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Immunochemical Characterization of Porphyridium Cruentum B-Phycoerythrin

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Immunochemical Characterization of Porphyridium Cruentum B-Phycoerythrin Immunochemical Characterization of Porphyridium cruentum B-Phycoerythrin: Proof of Cross-Reaction between Chromophore-Free Apoprotein and Holoprotein-Specific Antibodies Eva Sepp, Gerhard Wanner, Jörg Eder* and Hans-Peter Köst Botanisches Institut der Ludwig-Maximilians-Universität, D-8000 München 19, Menzinger Straße 67 Z. Naturforsch. 37c, 1146—1156 (1982); received July 21, 1982 Rhodophyta, Porphyridium cruentum, Phycobilisomes, B-Phycoerythrin, Cross-Reactivity of Holo- and Apoprotein Antibodies against phycobilisomes of Porphyridium cruentum immunoreact with B-phycoery- thrin from the same organism. A strong antigen-antibody complex is not only formed with the native B-phycoerythrin but also when the chromophore is chemically reduced, split or even degraded with chromic acid. Characteristics of precipitations and dissolutions in the presence of the chaotropic reagents urea and potassium rhodanide are discussed for the immunocomplex with native B-phycoerythrin. A sandwich-type immunoadsorbent column appropriate for antigen-isolation is described; its binding properties are investigated biochemically and by electron microscopy. Introduction In addition to spectroscopic investigations char­ acterizing the chromophores and chromophore- Biliproteins are bile pigment-chromophore com­ protein interactions of biliprotins (e.g. [33-36], for plexes with thioether-linked chromophores [1-5]. review see [37], immunological methods have been According to their occurence and spectral properties applied on a number of biliproteins from various they have been divided into C-, R-, Cryptomonad organisms [10, 26, 27, 38, 39], In some cases, im ­ phycocyanin and B-, R-, Allo-, Cryptomonad phyco- munological cross-reactions have been reported; for erythrin (for classification, see refs. [6-9]). They are example, antisera against C-PC of a red alga reacts part of the light-harvesting system of Cryptophytae with C-PC of a cyanobacterium [10], Cross-reaction and the so called phycobilisomes of cyanobacteria between different classes of biliproteins was not and red algae [12-31], Two types of phycobilisomes observed, e.g. antibodies against B-PE do not re- are described: the Porphyridium type found in Por­ cognice C-PC [10]. Till now investigations have been phyridium cruentum [20-27, 31] and the Rhode/la carried out exclusively with native biliproteins or type of Rhodella violacea [28-30] which is typical their subunits. It is unknown thus far whether for many cyanobacteria too, e.g. for Synechococcus specific antisera against the holoprotein cross-react 6301 [18]. The phycobilisomes of Porphyridium have with apoprotein containing chemically altered the geometry of the half of compressed rotational chromophore(s) or with the chromophore-free apo­ ellipsoids [31]. The Rhodella type phycobilisomes protein. This is mainly due to the difficulties of are disk-shaped and consist of rods of different apoprotein-preparation which usually results in lenghts. For phycobilisomes of Porphyridium cruen­ denaturation. In the special case of cytochrome c tum, (B + b)-PE is the major constituent biliprotein from Neurospora crassa, practically no cross-reac­ (84%) [32] besides R-PC and allo-PC. tivity was found between apo- and holoprotein [40]. In this communication we describe that holo- and apoprotein of B-PE appear strongly related im- munochemically and that their cross-reactivity is * Max-Planck-Institut für Biochemie, D-8033 Martinsried, Am Klopferspitz. little affected by alterations in the chromophore region. Various methods available for the modifica­ Abbreviations: B-PE, B-phycoerythrin from Porphyridium cruentum; PC, phycocyanin; PBS, phosphat buffered tion or cleavage of the chromophore make the saline. B-PE-system suitable for this type of study. In addi­ Reprint requests to Dr. H.-P. Köst. tion, we report experiments to design an immuno­ 0341-0382/82/1100-1146 S 01.30/0 adsorbent column appropriate for antigen isolation. Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. E. Sepp et al. ■ Immunochemical Characterization of Porphyridium cruentum B-Phycoerythrin 1147 Materials and Methods rhodanide solution (0, 0.5, 0.75, 1, 1.5, 2, 2.5, 3 m /1) and kept at 20 °C with occasional agitating. The Phycobilisomes of Porphyridium cruentum residual precipitate was pelleted by centrifugation Phycobilisomes of Porphyridium cruentum were and washed twice with PBS. An analogous experi­ prepared using a saccharose density gradient as ment was conducted in the presence of urea described by Gantt and Lipschultz 1972 [22]. The ( 0 - 9 m/1). The pellets were dissolved either in phycobilisome fraction was dialyzed against 0.75 m/1 200 |il of 96% formic acid and immediately diluted sodium phosphate buffer, pH 6.8 at 4 ° C and the to 1 ml with PBS, or in 1 m /1 acetic acid. The ab­ dialysate applied again on top of a saccharose sorption of the resulting solution was measured at density gradient. The phycobilisomes reached the 555 nm. The amount of B-PE was determined using equilibrium density after 16 h at 40000 x g. a standard reference curve for B-PE recorded under the same conditions. Antiserum preparation In precipitation experiments, 200 |il of antiserum and 50 fj.1 of B-PE solution (10 mg/ml) were mixed 1 ml of the phycobilisome fraction (^ 555 nrn = 0.3) with freshly prepared KSCN or urea solution (Final was emulgated with 1.5 ml complete Freund’s ad­ concentration 0-3 m/1 and 0 - 9 m/1 respectively) juvant (Difco). The emulsion was injected sub- and incubated at 20 °C (incubation time: 1 h for cutaneously into rabbits (600 jj.1 per rabbit). After 25 KSCN; 2h and 16 h for urea). After centrifugation days, a booster-injection was given. Serum was the pellets were analyzed for B-PE as above. obtained from blood drawn from the ear vein 52 resp. 63 days after the initial injection. The presence Determination of standard precipitation of antibodies was demonstrated by the double dif­ fusion method of Ouchterlony with electrophoret- The standard is defined as the amount of B-PE ically pure B-PE from Porphyridium cruentum [3]. contained in an immunoprecipitate formed from Ouchterlony plates were prepared as described [41]. 50 jil B-PE solution (10 mg/ml) and 200 |il of the resp. antiserum in a final volume of 1 ml. Kinetics of immunoprecipitation Determination of standard dissolution Increasing volumes (2-10|il) of a solution (1 mg The standard is defined as the percentage of B-PE/ml) of electrophoretically pure B-PE were diluted to 1 ml with PBS. The fluorescence of the precipitate that dissolves in a parallel control ex­ dilutions recorded at 585 nm (xexc = 380 nm, light periment using PBS without chaotropic reagent (amount dissolved = 0%; residual pellet = 100%). path 1 cm) was used as reference. For precipitation experiments, antiserum (100 jil, 150 |il, 200^1) was mixed with B-PE solution (10 nl, 10 mg/ml B-PE) Immunoprecipitation of B-phycoerythrin and made up to 1.000 ml with PBS (mixing = start­ with modified chromophore: preparation of B-PE ing time = zero). After 10, 20, 30, 40 min, the with reduced chromophore [35] precipitate was removed by centrifugation Solutions: a) urea-PBS: 48.0 g urea with PBS to (10 500xg; 5 min) and the fluorescence of the 100 ml; b) 10% sodium dithionite in urea-PBS. supernatant immediately determined. The amount 10 mg of B-PE were dissolved in buffer a. Buffer b of B-PE still present was calculated from the stan­ was added dropwise until the red color had com­ dard fluorescence curve and corrected for fluores­ pletely dissappeared (ca. 0.1 ml). Urea and salts cence due to antiserum components. were removed by chromatography on a Sephadex G 10 column (5 cm x 20 cm) and the nearly colorless Dissolution and formation of immunoprecipitates protein fraction was used for precipitation experi­ in solutions containing chaotropic reagents ments. Immunoprecipitates from 200 |il antiserum/50 |il Phycoerythrin with completely degraded chromophore B-PE solution (10 mg/ml), washed twice with PBS, were used throughout the experiments. The pre­ 10 mg B-PE were dissolved in 1 ml PBS contain­ cipitate was resuspended in 1 ml of potassium ing 6 m/1 urea. 100 |il chromic acid (2 g C r0 3, 1148 E. Sepp et al. ■ Immunochemical Characterization of Porphyridium cruenlum B-Phycoerythrin 100 ml 2n H2S04) were added. Excess chromium Immunofixation of whole phvcobilisomes (VI) was reduced by passing a stream of S02 The described sandwich-column was equilibrated through the reaction mixture and subsequent ad­ with 0.75 m/1 potassium phosphate buffer, pH 6.8 justment of the pH to 7.8 with 1 m NaOH. Chro­ and incubated with a phycobilisome solution in the mium (III) formed was removed by dialysis for 16 h same buffer. Free phycobilisomes were removed by against 6 m/1 urea-PBS or chromatography on Se­ extensive washing with equilibration buffer. phadex G10 (developed with 6 m/1 urea-PBS). Small precipitates formed during the work-up were Immunofixation of B-phycoervthrin removed by centrifugation. Precipitation experi­ ments were carried out with antiserum containing The sandwich-column was equilibrated with 6 m/1 urea. PBS. 0.5 ml of a 0.1% B-PE solution were applied and the unbound material was removed by several Preparation of immunoadsorbents rinses with PBS. The desorption was carried out a) Direct technique with polyspecific IgG with 8 m/1 urea-borate buffer (pH 8) or 8 m /1 urea- PBS. The amount of immunofixed B-PE was deter­ IgG was enriched by fractionated precipitation mined by subtracting the amount of B-PE recovered with ammonium sulphate (25% and 35% saturation) from the washings.
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