A microcalorimetric and electron spin resonance study of the influence of UV radiation on collagen

It was demonstrated microcalorimetrically that UV radiation cardinally changed the behavioral pattern of the temperature dependence of heat capacity of a collagen solution and decreased the enthalpy of collagen denaturation. It was discovered by electron spin resonance (ESR) that the primary products of UV-irradiated acid-soluble collagen were atomic hydrogen and the anion radical of acetic acid. The latter, when exposed to long-wavelength UV light, converted into the methyl radical, which interacted with acetic acid to produce the acetic acid radical. These free radicals interacted with the collagen molecule, leading to the appearance of seven superfine components with the split ΔH = 1.13 mT in the ESR spectrum. It was assumed that this spectrum is related to the free radical that occurred in the proline residue of the collagen molecule, which, in this particular case, was the major defect in the triple helix of collagen that caused instability of the macromolecule.     

A carbon-centered free radical intermediate in the prostaglandin synthetase oxidation of arachidonic acid. Spin trapping and oxygen uptake studies

The ESR spin-trapping technique has been used to identify a free radical involved in the oxygenation of arachidonic acid by ram seminal vesicle microsomes. The ESR spectrum of the radical adduct indicates that a carbon-centered arachidonic acid free radical has been observed. The formation of this species is inhibited by indomethacin, but not by phenol, and it is probably the first intermediate formed during the prostaglandin synthetase-catalyzed oxidation of arachidonic acid. The chemical identity of the trapped radical was substantiated with an independent synthesis of a closely related radical adduct.     

Direct detection of circulating free radicals in the rat using electron spin resonance spectrometry.

We developed a new technique for directly observing in vivo free radical formation in the circulating blood of living rats using electron spin resonance (ESR) spectrometry without any labeling or trapping agents. It was found that a doublet peak spectrum was obtained following ferric citrate and ascorbic acid injection. The signals were confirmed in different ways to be due to ascorbic acid radicals. These results provide evidence to support the involvement of free radical intermediates in iron-ascorbic acid reactions, and further confirm the suggested mechanisms of both the adverse and protective effects of ascorbic acid in biological systems. Furthermore, this method of direct observation is a new application of ESR spectrometry to living animals.     

The enzymatic one-electron reduction of porphyrins to their anion free radicals

The anaerobic enzymatic one-electron reduction of uroporphyrin I (in the absence of light) by the ferredoxin/ferredoxin:NADP+ oxidoreductase system was investigated using NADPH as the source of reducing equivalents. The porphyrin anion free radical metabolite formed by one-electron reduction of the parent molecule was detected with ESR spectroscopy. The ESR spectrum exhibited a singlet (g = 2.0021) with a 5.4-G peak-to-peak linewidth. The reduction process was also investigated under aerobic conditions. The reduction of molecular oxygen to superoxide anion radical by the porphyrin anion radical was demonstrated by using the ESR technique of spin trapping. The ESR spectra of the spin-trapped oxygen-derived radicals were superoxide dismutase-sensitive and catalase-insensitive, supporting the assignment of the trapped radical to the superoxide anion radical. These aerobic experiments demonstrate electron transfer from the porphyrin anion radical to molecular oxygen. The anaerobic reduction of Photofrin II by hepatic microsomes and the ferredoxin/ferredoxin:NADP+ oxidoreductase system to a porphyrin anion radical was also investigated. Free radical formation by ferredoxin: NADP+ oxidoreductase is totally dependent upon ferredoxin. The ESR spectrum of this porphyrin free radical also exhibited a singlet (g = 2.0026) with a 15-G peak-to-peak linewidth.     

ESR spin trapping of a carbon-centered free radical from agonist-stimulated human platelets

Several free radical intermediates formed during synthesis of prostaglandin H synthase (PGHS) catalyze the biosynthesis of prostaglandins from arachidonic acid (AA). We attempted to directly detect free radical intermediates of PGHS in cells. Studies were carried out using human platelets, which possess significant PGHS activity. Electron spin resonance (ESR) spectra showed a g = 2.005 signal radical, which was formed by the incubation of collagen, thrombin, AA, and a variety of peroxides with human platelets. The ESR spectra obtained using 5,5-dimethyl-1 pyrroline N-oxide (DMPO) and alpha-phenyl N-tert.-butylnitron (PBN) were typical of an immobilized nitroxide. Extensive Pronase digestion of both the DMPO and PBN adducts allowed us to deduce that it was a carbon-centered radical. The formation of this radical was inhibited by potassium cyanide and by desferroxamine. Peroxides stimulated formation of the g = 2.005 signal radical and inhibited platelet aggregation induced by AA. PGHS cosubstrates increased the intensity of the radical signal but inhibited platelet aggregation induced by AA. Both S-nitro-L-glutathione and reduced glutathione quenched the g = 2.005 radical but could not restore platelet aggregatory activity. These results suggest that the carbon-centered radical is a self-destructing free radical formed during peroxide-mediated deactivation of PGHS in human platelets.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

The free radical formed during the hydroperoxide-mediated deactivation of ram seminal vesicles is hemoprotein-derived

Prostaglandin synthase is a multi-enzyme complex which catalyzes the oxygenation of arachidonic acid to the various prostaglandins. During the oxygenation, the enzyme is self-deactivated and, on the basis of ESR data, it has been proposed to form a self-destructive free radical. The free radical was suggested to form from the oxygen lost from prostaglandin G2 during its reduction to prostaglandin H2, and the destructive species was therefore thought to be an oxygen-centered free radical, tentatively identified as the hydroxy radical. We have reinvestigated this ESR signal (g = 2.005) and have concluded, with the aid of the known ESR parameters for the hydroxy and other oxygen-centered free radicals, that the free radical formed during the oxygenation is neither a hydroxy nor any known oxygen-centred radical. Prostaglandin synthase is thought to be a hemoprotein, so this unknown ESR signal was compared with the previously observed free radical formed by the reaction of H2O2 with methemoglobin. This comparison indicates that the free radical formed by the reaction of prostaglandin G2 with ram seminal vesicles is hemoprotein-derived and may be formed by the oxidation of an amino acid(s) located near the iron of the heme.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37°C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is –OH>–OCH₃>–H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37 degrees C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is -OH>-OCH(3)>-H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Development of free radical products during the greening reaction of caffeic acid esters (or chlorogenic acid) and a primary amino compound.

ESR spectra were measured directly on a marked greening reaction mixture of Et-caffeate and a primary amino compound in alkali solution under aeration. A clear hyperfine structure was commonly detected early in the greening reaction with different amino compounds. Its hyperfine spectrum split into seven peaks was analyzed and found to be due to the oxidized free radical product of the Et-caffeate using an authentic sample system. Another type of hyperfine ESR spectrum was observed later in the reaction, and was altered with different amino compounds. The hyperfine structure for n-butylamine split into 12 lines. The latter type of free radical products were assumed to be a semiquinone type radical compound of the trihydroxy benzacridine derivative, which was identified as the principal structure of the green and yellow pigments formed by this greening reaction system. A formation mechanism of the green pigment and related products involving these free radical products is proposed.     

The formation of aminopyrine cation radical by the peroxidase activity of prostaglandin H synthase and subsequent reactions of the radical

The oxidation of aminopyrine to an aminopyrine cation radical was investigated using a solubilized microsomal preparation or prostaglandin H synthase purified from ram seminal vesicles. Aminopyrine was oxidized to an aminopyrine cation radical in the presence of arachidonic acid, hydrogen peroxide, t-butyl hydroperoxide or 15-hydroperoxyarachidonic acid. Highly purified prostaglandin H synthase, which processes both cyclo-oxygenase and hydroperoxidase activity, oxidized aminopyrine to the free radical. Purified prostaglandin H synthase reconstituted with Mn2+ protoporphyrin IX, which processes only cyclo-oxygenase activity, did not catalyze the formation of the aminopyrine free radical. Aminopyrine stimulated the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11-13-eicosatetraenoic acid. Approximately 1 molecule of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid was reduced for every 2 molecules of aminopyrine free radical formed, giving a stoichiometry of 1:2. The decay of the aminopyrine radical obeyed second-order kinetics. These results support the proposed mechanism in which aminopyrine is oxidized by prostaglandin H synthase hydroperoxidase to the aminopyrine free radical, which then disproportionates to the iminium cation. The iminium cation is further hydrolyzed to the demethylated amine and formaldehyde. Glutathione reduced the aminopyrine radical to aminopyrine with the concomitant oxidation of GSH to its thiyl radical as detected by ESR of the glutathione thiyl radical adduct.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light

When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl-1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy.     

胶原蛋白的电子活性与矽肺纤维化Ⅱ正常大鼠与实验性矽肺大鼠肺Ⅰ型胶原稳定自由基的研究

由正常大鼠肺撮取的酸溶性Ⅰ型胶原出现一个明显的ESR信号(g=2.006),而由矽肺大鼠提取的酸溶性Ⅰ型胶原较少或缺乏该ESR信号,该信号不是顺磁过渡元素产生,而是稳定自由基的反映.肺胶原可能存在两种稳定的自由基,一种易受紫外线辐照的激发并且在电场极化下自旋耦合而减弱,另一种自由基则相对稳定,对紫外线辐照和电场极化不敏感.矽肺胶原缺乏活泼性较高的稳定自由基.     

胶原蛋白的电子活性与矽肺纤维化Ⅱ正常大鼠与实验性矽肺大鼠肺Ⅰ型胶原稳定自由基的研究

由正常大鼠肺撮取的酸溶性Ⅰ型胶原出现一个明显的ESR信号(g=2.006),而由矽肺大鼠提取的酸溶性Ⅰ型胶原较少或缺乏该ESR信号,该信号不是顺磁过渡元素产生,而是稳定自由基的反映.肺胶原可能存在两种稳定的自由基,一种易受紫外线辐照的激发并且在电场极化下自旋耦合而减弱,另一种自由基则相对稳定,对紫外线辐照和电场极化不敏感.矽肺胶原缺乏活泼性较高的稳定自由基.     

Free radical signal of bile pigment in paraffin embedded liver tissue

A linear correlation have been found between the amplitude of the free radical signal of the electron spin resonance (ESR) spectrum of paraffin embedded liver blocks and the number of bile casts in the histological sections made from these blocks. It has been suggested that the major part of the ESR free radical signal arises from bile pigment, but the contribution of "age pigment" cannot be excluded. On the basis of model experiments the majority of these radical centers was assigned to protein bound bilirubin. In the course of histological processing more than 80% of various free radical centers arising in air dried liver tissue is extracted.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

Radical Intermediates and Antioxidants: An Esr Study of Radicals Formed on Carnosic Acid in the Presence of Oxidized Lipids

Carnosic acid, an antioxidant extracted from rosemary, is shown to produce radicals when in contact with oxidized methyl oleate in the absence of air above 50°C. Two radical species are formed: the first one, stable up to -110°C, is an hydroxy-phenoxy radical whose ESR spectrum was analyzed by studying its temperature dependence and its sensitivity to deuterium/proton exchange. The second species was observed above 110°C, its ESR spectrum was identical to the spectrum obtained when carnosol, another antioxidant extracted from rosemary, was heated at the same temperature in the presence of oxidized lipid. This observation is probably due to the transformation of carnosic acid into carnosol; the analysis of the corresponding ESR spectrum suggests the formation of a keto phenoxy radical exhibiting a great delocalization of the unpaired electron.     

Spin label studies of sulfhydryl environment in plasma fibronectin

The local environment of the free sulfhydryl groups in plasma fibronectin has been investigated by ESR techniques using a series of maleimide spin labels, varying in chain length between the maleimide and nitroxide free radical groups. Chemical modification with these analogs does not affect either the CD spectra or the cell adhesion activity of the protein molecule. The ESR results show that the free sulfhydryl group of plasma fibronectin is in a cleft about 10.5 A in length. The significance of this finding is discussed.     

Psoralen-photosensitized formation of free lipid radicals at 77 degrees K

It is shown by ESR method that 8-methoxypsoralen (8-MOP) under low temperature UV-irradiation has no influence on free radical formation of saturated fatty acids (FA). Under the same conditions the quantum yield of free radical photogeneration for unsaturated FA (oleate and limolenate) after addition of 8-MOP increased 7.5-15 times. The ESR spectrum of free radicals generated after interaction of 8-MOP with polyunsaturated FA, may be presented as superposition of signals-CH-(CH = CH)2-, -CH2CHCH2- and -CH2CH2. The molecular mechanism of photosensitized reactions is discussed.