Abstract-The excited state kinetics of trimeric C-phycocyanin from Masrigocladus /arnirzosu.c. has been measured as a function of the emission and excitation wavelength by the single-photon timing technique with picosecond resolution and simultaneous data analysis. A last decay componcnt of 22 ps (C-phycocyanin with linker peptides) and 36 ps (C-phycocyanin lacking linker peptides) is attributed to efficient energy transfer from sensitizing to fluorcscing chromophores. At long dctcction wavelengths the fast decay components are found to turn into a rise t a m . This finding further corroborates thc concept o f intramolecular energy transfer. Previous reports on the conformational heterogeneity of the chromophores and/or proteins in C-phycocyanin are confirmcd. Our data also provide indications for the importancc of the uncoloured linker peptides for this hctcrogeneity.

C-Phycocyanin (PC) and allophycocyanin (APC). as well as the a-subunit of PC. have been isolated from the blue-green alga (cyanobacterium). Spirulina platensis. The effects of partial thermal denaturation of PC and of its state of aggregation have been studied by ps time-resolved, polarized fluorescence spectroscopy. All measurements have been performed under low photon fluxes (< 10' ' photonsipulse x cm') to minimize singlet-singlet annihilation processes. A complex decay is obtained under most conditions, which can be fitted satisfactorily with a bi-exponential (7' = 70400 ps. T? = 1000-3000 ps) for both the isotropic and the polarized part, but with different intensities and time constants for the two decay curves. The data are interpreted in the frameworkof the model first developed by Teak and Dale (Biochern. J . 116, 161 (1970)], which divides the spectroscopically different chromophores in (predominantly) sensitizing (s) and fluorescing U, ones. If one assumes temperature dependent losses in the energy transfer from the s to the f and between f chromophores. both the biexponential nature of the isotropic fluorescence decay and the polarization data can be rationalized. In the isotropic emission (corresponding to the population of excited states) the short lifetime is related to the s-,f transfer. the longer one to the "free" decay of the final acceptor(s) (= f). The polarized part is dominated by an extremely short decay time. which is related to s+f transfer, as well as to resonance transfer between the f-chromophores.-<, . <, 22 1

The influence of aggregation and temperature on the excited state kinetics of C-phycocyanin from Mastiqocladus laminosus has been studied. Polarized fluorescence decay curves have been recorded using a synchronously pumped dye laser in conjunction with a synchroscan streak camera. The experimental data for all samples can be fit satisfactorily assuming a biexponential decay law. Fluorescence depolarization times have been interpreted in terms of energy transfer among the different chromophores. The influence of temperature is only moderate on the intra­molecular relaxation, but pronounced on the rates of energy transfer. Both are dependent on the size of the aggregate. The biexponential decay of the α-subunit containing only one chromophore, indicates the presence of different subsets of chromophores in these samples. The results are discussed in terms of variations of the chromophore arrangements upon temperature induced changes in the protein conformation.

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...

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