.

to

to be split by PCMB into at least two kinds of subunits, one characterized by the 500-rnp chromophore and the other by both the 545and 565-rnp chromophores. This report presents evidence on the dissociation of phycoerythrin isolated from P. cruentum into two distinct kinds of subunit when treated with PCMB. The separation and spectral characteristics of both subunits are reported upon. Results of the sedimentation studies of one of the subunits as well as its behavior in the presence and absence of PCMB are also presented. MATERIALS

AND

METHOD

Materials. Glutathione (reduced), p-chloromercuribenzoate, Na salt, and 2mereaptoethanol were obtained from the California Corporation for Biochemical Research. The Sephadex G-100 was obtained from Pharmacia Fine Chemicals, Inc. Algal cult#ures of P. cruentum (No. 755) were obtained from the Culture Collection of Algae at the University of Indiana. P. cruentum was grown in Sea Water Medium (4) at 25” while 1% carbon dioxide in air was continuously bubbled through the culture. Constant illumination was provided by a 499-W cool-white fluorescent lamp. The algal cells were harvested using a de Lava1 separator. 448

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The resulting dark red, jelly like masses were stored in plastic petri dishes at about 4” until required. Preparation oj phycoerylhrin. The dark red, jelly-like masses were homogenized for two minutes with 0.1 M phosphate buffer (pH 7.0) using a Waring Blendor. The suspension was allowed to stand overnight at 4”. Portions of the suspension (250 ml each) contained in a 600-ml beaker were insonated for 5 minutes each using a model S-110 Bronson Instruments, Inc. Sonifier equipped wit,h a W-inch solid stephorn. The suspension remained immersed in a dry ice-alcohol bath during the insonation which was carried out at the maximum int,ensity of the instrument. The insonate was allowed to macerate overnight at 4” and then centrifuged at 30,OOOg for 45 minutes at about 5” to remove the bulk of insoluble material. The supernate was recentrifuged at 1OO,C0Og for 60 minutes. The final supernate was intimately mixed with an equal amount of 1-butanol (5) and the resulting emulsion was centrifuged at 10,ooOg for 30 minlltes. After centrifugat,ion, two liquid phases appear, the upper being yellow and the lower red. At the interface, there appears an insoluble gelatinous blue layer of crude phycocyanin. The lower red aqueous phase was carefully removed, made 30% saturated with ammonium sulfate, and centifuged at 30,ooOg for 30 minutes. A compact red layer of crude phycoeryt,hrin present at the top of the centrifuge tube was transferred to a minimum amount of 0.1 M phosphate buffer (pH 7.0). The mixture was dialyzed against. two 5-liter changes of 0.1 M phosphate buffer (pH 7.0) for at least 8 hours each at 4”. After dialysis, the insoluble material remaining was removed by centrifuging at 10,000g for 15 minutes. The supernatant fraction was made 35yo saturated with ammonium sulfate and centrifuged at 30,OOOgfor 30 minutes. The red precipitate was dissolved in a minimum amount of 0.1 M phosphate buffer (pH 7.0) and dialyzed against the same buffer at 4”. In order to obtain highly concentrated solutions of phycoerythrin, we centrifuged t)he dialyzate at 198,000g for four hours. The supernatant liquid was discarded and to the precipitate of phycoerythrin, a minimum amount of 0.1 M phosphate buffer (pH 7.0) was added. The suspension was mixed int,imately and stored overnight at 4”, after which complete solution was effected. Phycoerythrin prepared in t,his manner consistently showed a ratio of absorbance at 545 rnp of 4.0 or greater. Measurement of profein concenlration. For concentrat,ion determinat.ions, aliquots of the protein solution were dialyzed against distilled water to remove buffer salts. The dialyzates were quantitatively transferred to tared, pretreated flasks and lyophilized. Residues were dried in a vacuum

449

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desiccator over Drierite to constant weight. Further drying overnight at 105” resulted in no significant weight change. Sedimentation analysis. Sedimentation st)udies were made at 20” and 50,740 rpm using a Spinco model E analytical ult racentrifuge. Schlieren optics and 30.mm cells were employed. Due to the intense red color of phycoerythrin, red-sensitive Kodak Type 1-N plates were used together with a double Kodak No. 25 red filter. Standard procedures lvere used for the determination of sedimentation coefficients. All distances were measured using a NikonModel6C Profile Projector. A partial specific volume of 0.75 as reported for phycoerythrin from Ceramium rubrum (6) \vas Ilsed in all of our calculations. Corrections drle to the presence of PCMB and/or 2-mercapt.oet,hanol were considered to be negligible and were therefore neglected when correcting s values to s20,~ values. St,udies were carried out using several different concentrations of protein. Optical measurements. Absorption spectra were measured with a Gary model 14 recording spectrophotometer. Gel filtration. A column (1.5 cm in diameter, 25 cm in length) of Sephadex G-100 was used. Eluted fractions of 1 ml each in 0.1 M phosphate buffer pH 7.0 were collected using a Research Specialties Co. model 1205 automatic fraction collector. Each fraction was diluted with t,he same buffer solution to 3.5 ml for the measurement, of absorption spectra. RESULTS

Excess PCMB was added to a 0.14 % soluof phycoerythrin in 0.1 M phosphate buffer (pH 7.0) and allowed to stand for several days at 4”. The resulting mixture was filtered through Pyrex glass wool to remove undissolved PCMB. Figure 1 presents absorption spectra of 1:20 dilutions of this treated phycoerythrin compared to that of native phycoerythrin. The complete disappearance of the 56;i-111~ maximum along with a concomitant diminution in absorbance at 54.5 and rjO0 mp is plainly evident. This decrease in absorbance at 545 and 500 rnti was not evident in earlier studies (2) where low concent,rat’ions of I’CMB were used. The increase in absorption at about 600 ml.r and below about 350 rng is worthy of notice. It will be shown later t’hat the disappearance of t’he 56S-rnp band along with t’he increase in absorption at about 600 mp is at’tributable to two separate spectral changes occurring tion

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spectra of diluted solutions FIG. 1. Absorption of phycoerythrin (--), phycoerythrin saturated wit,h PCMB (-----), and phycoerythrin saturated with PCMB, t,hen treated with 5 X 1OV M glutathione (----) in 0.1 M phosphate buffer, pH 7.0.

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FIG. 3. Absorption spectra of fraction Nos. 11, 12, and 18 from the gel filtration of phycoerythrin saturated with PCMB.

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FIG. 2. Sephadex G-100 gel filtration of phycoerythrin (X), phycoerythrin saturated with PCMB (0), and phycoerythrin saturated with PCMB, then treated with 1.5 X 10e2 M glutathione (0) in 0.1 M phosphate buffer, pH 7.0.

in distinctly different subunits. The effect of the addition of glutathione upon the treated phycoerythrin is also shown in Fig. 1. Glutathione gives rise to a partial regeneration of the 565-rnp band as well as an increase in absorbance of the 545 and 500 rnp bands. These three systems, namely, native phycoerythrin, phycoerythrin saturated with PCMB, and phycoerythrin saturated with PCMB and then treated with glutathione were subjected to Sephadex G-100 gel filtration. As shown in Fig. 2, native phycoerythrin moved as a single band, whereas phycoerythrin saturated with PCMB formed two bands, one purple and the other red. Figure 3 shows absorption spectra of the selected fractions 11, 12, and 18. The purple component, which appeared earlier than did the untreated phycoerythrin, exhibited a clear maximum at 500 rnp with two subsidiary ones at 558 and 593 mM. The presence

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FIG. 4. Absorption spectra of the purple subunit (----) and red subunit (--) separat,ed by centrifugation.

of this 593-rnp band appears to account for t,he observed increase at about 600 rnp in t.he PCMB-treated phycoerythrin shown in Fig. 1. The red component, appearing lat’er and in greater quantity, exhibit’ed a single maximum at 545 rnp. This unequivocally demonstrates that phycoerythrin, when saturated with PCMB, is split into two kinds of suba pigment whose unit, one containing principal absorption maximum is at 500 rnp and the other containing a pigment absorbing at 545 mp. Light-scattering effects are clearly present in the absorption spectra (Fig. 3) of fractions 11 and 12. This is indicative of the fact that this subunit exist’s in 0.1 M phosphate buffer (pH 7.0) as a suspension of insoluble particles. These heavy particles move fastest on the column, appearing in the early fractions of the gel filtration. It soon became obvious that the t’wo subunits could be easily separated from one another by centrifugation. Therefore, excess PCMB was added to a solution of phycoerythrin in 0.1 M phosphate buffer (pH 7.0) and allowed to stand for 2 weeks at 4”. The resulting mixture was filtered through glass wool and the filtrate was centrifuged at 198,000g for 2 hours. The purple precipitate t’hus recovered was washed several times

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FIG. 5. Absorption spectra of fraction Nos. 13, 14, 18 and 23 from the gel filtration of phycoerythrin saturated with PCMB, then treated wit)h 1.5 X 10-Z M glutathione.

with 0.1 M phosphate buffer (pH 7.0), centrifuging at 100,OOOgfor 30 minutes between washings, and resuspended in the same buffer. Figure 4 presents the absorption spectra of the isolated purple suspension as well as of the red supcrnate. These spectra are identical to those of the purple and red subunits separated by means of gel filtration as shown in Fig. 3. Figure 2 also shows that gel filtration of PCAIB-treated phycoeryt,hrin subsequently treated with glutathione gives rise to a species that appears in slightly later fractions than does IYXIB-treated phycoerythrin alone. This may be at,tributable to a removal of protein-bound mercury by the glutathione giving rise t’o a lighter species moving somewhat slower on the column. The spectra presented in Fig. 5 as well as the results of ultracentrifugal studies do not demonstrate complet’e reconstitution of ntttive phycoeryt)hrin upon removal of the PCNB from the subunits with glutathione. However, there does exist some indication of heterogeneous characteristics in the fraction

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pattern of Fig. 2 as evidenced by the broadness of the fraction peaks. This heterogeneity is further evidenced by examining the spectra of the initial and trailing fractions of the second band (red subunit) shown in Fig. 5. The initial fraction, 18, shows a better recovery of the 565-rnp maximum in the presence of glutathione than does the t,railing fraction, 23. The regenerat,ion of the 565.rnp peak will be better demonstrated when these fractions are treated with sodium hydrosulfite. This result suggests that the highly regenerated (565.rnp) species is formed separately from the poorly regenerated (565-mp) component. The fraction containing the former species moves faster than that of the latter component, indicating that after addition of glutathione, some of the red subunit undergoes association giving rise to its faster moving associated form characterized by the higher regeneration of the 565-rnp band. Since there is some overlap of the major absorption maximum at 54.5 rnp with that at 565 rnp, the regeneration of this 565-rnp maximum is effectively masked especially when the degree of regeneration is slight. We can show this premise to be so by using sodium hydrosulfite whose bleaching effect has been shown to be more selective on the 500- and 545-rnp peaks than on the 565-rnp peak (1, 2). That fraction obtained from the gel filtration of PCMB-treated phycoerythrin and characterized by the complete absence of the 565-rnp maximum (the red subunit) exhibited a uniform bleaching of its 545-rnp peak when treated with sodium hydrosulfite without any revelation of a 565rnp peak (Fig. 6b). As shown in Fig. 6a, when glutathione was added to the red subunit, a slight diminution of the 545-rnM peak along with a slight increase of absorbance in the longer wavelength regions was observed. Upon the addition of sodium hydrosulfite, we were able to follow the rapid bleaching of the 545-rnp maximum with time (Fig. 6a), but in this case the regenerated 565-rnp peak was revealed indicating that the pigment moiety absorbing at 565-rnp originates from this red subunit as has been preliminarily suggested in previous paper (3). We also used sodium hydrosulfite on selected initial and trailing fractions of the

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FIG. 6. Effect of sodium hydrosulfite (lo+ M) on the red subunit, fractions 20 and 21, obtained from the gel filtration of PCMB-treated phycoerythrin: (a) fraction 20, without glutathione (----) and with lo-$ M glutathione (-). (b) fraction 21, without glutathione.

red subunit to which glutathione had been added before passing it through the column. The spectra of the initial fraction, 18, are shown in Fig. 7a. It is evident that there has been considerable regeneration of the 565rnp peak. After adding sodium hydrosulfite, the 565-rnp peak becomes more prominent as the 545-rnp peak becomes bleached. Figure 7b shows the apparently single-peaked trailing fraction, 23, revealing a smaller amount of its 565-rnp peak upon the addition of sodium hydrosulfite. It should be noted that those fractions used in Figs. 6 and 7 had been stored at 4” for about 2 weeks before using. During this storage period, some bleaching had occurred along with a clouding of the solution. However, the general fea-

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FIG. 7. Effect of sodium hydrosulfite (10-z M) on the red subunit, fractions 18 and 23, obt,ained from the gel filtration of PCMB-treated phycoeryt,hrin to which 1.5 X 10m2M glutathione had been added prior to gel filt,rat,ion: (a) fraction 18; (b) fraction 23.

tures of the absorpt’ion spect’ra were not affected. Figure 8 shows a typical ultracentrifuge pattern of the red subunit, separat,ed by cent’rifugation from the purple subunit, as compared to that of native phycoerythrin. The sedimentation coefficient’ (s*~,~) vs. concentration plots for both native phycoerythrin and the red subunit are presented in Fig. 9. It is interesting to not,e that the plot exhibits a negative slope for native phycoerythrin, whereas the red subunit exhibits a positive slope. The s~~,~ value obtained by extrapolating the sZ,,,~values to zero concentration, was 11.5 S for nat.ive phycoerythrin and 2.3 S for the red subunit’. The s20,Wvalue of 10.4 X for a 1% solution of phycoerythrin is in excellent agreement with the value of 10.63 f 0.13 obtained by Leibo and Jones (5) for the same concentrat)ion of phycoerythrin from P. cruentum in 0.001 JI phosphate buffer (pH 6.6). The s!jo,Wvalue of 2.3 S for the red subunit agrees favorably with the

FIG. 8. Sedimentation patter11 of native phycoerythrin (0.26’3&, lower pattern) and the red subunit (O.l4a/ upper pattern). Solvent: 1% NaCl0.1 M phosphate buffer (pH 7.0). Photographs were taken 77 minutes after t,he centrifuge reached 50,740 rpm. Menisci are 011the left. Temperat,lxe: 20”; phaseplate angle: 00’.

value of about 2 S as reported for the dissociated component of phycoerythrin (about 0.1%) from C. ~ub~urn at. pH 11.4 and above (‘7). When the 2.3 S subunit was treated with 2-mcrcapt,oethanol, no significant differences betBweens?o,Wvalues were observed, 3.0 S for the treat,ed as opposed t’o 2.8 S for the untreated protein (0.14 %). Upon prolonged cent’rifugation, however, the sedimentation pattern peak of t’he treatred red subunit became much broader t,han that of the untreated one. This result’ is t’o be expect’ed since it has been shown from our gel-filtration studies that the red subunits associate amongst themselves to some extent giving rise to a heterogeneous mixture.

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FUJIMORI

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CONCENTRATION

n 8

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( mg/ml)

FIG. 9. A plot of sedimentation coefficient as a functionof concentrationfor native phycoerythrin (0) and the red subunit (X). DISCUSSION

The results presented here show that phycoerythrin isolated from P. cruentum is split. into two kinds of subunit when treated with PCMB. At pH 7.0 the purple subunit aggregates to the extent that it is no longer held in solution and is deposited as an insoluble precipitate which is not solubilized even after removing the PCMB with either 2-mercaptoethanol or glutathione. We have so far been unsuccessful in obtaining the purple subunit in a soluble form but work is continuing along these lines. The significance of the positive slope observed for the szo,Wvs. concentration plot as we observed for the red subunit is discussed by Schachmann (8) and is presumed to be due to association effect’s. Upon the addition of sulfhydryl compound to the red subunit, the tendency to associate is increased as evidenced by the results of our gel-filtration and sedimentation experiments. The associated form shows a better regeneration of the 565rnk band, suggesting that when the red subunit undergoes association, the pigment moiety may be specifically oriented in a

AND PECCI

position favorable for regenerating the 565rnp absorption. It has been concluded (9? 10) that phycoerythrin from C. rubrum contains two prosthetic groups, namely phycoerythrobilin and phycourobilin. The NO-rnp absorption band has been attributed to phycourobilin and the 545- and 565-rnp band to phycoerythrobilin. This indicates that in phycoerythrin from P. cruentum these two chromophores are present in two separate subunits: phycourobilin in the purple subunit and phycoerythrobilin in the red subunit. It was also suggest,ed that the phycoerythrobilin groups are locat’ed in two different protein environments and altered by noncovalent interactions with the proteins producing two separate absorption bands at 545 and 565 rnp. (9, 10). The 565-rnp chromophore in t’he red subunit is particularly sensitive to the effects of PCSIB. Its presence is also dependent upon the association state of the subunit. This implies that there is a specific relationship between the 565-rnp chromophore and the conformation of the protein. The nature of this conformation is contingent upon the spatial arrangement of the sulfhydryl groups. Since this highly fluorescent 565-rnp chromophore is known to accept excitation energy from the other 500- and 545-rnp chromophores (a), this specific interaction may play an important role in the efbcient transfer of energy to chlorophyll during photosynthesis. Further studies to elucidate the character of this interaction are contemplated. The dissociation of proteins by mercurials into subunits has recently been shown to occur in other proteins and enzymes (11, 12). Sulfhydryl groups which are essential for intact protein structure appear to be of importance in holding the subunits together. Although the nature of the intersubunit bonds is still unknown, there is substantial evidence available supporting the concept that the sulfhydryl groups are localized in hydrophobic regions within the protein molecule (13, 14). The total number of purple and red subunits contained in each molecule of phycoerythrin from P. cruentum as well as whet’her

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or not these two subunits represent the smallest subunits is still open to question. On the assumption that all the subunit’s of the molecule were identical, 6 hEocha and his co-workers (15) have recently shown that the values for the minimal molecular weight of phycoerythrin from C. I-ubrum as calculat,ed from total amino acid, C-, and Nterminal residue analyses (16, 17) are in reasonably good agreement. This indicated to them that the molecule contains about fourteen subunits. Vaughan (lS), however, reported that this phycoerythrin may con sist of two different kinds of subunit. The result of t’his work clearly demonstrates the presence of two differently-colored subunits in phycoerythrin from P. cmentuna and work is currently being carried out with phycoerythrin from 6. v&mm. ACKNOWLEDGMENT We wish to thank Mr. G. Hemerick of the Waltham Field Station, University of Massachusetts, Waltham, Massachusetts, for providing the algae cultures. REFERENCES 1. JONES, R. F., AND FUJIMORI, E., Physiol. Plantarum 14, 253 (1961). 2. FUJIMORI, E., AND QUINLAN, K., in “Photosynthetic Mechanisms of Green Plants,” p.

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3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17 LO.

18.

519. Nat,ional Academy of Sciences, National Research Cotmcil, Washington, D.C. (1963). FuJIMoRI,E.,X~~W~ 204, 1091 (1964). BRODY, M., AXD EMERSON, li., .4m. J. Botany 46, 433 (1959). LEIBO, S. I'., AND JONES, I?. F., Arrh. Hiochem. Biophys. 106, 58 (1964). SVEDBERG, T., AND LEWIS, N. B., J. Am. Chem Sot. 50, 525 (1928). ERIKSSON-QCENSEL, 1. B., Biochem. J. 32, 585 (1938). in SCHACHMAN, H. K., “Ultracentrifugation Biochemistry.” Academic Press, New York (1959). 6 HEOCHA, C., AND 6 CARRA,~., J. L4~n. Chem. sot. 83, 1091 (1961). 6 CARRA, P., 6 HEO~HA, C.. AND CARROLL, D. M., Biochemistry 3, 1343 (1964). GERHART, J.C., AND SCHACHMAN, H.K.,Biochemistry 4, 1054 (lYG5). Buccr, E., AND FRONTI~EI,I,I, C., J. Riol. Chem. 240, PC55 (1965). CECIL, R., AND TIIOMIAS, M. A. W., X’ature 206, 1317 (1965). GODSCHALK, IV., ~\ND VELDSTRA, H., ilrch. B&hem. Biophys. 111, 161 (1965). 6 HEOCHA, C., .Inn. Rev. Plant Physiol. 16, 415 (1965). RAE.TERY, M. A., AND Ci HEOCHA, C., Biochem. J. 94, 166 (1965). (5 CARRA, P., Biochem. J. 94, 171 (1965). VAUGHAN, M. H., Federation Proc. 22, 681 (1963).