CONCERNING THE PRODUCTION OF FREE RADICALS IN PROTEINS BY ULTRAVIOLET LIGHT

Abstract— Using electron paramagnetic resonance techniques, the response to u.v. light of several solid proteins and model compounds has been studied in vacuum and at low temperature. Emphasis has been placed on determining the response as a function of the wavelength (Λ 250 nm) and intensity of the incident radiation. Correlation of the parameters of radical production with sample luminescence, molecular amino-acid sequence and tertiary structure, light intensity and total irradiation time has allowed some insight into the mechanisms of free radical formation.
It is shown that the details of amino acid composition, sequence and the tertiary structure of a protein are important in determining both the rate of, and the mechanism for, radical production (two basic mechanisms are described), and in determining the conditions under which sulfur-type radicals can be produced. The results are related to enzyme inactivation and to the u.v. stability of proteins generally.     

DISTRIBUTIONS OF FREE RADICALS AMONG AMINO ACIDS FROM LYOPHILIZED ULTRAVIOLET-IRRADIATED PROTEINS

Abstract— The effects of u.v.-irradiation at 254 nm upon lyophilized ribonuclease, lysozyme, insulin, and chymotrypsinogen have been investigated by electron spin resonance (ESR). enzymatic assay, and labeling of free radical sites with tritiated hydrogen sulfide (HST). The ESR signal of the irradiated protein diminishes on exposure to HST, and tritium becomes covalently bound to carbon. The distribution of tritium among the amino acids of each protein. studied as an indicator of the carbon free radical distribution, differs markedly from those observed previously to result from exposure to gamma radiation, electrical discharge. or hydrogen atoms. However, the earlier observation that the tritium distribution is influenced by protein conformation holds true as well for u.v.-irradiation. Moreover, the distributions of tritium among the amino acids of u.v.-irradiated proteins indicate a broad scattering of free radicals. Tyrosine and phenylalanine, residues that absorb light energy in the region of the wavelength employed, are not particularly important as radical carriers. Thus, for ribonuclease, these residues incorporated 3.8 and 1.5 per cent of the total tritium, but they absorb 51 and 12 per cent of the light, respectively. These results, together with the observed low recoveries of methionine, an amino acid that does not absorb at 254 nm, add weight to the concept that a migration of energy ensues after the initial absorption of light energy and that photolytic damage may thus be due to destruction of amino acids other than those initially absorbing the u.v.-radiation.     

Radical protection by sunscreens in the infrared spectral range

One essential reason for skin ageing is the formation of free radicals by excessive or unprotected sun exposure. Recently, free radical generation in skin has been shown to appear not only after irradiation in the UV wavelength range but also in the infrared (IR) spectral range. Sunscreens are known to protect against radicals generated by UV radiation; however, no data exist for those generated by IR radiation. This paper has investigated four different, commercially available sunscreens and one COLIPA standard with regard to radical formation in the skin after IR irradiation, using electron paramagnetic resonance spectroscopy. The use of sunscreens has led to reduced amounts of radicals compared to untreated skin. Furthermore, absorption and scattering properties and the radical protection factor of the formulations were determined to investigate their influence on the radical protection of the skin. None of these formulations contained an optical absorber in the IR range. The protection efficiency of the sunscreens was shown as being induced by the high scattering properties of the sunscreens, as well as the antioxidants contained in the formulations.     

The role of hydroperoxides in photo-oxidative degradation of cis-1,4-polybutadiene

Hydroperoxides undergo various types of homolytic reactions on exposure to u.v. radiation. Free radicals formed from the photodecomposition of the hydroperoxide group (OOH) are oxy (HO.) and peroxy (HOO.) radicals which participate in further reactions. In cis-1,4-polybutadiene, they may initiate free radical oxidations. Cleavage of alkoxy (RO.) radicals and crosslinking of polymer radicals through polymer peroxides in the presence of air in solid film nearly balance. Most polymer radicals produced in the absence of oxygen undergo cross-linking but form peroxy radicals (POO.) in its presence. This paper presents results on the photodecomposition of tert-butyl hydroperoxide, cumyl hydroperoxide and 2,5-dimethyl-2,5-dihydroperoxyhexane in cis-1,4-polybutadiene in film and in solution.     

Electron donor-acceptor complexes and polymerization—V copolymerization of maleic anhydride and trans-stilbene under ultra-violet irradiation

Trans-Stilbene and maleic anhydride in THF or acetone solution undergo copolymerization upon u.v. irradiation to give soluble copolymers having an alternating structure. Even at ?78°, no cycloadducts are formed. Several photosensitizers had no effect on the rate of propagation, with the exception of benzil which generates free radicals upon irradiation. The copolymers obtained in the presence of benzil are crosslinked.     

Effect of ascorbic acid (vitamin C) on the ESR spectra of the red and black hair: pheomelanin free radicals are not always present in red hair

Increased incidence of melanoma in the population with red hair is conditioned by synthesis of pheomelanin pigments in the skin and their phototoxic properties. The recent research has shown that free radicals of pheomelanin are produced not only by the influence of UV irradiation, but also in UV‐independent pathways of oxidative stress. It has been ascertained, that the color of the hair is not always determinant of the amount of pheolemanin radicals in red hair. Therefore, in order to evaluate the risk of melanoma in different individuals, it is necessary to define the amount of free radicals of pheomelanin in red hair using ESR spectroscopy method. Besides, it is very important to find effective antioxidant, capable of neutralizing free radicals of pheomelanin. It was proved that ascorbic acid neutralizes free radicals of pheomelanin very effectively. The main goal of our research was to define the presumably optimal concentration of ascorbic acid as an antioxidant and study the kinetics of the influence of this concentration on red and black hair. It has been found out, that ascorbic acid influences the free radicals of red and black hair, and its appropriate optimal concentration is 10 mM. The obtained results can be considered in dermatology and cosmetology. Copyright © 2015 John Wiley & Sons, Ltd.     

DECAY KINETICS OF FREE RADICALS IN IRRADIATED PROTEINS

Abstract— A detailed analysis is presented of the decay kinetics of free radicals in irradiated biopolymers. Available data in the literature are critically evaluated and new data for irradiated keratin are presented. It is shown that a rigorous analysis of the data available in the literature leads to more detailed information on the decay mechanisms than the conclusions which have been previously given; in some cases the analysis leads to mechanisms contradicting those claimed in the literature.
Our results suggest that the free radicals in illuminated keratin consist of three distinct species: one of the species decays with a half life of approximately 20 hr, the second has a half life of about 4600 hr, while the half life of the third is so long that the free radical concentration is practically constant. A similar behaviour is obtained on assumption of second order decay.
The consistency of the kinetic constants obtained has been verified in kinetic experiments using samples prepared under very different experimental conditions.     

PHOTOCHEMISTRY OF 2-MERCAPTOPYRIDINES. PART 3. EPR STUDY OF PHOTOPRODUCTION OF HYDROXYL RADICALS BY N-HYDROXYPYRIDINE-2-THIONE USING 5,5-DIMETHYL-1-PYRROLINE N-OXIDE IN AQUEOUS SOLUTIONS*

Abstract— N-Hydroxypyridine-2-thione, 2-S-PyrNOH, a potent antimicrobial, antifungal and anticancer agent, is photochemically active and upon UV irradiation generates free radicals. We have employed EPR and the spin-trap 5,5-dimethyl-l-pyrroline TV-oxide (DMPO) to investigate the photochemistry in aqueous solutions of 2-S-PyrNOH (used here in the form of a sodium salt, 2-S-PyrNONa). We found that upon photoactivation 2-S-PyrNONa can follow two different pathways: it can produce hydroxyl radicals and/or it can act as a photoreducing agent. The capacity of 2-S-PyrNONa to produce “OH” radicals has been demonstrated by: (1) EPR detection of the DMPO/OH adduct in UV-irradiated samples; (2) inhibition of the DMPO/OH formation by OH scavengers such as methyl alcohol, formate and DMSO and (3) by detection of EPR signals of DMPO adducts with radicals derived from reaction of OH with these inhibitors. The photoreductive capacity of 2-S-PyrNONa was deduced from the observation that the amplitude of the EPR signal of the spin adduct DMPO/OH decreased on UV irradiation in air-free pH 7.0 buffers and that the signal recovered in the dark and after aeration. The ability to generate free radicals upon UV irradiation suggests that 2-S-PyrNONa can be regarded as a potential photocytotoxic agent. This feature may be relevant to the biological action of this compound. Our findings also emphasize that caution should be used when 2-S-PyrNOH is employed as a source of OH radicals in biological or chemical systems.     

EPR STUDY OF WOOL IRRADIATED WITH BLUE LIGHT

Abstract— Wool fibres were exposed to visible light in the blue range. The free radicals created by the illumination were studied using electron paramagnetic resonance. It is shown that by control of the light intensity and frequency it is possible to get considerable insight into the mechanism of the creation and the decay of the free radicals. The radical population under the above conditions appears to consist of an initial stable radical insensitive to radiation, and three other radicals, two of which follow decay kinetics considerably different from that of the remaining free radical. All the latter three radicals are probably caused by N–H or C–H bond scission.
The kinetics of radicals in irradiated proteins is discussed. It is shown that the mechanisms previously suggested in the literature are inadequate to account for the form of the experimental curve describing the number of radicals vs. time in irradiated protein. A new mechanism is suggested in which the decayed radicals form sites immune to further irradiation. Such a mechanism is shown to be consistent with the experimental curves. The kinetics of the decay of radicals after the cessation of illumination is examined in detail. It is found that in the case of irradiated keratin the mechanisms assuming first order decay are inconsistent with the experimental results, while the assumption of second order decay for two different species of radicals yields results consistent with the experimental evidence.     

Wavelength dependence of ultraviolet-induced DNA damage distribution: involvement of direct or indirect mechanisms and possible artefacts.

DNA damage profiles have been established in plasmid DNA using purified DNA repair enzymes and a plasmid relaxation assay, following exposure to UVC, UVB, UVA or simulated sunlight (SSL). Cyclobutane pyrimidine dimers (CPDs) are revealed as T4 endonuclease V-sensitive sites, oxidation products at purine and pyrimidine as Fpg- and Nth-sensitive sites, and abasic sites are detected by Nfo protein from Escherichia coli. CPDs are readily detected after UVA exposure, though produced 10(3) and 10(5) times less efficiently than by UVB or UVC, respectively. We demonstrate that CPDs are induced by UVA radiation and not by contaminating UVB wavelengths. Furthermore, they are produced at doses compatible with human exposure and are likely to contribute to the mutagenic specificity of UVA [E. Sage et al., Proc. Natl. Acad. Sci. USA 93 (1996) 176-180]. Oxidative damage is induced with a linear dose dependence, for each region of the solar spectrum, with the exception of oxidized pyrimidine and abasic sites, which are not detectable after UVB irradiation. The distribution of the different classes of photolesions varies markedly, depending on wavelengths. However, the unexpectedly high yield of oxidative lesions, as compared to CPDs, by UVA and SSL led us to investigate their production mechanism. An artificial formation of hydroxyl radicals is observed, which depends on the material of the sample holder used for UVA irradiation and is specific for long UV wavelengths. Our study sheds light on a possible artefact in the production of oxidative damage by UVA radiation. Meanwhile, after eliminating some potential sources of the artefact ratios of CPDs to oxidized purine of three and five upon irradiation with UVA and SSL, respectively, are still observed, whereas these ratios are about 140 and 200 after UVC and UVB irradiation.     

Gamma radiation effects on an amine antioxidant added in an ethylene-propylene copolymer

The aim of this work was to compare the gamma radiation induced effects on samples of an ethylene-propylene copolymer antioxidant free with samples loaded with an antioxidant characterised by the presence of an -NH functional group. The employed techniques were Electron Spin Resonance spectroscopy (ESR) and High Performance Liquid Chromatography (HPLC). Stable radicals R---NO° due to the interaction of free radicals produced in the irradiated polymer with the antioxidant have been observed by ESR at room temperature. The time evolution of the ESR signals following the irradiation was examined at different doses. The amount of antioxidant not involved in the oxidation reactions has been determined using HPLC.     

ESR study of the initial reaction of the photodegradation of nylon 6

The free radicals formed on irradiation of nylon 6 have been studied by means of electron spin resonance (ESR). The irradiation was performed at liquid nitrogen temperature with wavelengths in the region of solar radiation. The spectrum so obtained could best be fitted by assuming ? CH₂? ?H? CH₂? and ? CH₂? ??O to be the trapped radicals. The assignment of the spectrum supports the idea that the first step in photodegradation is the breaking of the amide bond. The splitting constant of the α proton of the alkyl radical has been found to be nearly the same for drawn and undrawn yarn. This strongly suggests that the low-energy radiation is capable of breaking bonds only in the unoriented amorphous regions.     

An EPR and Optical Spectroscopy Study of the Effect of Laser Radiation on Biological Tissues

The formation of radicals upon UV (248 nm) and IR (1.56 m) laser irradiation of some biological tissues (cartilage, bone tissue, fish scale) and their components (chondroitin sulfate, collagen) was studied by the EPR technique. The radical decay kinetics in bony tissue specimens after their irradiation with UV light were described. By the spin trapping technique, it was shown that radicals were not produced during IR (1.56 m) laser irradiation of cartilaginous tissue. A change in optical absorption spectra and the dynamics of optical density of cartilaginous tissue, fish scale, and a collagen film under exposure to laser radiation (248 nm) in an air, oxygen, and nitrogen atmosphere was studied.     

FLASH PHOTOLYSIS OF SEVERAL AROMATICS BY ULTRAVIOLET LIGHT AND VISIBLE LIGHT IN THE PRESENCE OF EOSIN Y*

Abstract— A flash photolysis investigation was made of the photo-oxidation of aqueous aniline, resorcinol, βnaphthol, p-sulfanilic acid, and p-bromophenol induced by ultraviolet and visible light irradiation in the presence of eosin Y. The transient spectra show that u.v. irradiation generates the hydrated electron (except in p-bromophenol) and the radical products of one-electron oxidation. The initial products of the eosin-sensitized oxidations are the dye semi-quinone and aromatic radicals which coincide with the u.v. photolysis products in at least several cases. The investigation of the reaction kinetics by rapid spectrophotometry with analog computer analysis shows that the aromatics quench the triplet state of eosin and also react with it in a slower electron-transfer process, in competition with ‘dye-dye’ quenching and electron-transfer reactions. The u.v. and dye-sensitized oxidations are discussed in terms of their energetics.     

Electrochemical behaviour of organolead compounds: Part I. Triphenyl-lead acetate

The electrochemical behaviour of triphenyl-lead acetate in 50% (v/v) ethanol has been investigated using various electrochemical techniques including polarography, cyclic voltammetry and controlled potential coulometry. It has been found to give three/four polarographic waves. The first normal wave involves an one-electron irreversible reduction of triphenyl-lead ions giving triphenyl-lead free radicals which are strongly adsorbed at the DME giving rise to an adsorption prewave. The triphenyl-lead free radicals produced in the normal reduction step immediately react with mercury of the DME producing phenylmercury radicals and metallic lead. Wave II has been ascribed to the simultaneous reduction of triphenyl-lead free radicals and phenylmercury free radicals. The metallic lead produced in these processes undergoes oxidation at the electrode and “distorts” the “normal” adsorption prewave of step I. A mechanism of reduction is proposed and polarographic methods have been developed for determining triphenyl-lead compounds at ordinary level and down to submicromolar level.     

The influence of UV irradiation on the properties of chitosan films containing keratin

The mechanical, thermal and surface properties of chitosan and chitosan containing keratin hydrolysates have been studied and the influence of UV irradiation on these properties has been compared. The surface properties of chitosan films containing 5%, 15% and 30% of keratin hydrolysate before and after UV irradiation (λ = 254 nm) were investigated by means of contact angle measurements allowing the calculation of surface free energy. The chemical and structural changes during UV irradiation were studied by UV-vis and FTIR-ATR spectroscopy.The changes in mechanical properties such as breaking strength, percentage elongation and Young’s modulus have been investigated. The results have shown that the mechanical properties of the chitosan/keratin films were greatly affected by UV irradiation, but the level of the changes of these properties was smaller in the blend than in pure chitosan and strongly dependent on the time of irradiation and composition of the samples. The contact angle and the surface free energy were altered by UV irradiation, which indicates photooxidation and an increase of polarity of specimens. The range of these changes point to greater susceptibility of chitosan to photooxidation in the presence of keratin.     

Comparison of photo- and radiation-induced radical formation in polyethylene by ESR spectroscopy

The behaviors of free radicals produced in polyethylene irradiated with ultraviolet light and electron beams were compared in connection with primary processes of radical formation and trapping regions of free radicals. In the case of irradiation with ultraviolet light, an ESR spectrum observed at ?196°C immediately after irradiation is an eight-line spectrum due to alkyl radicals of the type ? CH₂? ?H? CH₃, while in the case of ionizing radiation, a six-line spectrum due to ? CH₂? ?H? CH₂? was observed. The former radicals are produced by the Norrish type I reaction of the carbonyl groups contained in the polymer, followed by radical rearrangement; and the latter are formed by dissociation of hydrogen atom from the excited state of the polymer or ion-molecular reactions. From the sensitivity to oxygen molecules, it was deduced that free radicals are trapped in amorphous regions after ultraviolet irradiation, but mainly in crystalline regions after irradiation with electron beams. Saturation studies of ESR spectra seem to support this conclusion.     

U.V. INDUCED CONFORMATIONAL CHANGES IN TRANSFER RNA

Abstract— The quantum yields for the u.v. inactivation of the amino acid acceptor function of E. coli transfer RNA (for val, phe and lys) and for the loss of its conformation, as a function of exposure, have been determined following irradiation at 280, 265 and 254 nm. Our results suggest that u.v. damage produces a change in the conformation of transfer RNA which in turn inactivates it, and that the anticodon is not the u.v. sensitive site. Calculations indicate that a small number of photoproducts inactivate the transfer RNA.     

FREE RADICALS PRODUCED IN A RIGID SOLUTION OF TRYPTOPHAN BY A BIPHOTONIC PROCESS

Abstract— Free radicals were trapped by irradiation of tryptophan in an ethylene glycol/water glass with 250 nm U.V. light at 77°K. The ESR-spectrum was attributed to approximately equal amounts of trapped electrons and ethyl radicals. The electrons could be transformed into the ethyl type radicals by heating or bleaching with near u.v. or visible light. The quantum yield of trapped radicals increased with increasing intensity of the irradiation by more than one order of magnitude over the range of intensities studied. A kinetic model was formulated on the assumption that the radicals were produced by the successive absorption of two light quanta, the absorption of the first giving rise to a transient intermediate which subsequently absorbs a second quantum to produce the radicals. The experiments confirmed the applicability of this model to the present case. The decay time obtained for the intermediate was 6.8 sec which equals the phosphorescence decay time of tryptophan under similar conditions, indicating that the intermediate involved was the lowest triplet level of this molecule. The absorption cross section of this intermediate at 250 nm with regard to the formation of trapped radicals was found to be 8000 l./mol cm.     

UV-generated free radicals (FR) in skin: their prevention by sunscreens and their induction by self-tanning agents

In the past few years, the cellular effects of ultraviolet (UV) irradiation induced in skin have become increasingly recognized. Indeed, it is now well known that UV irradiation induces structural and cellular changes in all the compartments of skin tissue. The generation of reactive oxygen species (ROS) is the first and immediate consequence of UV exposure and therefore the quantitative determination of free radical reactions in the skin during UV radiation is of primary importance for the understanding of dermatological photodamage. The RSF method (radical sun protection factor) herein presented, based on electron spin resonance spectroscopy (ESR), enables the measurement of free radical reactions in skin biopsies directly during UV radiation. The amount of free radicals varies with UV doses and can be standardized by varying UV irradiance or exposure time. The RSF method allows the determination of the protective effect of UV filters and sunscreens as well as the radical induction capacity of self-tanning agents as dihydroxyacetone (DHA). The reaction of the reducing sugars used in self-tanning products and amino acids in the skin layer (Maillard reaction) leads to the formation of Amadori products that generate free radicals during UV irradiation. Using the RSF method three different self-tanning agents were analyzed and it was found, that in DHA-treated skin more than 180% additional radicals were generated during sun exposure with respect to untreated skin. For this reason the exposure duration in the sun must be shortened when self-tanners are used and photoaging processes are accelerated.