Comparison of photosensitized plasma membrane damage caused by singlet oxygen and free radicals

Abstract

The efficiency and selectivity of photosensitized damage to membrane functions may be influenced strongly by the identity of the initial reactive species formed by the photosensitizer. To test this possibility, a photosensitizer, rose bengal (RB), was used that resides in the plasma membrane and which generates singlet molecular oxygen ( 1O 2 ∗) upon excitation with visible light, and radicals plus 1O 2 ∗ upon excitation with UV radiation. With this approach, 1O 2 ∗ and radicals are formed at the same locations in the plasma membrane. The response of three plasma membrane functions, namely, proline transport, membrane potential, and membrane impermeability to charged dye molecules, was assessed. The efficiencies of the responses in the presence and absence of oxygen were compared per photon absorbed by RB at two wavelengths, 355 nm (UV excitation) and 532 nm (visible excitation). The efficiency of oxygen removal before irradiation was assessed by measuring the RB triplet lifetime. The three membrane functions were inhibited more efficiently at 355 nm than at 532 nm in the presence of oxygen indicating that the radicals are more effective at initiating damage to membrane components than 1O 2 ∗. The ratio of photosensitized effects at the two wavelengths in the presence of oxygen was the same for two membrane functions but not for the third suggesting that 1O 2 ∗ and radicals initiate a common mechanistic pathway for damage to some membrane functions but not to others. Removing oxygen reduced the efficiency of 355 nm-induced photosensitization by factors of 1.4 to 7. The sensitivity of the three membrane functions to 1O 2 ∗-initiated damage varied over a factor of 50 whereas radical initiated damage only varied by a factor of 15. In summary, these results indicate that radicals and 1O 2 ∗ formed at the same locations in the plasma membrane vary in their efficiency and specificity for membrane damage but may, in some cases, operate by a common secondary damage mechanism in the presence of oxygen.