Károly Csatorday

Chromatographic and spectroscopic analysis of phycoerythrin 545 and its subunits

Phycoerythrin 545 is a light-harvesting biliprotein isolated from the cryptomonad Rhodomonas lens. Although the absorption spectrum of the native protein suggests that this protein has only phycoerythrobilins for its chromophores, the denaturated protein shows a small near ultraviolet absorption band with a maximum at 333 nm which is not present in phycoerythrobilin. Two methods were employed to separate the α and β subunits of this protein: chromatography with Sephacryl S-200 in acidic urea or centrifugation in a sucrose density gradient at pH 3.0. The chromatography experiments yielded two bands, which were shown to be pure α or β subunits by sodium dodecyl sulfate gel electrophoresis. The absorption spectrum of β showed only phycoerythrobilins, but the spectrum of α was not like that of any known bilin chromophore. Its absorption spectrum could be constructed by a combination of phycoerythrobilin and cryptoviolin. The β subunit separated on the sucrose density gradient was highly aggregated. Circular dichroism and fluorescence polarization spectroscopy indicated that this aggregated β subunit has chromophores in atypical environments. Comparison of the absorption, spectra of native and denatured phycoerythrin 545 suggests that chromophores in the native state are held by the protein in a more linear confirmation.

The route of exciton migration in phycocyanin 612

Abstract The biliprotein, phycocyanin 612, was purified from the cryptomonad, Hemiselmis virescens , and its chromophore content confirmed by an analysis of its tryptic peptides. Chromatography of a tryptic digest indicated that the isolated protein has three different phycocyanobilin chromopeptides and a single cryptoviolin chromopeptide. Spectroscopic data indicated that each different chromopeptide occurs twice for a total of eight chromophores on each oligomer. The tryptic digest data and the results from earlier experiments suggest that the protein may have two identical sets of four chromophores that comprise an energy-transfer unit within the protein. CD spectroscopy on phycocyanin 612 in the visible region of the spectrum was used to obtain information on the topography of these chromophores. Two chemicals, potassium permanganate and sodium thiocyanate, were used to alter the CD spectrum. The effects of both these chemicals suggested a close relationship between two of the four observed components in the visible CD spectrum of this biliprotein. This chromophore relationship is assigned to strong coupling of the dipoles of the two lowest-energy chromophores and produce exciton delocalization between these chromophores. Each dimeric protein has two identical delocalized pairs that produce spectral splitting and are the fluorescence emitters of the isolated protein. Based on this evidence and data from the literature, a tentative model is presented that shows the exciton migration route for phycocyanin 612. After excitation of the highest-energy chromophore, cryptoviolin, excitons are transferred by very weak dipole coupling to the highest-energy phycocyanobilin and from there by the same mechanism to the strongly coupled pair of phycocyanobilins. The identical process occurs on both halves of the protein.

Synchronization of Anacystis nidulans. Oxygen evolution during the cell cycle.

A new technique of short alternating lightdark periods was successfully used to synchronize the blue-green alga Anacystis nidulans. Oxygen evolution during the cell cycle is characterized by a maximum in the middle of the cycle and by a minimum at the time of division, a pattern very similar to that found in synchronized green algae.

Two simple procedures for isolation of allophycocyanin II from anacystis nidulans

Allophycocyanin II purification using initial extraction of phycobiliproteins by acetone treatment is introduced. An additional fast method using Al2O3 is described. Both extraction procedures are followed up by conventional hydroxylapatite chromatography.

Fluorescence from sensitizing phycobilin chromophores in the blue-green alga Anacystis nidulans.

Regeneration of the pigment system of Anacystis nidulans was studied following nitrate starvation. Three new, distinct fluorescence bands, at 596, 615 and 636 nm attributed to sensitizing phycobilin chromophores were detected. They each possess a separate excitation band at 425, 395 and 410 nm, respectively.

Detection of pigment forms in Anacystis nidulans by fluorescence reabsorption spectroscopy

Pigment forms in chloroplasts and unicellular photosynthetic organisms are not unequivocally resolved by absorption and fluorescence spectroscopy [1-7]. When obtaining true fluorescence spectra fluorescence reabsorption is a phenomenon usually corrected for (or avoided by preparing very dilute samples). In the present study, however, in contrast to common practice, the reabsorption of fluorescence was intentionally enhanced with the assumption that pigment forms involved in the reabsorption of fluorescence can be readily detected and they are the very ones the absorption of which constitute the conventional absorption spectra. The results presented show that fluorescence reabsorption spectroscopy provides hitherto unexploited advantages in discerning weakly absorbing components in complex pigment systems even if they scatter light.

Metal-Ion Induced Tetrapyrrole Synthesis in Algae

In suspensions of Anacystis cells regenerating after nitrate starvation the fluorescence from several tetrapyrroles was detected (Csatorday, 1978). To investigate this further the influence of metal ions on the biosynthesis of tetrapyrrole pigments was studied in the cyanobacterium Anacystis nidulans and mutant GGB of the rhodophyte Cyanidium caldarium.