J. Biochem. 89, 79-86 (1981)
Absorption Spectrum of Allophycocyanin Isolated from Anabaena cylindrica: Variation of the Absorption Spectrum Induced by Changes of the Physico-Chemical Environment1
Akio MURAKAMI, Mamoru MIMURO,2 Kaori OHKI , and Yoshihiko FUJITA
Ocean Research Institute, The University of Tokyo, Nakano-ku, Tokyo 164
Received for publication, May 29, 1980
The absorption spectrum of allonhvcocvanin of Anabaena cylindrica was studied. The extinctions of the main absorption bands (650 and 620nm) varied depend ing on the protein concentration, ionic strength, and pH. At higher protein concen trations or higher ionic strength, the 650nm band became stronger and the 620nm band became weaker. At pH values lower than 6.0, reverse changes occurred in association with protein dissociation into monomer. Similar spectral variation was also induced by sugars and polyols. Glucose, sucrose, or glycerol (1-5M) induced an increase in the 650nm band and a decrease in the 620nm band without causing any changes in protein conformation. Propylene glycol and ethylene glycol showed a reverse effect and caused protein dissociation into monomer. The differ ence spectra of all spectral changes were identical, consisting of a sharp and strong peak at 650nm and a broad and weak one in the reverse direction at a wavelength below 620 run. The spectral variation probably results from shifts of the electronic state of phycocyanobilin. We postulated that a protein field favorable to the state producing the 650nm band is established around phycocyanobilin when the protein takes a "tight state" through protein association or by the action of sugar in aqueous environment; in a "relaxed state" in the monomer, the state of phycocyanobilin similar to that in phycocyanin becomes dominant.
Phycobilins (phycocyanobilin (PCB) and phyco are present as chromoproteins. The absorption erythrobilin) are the major light-harvesting pig spectra of phycobilins in chromoproteins are very ments in the photosynthetic systems of cyano different from those in the free forms; the main phycean, rhodophycean, and some cryptophycean band of PCB in the free form is located at around algae (1). They are tetrapyrrole derivatives, and 655nm in the visible region, while the band is
1 This work was supported in part by a Grant-in-Aid for Special Project Research on Photobiology (No. 411204) from the Ministry of Education Science and Culture. of Japan. 2 Present address: National Institute for Basic Biology, Okazaki, Aichi 444. Abbreviations: APC, allophycocyanin; PC, phycocyanin; PCB, phycocyanobilin.
Vol. 89, No. 1, 1981 79 80 A . MURAKAMI, M. MIMURO, K. OHKI, and Y. FUJITA
shifted to around 620nm in phycocyanin (PC) or tronic state that are probably similar to those of to 650nm in allophycocyanin (APC) with an PCB in PC. increase in extinction (2). The electronic states of
phycobilins are thus largely determined by the MATERIALS AND METHODS protein field; these states are so strongly fixed that
the energy absorbed by phycobilins can be con Algal Culture-Anabaena cylindrica (M-1, verted to fluorescence without loss in vitro and Algal Collection at the Institute of Applied Micro efficiently transferred to chlorophyll a by a dipole biology, The University of Tokyo) was grown dipole transition mechanism in vivo (3). autotrophically in a modified Detmer's medium
Phycobilins are covalently bound to the pro (cf. 12) as described previously (13). Cells at the tein moiety through a thio-ether linkage between linear growth phase were harvested, washed with the vinyl group of ring I and the cysteinyl residue deionized water and stored in pellet form below of a protein (4). However, the electronic state -20•Ž . of phycobilin in a chromoprotein will not be APC Preparation-Cells were suspended in a determined by this covalent bonding, but will be K-K phosphate buffer (10mM, pH 6.0) and broken largely established by non-covalent bondings which by sonication at 20 kHz for 5min. After removal depend on the field formed by the higher-order of cell debris by centrifugation, the crude extracts protein conformation. Indeed, the absorption were first fractionated by ammonium sulfate spectra of chromoproteins are dramatically precipitation (20%. saturation) so as to remove changed to those of free phycobilin when the small cell fragments, then the proteins were pre higher-order protein conformations are destroyed cipitated at 60%. saturation. Precipitated proteins with chaotropic reagents (cf. 5). Therefore, the were dissolved in K-K phosphate buffer (10mM,
higher-order protein conformation will be one of pH 6.0) and dialyzed against the same buffer. the most important objectives for studies of the They were fractionated by DEAE-cellulose column molecular characteristics of phycobiliproteins as chromatography. Samples equivalent to 500mg light-harvesting pigments in photosynthesis. Most of proteins were charged onto the column (3 •~ investigations along this line have been made with 30cm; equilibrated with the same buffer), and the PC, focusing on the relationship between the column was developed with a linear gradient of absorption spectrum in the red region and the phosphate buffer from 0.01 to 0.2M. PC ran quaternary structure of the protein (6-8) or on the faster than APC. APC-rich fractions were eluted PCB conformation in the protein (9, 10). How at 0.1-0.15M phosphate buffer. The proteins were ever, the absorption spectrum of PC in the red concentrated by ammonium sulfate precipitation region consists of multiple bands. Thus, PC is (60% saturation) and fractionated again by the not necessarily suitable for an analysis of this same column chromatography. Two or three kind, in which the absorption spectrum is used repetitions were needed to obtain sufficiently as an index of the electronic state of the chro purified APC. The APC preparation thus ob mophore. Both PC and APC share a common tained showed the absorption spectrum presented phycobilin, PCB (11). APC has a characteristic in Fig. 1. The pattern upon polyacrylamide gel absorption band at 650 nm as well as a band electrophoresis (cf. 14, 15) showed that PC con similar to the main band of PC. Thus, a com tamination in the preparation was not detectable . parison of the electronic states of PCB in PC and All treatments were done below 4•Ž. APCCmay provide useful information. Spectrophotometry-All absorption spectra As a step in the analysis of the phycobilin were measured with a Hitachi 340 spectropho protein interaction, we studied the absorption tometer. Unless otherwise indicated, measure spectrum of APC isolated from Anabaena cylindrica ments were done at room temperature with a cell under various physico-chemical conditions which of 1.0cm light path. For temperature-controlled modify the protein conformation. We found that measurements, a cell holder having a large heat the main 650nm band characteristic of APC is sink was used, and the temperature of the sample shifted reversibly to below 620nm, and that this was measured with a Cu-constantan thermocouple . change can be attributed to changes in the elec APC concentration was determined from the
J. Biochem. ABSORPTION SPECTRUM OF ALLOPHYCOCYANIN 81
absorption coefficient of Hattori and Fujita (6.54 location. A blue-shift also occurred in the side ml-mg-1-cm-1 at 650nm, 16). The spectral bands. At a concentration of 4.1 •~ 10-5mM, the measurements for this purpose were made under side band became more intense than the main the same physico-chemical conditions as those band, so that the pattern became similar to that used for determination of the absorption coefficient of PC (Fig. 1, d). -by Hattori and Fujita. Thus, the value estimated The difference spectrum for 4.1 •~ 10-5mM should be reasonably reliable, though the extinc concentration minus 8.2 •~ 10-2mM concentration tion of 650nm absorption is variable depending is shown in Fig. 2. The decrease in the absorption on the physico-chemical environment. at 650 nm was far stronger and sharper than the Gel-Filtration Chromatography-For determi absorption increase at below 620 nm. The trough
nation of the molecular size of APC, Sepharose at 650 nm appears almost symmetric. However,
CL-6B gel-filtration chromatography was carried the changes do not seem to have an isosbestic out in media with and without a non-polar solute point, and the spectrum at below 620nm consists
(glucose or propylene glycol). Sepharose CL-6B
gel was equilibrated with phosphate buffer (10 mM, pH 6.0) for 3 days; when necessary, buffer
containing non-polar solute (2M glucose or 5M
propylene glycol) was used. The column (1.6•~ 90cm) was kept at constant temperature by
running thermostated water through a water
jacket. Samples were applied to the top of the column, and a constant flow rate (3ml/h) was
maintained by a peristaltic pump attached to the bottom of the column. The column was calibrated
by using fungal glucose oxidase (molecular weight; 160,000), bovine serum albumin (68,000), oval
bumin (45,000), and horse heart cytochrome c
(12,500). Fig. 1. Absorption spectra of APC at various concen trations. Curve a: 8.2 •~ 10-2mM APC in 10 mM phos RESULTS phate buffer (pH 6.0), curve b: 4.1 •~ 10-3mM APC, curve c: 4.1 •~ 10-4mM APC and curve d: 4.1 •~ 10-5mM Absorption Spectrum at Various APC Concen APC. Curve a was measured with an 0.1 cm cell, and trations-The absorption spectrum of APC con curve d with a 10cm cell. Absorption spectra b, c, and sists of a characteristic and sharp absorption d are enlarged •~2, •~ 20, and •~ 20, respectively. band3 at 650nm and side bands at around 620 Measurements were made at 25•Ž. nm in the red wavelength region. The extinction of the former is so strong that the latter bands always appear as a shoulder (cf. Fig. 1, a). How ever, the ratio of the two was found to be variable. At low concentrations of APC, the extinction of the 650nm band became weaker, and the side bands became stronger (Fig. 1). Reduction in the extinction of the main band was always accompanied by a slight blue-shift of the band
3 We call this band the 650nm band in this report. However, this does not imply the exact location of the Fig. 2. Difference spectrum of spectral changes in absorption band. Further, preliminary analysis indi duced by APC concentration change (curve d minus a cated that the absorption peak consists of multiple absporption bands. in Fig. 1).
Vol. 89, No. 1, 1981 82 A. MURAKAMI, M. MIMURO, K. OHKI, and Y. FUJITA of several bands. These features indicate that at pH, most of the APC molecules take monomer lower APC concentrations the electronic state of form (17). Phycobiliproteins generally associate PCB responsible for the 650 nm band changes in to higher oligomers at high protein concentration such a way as to produce the bands at below or at high ionic strength (6, 7, 18, 19). Thus, 620 run. These changes are reversible. The spec the spectral changes observed in our experiments trum, once altered by dilution of APC, recovered may result from changes in the dissociation to the original one when the solution was concen association state. The electronic state of PCB's trated in a collodion bag under reduced pressure that produces the 650nm band may be stable (Fig. 3). when the protein takes a highly associated form, Effects of Physico-Chemical Environment and it may shift to give rise to the bands at below - Similar spectral changes also occurred when the 620nm in the protein dissociated into monomer. ionic strength or pH of the medium was changed. Hydrophobic bonding may be a main mono The extinction at 650 nm relative to that at 610 mer-monomer interaction in oligomer molecules. nm was enhanced at higher ionic strength by an As shown in Table I, chaotropic anions can induce increase in the concentration of phosphate buffer spectral changes similar to those caused by protein
(pH 6.0) (Fig. 4-A). The APC concentration was dissociation, and the effect is stronger in the order 4.1 •~ 10-4MM in 1.0M phosphate buffer. How of their chaotropic action (Cl- Fig. 4. Changes in the ratio of absorptions at 650 and Fig. 3. Reversibility of spectral changes induced by 610nm (A650/A610) induced by concentration (A) and APC concentration change. Curve a: 3.2 •~ 10-9mM pH (B) changes of buffer and change of the measuring APC in 10mM phosphate buffer (pH 6.0), curve b: temperature (C). Before measurements, APC (4.1 •~ diluted 50 times with 10mM phosphate buffer and, curve 10-4mM) was incubated for 5min in the dark under the c: concentrated 50 times in a collodion bag under indicated conditions. In A and C, phosphate buffer reduced pressure. The absorption spectrum of b was (pH 6.0) was used (10mM in C), and in B, Tris-maleate measured with a 5cm cell and is enlarged •~ 10. Meas buffer (ƒÊ=7 •~ 10-3). Except for C, all measurements urements were made at 25•Ž. were made at 25•Ž. J. Biochem. ABSORPTION SPECTRUM OF ALLOPHYCOCYANIN 83 TABLE 1. Effects of chaotropic anions on the ratio of absorptions at 650 and 610nm (A650/A610) at 20•Ž . a APC (4 .1 •~ 10-4mM) in 10mM phosphate buffer (pH Fig. 6. Changes in the ratio of absorptions at 650 and 6.0) gave 1.21 A650/A610. Anions were added to the 610nm (A650/A610)induced by glucose (closed circles) above APC solution at the indicated concentrations . and sucrose (open circles). b The value in parenthesis was obtained after removal of 0.25M SCN by dialysis. Fig. 7. Gel-filtration patterns (A) of APC in the presence or absence of glucose (2M) and absorption spectra of the eluates (B). Phosphate buffer (10mM, Fig. 5. Spectral changes induced by glucose (1.8 M, pH 6.0) without (A-1, B-1) or with 2M glucose (A-2, A) and difference spectrum of glucose minus glucose B-2) was used for development. As molecular weight free medium (B). In A, curve a (solid line) : 3.7 •~ 10-4 markers, fungal glucose oxidase (a), bovine serum mM APC in 10mM phosphate buffer (pH 6.0) and curve albumin (b), and ovalbumin (c) also run. Gel-filtration b (dotted line) : a plus 1.8M glucose. Measurements and spectral measurements were made at 25•Ž. For were made at 25•Ž. details, see the text. non-electrolytic solutes, sugars and polyols in the sucrose. This ratio increased to 1.54 in the 1.4M medium. In the presence of 1.8 M glucose, the sucrose medium. If these changes are ascribed 650 nm band was intensified, and the extinction to the increase in APC concentration, the APC below 620nm was reduced (Fig. 5). The changes concentration in the sucrose medium would have were again reversible, e.g., after removal of glucose to be 30 times higher than the concentration in by dialysis, the spectrum recovered its original the medium without sucrose. However, the form. Sucrose had a similar effect. The effect reduction of water concentration in the sucrose appeared at very high concentration of sugars medium was only 30%. (cf. Fig. 6). Thus, one (Fig. 6). In such solutions, the concentration of molecule of sucrose must be hydrated with about active water must be significantly reduced, so that 30 molecules of water. According to the data the APC concentration in active water may be derived from isopiestic vapor pressure measure increased, and APC may take a more associated ments (20, 21), or ultrasonic interferometry (22), form which gives a more intense 650nm band. however, the hydration number of sucrose is in In Fig. 6, A650/A610was 1.23 in the medium without the range between 1.1 and 9.7 mol per mol of Vol. 89, No. 1, 1981 84 A . MURAKAMI, M. MIMURO, K. OHKI, and Y. FUJITA sucrose. Thus, the above sugar effect is unlikely The spectral changes induced by sugar at high to be the major factor. Indeed, molecular size concentrations thus may not be due to changes analysis by Sepharose CL-6B gel-filtration was in the dissociation-association equilibrium. not significantly affected by glucose, though the Glycerol had the same effect as sugars. spectrum was changed markedly (Fig. 7-A vs. -B); However, diols (ethylene glycol and propylene the size (ca. 100,000) corresponded to the trimer. glycol) had an opposite effect. In the presence of propylene glycol, the 650 nm band was greatly reduced and the bands at around 620nm became more intense (Fig. 8). Difference spectra were identical with those for the changes due to protein dissociation-association. The changes were again reversible. These diols caused changes not only in the absorption spectrum but also in the molec ular size of APC. APC dissociated to the mono mer in the medium with propylene glycol (Fig. 9). DISCUSSION The absorption spectrum of APC is characterized by a 650nm band and side bands around 620nm. The extinction of the 650nm band relative to that of the 620nm band or protein band has been Fig. 8. Spectral changes induced by propylene glycol commonly used as an index of APC purity. How (4.5;mt, A) and difference spectrum of propylene glycol ever, our results, as well as those reported by ( minus propylene glycol-free medium (B). In A, curve previous investigators (23, 24), indicate that the a(solid line): 4.1 •~ 10-4mM APC in 10mM phosphate extinction of the 650nm band is variable, depend buffer (pH 6.0) and curve b (dotted line): a plus 4.5M ing on the physico-chemical environment. Our propylene glycol. Measurements were made at 25•Ž. results clearly show that the extinction is strongly affected by APC concentration (Fig. 1) and by the ionic strength and pH of the medium (Fig. 4-A, -B). Thus, when an APC preparation is characterized spectroscopically, it is extremely important to specify the physico-chemical param eters of the APC solution in question. Except for the sugar effect, all other environ mental factors affecting the absorption spectrum of APC are modifiers of the dissociation-associa tion state of phycobiliprotein (25). Variation of the absorption spectrum induced by such factors indicates that the electronic state of PCB respon sible for the 650nm band becomes unstable and shifts to states that give bands at below 620nm, Fig. 9. Gel-filtration pattern of APC in the presence of when APC dissociates to the monomer. This is 5M propylene glycol (A) and absorption spectrum of the supported by our experiments with propylene eluates (B). Phosphate buffer (10mM, pH 6.0) with glycol and by those of Ohad et al. on the pH 5M propylene glycol was used for development. Gel effect (17). The CD spectrum of APC has been filtration and spectral measurement were done at 25•Ž. reported to have two positive-ellipticity maxima As molecular weight markers, fungal glucose oxidase at around 650nm and 620nm in the red spectral (a), bovine serum albumin (b), ovalbumin (c), and horse heart cytochrome c (d) were also run. For details, see region, which correspond well to the two absorp the text. tion bands (23, 24, 26). These simple features J. Biochem. ABSORPTION SPECTRUM OF ALLOPHYCOCYANIN 85 may Indicate that the electronic state of PCB in environment. The main band of the former is APC is mainly determined by chromophore-protein located at around 653nm, and is characteristic interaction. If so, the electronic state of PCB must of APC, while the main band of the latter appears be affected strongly by changes in the tertiary at around 620nm, showing that the electronic structure of the protein moiety, especially the state is very similar to that in PC. APC of structure around PCB. When APC takes an Anabaena cylindrica was found to have two differ associated form, each monomer molecule involved ent N-terminal amino acids (Hase, T. & Matsu will have its tertiary structure slightly modified bara, H., personal communication) indicating that from that of the free form. Since the electronic APC monomer consists of two different peptides. state corresponding to the 650nm band is more Thus, the two groups of absorption bands may stable in the associated form than those responsible be attributed to the PCB's present in the different for the bands at below 620nm, such modification peptides. Details of our analysis along this line must provide a tertiary structure around PCB will be reported in a separate paper. favorable to the 650nm form. Sugars at high concentrations also caused enhancement of the The authors wish to express their thanks to Prof. H. 650 urn band. However, the molecular size of Suzuki of Waseda University and to Prof. H. Matsubara of Osaka University for their helpful criticism and advice APC remained unaltered; APC remained in a during this study. trimer state even in a medium with glucose at a high concentration. The sugar effect indicates that the tertiary structure favorable to the PCB REFERENCES state responsible for the 650nm band can be 1. Bogorad, L. (1975) Ann. Rev. Plant Physiol. 26, established without changes in the dissociation 369-401 association state. Such action of sugar or glycerol 2. Glazer, A.N. (1976) in Photochemical and Photo has been reported for yeast alcohol dehydrogenase biological Reviews (Smith, K.C., ed.) Vol. 1, pp. (27) and E. colt aspartase (28). The conformation 71-115, Plenum Press, New York around the active site is . presumably modified 3. Searle, G.F.W. & Tredwell, C.3. (1979) in Chloro independently of the dissociation-association state. phyll Organization and Energy Transfer in Photo When the tertiary conformation of APC is thus synthesis (Ciba Foundation Symposium 61) pp. modified, the resulting "tight state" of monomer 257-281, Excerpta Medica, Amsterdam structure will generally provide a structure favor 4. Crespi, H.L. & Smigh, U.H. (1970) Phytochemistry 9,205-212 able to the PCB state responsible for the 650nm 5. O'hEocha, C. (1965) in Chemistry and Biochemistry band. The absorption spectrum of monomer of Plant Pigments (Goodwin, T.W., ed.) pp. 175 APC closely resembles that of PC. Further, mild -196, Academic Press, London treatment with urea (1.8M) also gave a spectrum 6. Hattori, A., Crespi, H.L., & Katz, J.J. (1965) Bio similar to that of PC (data not shown). There chemistry 4, 1225-1238 fore, when the protein conformation is "relaxed" 7. Scott, E. & Berns, D.S. (1965) Biochemistry 4, by dissociation to monomer, the electronic state 2597-2606 of PCB will become very similar to or identical 8. Glazer, A.N., Fang, S., & Brown, D.M. (1973) J. with that in PC. Though the PCB state in PC is Biol. Chem. 248, 5679-5685 also determined by a strong chromophore-protein 9. Scheer, H. & Kufer, W. (1977) Z. Naturforsch. 32c, interaction (9), the PCB state responsible for the 513-519 10. Kufer, W. & Scheer, H. (1979) Hoppe-Seyler's Z. 650nm band in APC must be fixed more strongly Physiol. Chem. 360, 93 5-956 and thus more rigidly by the electronic filed formed 11. Chapman, D.J., Cole, W.J., & Siegelman, H.W. by the protein. This may be why the 650nm (1967) Biochem. J. 105, 903-905 band is so sharp. 12. Watanabe, A. (1960) J. Gen. Appl. Microbial. 6, Our preliminary analysis indicated that me 283-292 absorption spectrum of APC consists of at least 13. Fujita, Y. & Suzuki, R. (1971) Plant Cell Physiol. 12, two groups of component bands; one causes the 641-651 14. Williams, D.E. & Reisfeld, R.A. (1964) Ann. N.Y. spectral changes observed here, and the other is Acad. Sci. 121, 373-381 not affected by changes in the physico-chemical Vol. 89, No. 1, 1981 86 A. MURAKAMI, M. MIMURO, K. OHKI, and Y. FUJITA 15. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 23. Brown, A.S., Foster, J.A., Voynow, P.V., Franzblau, 16. Hattori, A. & Fujita, Y. (1959) J. Biochem. 46, C., & Troxler, R.F. (1975) Biochemistry 14, 3581 633-644 -3588 17. Ohad, I., Schneider, H.A.W., Gendel, S., & Bogorad, 24. Brown, A.S. & Troxler, R.F. (1977) Biochem. J. 163, L. (1980) Plant Physiol. 65, 6-12 571-581 18. Neufeld, G.J. & Riggs, A.F. (1969) Biochim. Bio 25. Chapman, D.J. (1973) in The Biology of Blue-Green phys. Acta 181, 234-243 Algae (Carr, N.G. & Whitton, B.A., eds.) pp. 162 19. MacColl, R., Lee, J.J., & Berns, D.S. (1971) -185, Blackwell Sci. Pub., London Biochem. J. 122, 421-426 26. Canaani, O.D. & Gantt, E. (1980) Biochemistry 19, 20. Robinson, R.A. & Stokes, R.H. (1959) Electrolyte 2950-2956 Solutions 2nd Ed., pp. 174-222, Butterworths, 27. Myers, J.S. & Jakoby, W.B. (1975) J. Biol. Chem. London 250,3785-3789 21. Miller, D.G. (1956) J. Phys. Chem. 60, 1296-1299 28. Tokushige, M. & Mizuta, K. (1976) Biochem. Bio 22. Shiio, H. (1958) J. Am. Chem. Soc. 80, 70-73 phys. Res. Commun. 68,1082-1087 J. Biochem.