A. Paul Schaap

Chemical and enzymatic triggering of 1,2-dioxetanes. 3: alkaline phosphatase-catalyzed chemiluminescence from an aryl phosphate-substituted dioxetane

Abstract We describe herein the preparation of an aryl phosphate-substituted 1,2-dioxetane and the enzymatic cleavage of this chemiluminescent dioxetane in aqueous buffer with alkaline phosphatase.

Reactivity of singlet molecular oxygen with cholesterol in a phospholipid membrane matrix. A model for oxidative damage of membranes

Abstract Photooxidation of cholesterol in liposomes with hematoporphyrin sensitization has been studied. With liposomal samples in which the hematoporphyrin is incorporated in the membrane, the yield of the characteristic singlet oxygen product, 3β-hydroxycholest-6-ene 5α-hydroperoxide, was approximately 6 times greater than that observed in the samples in which the hematoporphyrin was outside the membrane. Small amounts of 3β-hydroxycholest-5-ene 7α- and 7β-hydroperoxides, radical autoxidation products, were formed in both samples. Photolysis of a dispersion of cholesterol in an aqueous solution of hematoporphyrin gave no singlet oxygen products. It is concluded from these results that endogenous singlet oxygen when formed in the phospho-lipid membrane has a sufficiently long lifetime to effect oxygenation of cholesterol; whereas exogenous singlet oxygen generated outside the membrane is quenched by solvent before appreciable diffusion into the membrane can occur.

Chemical and enzymatic triggering of 1,2-dioxetanes. 1: Aryl esterase-catalyzed chemiluminescence from a naphthyl acetate-substituted dioxetane

Abstract A thermally stable 1,2-dioxetane bearing a naphthyl acetate group is enzymatically cleaved in aqueous buffer to generate chemiluminescence at ambient temperature.

An evidence of the peroxidase-dependent oxygen transfer from hydrogen peroxide to sulfides

Horseradish- and chloro-peroxidase catalyzed oxidation of sulfides have been investigated. Thioanisoles were oxygenated to the corresponding sulfoxides by such peroxidases at the expense of H2O2. Dealkylation was observed only in the chloroperoxidase-dependent oxidations of p-methoxy- and p-iso-propoxy-thioanisoles. The experiments with 18O-labeled H2O2 indicated that an oxygen atom of H2O2 is incorporated into the sulfoxides. These research lead to the conclusion that compound I or II is capable of acting as an oxygen donor as well as an electron acceptor.

MECHANISMS OF 1,2-DIOXETANE DECOMPOSITION: THE ROLE OF ELECTRON TRANSFER

Abstract— The chemiluminescence from the cleavage of a number of 1,6‐diaryl‐2,5,7,8‐tetraoxabicyclo[4.2.0]octanes 1 has been examined. The ease of oxidation of (object) the aryl moiety strongly influences both the stability and chemiluminescence efficiency of these 1,2‐dioxetanes. When Ar is difficult to oxidize, 1 is comparable in stability to simple. alkyl‐substituted 1,2‐dioxe‐tanes and affords triplet excited states in moderate yield. Both biradical and concerted cleavage mechanisms have been suggested to explain this behavior. However, when Ar is a readily oxidized group, 1 is substantially destahilized and gives excited singlet states in high yield. In this instance 1 is analogous to a number of bioluminescent systems. Cleavage mechanisms involving intramolecular electron transfer are proposed to account for this observation. In certain cases thermolysis of 1 occurs by both types of mechanism in competition, and the electron transfer mechanism may be selectively catalyzed by polar, protic media.

Cytochrome c enhancement of singlet molecular oxygen production by the NADPH-dependent adrenodoxin reductase-adrenodoxin system: the role of singlet oxygen in damaging adrenal mitochondrial membranes.

Abstract In the presence of NADPH, cytochrome c stimulates approximately a 200-fold increase in the production of singlet oxygen by the bovine adrenodoxin reductase-adrenodoxin system. The formation of singlet oxygen, which was monitored by the attending chemiluminescence, was markedly inhibited by the addition of superoxide dismutase or 1,4-diazabicyclo[2.2.2]octane. The adrenal system, in the presence of cytochrome c, peroxidized adrenal mitochondrial lipids, as indicated by the formation of malondialdehyde. This oxidation is also inhibited by the addition of dismutase and 1,4-diazabicyclo[2.2.2]octane.

REACTION OF SINGLET OXYGEN WITH CHOLESTEROL IN LIPOSOMAL MEMBRANES. EFFECT OF MEMBRANE FLUIDITY ON THE PHOTOOXIDATION OF CHOLESTEROL

Abstract— The photosensitized oxygenation of cholesterol in liposomal membranes results in the formation of the characteristic singlet oxygen product, 3β‐hydroxy‐5α‐cholest‐6‐ene‐5‐hydroperoxide. The yield of this product was found to be strongly temperature dependent with 6‐ to 7‐fold increase above the transition temperature of the membrane using 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphorylcholine or 1.2‐dimyristoyl‐sn‐glycero‐3‐phosphorylcholine. In contrast, the formation of the radical autoxidation products of cholesterol was not significantly enhanced in these systems by higher temperatures. We conclude from the results that the oxidation of cholesterol by singlet oxygen in the artificial membranes is affected by both membrane fluidity and amount of hematoporphyrin incorporated into the membrane. The ratio of cholesterol to phospholipid which affects the morphology of membranes is a crucial factor in determining the yield of the singlet oxygen product.

Comparison Between Acridan Ester, Luminol, and Ruthenium Chelate Electrochemiluminescence

Transparent indium tin oxide (ITO) electrodes are attractive for use in electrochemiluminescence (ECL). In this work the ECL of an acridan ester (2′,3′,6′-trifluorophenyl 10-methylacridan-9-carboxylate), luminol, and a ruthenium chelate (ruthenium tris 2,2′-bipyridyl) are compared using ITO electrodes. The electrochemistry of all the compounds is described and related to their ECL mechanisms; three possible mechanisms for luminol ECL are described, and it is suggested that the currently accepted mechanism involving luminol and superoxide radicals may not always be correct. Maximum light emission in ruthenium chelate ECL occurs at 1.5 V (vs. Ag/AgCl), but potentials of more than 1 V have a corrosive effect on ITO. The effect of this limitation on ruthenium chelate ECL is discussed as part of a wider assessment of the advantages and disadvantages of ITO as an electrode material for ECL. The effects of pH and H2O2 concentration on ECL detection of the acridan ester and luminol in a planar flow cell are described. The limits of detection of the acridan ester and luminol were 65 pM and 72 pM, respectively.

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450s are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.

Singlet molecular oxygen in biological systems: Non-quenching of singlet oxygen-mediated chemiluminescence by superoxide dismutase

Summary Chemiluminescence measurements indicate that superoxide dismutase does not significantly quench singlet molecular oxygen. 1-Phospha-2,8,9-trioxaadamantane ozonide is used as a singlet oxygen source. However, superoxide dismutase effectively inhibits the chemiluminescence produced by xanthine-xanthine oxidase or potassium superoxide. It is, therefore, likely that superoxide dismutase produces triplet molecular oxygen from its copper-containing active site during the dismutation of superoxide anion radicals, unlike the non-enzymatic dismutation reaction which yields singlet oxygen.