Involvement Of Both Type I And Type Ii Mechanisms In Gram-positive And Gram-negative Bacteria Photosensitization By A Meso-substituted Cationic Porphyrin

摘要

A meso-substituted cationic porphyrin (TMPyP) showed a photocytotoxicity against Gram-positive and Gram-negative bacteria. In order to determine the mechanism involved in the phototoxicity of this photosensitizer, electron paramagnetic resonance (EPR) experiments with 2,2,6,6-tetramethyl-4-piperidone (TEMP), a specific probe for singlet oxygen, and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were carried out with illuminated TMPyP. An EPR signal characteristic of TEMP-singlet oxygen (TEMPO) adduct formation was observed, which could be ascribed to singlet oxygen (~1O_2) generated by TMPyP photosensitization. The signal for the DMPO spin adduct of superoxide anion (DMPO-OOH) was observed in DMSO solution but not in aqueous conditions. However, an EPR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-OH) was observed in aqueous conditions. The obtained results testify a primary hydroxyl radical (~·OH) generation probably from superoxide anion (O_2~(·‐) via the Fenton reaction and/or via Haber-Weiss reaction. Gram-positive and Gram-negative bacteria inactivation by TMPyP photosensitization predominantly involved Type II reactions mediated by the formation of ~1O_2, as demonstrated by the effect of quenchers for ~1O_2 and scavengers for ~·OH (sodium azide, thiourea, and dimethylsulphoxide). Participation of other active oxygen species cannot however be neglected since Type I reactions also had a significant effect, particularly for Gram-negative bacteria. For Gram-negative bacteria the photoinactivation rate was lower in the presence of superoxide dismutase, a specific O_2~(·‐) scavenger, and/or catalase, an enzyme which specifically eliminates H_2O_2, but was unchanged for Gram-positive bacteria. The generation of ~1O_2, O_2~(·‐) and ~·OH by TMPyP photosensitization indicated that TMPyP maintained a photodynamic activity in terms of Type I and Type II mechanisms.