UV photolysis of catalase revisited: a spectral study of photolytic intermediates

The 365-nm irradiation of 4.6 microM (approximately equal to 1.1 mg/ml) catalase solutions in pH 7.4 phosphate buffer induces spectral modifications. Difference spectra show maxima at 434, 555, 584 nm at the beginning of the irradiation, then a final spectrum with a maximum at 568 nm and a shoulder at 530 nm is observed. These results suggest the formation of compound III (oxyferrous catalase) and compound II, respectively. In deaerated 0.1 M, pH 8.7 borate buffer, the ferrous catalase is characterized by maxima at 563 and 594 nm. Hydrogen donors such as ethyl alcohol, formate and p-cresol inhibit, but citrate ions enhance the formation of these intermediates. A mechanism involving Fe(III) reduction according to an internal electron transfer is proposed.     

ULTRAVIOLET AND N-FORMYL-KYNURENINE-SENSITIZED PHOTOINACTIVATION OF BOVINE CARBONIC ANHYDRASE: AN INTERNAL PHOTODYNAMIC EFFECT

Abstract —Direct photoinactivation by UV light of bovine carbonic anhydrase, as well as its photosensitization by N -formyl-kynurenine, a tryptophan photooxidation product, have been investigated. In the presence of oxygen both methods lead to similar results: the enzyme loses its activity, the tryptophanyl, histidyl and, to a lesser extent, tyrosyl residues being destroyed. In nitrogen-saturated solutions, a dramatic drop is observed in the photoinacitivation yield under the direct action of ultraviolet light, whereas histidyl residues remain intact. Evidence indicates an internal photodynamic action of N -formyl-kynurenine in the protein core produced by the UV photooxidation of tryptophanyl residues. Photoinactivation of oxygenated enzyme solutions by external and internal photodynamic action correlates with histidyl residue destruction via singlet oxygen. The possible importance of the photodynamic ability of N -formyl-kynurenine in the photochemistry of proteins, DNA, and cells is discussed.     

THE KINETICS OF REDUCTION OF CYTOCHROME c BY SEMI-REDUCED FLAVIN

Abstract— Flavin mononucleotide radicals, FMNH', generated by laser flash photolysis of FMN in the presence of the electron donors, histidine, guanosine monophosphate or EDTA, were found to reduce cytochrome c with an apparent rate constant of 6 ± 10⁷ M ⁻¹ s⁻¹. These flash photolysis results were, however, complicated by the electron donor radicals formed simultaneously which, particularly with EDTA, also lead to reduction of cytochrome c. Pulse radiolysis of a nitrous oxide saturated aqueous solution of FMN containing a high concentration of HCOONa, leads to the exclusive formation of FMNH'. By adding small concentrations of cytochrome c to this solution, a rate constant of 4.0 ± 10⁻¹ M ⁻¹ s⁻¹ was obtained for the reduction of cytochrome c by FMNH'. Replacement of the HCOONa by EDTA in such solutions leads to further routes for reduction of cytochrome c on radiolysis. as in the photolytic situation. The relevance of these results to flavin-photosensitised reduction of cytochrome c and other components of the mitochondrial electron transport chain is discussed.     

PHOTOSENSITIZATION BY PORPHYRINS DELIVERED TO L CELL FIBROBLASTS BY HUMAN SERUM LOW DENSITY LIPOPROTEINS. A MICROSPECTROFLUOROMETRIC STUDY

Human serum LDL were used as vehicles to deliver protoporphyrin and hematoporphyrin dimer to L cell mouse fibroblasts. Topographic analysis by microspectrofluorometry on single living cells shows that after digestion of LDL, protoporphyrin is localized in cytoplasmic areas. Protoporphyrin and hematoporphyrin dimer are readily bleached by 420 ± 60 nm radiations at the high fluence rate used. Complex bleaching kinetics are observed. Spectral studies using the same technique demonstrate that an intense fluorescent emission (λ_max= 450 nm) is produced immediately after the onset of irradiation with 365 ± 2 nm or 420 ± 60 nm radiations using LDL loaded with protoporphyrin or Photofrin II. These fluorescent products have been previously identified as lipofuscin-like pigments formed by reaction of lipid photoperoxides with amino groups. The permeation of lysosomal membranes is also induced after delivery of the porphyrins by LDL. This permeation can be strongly inhibited not only by the lysosomal inhibitors chloroquine and monensin but also by a-tocopherol. On the other hand, neither α-tocopherol nor chloroquine or monensin inhibit the lipofuscin-like pigment formation.     

SUPEROXIDE DISMUTASE AS AN AMPLIFIER OF THE CHEMICAL REACTIVITY OF PORPHYRIN RADICAL-CATIONS

Abstract— Porphyrin radical-cations have been produced using laser flash photolysis via oxidation of the porphyrin triplets by metronidazole. This radical-cation reacts with OJ as shown by its increased half-life in the presence of native superoxide dismutase. Comparable results are obtained when porphyrin radical-cations are formed by Br₂^-O₂^-oxidation of porphyrins produced in pulse radiolysis of oxygen-saturated aqueous solutions containing 20 mM Br^-O^-. These results provide an explanation for the enhancement by superoxide dismutase of the photosensitizing capacity of porphyrins in the presence of electrophilic nitroimidazoles (Bazin and Santus, 1986). They may also apply to porphyrin radical-cations formed by monophotonic or biphotonic photoionization processes.     

A laser flash photolysis and pulse radiolysis study of primary photochemical processes of flumequine

The 355 nm laser flash photolysis of argon-saturated pH 8 phosphate buffer solutions of the fluoroquinolone antibiotic flumequine produces a transient triplet state with a maximum absorbance at 575 nm where the molar absorptivity is 14,000 M(-1) cm(-1). The quantum yield of triplet formation is 0.9. The transient triplet state is quenched by various Type-1 photodynamic substrates such as tryptophan (TrpH), tyrosine, N-acetylcysteine and 2-deoxyguanosine leading to the formation of the semireduced flumequine species. This semireduced form has been readily identified by pulse radiolysis of argon-saturated pH 8 buffered aqueous solutions by reaction of the hydrated electrons and the CO2*- radicals with flumequine. The absorption maximum of the transient semireduced species is found at 570 nm with a molar absorptivity of 2,500 M(-1) cm(-1). In argon-saturated buffered solutions, the semireduced flumequine species formed by the reaction of the flumequine triplet with TrpH stoichiometrically reduces ferricytochrome C (Cyt Fe3+) under steady state irradiation with ultraviolet-A light. In the presence of oxygen, O2*- is formed but the photoreduction of Cyt Fe3+ by O2*- competes with an oxidizing pathway which involves photo-oxidation products of TrpH.     

An insight into the mechanisms of the phototoxic response induced by cyamemazine in cultured fibroblasts and keratinocytes

The phototoxicity of cyamemazine (CMZ, Tercian), a neuroleptic of the phenothiazine family, has recently been reported in humans. CMZ has an absorbance maximum at 267 nm (molar absorptivity, 25,800 M(-1) cm(-1)) but a weaker molar absorptivity in the ultraviolet A (UV-A) region. CMZ exhibits a fluorescence with maximum emission at 535 nm and a quantum yield of 0.11. CMZ is a powerful photosensitizing agent toward HS 68 human skin fibroblasts and NCTC 2544 keratinocytes. At a UV-A radiation dose of 10 J/cm2, innocuous to cells in the absence of CMZ, the LD50 (lethal dose corresponding to 50% killing) are 0.5 and 1 microM for the fibroblasts and the keratinocytes, respectively, after overnight incubation with the drug. Short incubation times do not significantly alter the LD50. The CMZ-induced phototoxicity is accompanied by lipid membrane peroxidation consistent with the amphiphilic character of this photosensitizer. Keratinocytes are an order of magnitude less sensitive to the photosensitized lipid peroxidation than fibroblasts. Microspectrofluorometry reveals that lysosomal membranes are major sites of CMZ incorporation into the two cell lines because a Forster-type resonance energy transfer process occurs from CMZ to LysoTracker Red DND99 (LTR), a specific fluorescent probe of lysosomal membranes. The CMZ-photosensitized destruction of LTR demonstrates that CMZ retains its photosensitizing capacity after its lysosomal uptake.     

IS RHODAMINE 123 A PHOTOSENSITIZER?

Because of conflicting reports on the photoxicity of rhodamine 123 (Rh 123), we have undertaken a study of Rh 123 photosensitization in several in vitro systems. First, Rh 123 is not a photodynamic agent and does not react with singlet oxygen. Second, when bound to cytochrome c (Cyt c), Rh 123 photosensitizes ferro Cyt c but not ferri Cyt c degradation by an oxygen-independent process. When delivered to skin fibroblasts where it specifically stains mitochondria, Rh 123 photosensitizes membrane damage. These results are consistent with the hypothesis that Rh 123 is a phototoxic stain. The lack of photosensitivity of Rh 123-stained mitochondria in some cell lines might therefore be due to specific structural features.     

One-electron reduction of superoxide radical-anions by 3-alkylpolyhydroxyflavones in micelles. Effect of antioxidant alkyl chain length on micellar structure and reactivity

In micellar solutions, one-electron reduction of (*)O 2 (-) radical-anions by 3-alkylpolyhydroxyflavones (FnH) with alkyl chains of n = 1, 4, 6, 10 carbons produces phenoxyl radicals ( (*)Fn) identical to those obtained by one-electron oxidation by (*)Br 2 (-) radical-anions or by repair of tryptophan radicals. In cetyltrimethylammonium bromide (CTAB), F1H localizes in the Stern layer, and alkyl chains of other FnH solubilize in the hydrophobic interior, interacting with cetyl tails. This interaction produces more compact micelles with lower intramicellar fluidity, as suggested by the increase in the pseudo-first-order rate constant of (*)Fn formation ( k 1) from approximately 390 s (-1) for n = 1 to 610 s (-1) for n = 10, leading to an intramicellar bimolecular rate constant of 1 x 10 (5) M (-1) s (-1). Additionally, (*)F1 and (*)F4 decay by intermicellar bimolecular reaction (2 k = 20 and 2 x 10 (5) M (-1) s (-1), respectively) whereas other (*)Fn radicals are stable over seconds due to increased localization with regards to the Stern layer. In contrast, the thick uncharged hydrophilic palisade layer and the compact hydrophobic core of Triton X100 micelles are responsible for a much higher microviscosity resulting in a decrease in k 1 from approximately 15.6 s (-1) for n = 1 to 9.6 s (-1) for n = 10.