Singlet oxygen quenching by beta-carotene in HeLa cells

GABRIELA BOSIO; THOMAS BREITENBACH; FRANCES BLAIKIE; BRIAN PEDERSEN; DANIEL MARTIRE; PETER OGILBY

Mendoza

Congreso; 21st IAPS Conference; 2011

Whether the lifetime of O2(a1Δg) in cells is shortened by reactions with cellular molecules or reaches the inherent maximum value is still unclear. However, even with the maximum lifetime, the diffusion radius is only approximately 220 nm during three lifetimes (-5% O2(a1Δg) remaining), much shorter than cellular dimensions indicating that the primary reactions of O2(a1Δg) will be subcellularly localized near the site of O2(a1Δg) formation. [1]              Carotenoids in light-harvesting complexes quench O2(a1Δg) and the D1 protein in the PSII reaction centers in chloroplasts appear to protect plant tissues from O2(a1Δg) produced by  chlorophyll photosensitization suggesting that proximity to the site of O2(a1Δg) formation, rather than their average concentration, allows effective quenching. [2,3]          In our work we investigated the b-carotene quenching of singlet oxygen, O2(a1Δg), sensitized by different dyes with different subcellular localization.          Fluorescence and Resonance Raman Microscopy were used as non-invasive methods to investigate the subcellular localization of b-carotene in HeLa cells. To improve the fluorescence emission of the carotene, it was also synthesized carotene moiety covalently bonded to a fluorescein derivative.          Using a variety of different sensitizers, such as TMPyP, PPa, etc, the O2(a1Δg) phosphorescence decay was monitored in cells suspensions, both in the absence and presence of b-carotene. The results were compared to those obtained with other singlet oxygen quenchers (bovine serum albumin (BSA), and sodium azide. The ability of b-carotene to quench the O2(a1Δg) signal depended on the sensitizer used. For example when using a hydrophilic sensitizer, the hydrophobic b-carotene had little effect on the O2(a1Δg) signal whereas hydrophilic sodium azide had a large effect. However, for sensitizers localized in thin hydrophobic membranes, b-carotene and sodium azide were both efficient quenchers of the O2(a1Δg) signal. These data are consistent with the notion that O2(a1Δg) formed in the bilayer can diffuse out into hydrophilic domains. Quenching of O2(a1Δg) by BSA, which is localized in the extracellular domain, indicates that O2(a1Δg) can also diffuse across the plasma membrane. We can conclude that b-carotene is not only effective as an intracellular singlet oxygenquencher, but also serves as molecular probeto detect the location of the Singlet Oxygen signal which has an important application, for example, in the effectiveness of curingdiseases based onphotodynamic therapy.