A Multistate Model for the Fluorescence Response of R‐Phycoerythrin

ABSTRACT Although strong fluorescence makes the R‐phycoerythrin (R‐PE) proteins increasingly useful in biological and clinical assays, they are subject to nonlinear effects including transitions to collective dark states and photodegradation, which complicate quantitative applications. We report measurements of R‐PE fluorescence intensity as a function of incident power, duration of illumination and temperature. Emission intensity in the band at 570 nm is proportional to incident power for low power levels. At higher incident power, the emission at 570 nm is smaller than expected from a linear dependence on power. We propose that R‐PE undergoes both reversible emission cessation on a millisecond time scale attributed to transitions to a collective dark state, and irreversible photodegradation on a time scale of minutes. Singlet oxygen scavengers such as dithiothreitol and n‐propyl gallate have protective effects against the latter effect but not the former. Electrophoretic analysis of irradiated solutions of R‐PE indicates that significant noncovalent aggregation is correlated with photodegradation. A multistate model based on fluorescence measurements and geometric analysis is proposed for the fluorophores in R‐PE. The phycobilin fluorophores are partitioned into three groups: the phycourobilins (PUB) absorbing at 490 nm, one group of phycoerythobilins (PEB) absorbing at 530 nm (PEB‐530) and another group of PEB absorbing at 560 nm (PEB‐560). The two processes that result in the loss of fluorescence intensity are most likely associated with the PEB‐560 group.