The Protein-Chromophore Bond in B Phycoerythrin from Porphyridium cruentum. Radiosulfur Labeling Experiments

Abstract

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.

Phycoerythrins of the Red Alga Callithamnion

“…Consequently, it is unlikely that the spectroscopic differences resulted from proteolytic degradation in the 2Abbreviations: PEB, phycoerythrobilin; PUB, phycourobilin; DMSO, dimethylsulfoxide. of PEB to the protein (2,19,20,23), but the mode of PUB attachment has yet to be established. The number of half-cystine residues, present either as cysteinyl residues or in disulfide linkage, was determined as S-carboxymethylcysteine by the procedure of Crestfield et al (3).…”

“…Hydroxylapatite was prepared as described by Siegelman et at. (26) and stored at 4 C. The mol wt and sources of the polypeptide standards for SDS polyacrylamide gel electrophoresis were as follows: BSA, 66,000 (Sigma Chemical Co.,); ovalbumin, 43,000 (Nutritional Biochemicals Co., Cleveland, OH) and C-phycocyanin (a-subunit, 17,000; fl-subunit, 19,000) from Synechococcus 6301 (10).…”

Chromophores in Photomorphogenesis

“…Because the blue color was extracted at that Edman degradation step which also removed cysteine, the thiol group was considered as the chromophore-binding function of the protein (LAGARIAS and RAPOPORT 1980). This situation is the same as in PC (FRANK et al 1978, LAGAR-IAS et al 1979, WILLIAMS and GLAZER 1978, BRYANT et al 1978 and PE (KÖST-REYES et al 1975, MÜCKLE et al 1978, KÖST-REYES and KÖST 1979. …”

Immunochemical Characterization of Porphyridium cruentum B-Phycoerythrin: Proof of Cross-Reaction between Chromophore-Free Apoprotein and Holoprotein-Specific Antibodies

“…To test whether any im m unoreactivity is contrib uted by the chromophore, chrom opeptides prepared by digestion with pepsin [1,3] were used. These chromopeptides are of low m olecular weight (e.g.…”

“…63 days after the initial injection. The presence of antibodies was dem onstrated by the double dif fusion method o f Ouchterlony with electrophoretically pure B-PE from Porphyridium cruentum [3]. Ouchterlony plates were prepared as described [41].…”