Immunochemistry on Cryptomonad Biliproteins

Abstract

A survey is made of the immunochemical behavior of four of the six known types of cryptomonad biliproteins: phycocyanins 612 and 645 and phycoerythrins 545 and 566. They were compared both among themselves and to selected biliproteins isolated from blue-green and red algae. All the cryptomonad biliproteins were shown to be closely related to each other by Ouchterlony double diffusion technics. An antigenic relationship among all the cryptomonad biliproteins and B-phycoerythrin (red alp) and C-phycoerythrin (blue-green alga) was established. Only a very marginal cross-reactivity was found between C-phycocyanin (blue-green algae) and the cryptomonad biliproteins. These results suggest a common ancestor for the photosynthetic units of all three biliprotein-containing phyla. Biliproteins are light harvesting and excitation energy transfer chromoproteins that function as accessory pigments for PSII in the photosynthesis of blue-green (cyanobacterial), red, and cryp-tomonad algae. Cryptomonad biliproteins are unusual in that they apparently do not form phycobilisomes and in the crypto-monads no analog of allophycocyanin has yet been detected. There are six biliproteins in the cryptomonads, phycocyanins 612, 630, 645, and phycoerythrins 545, 555, 566, with only one type usually found in each alga. Immunochemical studies on biliproteins have greatly emphasized blue-green and red algal examples. By 1967 four publications had established in a very extensive and decisive manner three basic rules governing the immunochemical behavior of these two types of algal biliproteins (1-3, 29). These three generalizations are: all phycocyanins from both blue-green (procar-yotes) or red (eucaryotes) algae are immunochemically very similar; all phycoerythrins of all spectral types (C-, R-, Bare re immunochemically closely related whether they are from blue-green or red algae; no phycocyanin is related immunochemically to any phycoerythrin. This last rule holds even when the phy-cocyanin and phycoerythrin are isolated from the same alga. These first two results were most important because of the great differences in structure between procaryotic blue-green algae and eucaryotic red algae. The immunochemical results then established the principle that, although major cellular changes have occurred in the evolution from procaryotes to eucaryotes, the individual proteins could remain virtually unaltered. Subsequent research has shown that the two subunits of phycoerythrin are immunochemically related (27) and the properties of allophy-cocyanin have been investigated (12). Research on the immunochemistry of cryptomonad bilipro-teins, has been much less extensive and then usually only a fragment of a larger study. It is therefore not too surprising that unlike the comparison of blue-green and red algal biliproteins, cryptomonad results have been so far perhaps inconclusive and controversial (2, 4, 12, 13, 16, 26, 29). We, therefore, undertook an immunochemical investigation using Ouchterlony double-diffusion and four different cryptomonad biliproteins. Two different phycoerythrins and two different phycocyanins were compared among themselves using several different antisera. For one, phycocyanin 612, this is the first immunochemical study. They were then tested against selected biliproteins from blue-green (C-phycocyanin and C-phycoerythrin) and red algae (B-phycoery-thrin). EXPERIMENTAL Biliproteins were isolated and purified from crytomonads, phycocyanin 612-Hemiselmis virescens, phycocyanin 645-Chroomonas species, and phycoerythrin 545-Rhodomonas lens, and phycoerythrin 566-Cryptomonas ovata, as described previously (14). The cryptomonad proteins were extensively purified by (NH4)2SO4 fractionation and gel filtration on Sepharose 4B and Ultrogel AcA54. The protein purity was established by the ratio of the absorbances of visible absorption maximum to 280 nm and by SDS gel electrophoresis. C-Phycocyanin was isolated by lysozyme treatment of Phormidium luridum and purified by fractionation using (NH4)2SO4. C-Phycoerythrin (P. persicinum and Calothrix membranacea and B-phycoerythrin, and allophy-cocyanin (Porphyridium cruentum) were purified by (NH4)2SO4 fractionation followed by chromatography on hydroxyapatite (Bio-Rad, Richmond, CA) using a phosphate gradient. The purity of the biliproteins from cyanobacteria and red algae was monitored by the ratio of absorption at the visible maximum to absorption at 280 nm. Antiserum was derived by injecting purified biliproteins together with Freund's complete adjuvant (Difco, Detroit, MI) in the neck area of rabbits. Prior to the first injection some blood was taken from a vein in the ear to serve as a control. All such prebleeds ultimately proved to be negative in tests versus bilipro-teins. Our immunization protocol called for two injections of 2 to 4 mg of protein each at a 1 month interval. A week following the second exposure the animals were anesthetized with ketamine hydrochloride (Bristol, Syracuse, NY), and a maximal amount of blood withdrawn via cardiac puncture. Sera were prepared by centrifugation and stored frozen. In some cases the serum was fractionated by (NH4)2SO4 precipitation prior to use. Antisera were prepared against the following: phycocyanin 612 (H. vires-cens); phycoerythrin 545 (R. lens), phycocyanin 645 (Chroom-onas sp.); phycoerythrin 566 (C. ovata); B-phycoerythrin (P. cruentum); C-phycocyanin (Synechococcus lividus); C-phycoer-ythrin (P. persicinum). Several rabbits were used for each type of antigen. Ouchterlony double diffusion experiments were performed in 38