Construction Of B-phycoerythrin-R-phycocyanin Conjugate And Comparative Study On Properties Of The Conjugate And Natural Phycobilisome From Porphyridium Cruntum

Intact phycobilisomes were isolated and purified from Porphyridium cruentum,a unicell red algae by sucrose density gradient supercentrifugence. Spectra analysis indicates that it has four absorption peaks(545nm,565nm,618nm and 650nm) and a aborption shoulder(498nm) in visible light region.Its fluorescence emission peak in room tempretrue is 68Onni and 595nm respectively,and the ratio of 680nm and 595nm can be up to more than 1 0,which indicated we have got very intact phycobili- some.Isolated phycobilisome contains at least three bands(B-PE,b-PE snd R-PC) on non-denature PAGE electrophoresis,APC is not found accounting for its small content.At least three clear bands were obtained by sodium dodecyl sulfate polyacry- lamide gel electrophoresis, a subunit and 13 subunit of phycoerythrin (overlapping), y subunit of phycoerythrin and one subunit of R-phycocyanin. B-PE, b-PE and R-PC with high purity were obtained by the Sephadex G-200 gel chromatography. B-PE had two absorption maxima (545nm, 565nm) and one absorption shoulder (498nm), with the A545,,/A498,, radio about 2.45. b-PE had its principal absorption maxima at 543nm and 563nm, but it did not have the absorption shoulder at 498nm, which is the main difference between B-PE and b-PE. The principal maxima of PC lied in 555nm and 618nm. Fluorescence emission peaks of PE and PC were at S8Onm and 640nm respectively. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed two bands of B-PE, standing for molecular weights of 2OKD and 31 KD. The former band was rather wide because overlapping due to molecular weight similarity of subunit a and subunit f3. The latter band was subunit y. b-PE only showed one 20 KD band, while not possess y subunit. Two clear bands of PC were also shown with molecular weights of 1 6KD and 20 KD. B-PE-R-PC conjugate was obtained using glutaraldehyde. Conguj ate had an aborption maximum at 268nm while the control showed a peak at 278nm which was chiefly attributed to aromatic amino acid residues.Conjugate showed two fluorescence emision pcaks,588nni and 640nm,attributed to free B-PE and R-PC in the conjugate respectively,while the control had only a fluorescence emmsion of 590nm orginating from B-PE.Furthermore,the 588nm peak of conjugate is far lower than that of the control. New bands appeared on non-denatured PAGE electrophoresis except for the B-PE and R-PC bands.All indicated that the phycobiliproteins energy transfer model is successfully covalently coupled. Artificial conjugate is more stable than natural phycobilisome in low ion strength and low temperature(40C),but efficiency of energy transfer is comparatively lower.