Fast preparative isoelectric focusing of phycocyanin subunits in layers of granulated gels

Abstract

A new method is presented for the fast preparative separation of the light-harvesting photosynthetic pigment C-phycocanin into its a and p subunits, which is based on isoelectric focusing in layers of granutaled gels containing 7 M urea. The method has been successful in cases where other separation procedures failed. The recovery of the separated chains of the light -sensitive biliprotein amounts to 70 10 % when the separation is carried out under light exclusion and in an argon atmosphere. A simple and inexpensive setup for work under an atmosphere of protective gas is described.Rhodophyta, Cryptophyta and cyanobacteria contain phycobilins, a special class of light-harvesting pigments 11-31. Phycobilins are biliproteins, composed of subunits with covalently linked bile pigment chromophores (e. g. . Especially interesting is the blue phycocyanin (PC) which is composed of two subunits, the a-chain and the f3-chain. For many research applications a separation of these subunits is required. The methods applied so far for isolation are: (i) Ion exchange chromatography on Biorex-70 with an 8 to 9 Murea gradient according to Glazer and Fang 161.6) Gel chromatography on Bio-Gel P-60 with 63 mM formic acid as solvent system [ 71. (iii) Preparative gel electrophoresis in 7 Murea 181. Problems arise due to the lability of the phycocyanobilin chromophores. Bleaching is observed especially when biliprotein solutions of low concentrations are used and long separation times are required due to a high dilution factor. We present a new method which combines some advantages of the above separation procedures, especially speed and resolution, while avoiding the disadvantage of excessive dilution inherent to most chromatographic procedures 191. Basically, the method described is preparative isoelectric focusing in a granulated gel [ 101 containing 7 M urea. For gel preparation, 6 g Sephadex G-75 (Pharmacia, Uppsala, Sweden) are allowed to swell for 3-4 h at 80 "C in 200 mL of double distilled water. Then 100 g of analytical grade urea (Serva, Heidelberg, FRG) are slowly dissolved while stirring carefully with a glass rod. The gel is allowed to settle and the supernatant is carefully decanted, leaving a residueof 1 13 mL which is carefully degassed in the aspirator vacuum for 5 min, followed by addition of 2 mL Servalyt (analytical grade, pH 3-7, Serva). A gel layer is prepared by pouring the gel suspenCorrespondence: E. Kost-Reyes and S. Schneider. Technische Universitat Munchen, Lichtenbergstr. 4, D-8046 Garching. Federal Republic of Germany sion into the trough followed by water evaporation to about 20 % weight loss using an infrared lamp (Philips Infrared Lamp R 95 E, 220 V, 150 W, distance 35 cm) and a fan.The electrophoresis apparatus consisted of an LKB basis unit fitted with gel trough, electrode strips and lid (LKB 21 17; LKB productors, Bromma, Sweden). For light exclusion, the electrofocusing bed has to be kept in a dark room or covered by a hood. To avoid chromophore losses through oxidation, all work is carried out un...

Solution‐State 15N NMR Spectroscopic Study of α‐C‐Phycocyanin: Implications for the Structure of the Chromophore‐Binding Pocket of the Cyanobacterial Phytochrome Cph1

“…In contrast, the a and b subunits of C-PC in the thermophilus cyanobacterium Mastigocladus laminosus differ clear- www.chembiochem.org ly in their isoelectric points (pI a = 6.1, pI b = 5.0), and a protocol for the Mastigocladus PC subunit separation by preparative isoelectric focusing already existed. [43] For labeling purposes, we modified the composition of the inorganic Castenholz medium to use a single nitrogen source (NaA…”

Picosecond time-resolved fluorescence of phycobiliproteins: Subunits of phycocyanin from Mastigocladus laminosus

“…The subunits of PC were obtained by an improved isoelectric focusing procedure in 7 M urea. The modifications of the previously used procedure [25] were as follows. The dextran gel was presaturated with argon, and the electrofocusing done at 2 "C under argon and in darkness.…”

“…The subunit preparation followed essentially the procedure of KiistReyes et al [25], but involved several additional safeguards. The original procedure generally yielded subunits which on SDS-PAGE showed one or more bands moving ahead of the main colored band.…”

PHOTOCHEMISTRY OF PHYCOBILIPROTEINS: RECIPROCITY OF REVERSIBLE PHOTOCHEMISTRY AND AGGREGATION IN PHYCOERYTHROCYANIN FROMMastigocladus laminosus

“…After separation of the biliproteins by chromatography on DEAE cellulose, PEC was concentrated by ammonium sulfate precipitation. The subunits of PEC were prepared by isoelectric focusing as used before for PC from the same organism (Kost-Reyes et al, 1987). After elution from the gel, the subunits were renatured by dialysis against potassium phosphate buffer (100 mMipH 7.0) at first at room temperature and subsequently at 4 C.…”