Absorption Spectrum of Allophycocyanin Isolated from Anabaena Cylindrica: Variation of the Absorption Spectrum Induced by Changes of the Physico-Chemical Environment1
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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.