Abstract

Phycobiliproteins are important photodynamic pigmental proteins. The photosensitized generation of superoxide radical anion (O 2 ⋅−), hydrogen peroxide (H 2O 2), hydroxyl radical ( ⋅OH) and singlet oxygen ( 1O 2) from three phycobiliproteins, R-phycoerythrin (R-PE), C-phycocyanin (C-PC) and allophycocyanin (APC) has been investigated throughout irradiation of these proteins with visible light of wavelengths longer than 470 nm. Upon irradiation of an oxygen-saturated aqueous solution of C-PC, R-PE or APC, the generation of O 2 ⋅− and ⋅OH was clearly observed by ESR-spin trapping with 5,5-dimethyl-1-pyrroline- N-oxide as the spin trap. The formation of ⋅OH can be significantly inhibited by catalase, indicating that H 2O 2 is also generated in the system and acts as a potential source of ⋅OH. Irradiation of the oxygen-saturated aqueous solutions of the phycobiliproteins can also result in the photobleaching of these proteins. Inhibition of the bleaching by superoxide dismutase (SOD) and catalase confirms further the generation of O 2 ⋅−, H 2O 2 and ⋅OH, and their involvements in the photobleaching of phycobiliproteins. Furthermore, the bleaching can be significantly inhibited by histidine and NaN 3, two specific inhibitors of 1O 2, suggesting that 1O 2 is also generated from C-PC, R-PE or APC on photosensitization and participates in the photobleaching of these proteins. The photobleaching rates of these proteins are sharply promoted in oxygen-saturated 2H 2O, further verifying the generation of 1O 2. Quantitative analyses of the photobleaching rates ( K d) are presented on visible-light-induced ( λ>470 nm) damage to these phycobiliproteins under some inhibitors of reactive oxygen species, which further confirm that the generation of reactive oxygen species and their participation in the photobleaching of these proteins. Since the fluorescences of the phycobiliproteins are not quenched by oxygen, it seems probably that the triplet states of these proteins are the inductors of 1O 2 and O 2 ⋅− generated. Therefore, the photodynamic action of phycobiliproteins proceeds by both free radical and singlet oxygen pathways which could contribute to the reported phototoxicity of phycobiliproteins in vivo.

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