[14] Phycoerythrin fluorescence-based assay for reactive oxygen species

TLDR: The assay for reactive oxygen species depends on the detection of chemical damage to phycoerythrin through the decrease in its fluorescence emission, and the effect of added compounds on the rate of this fluorescence loss is a measure of their ability to protect the protein.

Abstract: Publisher Summary This chapter describes the phycoerythrin fluorescence-based assay for reactive oxygen species. Phycobiliproteins are photosynthetic accessory proteins in cyanobacteria (blue-green algae) and in two groups of eukaryotic algae, the red algae and the cryptomonads. Among these proteins, B- and R-phycoerythrins are the most remarkable with respect both to the stability of their quaternary structure and to their spectroscopic properties. Both these phycoerythrins occur in numerous red algae and can be readily purified by conventional procedures. B- and R-phycoerythrins have molecular weights of 240,000 and carry 34 covalently attached tetrapyrrole prosthetic groups. Both are multisubunit proteins with the structure (αβ)6γ. The complete amino acid sequences of the α and β subunits of Porphyridium cruentum B-phycoerythrin are known, and the sequences about the tetrapyrrole attachment sites in the α, β, and γ subunits of Gastroclonium coulteri R-phycoerythrin are determined in the chapter. The assay for reactive oxygen species depends on the detection of chemical damage to phycoerythrin through the decrease in its fluorescence emission. The fluorescence of phycobiliproteins is highly sensitive to the conformation and chemical integrity of the protein and of the prosthetic groups. Under appropriate conditions, in the presence of reactive oxygen species, the rate of loss of phycoerythrin fluorescence is an index of free radical damage. The effect of added compounds on the rate of this fluorescence loss is a measure of their ability to protect the protein.