Biomedical subjects

A W Girotti

Publications and source records attributed to A W Girotti.

At least 55 records · Page 3Linked to original sources

PubMed1992

Photosensitized oxidation of cholesterol in biological systems: reaction pathways, cytotoxic effects and defense mechanisms.

Cholesterol resembles other unsaturated lipids in being susceptible to peroxidative degradation when exposed to a sensitizing agent, exciting light of suitable wavelength and molecular oxygen. Selected hydroperoxides of cholesterol can be used as relatively convenient and reliable indicators of primary photochemical mechanisms, allowing a distinction to be made between free radical-mediated and singlet oxygen-mediated reactions. When generated in cell membranes, hydroperoxides of cholesterol and other lipids can have deleterious effects on membrane structure and function. Such damage may be exacerbated if these photoproducts undergo one-electron reduction to oxyl radicals which in turn initiate chain peroxidation reactions. Cells can resist these effects by using a membrane-based glutathione peroxidase to catalyze the two-electron reduction and detoxification of lipid hydroperoxides. Recent advances in our understanding of cholesterol photo-oxidation from the standpoints of (a) mechanistic information, (b) cytotoxicity and (c) cytoprotection are discussed in this article.

Animals↗

PubMed1992

Photoperoxidation of cholesterol in homogeneous solution, isolated membranes, and cells: comparison of the 5 alpha- and 6 beta-hydroperoxides as indicators of singlet oxygen intermediacy.

Singlet oxygen (1O2) can react with cholesterol (Ch) to give three possible ene-addition hydroperoxides: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH), and 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH). The rates of dye-sensitized photogeneration and also the fates of 5 alpha-OOH and 6 beta-OOH in membrane bilayers have been studied and compared. Irradiation of unilamellar [14C]Ch/phospholipid vesicles in the presence of aluminum phthalocyanine tetrasulfonate or merocyanine 540 resulted in formation of 5 alpha-OOH and 6 beta-OOH, as determined by high performance liquid chromatography with radiochemical or electrochemical detection. The initial rate of 6 beta-OOH formation was 30-35% that of 5 alpha-OOH in a variety of liposomal systems. However, after a lag, 5 alpha-OOH invariably decayed via allylic rearrangement to 7 alpha-OOH (also known to be a free radical product), whereas 6 beta-OOH accumulated in unabated fashion until Ch depletion became limiting. Photooxidation of Ch in an isolated natural membrane (erythrocyte ghost) or in L1210 leukemia cells gave similar results. When the reaction was carried out in pyridine or methanol, the rate of 6 beta-OOH formation relative to 5 alpha-OOH was reduced by approximately half, with essentially no isomerization of the latter to 7 alpha-OOH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Photodynamic action of merocyanine 540 on erythrocyte membranes: structural perturbation of lipid and protein constituents.

erocyanine 540 (MC540) is a membrane-directed photosensitizing dye with antileukemic and antiviral properties. In this study, biophysical and biochemical techniques have been used to examine MC540-sensitized photooxidative damage in the lipid and protein compartments of a test membrane, the human erythrocyte ghost. Irradiation of MC540-sensitized ghosts with white light resulted in oxidative damage to proteins, as manifested by (i) loss of sulfhydryl groups; (ii) intermolecular cross-linking of major polypeptides; and (iii) loss of Mg(2+)-ATPase and Na+,K(+)-ATPase activities. Photooxidation also produced a rapid and progressive increase in general protein motion, as measured by electron paramagnetic resonance spectrometry (EPR) with the sulfhydryl spin label MAL-6. In addition to these effects, ghosts exposed to MC540 and light underwent lipid peroxidation. EPR with two lipophilic spin probes, 5-doxylstearate and 16-doxylstearate, showed that lipid peroxidation is accompanied by a progressive decrease in bilayer fluidity (motional freedom). At a given dye concentration, structural perturbations of proteins were detected at much lower light fluences than those of lipids. When photoreactions were carried out in the presence of ascorbate and iron, there was a strong stimulation of lipid peroxidation (attributed to free radical chain reactions), with a concomitant greater decrease in lipid mobility. Thus, the deleterious effects of photoperoxidation on lipid structure and motional freedom were greatly exacerbated by ascorbate and iron. Membrane damage similar to that described here may play a role in the phototherapeutic activity of MC540.

Adenosine Triphosphatases↗

PubMed1991

Chromatographic separation and electrochemical determination of cholesterol hydroperoxides generated by photodynamic action.

Reverse-phase HPLC with electrochemical detection (HPLC-EC) was used to separate and quantitate photochemically generated cholesterol hydroperoxides. The EC measurements were performed in the reduction mode under anaerobic conditions. When cholesterol-containing liposomes were irradiated in the presence of a phthalocyanine dye, at least four major oxidation products of cholesterol were detected by HPLC-EC:5 alpha-hydroperoxide (5 alpha-OOH), 6 beta-hydroperoxide (6 beta-OOH), 7 alpha-hydroperoxide (7 alpha-OOH), and 7 beta-hydroperoxide (7 beta-OOH). The detection limit for each compound was found to be approximately 25 pmol. Product identification was based on matching HPLC and TLC behavior of standards and on physical indicators (melting points and NMR chemical shifts). The cholesterol hydroperoxides were barely separated from EC-silent diol derivatives, which could be detected by 210 nm absorbance after reduction of the hydroperoxides with triphenylphosphine. Irradiation of a dye-sensitized natural membrane, the human erythrocyte ghost, also resulted in formation of 5 alpha-OOH, 6-OOH, and 7-OOH, as evidenced by HPLC-EC. Under the chromatographic conditions used, these species were well separated not only from one another but also from a family of at least six phospholipid hydroperoxides. These results illustrate the strengths of HPLC-EC as a relatively convenient, sensitive, and selective means of analyzing cholesterol hydroperoxides in biological samples.

Cholesterol↗

PubMed1991

Lethal damage to murine L1210 cells by exogenous lipid hydroperoxides: protective role of glutathione-dependent selenoperoxidases.

The effect of selenium deprivation on the viability of murine L1210 cells exposed to various exogenous lipid hydroperoxides has been investigated. Selenoperoxidase activities of cells grown for longer than 1 week in 1% serum with no added selenium [Se(-) cells] were less than 10% of the activities of selenium-satisfied controls [Se(+) cells] or selenium-repleted counterparts [Se(-/+) cells]. The enzymes measured were classical glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). Se(-) cells exhibited a compensatory increase in catalase activity. Dye exclusion and clonal survival assays indicated that Se(-) and Se(+) cells were relatively insensitive to photochemically generated phospholipid hydroperoxides in liposomal form. However, both cell types were sensitive to liposomal cholesterol hydroperoxides, e.g., 7-hydroperoxycholesterol (7-OOH), Se(-) being much more so (LD50 approximately 10 microM) than Se(+) (LD50 approximately 75 microM). By contrast, 7-hydroxycholesterol over a comparable concentration range was minimally toxic to Se(-) and Se(+) cells. Cell killing by 7-OOH was inhibited by desferrioxamine and by butylated hydroxytoluene, suggesting that iron-mediated free radical reactions are involved. The involvement of glutathione in cytoprotection was confirmed by showing that Se(+) cells were more sensitive to 7-OOH after treating with buthionine sulfoximine, an inhibitor of GSH synthesis. Cellular detoxification of 7-OOH is provisionally attributed to PHGPX rather than GPX, since 7-OOH and other cholesterol hydroperoxides were found to be good substrates for PHGPX in a cell free system, but were unreactive with GPX.

Animals↗

PubMed1991

Phthalocyanine-sensitized lipid peroxidation in cell membranes: use of cholesterol and azide as probes of primary photochemistry.

Various phthalacyanine (Pc) derivatives of phototherapeutic interest have been shown to be efficient type II (singlet oxygen, 1O2) sensitizers in aqueous and non-aqueous solutions. However, primary Pc photochemistry in biological environments, e.g. cell membranes, has not been studied in a definitive manner. To address this question, we used endogenous cholesterol in the erythrocyte ghost as a mechanistic reporter lipid Membranes sensitized with chloroaluminum Pc tetrasulfonate (AlPcS) and exposed to white light at 10 degrees C underwent lipid peroxidation, as indicated by the accumulation of hydroperoxides and thiobarbituric acid reactivity. Specific analysis of cholesterol photo-products by thin layer chromatography and high performance liquid chromatography revealed the presence of 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), with much smaller amounts of 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH) and 5 alpha-cholest-6-en-3 beta, 5-diol and cholest-5-en-3 beta, 7 alpha-diol (5 alpha-OH and 7 alpha-OH). Identification of 5 alpha-OOH as a major photoproduct provides unambiguous evidence for large scale 1O2 intermediacy. Azide inhibited lipid peroxidation in a dose-dependent fashion, providing additional support for a type II mechanism. However, the 1O2 quenching constant from Stern-Volmer analysis was approximately 50 times lower than that determined for a non-membrane probe, lactate dehydrogenase. The latter value agreed with literature values. A probable explanation is that membrane-bound dye generates most of the 1O2 involved in lipid peroxidation. Although azide can intercept any 1O2 escaping into (or formed in) the medium, it has limited access to 1O2 generated on the membrane and reacting (or being quenched) near its site of origin.

Azides↗

PubMed1991

Photosensitized lipid peroxidation and enzyme inactivation by membrane-bound merocyanine 540: reaction mechanisms in the absence and presence of ascorbate.

The lipophilic photosensitizing dye merocyanine 540 (MC540) is being studied intensively as an antitumor and antiviral agent. Since plasma membranes are believed to be the principal cellular targets of MC540-mediated photodamage, we have studied membrane damage in a well characterized test system, the human erythrocyte ghost. When irradiated with white light, MC540-sensitized ghosts accumulated lipid hydroperoxides (LOOHs derived from phospholipids and cholesterol) at a rate dependent on initial dye concentration. Neither desferrioxamine nor butylated hydroxytoluene inhibited LOOH formation, suggesting that Type I (iron-mediated free radical) chemistry is not important. By contrast, azide inhibited the reaction in a dose-dependent fashion, implicating a Type II (singlet oxygen, 1O2) mechanism. Stern-Volmer analysis of the data gave a 1O2 quenching constant approximately 50 times lower than that determined for an extramembranous target, lactate dehydrogenase (the latter value agreeing with literature values). This suggests that 1O2 reacts primarily at its membrane sites of origin and that azide has limited access to these sites. Using [14C]cholesterol-labeled membranes and HPLC with radiodetection, we identified 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide as the major cholesterol photoproduct, thereby confirming 1O2 intermediacy. Irradiation of MC540-sensitized membranes in the presence of added iron and ascorbate resulted in a large burst of lipid peroxidation, as shown by thiobarbituric acid reactivity and appearance of 7-hydroperoxycholesterol and 7-hydroxycholesterol as major oxidation products. Amplification of MC540-initiated lipid peroxidation by iron/ascorbate (attributed to light-independent reduction of nascent photoperoxides, with ensuing free radical chain reactions) could prove useful in augmenting MC540's phototherapeutic effects.

Ascorbic Acid↗

PubMed1991

Photodynamic action of merocyanine 540 in artificial bilayers and natural membranes: action spectra and quantum yields.

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully-saturated dimyristoylphosphatidylcholine vesicles, mono-unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane-bound MC540 in aqueous buffer was determined to be 0.065 +/- 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer-dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.

Cell Membrane↗

PubMed1991

Reactivity of phospholipid hydroperoxide glutathione peroxidase with membrane and lipoprotein lipid hydroperoxides.

A comparative study has been carried out on the general reactivity of lipid hydroperoxides in liposomes, biological membranes and lipoproteins with two Se-dependent peroxidases: Glutathione Peroxidase (GPX) and Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPX). While PHGPX reduces all hydroperoxides derived from phospholipids, cholesterol and cholesterol esters, GPX reduces only fatty acid hydroperoxides released after treatment of phospholipid hydroperoxides with phospholipase A2. These findings highlight the role of PHGPX in protecting biomembranes from peroxidative damage and add new insight into how cholesterol hydroperoxides are detossified in cells.

Cholesterol↗

PubMed1990

Enzymatic reduction of phospholipid and cholesterol hydroperoxides in artificial bilayers and lipoproteins.

Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and deactivated by phospholipid hydroperoxide glutathione peroxidase (PHGPX), the second selenoperoxidase to be identified and characterized. Coupled spectrophotometric analyses in the presence of NADPH, glutathione (GSH), glutathione reductase and Triton X-100 indicated that photochemically generated LOOHs in small unilamellar liposomes are substrates for PHGPX, but not for the classical glutathione peroxidase (GPX). PHGPX was found to be reactive with cholesterol hydroperoxides as well as phospholipid hydroperoxides. Kinetic iodometric analyses during GSH/PHGPX treatment of photoperoxidized liposomes indicated a rapid decay of total LOOH to a residual level of 35-40%; addition of Triton X-100 allowed the reaction to go to completion. The non-reactive LOOHs in intact liposomes were shown to be inaccessible groups on the inner membrane face. In the presence of iron and ascorbate, photoperoxidized liposomes underwent a burst of thiobarbituric acid-detectable lipid peroxidation which could be inhibited by prior GSH/PHGPX treatment, but not by GSH/GPX treatment. Additional experiments indicated that hydroperoxides of phosphatidylcholine, cholesterol and cholesteryl esters in low-density lipoprotein are also good substrates for PHGPX. An important role of PHGPX in cellular detoxification of a wide variety of LOOHs in membranes and internalized lipoproteins is suggested from these findings.

Cholesterol↗

PubMed1990

Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides.

The general reactivity of membrane lipid hydroperoxides (LOOHs) with the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) has been investigated. When human erythrocyte ghosts (lipid content: 60 wt % phospholipid; 25 wt % cholesterol) were treated with GSH/PHGPX subsequent to rose bengal-sensitized photoperoxidation, iodometrically measured LOOHs were totally reduced to alcohols. Similar treatment with the classic glutathione peroxidase (GPX) produced no effect unless the peroxidized membranes were preincubated with phospholipase A2 (PLA2). However, under these conditions, no more than approximately 60% of the LOOH was reduced; introduction of PHGPX brought the reaction to completion. Thin layer chromatographic analyses revealed that the GPX-resistant (but PHGPX-reactive) LOOH was cholesterol hydroperoxide (ChOOH) consisting mainly of the 5 alpha (singlet oxygen-derived) product. Membrane ChOOHs were reduced by GSH/PHGPX to species that comigrated with borohydride reduction products (diols). Sensitive quantitation of PHGPX-catalyzed ChOOH reduction was accomplished by using [14C]cholesterol-labeled ghosts. Kinetic analyses indicated that the rate of ChOOH decay was approximately 1/6 that of phospholipid hydroperoxide decay. Photooxidized ghosts underwent a large burst of free radical-mediated lipid peroxidation when incubation with ascorbate/iron or xanthine/xanthine oxidase/iron. These reactions were only partially inhibited by PLA2/GSH/GPX treatment, but totally inhibited by GSH/PHGPX treatment, consistent with complete elimination of LOOHs in the latter case. These findings provide important clues as to how ChOOHs are detoxified in cells and add new insights into PHGPX's protective role.

Animals↗

PubMed1990

Photodynamic lipid peroxidation in biological systems.

Oxidative degradation of cell membrane lipids in the presence of molecular oxygen, a sensitizing agent and exciting light is termed photodynamic lipid peroxidation (photoperoxidation). Like other types of lipid peroxidation, photoperoxidation is detrimental to membrane structure and function, and could play a role in many of the toxic as well as therapeutic effects of photodynamic action. Recent advances in our understanding of photoperoxidation and its biomedical implications are reviewed in this article. Specific areas of interest include (a) reaction mechanisms; (b) methods of detection and quantitation; and (c) cellular defenses (enzymatic and non-enzymatic).

Animals↗

PubMed1989

Role of lipid peroxidation in hematoporphyrin derivative-sensitized photokilling of tumor cells: protective effects of glutathione peroxidase.

The ability of cells to detoxify lipid hydroperoxides (LOOHs) generated by hematoporphyrin derivative (HPD)-sensitized photooxidation was investigated for the first time. The general importance of glutathione in cytoprotection was confirmed by showing that murine L1210 cells were more sensitive to the lethal effects of HPD plus red light after being treated with buthionine sulfoximine. The specific role of Se-dependent glutathione peroxidase was investigated by using L1210 cells that were grown in Se-deficient media. Glutathione peroxidase activity of such cells was typically less than 5% of that exhibited by Se-replete cells. When examined by means of dye exclusion or clonogenic assay, Se-deficient cells were dramatically more sensitive to HPD-mediated photokilling than normal counterparts. Impaired metabolism of hydrogen peroxide was ruled out as a possible cause of enhanced photokilling, since added catalase had no protective effect on Se-deficient cells. Iodometric analysis of lipid extracts from photooxidized cells indicated a significantly greater rate of LOOH accumulation as a result of Se depletion. Moreover, when depleted cells were incubated in the dark after a short period of photo-peroxidation, LOOH decay was markedly slower than in controls. Similar results were obtained with human CaSki cells derived from cervical carcinoma. It is apparent from these results that lipid peroxidation plays an important role in tumor cell eradication by HPD/phototherapy, and that glutathione peroxidase serves as a natural protectant against photokilling by catalyzing the reduction of LOOHs.

Animals↗

PubMed1989

Reactivity of photochemically-generated lipid hydroperoxides in cell membranes with glutathione peroxidase.

The ability of glutathione peroxidase (Gpx) to catalyze the reductive inactivation of photochemically-generated lipid hydroperoxides (LOOHs) was investigated, using hematoporphyrin derivative (HPD) as a photosensitizing agent and erythrocyte ghosts as membrane targets. Glutathione peroxidase was reactive toward photoperoxidized membranes only after their exposure to phospholipase A2 (PLA2). Iodometrically-determined LOOH values were typically 30-40% greater than values measured by enzymatic assay using Gpx and glutathione reductase. A consistent result was obtained when photooxidized membranes were treated with PLA2 and GSH/Gpx followed by iodometric assay, viz. persistence of approximately 40% of the starting LOOH. Whereas photooxidized egg phosphatidylcholine liposomes underwent total LOOH loss when incubated with PLA2 and GSH/Gpx, no net loss was observed with photooxidized cholesterol/dimyristoyl-phosphatidylcholine liposomes. The results suggest that cholesterol hydroperoxides in ghost membranes account for the Gpx-resistant fraction of LOOHs.

Erythrocyte Membrane↗

PubMed1989

Glucose administration augments in vivo uptake and phototoxicity of the tumor-localizing fraction of hematoporphyrin derivative.

Rhabdomyosarcoma tumors in rats made hyperglycemic by multiple injections of glucose exhibited a transient decrease in pH and an increased ability to accumulate derivatives of hematoporphyrin (HPD). Photoradiation of tumors in glucose/HPD-treated animals produced a greater cell kill than in galactose/HPD-treated controls. A therapeutic benefit of glucose administration in conjunction with HPD-phototherapy is suggested.

Animals↗

PubMed1988

Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase.

The susceptibility of photodynamically-generated lipid hydroperoxides to reductive inactivation by glutathione peroxidase (GPX) has been investigated, using hematoporphyrin derivative as a photosensitizing agent and the human erythrocyte ghost as a target membrane. Photoperoxidized ghosts were reactive in a glutathione peroxidase/reductase (GPX/GRD)-coupled assay only after phospholipid hydrolysis by phospholipase A2 (PLA2). However, enzymatically determined lipid hydroperoxide values were consistently approx. 40% lower than iodometrically determined values throughout the course of photooxidation. Moreover, when irradiated ghosts were analyzed iodometrically during PLA2/GSH/GPX treatment, a residual 30-40% of non-reactive lipid hydroperoxide was observed. The possibility that cholesterol product(s) account for the non-reactive lipid hydroperoxide was examined by tracking cholesterol hydroperoxides in [14C]cholesterol-labeled ghosts. The sum of cholesterol hydroperoxides and GPX/GRD-detectable lipid hydroperoxides was found to agree closely with iodometrically determined lipid hydroperoxide throughout the course of irradiation. Thin-layer chromatography of total lipid extracts indicated that cholesterol hydroperoxide was unaffected by PLA2/GSH/GPX treatment, whereas most of the phospholipid peroxides were completely hydrolyzed and the released fatty acid peroxides were reduced to alcohols. It appears, therefore, that the GPX-resistant lipid hydroperoxides in photooxidized ghosts were derived primarily from cholesterol. Ascorbate plus Fe3+ produced a burst of free-radical lipid peroxidation in photooxidized, PLA2-treated ghosts. As expected for fatty acid hydroperoxide inactivation, the lipid peroxidation was inhibited by GSH/GPX, but only partially so, suggesting that cholesterol hydroperoxide-derived radicals play a major role in the reaction.

Ascorbic Acid↗

PubMed1988

Ascorbate-enhanced lipid peroxidation in photooxidized cell membranes: cholesterol product analysis as a probe of reaction mechanism.

Cholesterol was used as an in situ probe for studying mechanisms of lipid peroxidation in isolated erythrocyte membranes subjected to different prooxidant conditions. The membranes were labeled with [14C]cholesterol by exchange with prelabeled unilamellar liposomes and photosensitized with hematoporphyrin derivative. Irradiation with a dose of blue light resulted in thiobarbituric acid-detectable lipid peroxidation that was increased markedly by subsequent dark incubation with 0.5-1.0 mM ascorbate (AH-). Ascorbate-stimulated lipid peroxidation was inhibited by EDTA, desferrioxamine (DOX) and butylated hydroxytoluene (BHT), suggesting that the process is free radical in nature and catalyzed by membrane-bound iron. Thin layer chromatography and radiometric scanning of extracted lipids from photooxidized membranes revealed that the major oxidation product of cholesterol was the 5 alpha-hydroperoxide (5 alpha-OOH), a singlet oxygen adduct. Post-irradiation treatment with AH-/Fe(III) resulted in an almost-total disappearance of 5 alpha-OOH and the preponderance of free radical oxidation products, e.g. 7-ketocholesterol, the epimeric 7 alpha-/7 beta-hydroperoxides (7 alpha-/7 beta-OOH) and their respective alcohols (7 alpha-/7 beta-OH). EDTA, DOX and BHT inhibited the formation of these products, while catalase and superoxide dismutase had no effect. These results are consistent with a mechanism involving 1-electron reduction of photogenerated hydroperoxides to oxyl radicals, which trigger bursts of free radical lipid peroxidation. Though generated in this system, partially reduced oxygen species, viz. superoxide, hydrogen peroxide and hydroxyl radical, appear to be relatively unimportant in the autoxidation process.

Ascorbic Acid↗

PubMed1988

Light-stimulated formation of hydrogen peroxide and hydroxyl radical in the presence of uroporphyrin and ascorbate.

Blue light irradiation of 2-deoxyribose (DOR) in the presence of uroporphyrin I (UP), ascorbate (AH-), trace iron, and phosphate buffer resulted in a strong stimulation of hydroxyl radical (OH.)-dependent oxidation of DOR. Photostimulated generation of H2O2 was monitored after removal of residual AH- (i) by ascorbate oxidase treatment, or (ii) by anion exchange on mini-columns of DEAE-Sephadex. Irradiation of the above mixture produced a strong burst of H2O2 which was intensified by desferrioxamine and suppressed by catalase or EDTA. The mechanism suggested by these observations is one in which photoreduction of UP to the radical anion initiates the formation of H2O2, which gives rise to OH. via Fenton chemistry. This is the first known investigation of H2O2 fluxes in a Type I (free radical) photoreaction involving AH- as the electron donor.

Ascorbate Oxidase↗