Red algae of the species Porphyridium cruentum were grown in a minimum sulfate medium containing 3sS02-. 35S-labeled phycoerythrin was extracted. B Phycoerythrin, b phycoerythrin and R phycocyanin could be separated from other proteins by using a carrier-free electrophoresis on columns. The final ratio A s~s / A~~o of B phycoerythrin thus obtained was >, 5.3sS-labeled B phycoerythrin was digested proteolytically with trypsin and pepsin. The resulting 3sS-containing bilipeptides were separated by isoelectric focusing. Zones of enhanced chromophore concentration always showed an enhanced radioactivity.Peptide fractions with a low molar ratio sulfur/chromophore (1.1 -1.8) were purified to remove sucrose and the carrier ampholyte. A modified, optimized Edman degradation followed. A butylacetate-soluble, red Edman product was obtained that contained most of the chromophore and the bulk of the radioactivity. This product was purified by two-dimensional thin-layer chromatography. The main spot of the chromatogram was subjected to acidic hydrolysis. The major part of the radioactivity in the hydrolysate cochromatographed with cysteine. That proves cysteine to be the binding amino acid in all cases investigated.

B‐Phycoerythrin aus Porphyridium cruentum wird mit Pepsin zu Bilipeptiden abgebaut. Aus einem Gemisch kurzkettiger Bilipeplide (Fraktion I) wird der Galienfarbstoff zusammen mil dem N‐terminalen Cystein beim Edman‐Abbau abgespalten. Der weitere Pepsin‐Abbau führt zu einem Tripeptid (Fraktion 2), das den Gallenfarbstoff, Cystein, Valin und Leucin enthält. Beim Edman‐Abbau entsteht das rote Produkt 1. Als Bindungstelle an der Seiten‐ kette von Ring A des Farbstoffs wurde die Thiolgruppe von Cystein ermittelt.

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...

The circular dichroism of bilipeptides from Spirulina geitleri phycocyanin is strongly solvent and pH dependent. Maximum optical activity has been observed in aqueous solutions containing urea (8 M). In aqueous buffer, a sign reversal occurred upon the change from neutral to acidic pH; in methanolic solutions shows the optical activity a strong pH dependence both with respect to sign and magnitude. These findings have been rationalized by the presence o f chrom ophorepeptide interactions, which are minim ized in the presence of urea. M olecular orbital calculations indicate that the observed sign reversal is not necessarily due to a reversal o f the chirality o f the entire chromophore, but may also result from more localized conform ational changes.

Four different chromopeptides were isolated after digestion of C‐phycocyanin with pepsin. Their UV‐vis absorption and circular dichroism spectra were measured at two different pH values without and with urea present in the buffered solutions. For one chromopeptide, fluorescence spectra and the kinetics of fluorescence decay were studied in more detail. The results are discussed in comparison with quantum‐mechanical model calculations. It is concluded that due to the interaction between the oligopeptide chain and the chromophore, the latter is present as a mixture of helical and semi‐extended forms, respectively.