Amino acid-pyrrole N-conjugates; a new class of antioxidants II. Effectiveness of singlet oxygen quenching by luminescence measurements

The pyrrole-amino acid and peptide N-conjugates synthesized from tyrosine, histidine and glutathione very effectively quench the 1270 nm singlet oxygen luminescence, at rates ranging from 10⁸ to 10⁹ M⁻¹ s⁻¹. Nuclear magnetic resonance spectroscopy suggests that the electron-donating properties of the methyl groups after 2,5-dimethyl substitution on the pyrrole ring are probably an important determinant of the reactivity of singlet oxygen with the N-conjugate of glutathione. However, intramolecular interactions between the pyrrole ring and the side chain may also modulate the reactivity of the antioxidant as suggested by absorption and fluorescence spectroscopies carried out on tyrosine derivatives. Efficient fluorescence quenching of the phenol ring by the pyrrole ring occurs in the tyrosine derivatives. The reactivities of these antioxidants with ¹O₂ are comparable in methanol, ethanol and D₂O.     

Solvent Polarity and pH Effects on the Spectroscopic Properties of Neutral Red: Application to Lysosomal Microenvironment Probing in Living Cells

Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRW) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of ly-sosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal rnicroenvironrnents.     

SUBCELLULAR LOCALIZATION OF AND PHOTOSENSITIZATION BY PROTOPORPHYRIN IX IN HUMAN KERATINOCYTES AND FIBROBLASTS CULTIVATED WITH 5-AMINOLEVULINIC ACID

Abstract— The subcellular localization of protoporphyrin (PP) has been studied by microspectrofluo-rometric techniques in NCTC 2544 keratinocytes incubated with 5-aminolevulinic acid (ALA) for times up to 42 h. Whereas the plasma membrane shows strong staining, fluorescent spots are observed within the cytoplasm especially in the perinuclear region. Although the topographic pattern of the PP distribution does not change much with the incubation time with ALA, the fluorescence spectra suggest that the PP microenvironments are quite different at short and long incubation times. Addition of 18 uJW desferoxamine almost doubles the ALA-induced PP concentration. Colocalization experiments with rhodamine 123, a mitochondrial probe, and lucifer yellow (LY) or neutral red (NR), two lysosome probes, demonstrate that at least some of these spots are of lysosomal origin. Study of the time evolution of the NR fluorescence under irradiation with visible light in the presence and absence of ALA demonstrates that lysosomes are damaged in cells that have synthesized PP. No PP fluorescence can be detected in mitochondria after incubation with ALA. However, photosensitization of mitochondria occurs under irradiation with visible light. Very little formation of lipofuscins by photosensitization with exogenous PP or ALA-induced PP is observed with the NCTC 2544 keratinocytes, as compared to normal human fibroblasts.