tion around 440 nm where chlorophyll a absorbs max- imally. The red-shifted 730 nm .... Betzel C, Wilson KS, Witt HT and Saenger W (1993) Three- dimensional ...
Photosynthesis Research 45: 41--49, 1995. (~) 1995 Kluwer Academic Publishers. Printed in the Netherlands.
Regular paper
Rapid isolation of Photosystem I chlorophyll-binding proteins by anion exchange perfusion chromatography Staffan E. Tjus 1, Margrit R o o b o l - B o z a 1, Lars O l o f P~lsson 2 & Bertil A n d e r s s o n 1
1Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden; 2Department of Physical Chemistry, University of Ume&,S-901 87 Ume&,Sweden Received 16 December 1994; accepted 20 June 1995
Key words: fluorescence, LHC 1-680, LHC 1-730, light-harvesting complex I (LHC I), PS I
Abstract
With the new method of anion exchange perfusion chromatography we have devised an extremely rapid technique to subfractionate spinach Photosystem I into its chlorophyll a containing core complex and various components of the Photosystem I light-harvesting antenna (LHC I). The isolation time for the LHC I subcomplexes following solubilisation of native Photosystem I was reduced from 50 h using traditional density centrifugation procedures down to only 10-25 min by perfusion chromatography. Within this very short period of isolation, LHC I has been obtained as subfractions highly enriched in Lhca2+3 (LHC 1-680) and Lhca 1+4 (LHC 1-730). Moreover, other highly enriched subfractions ofLHC I such as Lhca2, Lhca3 and Lhcal +2+4 were obtained where the later two populations have not previously been obtained in a soluble form and without the use of SDS. These various subfractions of the LHC I antenna have been characterised by absorption spectroscopy, 77 K fluorescence-spectroscopy and SDS-PAGE demonstrating their identities, functional intactness and purity. Furthermore, the analyses located a chlorophyll b pool to preferentially transfer its excitation energy to the low energy F735 chromophore, and located specifically the origin of the 730 nm fluorescence to the Lhca4 component. It was also revealed that Lhca2 and Lhca3 have identical light-harvesting properties. The isolated Photosystem I core complex showed high electron transport capacity (1535 #moles 02 mg Ch1-1 h -1) and low fluorescence yield (0.4%) demonstrating its high functional integrity. The very rapid isolation procedure based upon perfusion chromatography should in a significant way facilitate the subfractionation of Photosystem I proteins and thereby allow more accurate functional and structural studies of individual components.
Abbreviations: a.u.- arbitrary units; DCIP-2.6-dichlorophenol indophenol; LHC-light harvesting complex Introduction
Photosystem I entraps light energy by a heterogeneous pigment-protein antenna which funnels excitation energy into the reaction centre chlorophyll P700. Unlike Photosystem II, Photosystem I was long considered to lack an outer chlorophyll alb antenna. However, primary indications of a specific light-harvesting chlorophyll a/b complex (LHC I) associated with Photosystem I in higher plants (Anderson et al. 1978; Thornber et al. 1979) soon led to its definite identification (Mullet et al. 1980a, b). Later this higher plant LHC
I could be isolated by sucrose density gradient centrifugation following solubilisation of native Photosystem I complexes with dodecyl maltoside and zwittergent (Mullet et al. 1981; Haworth et al. 1983). A similar LHC I complex was also purified from green algae (Wollman and Bennoun 1982; Ish-Shalom and Ohad 1983). The Photosystem I core complex of cyanobacteria and higher plants consists of at least 15 polypeptides ranging between 84 and 3 kDa (Andersson and Franz6n 1992; Ikeuchi 1992). These are designated PsaA-PsaO according to their corresponding genes
42 (Golbeck 1992). The two reaction centre proteins, PsaA and PsaB carry an inner antenna with approximately 100 chlorophyll a molecules and bind together with the PsaC subunit all the cofactors required for stable charge separation (Golbeck and Bryant 1991; Andersson and FranzEn 1992). Recently, the structure of a cyanobacterial Photosystem I core complex was determined at 6 ]k resolution by X-ray crystallography (Witt et al. 1992; Krauss et al. 1993). LHC I is composed of 4 polypeptides with molecular weights in the range of 21-24 kDa (Ikeuchi et al. 1991; Knoetzel et al. 1992) which together bind approximately 120 chlorophyll molecules (Bassi and Simpson 1987). The chlorophyll a/b ratio is approximately 3.5 (Haworth et al. 1983). Electron microscopy studies (Boekema et al. 1990) suggest that 8 LHC I polypeptides surround each Photosystem I core complex in a monolayer. Derived amino acid sequences (Ikeuchi et al. 1991; Knoetzel et al. 1992) have assigned these four polypeptides to specific genes designated L h c a l - 4 (Jansson et al. 1992). The corresponding proteins have accordingly been named L h c a l - 4 (Jansson 1994). LHC I can be further fractionated into two chlorophyll a/b binding subcomplexes by sucrose density centrifugation (Lam et al. 1984; Bassi and Simpson 1987) or mild SDS-PAGE (Bassi et al. 1985; Knoetzel et al. 1992). One subfraction shows 77 K fluorescence at 680 nm and is composed of the Lhca2+3 polypeptides with molecular weights of 23 and 24 kDa, respectively. The other subcomplex gives maximal 77 K emission at 730 nm and is composed of the Lhcal+4 polypeptides, often resolved by SDS-PAGE as a double band at 21-21.5 kDa. These two subcomplexes have been termed LHC 1-680 and LHC 1-730, respectively (Bassi et al. 1985; Bassi and Simpson 1987). Knoetzel et al. (1992) were able to further fractionate the LHC 1-680 complex into subcomplexes LHC 1-680A (Lhca3) and LHC 1-680B (Lhca2) by repetitive mild SDS-PAGE, both chlorophyll a/b binding. It should be stressed however that none of these earlier described subfractions are completely free of cross contamination of other LHC I components (Lam et al. 1984; Bassi et al. 1985; Bassi and Simpson 1987; Ikeuchi et al. 1991; Knoetzel et al. 1992). The available procedures for subfractionation of native Photosystem I into its core complex and LHC I subfractions are very time-consuming and usually require several over-night sucrose gradient ultracentrifugation steps. This may lead to structural and functional impairment, including release of pigments
and/or dissociation of protein subunits. This in turn may influence the energy transferring properties of the isolated complexes. Moreover, the LHC 1-680 and -730 subcomplexes are difficult to obtain wellseparated by density centrifugation since their densities are very similar and they consequently migrate very closely in the gradient. Other previous methods have involved electrophoresis and required the use of highly denaturating SDS (Ikeuchi et al. 1991; Knoetzel et al. 1992). Efficient and rapid isolation of hydrophobic membrane proteins is not easily achieved. We present in this work a novel and extremely rapid chromatographic method for subfractionation of the spinach Photosystem I holocomplex into its Photosystem I core complex and various LHC I components after solubilisation by mild detergents. The procedure is based upon a new kind of chromatographic beads with pores lined inside with ion-exchange groups (POROS). This method, named perfusion chromatography (Afeyan et al. 1990; Afeyan and Fulton 1991; Regnier 1991; Fulton et al. 1992; Lehman et al. 1993), combines a high reactive surface area per bead volume with extremely high elution flow. Perfusion chromatography thus allows very rapid and efficient separation even of quite complex mixtures of proteins. It has recently been applied to the isolation of PS II reaction centres (Roobol-Boza et al. 1993) but has otherwise not been used for the isolation of any hydrophobic membrane proteins. With this rapid technique of ion-exchange perfusion chromatography, the time to obtain LHC I subfractions from native Photosystem I complexes has been drastically reduced from approximately 50 h to only 10-25 min. All isolated subpopulations of Photosystem I, as characterised by SDS-PAGE, absorption spectroscopy and steady-state fluorescence at 77 K, show very high purity and posess high functional integrity.
Materials and methods
Isolation procedures Spinach (Spinacia oleracia L.) was grown under artificial light in nutrient solution as described by Andersson et al. (1976). Native Photosystem I complexes used as starting material were isolated after solubilisation of thylakoid membranes with Triton X-100 followed by overnight sucrose gradient centrifugation according to Mullet
43 et al. (1980a). Thylakoid membranes were prepared, destacked by low salt treatment, diluted with water and solubilised with Triton X-100 (0.75% w/v) at 0.8 mg Chl/ml (detergent:chlorophyll = 0.94:1) and pH = 7.5, for 30 min at room temperature. The solubilised thylakoid membranes were subjected to sucrose density centrifugation for 20 h at 90 000 x g. The Photosystem I fraction was collected from the bottom of the gradient, diluted three times with water and centrifuged for 60 min at 120000 × g. The pellet containing native Photosystem I was dissolved in a buffer containing 50 mM sorbitol and 5 mM EDTA (pH 7.8). For further fractionation of the native Photosystem I complex by perfusion chromatography, aliquots of 1 mg chlorophyll were solubilised as in Haworth et al. (1983) with minor modifications as specified below. The photosystem I particles were diluted to 0.5 mg chlorophyll/ml in 50 mM sorbitol and 5 mM EDTA./3-D-dodecyl maltoside (2.5 mg/ml) and zwittergent-16 (2.8 mg/ml) were added to yield a total detergent:chlorophyll ratio of 10.6:1 and finally the pH was adjusted to 7.5. The solubilisation was performed in darkness for 80 min at 10 °C under slow stirring.
a stacking gel of 6%. SDS-PAGE was performed without urea as this clearly separates the LHC I subunits from the polypeptides of the PS I core complex in contrast to urea systems where LHC I is migrating closely together with PsaD. Solubilisation of samples was carried out with SDS and mercapto-ethanol in Trisbuffer (pH 6.8) at room temperature for 10 min and the electrophoresis was run over-night at 0°C. Silver staining was performed according to Wray et al. (1981). In order to identify the photosystem I polypeptides, immunoblotting was carried out as described in Towbin et al. (1979). Antibodies used were directed against the PsaC, PsaD, PsaE, PsaF, PsaG, PsaH and PsaL polypeptides of the Photosystem I core complec and against the Lhca2 and Lhca3 subunits of LHC I. Photosystem I electron transport from ascorbate/DCIP to methylviologen was measured as oxygen consumption in a Clark type oxygen electrode at saturating light. The assay medium contained; 40 mM Na-phosphate (pH 7.4), 1.0 mM NaC1, 10 mM sucrose, 10 #M DCMU, 5 mM Na-azide, 1.0 mM ascorbate, 0.1 mM DCIP, 0.12 mM methylviologen and 5/lg of chlorophyll/ml.
Perfusion chromatography
Absorption and fluorescence spectral analyses
The solubilised photosystem I particles were diluted 5 times with water and their pH was raised to 8.1 before loading onto a small (1 ml) anion exchange column (20 POROS Q, PerSeptive Biosystems Inc., USA) connected to a FPLC-system (Pharmacia, Sweden). The column was pre-equilibrated using a buffer composed of 50 mM Tricine (pH 8.1) supplemented with 0.02% zwittergent and 0.03% dodecyl maltoside. After loading of the solubilised Photosystem I particles, the column was washed with equilibration buffer. Elution was performed by applying a 0--400 mM NaC1gradient (Fig. 1) at a flow rate of 1 ml/min. The salt gradient was halted at fixed concentrations during the appearance of elution peaks. Eluted protein fractions were detected at 280 nm (UV-1, Pharmacia, Sweden) and collected in small (1 ml or less) aliquots.
Absorption spectra were recorded on a GBC 920 UV-VIS spectrophotometer prior to the fluorescence mesurements and the maximal absorbance at 670 nm for the samples used was approximately 0.1/cm. Steady-state fluorescence measurements were carried out on a Spex fluorolog 21. A reference substance, Oxazine 1 (Lambda Physik) whose quantum yield was determined to 0.14, was used to calculate quantum yields integrated over the emission spectra (P~lsson et al. 1995). The fluorescence measurements were performed in a medium of 20 mM Tricine (pH 7.8), 40 mM sucrose and 4 mM NaC1. Native Photosystem I and Photosystem I core fractions were supplemented with 20 mM Na-ascorbate and 20 #M DCIP in order to keep P700 in a reduced photochemically active state. For fluorescence measurements at 77 K, glycerol (60% ~v/w) was added to the samples and the temperature was controlled by an Oxford cryostat (DN 704).
Biochemical analyses The fractions obtained were analysed for their chlorophyll concentration and chlorophyll alb ratio according to Arnon (1949). The polypeptide composition was analysed by SDS-PAGE using the buffer system of Laemmli (1970), a separation gel of 12-22.5% acrylamide and
44
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Fig. 1. Elution profile (A280, 0--400 mM NaCi) showing peaks I-VI from anion exchange perfusion chromatography of Photosystem I proteins. Native Photosystem I particles (0.5 mg Chl/ml) were soinbilised with 2.5 mg dodecyl maltoside and 2.8 mg zwittergent/ml for 80 min and applied to a POROS Q column equilibrated with 50 mM Tricine (pH 8.1), 0.03% dodecyi maltoside and 0.02% zwittergent.
Fig. 2. SDS-PAGE of native Photosystem I and fractions isolated by anion exchange perfusion chromatography. (1) Native Photosystetn I. (2) Column void. (3--4) Peak I. (5) Peak II. (6) Peak III. (7-8) Peak IV. (9) Peak V. (10) Peak VI. Identification of the polypeptides in lane I was achieved by immunological detection and analysis of electrophoretic migration.
Results and discussion Rapid chromatographic isolation of LHC I subfractions and the Photosystem I core complex The native Photosystem I complex used as starting material for the subfractionation studies was shown to
contain the PsaA-H, PsaL and Lhcal-4 polypeptides and unidentified low molecular weight polypeptides around 5 kDa (Fig. 2, lane 1). The identification was achieved by immunological detection combined with analysis of electrophoretic migration. The PsaN sub-
45 unit was not identified but most likely is included in the polypeptide cluster around 9-10 kDa while the 5 kDa protein most likely corresponds to PsaK. In order to further dissociate the protein complex, isolated native Photosystem I was solubilised with dodecyl maltoside and zwittergent. To load the solubilised material, corresponding to 1 mg chlorophyll, onto the anion-exchange column for perfusion chromatography, the optimal pH was found to be 8.1. Subsequent to loading, some PsaE migrated through the column following the void volume (Fig. 1, peak I, Fig. 2, lanes 3-4) together with dissociated chlorophylls and detergents while the other polypeptides became attached to the POROS beads. By applying a NaCl-gradient (0-400 mM) the remaining components of Photosystem I could be eluted from the column in 5 fractions containing Lhca2+3, Lhca2(+l), Lhcal+4, the Photosystem I core complex and finally unsolubilised native Photosystem I complexes. The elution was completed in 40 min. The salt gradient was first halted at 40 mM NaC1 until full appearance of peak II (Fig. 1). This protein fraction with a chlorophyll alb ratio of 1.9 was composed of the Lhca2 and Lhca3 polypeptides of the LHC 1-680 complex as judged by SDS-PAGE (Fig. 2, lane 5). Lhca3 was clearly enriched as compared to the equal ratio between the two components in native Photosystem I (Fig. 2, lane 1). In addition, peak II contained the remainder of the 9 kDa PsaE polypeptide that did not flow through the column during the loading as well as some minor contamination of PsaF and PsaH. As PsaE, PsaF and PsaH in our second polypeptide peak are present in substoichiometric amounts they are likely to be comigrants of LHC I. PsaE has previously been shown to comigrate with LHC I during subfractionation (Knoetzel et al. 1992). An 11 kDa polypeptide that might correspond to PsaE or PsaH has also been identified as a 'green band' during mild electrophoresis (DERIPHAT-PAGE) of LHC I (Preiss et al. 1993). PsaH has earlier been proposed a role of binding LHC I to the Photosystem I core complex as it is lacking in cyanobacteria which are devoid of LHC I (Scheller and M¢ller 1990). We have also, by perfusion chromatography, isolated pure PsaF together with chlorophyll a and b (to be presented in a separate publication). This is in line with earlier work where PsaF has been isolated together with LHC I and been proposed to bind chlorophyll (Anandan et al. 1989; Vainstein et al. 1989; Preiss et al. 1993). Thus, it is possible that PsaF functions as an internal antenna polypeptide in
addition to its docking of plastocyanin to the thylakoid lumen side (Golbeck and Bryant 1991). The NaCl-gradient was continued to 135 mM (Fig. 1) where it was kept constant to finalise the elution of a small fraction (peak III) followed by a larger one (peak IV). The small third peak contained mainly the 23 kDa Lhca2 protein together with some minor contribution from Lhcal (Fig. 2, lane 6). Peak IV was composed of the Lhcal and Lhca4 polypeptides closely migrating at 21.5 and 21 kDa, respectively, and showed a chlorophyll a/b ratio of 2.9 (Fig. 1; lanes 7 and 8) thereby representing the LHC 1-730 complex. The Photosystem I core complex was eluted as a large fraction at 210 mM NaC1 (Fig. 1, peak V) with a chlorophyll a/b ratio of 15.5. This complex contained stoichiometric amounts of the reaction centre PsaA and PsaB subunits in the high molecular weight region and the PsaC, -D, -G, and -L subunits below 20 kDa (Fig. 2, lane 9). Compared to native Photosystem I (Fig. 2, lane 1) PsaE and PsaH were partially depleted while the 4 LHC I polypeptides and the 17 kDa PsaF subunit were virtually absent (Fig. 2, lane 9). Finally, a large fraction was eluted at 315 mM NaC1 (Fig. 1, peak VI). It had a chlorophyll a/b ratio of 7.4 and was by SDS-PAGE shown to contain remaining undissociated native Photosystem I (Fig. 2, lane 10). Basically, no chlorophyll remained bound to the column after termination of the elution indicating close to a total recovery of chlorophyll-binding proteins from the starting native Photosystem I preparation. In conclusion, peaks II, III and IV represented the Lhca2+3 (LHC 1-680), Lhca2(+l) and Lhcal+4 (LHC 1-730) polypeptides, respectively. The Photosystem I core complex and native Photosystem I were recovered in peaks V and VI, respectively. This pattern of subfractionation was clearly reproducible as seen in approximately ten similar chromatographic experiments. In another set of experiments, a somewhat milder solubilisation of native Photosystem I was performed using 2.0 mg dodecyl maltoside and 2.5 mg zwittergent per ml for 70 min. The sample pH was set to 8.0 before loading onto the POROS Q anion exchange column. During washing with 50 mM Tricine (pH 8.0) and 0.03% dodecyl maltoside a first fraction migrated through the column following the void volume. It was clearly dominated by the Lhca3 protein in addition to polypeptides at 9 (PsaE), 10 (PsaH) and 17 kDa (PsaF) (Fig. 3, lane 2) and showed a chlorophyll a/b
46 1.0
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Absorption spectra at room temperature of peaks II-VI isolated by anion exchange perfusion chromatography. The spectra were normalised with respect to their maximal absorbtion around 440 nm.
ratio of 3.6. A second fraction was then eluted at 30 mM NaCI which contained the Lhcal+2+4 polypeptides (Fig. 3, lane 3) with a chlorophyll a/b ratio of 3.1. This separation was completed within only 10 min and resulted in a complementary set of LHC I subfractions as those described above (Figs. 1 and 2).
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47 77 K fluorescence emission spectra (Fig. 5) gave further evidence for the identities of the various fractions. Peaks II (Lhca2+3) and IV (Lhcal+4) were dominated by 680 and 730 nm emission, respectively. These data, together with the polypeptide patterns (Fig. 2, lanes 5 and 8), showed that peaks II and IV indeed corresponded to the so-called LHC 1-680 and LHC 1-730 complexes (Bassi and Simpson 1987), respectively. Peak III was dominated by the Lhca2 polypeptide (Fig. 2, lane 6) and showed at room temperature maximal fluorescence at 680 nm without red-shifted emission, very similarly to that of the Lhca2+3 complex (not shown). This observation, combined with the similar absorption data of fractions II and III (Fig. 4), indicates that Lhca2 and Lhca3 have the same lightharvesting properties. In addition to Lhca2 peak III contained minor amounts of Lhcal but not the Lhca4 polypeptide of LHC 1-730• Thus, the lack of 730 nm fluorescence in fraction III (Fig. 5) suggests that the red-shifted emission of the LHC 1-730 complex either eminates solely from the Lhca4 component or else requires physical interaction between the Lhcal and -4 subunits. The Photosystem I core complex eluted in peak V and native Photosystem I of peak VI showed typical red-shifted maximal fluorescense at 724 and 735 nm, respectively. Fluorescence excitation spectra of the material recovered from peaks II, IV, V and VI were recorded at wavelengths according to their maximal emission, F680, F730, F724 and F735, respectively (Fig. 6). All fractions fluoresced most intensely with excitation around 440 nm where chlorophyll a absorbs maximally. The red-shifted 730 nm emission from both peaks IV (Lhcal+4 = LHC 1-730) and VI (native Photosystem I) was clearly increased by excitation at 470 and 650 nm, representing chlorophyll b absorption. This corroborates that the low-energy chromophore responsible for the 730 nm fluorescence preferentially becomes excited by a chlorophyll b pool (Mukerji and Sauer 1990). For the first time, we here demonstrate this chlorophyll b species to be specifically associated with the Lhca4 subunit of the LHC 1-730 subcomplex. Furthermore, the red-shifted fluorescence of these fractions was considerable with excitation at 470-510 nm due to absorption by carotenoids. The close similarities between the excitation spectrum of the Lhcal+4 fraction and that of the undissociated native Photosystern I (peak VI) provide evidence that Lhcal+4 was isolated in a functionally intact state in peak IV. The excitation spectrum concerning the 680 nm fluores-
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Wavelength [nm] Fig. 6. Fluorescence excitation spectra at 77 K of fractions isolated by anion exchange perfusion chromatography. The emission wavelength detected was chosen from the fluorescence emission maximum of each peak. (Peak II) emission at 680 nm. It should be noted that the high intensity at 680 nm is due to detection of excitation light. (Peak IV) emission at 730 nm. (Peak V) emission at 724 nm). (Peak VI) emission at 735 nm. The spectra were normalised with respect to their maximal emission intensities around 440 nm.
cence from Lhca2+3 (LHCI-680) of peak II showed very little contribution from absorption by chlorophyll b and carotenoids. This low fluorescence yield by chlorophyll b and carotenoids was also seen for the 724 emission by the Photosystem I core complex of peak V. Upon 77 K fluorescence measurements, the fractions enriched in Lhca3 and Lhcal+2+4, from the milder solubilisation procedure, emitted with single maxima at 680 nm and 730 nm, respectively (not shown). This demonstrated that after removal of Lhca3 there was still efficient exciton transfer between Lhcal, -2, and -4 towards the low-energy chromophore yielding 730 nm fluorescence. Knoetzel et al. (1992) also isolated the Lhcal+2+4 complex by repetitive mild SDS-PAGE demonstrating the close interaction between these components of LHC I in accordance with our present chromatographic study.
48
Activities of native Photosystem I and Photosystem I core complexes The two reaction centre containing fractions were assayed for electron transport activity and fluorescence quantum yield. Using ascorbate/DCIP as electron donor and methylviologen as electron acceptor, the native Photosystem I complex of peak VI consumed oxygen with a rate of 915 #moles 02 mg Chl-lh -1. The Photosystem I core complex of peak V showed a 168% higher oxygen consumption rate of 1535 #moles 02 mg Chl- 1h- 1. The fluorescence quantum yield was as low as 0.4% for the Photosystem I core complex and 0.6% for the native Photosystem I which gives evidence for a high level of intactness of both preparations. Notably, this was the case especially for our present photosystem I core complex since its fluorescence quantum yield was much lower compared to preparations obtained by very time-consuming conventional procedures based upon sucrose density gradient centrifugation (P~ilsson et al. 1995). In conclusion, the above results demonstrate that perfusion chromatography is a very rapid and still mild method in order to subfractionate Photosystem I. It will dramatically facilitate the availability of the Photosystem I core, LHC 1-680 and LHC 1-730 complexes as well as yield new subpopulations of the LHC I antenna under conditions where only mild detergents are required for subfractionation. The unique potentials of the technique should also be applicable for isolation of other hydrophobic proteins of photosynthetic membranes.
Acknowledgements This project was supported by the Swedish Natural Science Research Council and the G6ran Gustafsson Foundation for Research in Natural Sciences and Medicine. Drs Birger Lindberg M¢ller and Stefan Jansson are thanked for their generous gift of antibodies.
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