Properties and Ultrastructure of Phycoerythrin From Porphyridium cruentum " 2

Phycoerythrin, a photosynthetic accessory pigment, was isolated from Porphyridium cruentum and examined by eleotron microscopy and disc gel electrophoresis. The absorption monomer, with maxima at 563, 545, and a shoulder at 500 nm, has a molecular weight of about 300,000. With phosphatungstic acid staining it appears as a tightly structured disc-shaped particle possessing a mean diameter of 101 0.4A and height of 54 + 0.7A. The absorption maxima remained the same in glutaraldehyde fixed material, and in dimer and trimer aggregates. Treatment with sodium dodecyl sulfate caused a breakdown into smaller units acoompanied by a loss of the 563 nm peak. It is suggested that this absorption monomer is the in vivo functional species and comparable to the phycocyanin hexamer, but structurally distinguishable at the ultrastructural level. It has been oalculated that about 35 phycobiliprotein molecules can be contained within each phycobilisome. There are 1.4 X 103 chlorophyll molecules per phyoobilisome, but not contained within it. Our previous work (10) has shown that the photosynthetic accessory pigments in Porphyridium cruentum are located on the lamellae in the form of phycobhilisomes (in vivo phycobilin aggregates). Absorption spectra of isolated phycobilisomes indicate that phycoerythrin as well as phycocyanin are presen,t. Because only 1 type of phycobilisome is present in P. cruentum, and since phycocyanin is a necessary intermediate in the effective energy transfer between phycoerythrin and chlorophyll (6, 8) the 2 phycobilins must be very closely associated. Both pigments are believed to be within each individual aggregate. To understand the in vivo relationship of the 2 phycobiliproteins they must be distinguished at the ultrastructural level. Therefore, phycoerythrin was isolated from the red alga P. cruentum to study its structure in order to be able to compare it with phycocyanin. The work of Berns and Edwards (2) indicates that the purified C-phycocyanin, isolated from Plectonema calothricoides, in its most stable form exists as a hexameric ring with a central hole, an overall diameter of 130A and a molecular weight of ab-outt 200,000. It remains to be shown whether or not phycoerythrin has a similar shape and appearance as the phycocyanin hexamer, and in what way, if any, it differs. To permit a meaningful comparison of the 2 phycobilins in the present work, 1 Research was supported by the United States Atomic Energy Commission Contract No. AT(30-1)3913, and the Smithsonian Institution. 2 Published with the approval of the Secretary of the Smithsonian Institution. preparation.s of phycoerythrin were monitored by electron microscopy, absorption spectrophotometry, and the molecular weight of the smallest stable particle was determined. Materials and Methods Phycoerythrin was obtained from Porphyridium cruentum which had been grown in axenic cultures in an artificial sea water medium (14) at room temperature. The liquid cultures, aerated with 1 % carbon dioxide and 99 % air, were continuously agitated and illuminated with cool white fluorescent lamps (incident intensity 350 ft-c). Cells were harvested by centrifugation (10,000g) and rinsed twice in 0.01 M phosphate buffer (sodium and potassium) pH 6.8. The pellets were frozen in polyethylene tubes in an acetone-dry ice bath and stored in a freezer until used. For use the pellets were thawed and the slurry of cells was passed several times through a No. 20 syringe needle in order to break the cell clumps prior to breaking in an An4nco-French pressure cell at 8 to 12,000 psi. The phycoerythrin isolation method, involving n-butanol and ammonium sulfate fractionation, described by Leibo and Jones (15) was followed in most essentials. Throughout the entire isolation the preparation was kept at about 4°. For the precipitations a solution of supersaturated ammonium sulfate was used in decreasing concentrations (35-20 % of saturation) and repeated until a 545/275 nm absorbance ratio of 5 or higher was obtained. Final crystallization of phycoerythrin was accomplished by addition of recrystallized ammonium sulfate powder (15-20 % of saturation). 1629 www.plantphysiol.org on August 15, 2017 Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.