Efficient purification and excitation energy transfer characterization of phycoerythrin 545 from<i> Rhodomonas</i> sp.

Abstract

Cryptomonad phycoerythrin 545 (PE545) is an important type of phycobiliprotein in basic research and technological innovations. Herein, we report a minimalistic hydrophobic chromatography method for its purification. High purity was achieved, with a purity ratio (A545/A280) of 13.66 and a recovery ratio of 78.63 %. Following SDS-PAGE, Coomassie Brilliant Blue staining revealed three bands at 9 kDa, 10 kDa, and 20 kDa, corresponding to alpha 1, alpha 2 and beta subunits. Multiple spectral characteristics were analyzed to ensure that optical activity was consistent with that of the natural protein. Absorption and fluorescence spectroscopies of purified PE545 displayed a strong absorption peak at 545 nm, a shoulder peak at 564 nm, and a fluorescence emission peak at 587 nm, which confirmed unchanged energy transfer properties. Furthermore, the structural and functional integrity, especially the existence of strongly coupled central chromophore pairs with excitation delocalization, was verified by circular dichroism and ultrafast absorption spectroscopy. From the studies of ultrafast absorption spectroscopy of excitation energy transfer (EET) of PE545, four decay components with lifetimes at 0.5 ps, 2.2 ps, 63 ps, and 3000 ps were obtained. In addition, the dynamics of these components confirmed the EET pathways from the central PEB chromophore pairs to the peripheral pigments and localized in the lowest state. Our work will be of considerable value for both fundamental research and applications of PE545