Protein-encapsulated bilirubin: paving the way to a useful probe for singlet oxygen

Abstract

When dissolved in a bulk solvent, bilirubin efficiently removes singlet molecular oxygen, O₂(a¹Δ_g), through a combination of chemical reactions and by promoting the O₂(a¹Δ_g)→O₂(X³Σ_g⁻) nonradiative transition to populate the ground state of oxygen. To elucidate how such processes can be exploited in the development of a biologically useful fluorescent probe for O₂(a¹Δ_g), pertinent photophysical and photochemical parameters of bilirubin encapsulated in a protein were determined. The motivation for studying a protein-encapsulated system reflects the ultimate desire to (a) use genetic engineering to localize the probe at a specific location in a living cell, and (b) provide a controlled environment around the chromophore/fluorophore. Surprisingly, explicit values of oxygen- and O₂(a¹Δ_g)-dependent parameters that characterize the behavior of a given chromophore/fluorophore encased in a protein are not generally available. To the end of quantifying the effects of such an encasing protein, a recently discovered bilirubin-binding protein isolated from a Japanese eel was used. The data show that this system indeed preferentially responds to O₂(a¹Δ_g) and not to the superoxide ion. However, this protein not only shields bilirubin such that the rate constants for interaction with O₂(a¹Δ_g) decrease relative to what is observed in a bulk solvent, but the fraction of the total O₂(a¹Δ_g)–bilirubin interaction that results in a chemical reaction between O₂(a¹Δ_g) and bilirubin also decreases appreciably. The rate constants thus obtained provide a useful starting point for the general design and development of reactive protein-encased fluorescent probes for O₂(a¹Δ_g).