E.s.r. of spin-trapped radicals in aqueous solutions of amino acids. Reactions of the hydroxyl radical.

The radicals produced by reactions of hydroxyl radicals with amino acids in aqueous solutions have been investigated. Hydroxyl radicals were formed by U.V.-photolysis of hydrogen peroxide and the short-lived amino acid radicals were spin-trapped by tert-nitrosobutane and identified by electron spin resonance spectroscopy. Nineteen amino acids were studied, and several radicals were identified which have not been observed previously by other methods. Only side-chain radicals were identified for alanine, threonine, aspartic acid, asparagine, lysine, phenylalanine, tyrosine, proline and hydroxyproline; whereas for glycine the C(2) carbon radical was spin-trapped. Both C(2) carbon radicals and side-chain radicals were assigned to valine, leucine, isoleucine, serine, glutamic acid, glutamine, arginine and methionine.     

E.s.r. of spin-trapped radicals in aqueous solutions of amino acids. Reactions of the hydrated electron.

The reactions of hydrated electrons (eaq-) with amino acids were investigated by the spin-trapping method and by electron spin resonance. Tertiary nitrosobutane was used as a spin-trap to stabilize the short-lived radicals. Hydrated electrons were produced by gamma-radiolysis of de-aerated aqueous solutions of amino acids in the presence of sodium formate or tertiary butanol to scavenge OH. Radicals produced by reductive deamination of 19 amino acids were identified. Radicals formed by scission of the CH3-S- and -S-CH2- bonds of methionine as well as by deamination were observed. In the case of phenylalanine the radical formed by electron addition followed by proton transfer was identified. The reaction of proline and of hydroxyproline with eaq- resulted in the opening of the cyclic structure.     

E.s.r. of spin-trapped radicals in gamma-irradiated aqueous solutions of nucleic acids and their constituents.

The gamma-radiolysis of de-aerated neutral aqueous solutions of uracil, thymine, cytosine and of the corresponding nucleosides and nucleotides and of calf-thymus DNA was investigated. For uracil and thymine, the U.V. photolysis of aqueous solutions containing H2O2 was also studied. The short-lived radicals were spin-trapped by tert-nitrosobutane and identified by electron-spin-resonance spectroscopy. For all compounds two or more radicals were observed, and these could be distinguished by following the thermal decay of the spin adducts. Radicals formed by the addition of H or OH at the C(5) or C(6) positions of the pyrimidine derivatives were observed in all cases. Sodium formate was used as a scavenger for H and OH to identify the radicals formed by eaq-. Spin-trapped radicals in gamma-irradiated aqueous solutions of polynucleotides exhibited broad e.s.r. lines. For DNA gel, additional narrow lines due to scission products were also found.     

E.s.r. study of the post-radiolysis growth of spin-trapped radicals in gamma-irradiated aqueous solutions of thymine.

The post-irradiation growth of the spin-adduct nitroxide radical produced by the addition of the thymine--OD radical to t-nitrosobutane (tNB) in gamma-irradiated, de-aerated D2O solutions was investigated by e.s.r. The thymine--OD radical was formed by the addition of an OD radical to the C(5) position of thymine. Growth reached a greater maximum value and was more rapid with increasing dose. At a fixed dose, growth was also greater and more rapid if oxygen was present after gamma-radiolysis. The addition of a second radical to the spin-adduct nitroxide during radiolysis to give a diamagnetic intermediate, which can regenerate the spin-adduct radical during storage in air-free and in air-saturated solutions at room temperature, was inferred to be responsible for post-irradiation growth. U.V. photolysis at 260-280 nm of a solution containing the diamagnetic intermediate rapidly regenerates the spin-adduct nitroxide. The longer lifetime of the diamagnetic intermediate in oxygen-free solutions may be relevant to an understanding of the anoxic sensitization by nitroxides in cellular systems.     

E.s.r. of spin-trapped radicals in gamma-irradiated polycrystalline DL-alanine. A quantitative determination of radical yield

The quantitative aspects of determining free radicals in polycrystalline amino acids gamma-irradiated at room temperature and subsequently dissolved in spin-trap solutions were investigated. The deamination radical in DL-alanine was used for detailed studies and 2-methyl-2-nitrosopropane (MNP) was employed as the spin-trap. The spin-trapping efficiency (the number of radicals spin-trapped in solution divided by the number of radicals initially present in the gamma-irradiated solid) was found to be in the range 1 to 10 per cent for aqueous solutions depending on the experimental conditions. The effects of dose, particle size, pH, spin-trap concentration, age of spin-trap solution, MNP monomer to dimer ratio and the presence of organic solvents were investigated. Several reactions were found to decrease the spin-trapping efficiency; radical-radical recombination, the competition between the spin-adduct and the spin-trap for radicals and the reaction of radicals with the MNP dimer. The reaction of intact DL-alanine molecules with deamination radicals to produce H-abstraction radicals which are not spin-trapped does not significantly lower the spin-trapping efficiency. The results obtained with compounds such as glycine, glycylglycine, L-valine and L-proline suggest that the low spin-trapping efficiency found for DL-alanine may be representative of polycrystalline amino acids.     

E.s.r. study of spin-trapped radicals formed during the photolysis of aqueous solutions of acid amides and H2O2.

Free radicals formed by the reactions of OH radicals with amides and their N-methylated derivatives in aqueous solutions have been studied. The OH radicals were produced by U.V.-photolysis of H2O2, and the short-lived amide radicals were converted to more stable nitroxide radicals by addition to a spin-trap, tert-nitrosobutane. The spin-trapped radicals were identified by e.s.r. spectroscopy. For acetamide, chloroacetamide, malonamide, succinamide and propionamide, the observed radicals were formed by H-abstraction from the carbon atoms attached to the carbonyl group. The H atom attached to the carbonyl group was abstracted in formamide. For N-methyl acetamide, N,N-dimethyl acetamide and the corresponding formamide derivatives, H-abstraction occurred only from the N-methyl group. The non-equivalency of the amide protons was observed in the spin-trapped radicals for acetamide, formamide, malonamide, succinamide and propionamide. The identification of the site of OH attack on N-methyl amides is helpful for the study of radical formation in peptides and proteins.     

E.s.r. of free radicals in aqueous solutions of substituted pyrimidines.

Reactions of OH radicals with methyl and ethyl derivatives of uracil, cytosine and thymine in aqueous solutions have been investigated. Photolysis of H2O2 was used to generate OH radicals and the radicals on the base derivatives were spin-trapped using t-nitrosobutane and identified with the help of e.s.r. spectroscopy. Addition of OH radicals was found to take place predominantly to the C(5)--C(6) double bond of the bases. H-abstraction from the methyl group occurred in the N(1) methyl derivatives of uracil, cytosine and thymine. Radicals formed by H-abstraction from the methyl group were also detected for 3-methyluracil, thymine, 1-methylthymine and 1-ethylthymine. Introduction of a methyl or ethyl group at the N(1) position of uracil, cytosine and thymine causes an increase in the C(6) proton coupling and a decrease in the N(1) splitting for radicals formed by OH addition at the C(5) position.     

E.s.r. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids, N-acetyl amino acids and dipeptides

The radicals produced in several polycrystalline amino acids, N-acetyl amino acids and dipeptides by gamma-radiolysis at room temperature were investigated by spin-trapping. After irradiation in the solid state, the samples were dissolved in aqueous solutions f t-nitrosobutane and the trapped radicals identified by e.s.r. For alpha-amino acids, deamination radicals were found, and in some cases H-abstraction radicals were also observed. No decarboxylation radicals could be detected. For N-acetyl amino acids, except for N-acetylglycine, the major radical was the decarboxylation radical. For N-acetyglycine the H-abstraction radical from the glycine residue was observed. For dipeptides of the x-glycine, the radical formed by removal of H from the alpha-carbon of the carboxyl-terminal residue was always spin-trapped. Some primary deamination radicals and minor amounts of decarboxylation radicals could also be observed. For dipeptides of the type x-alanine, glycine-x and alanine-x, the decarboxylation radical was always the major spin-trapped radical. Some primary and secondary deamination radicals were also detected.     

E.S.R. of spin-trapped radicals in aqueous solutions of 5-halo derivatives of nucleic acid constituents: reactions of hydrated electrons, hydroxyl radicals and U.V. photolysis

In order to obtain information concerning the mechanism of radio- and photosensitization due to 5-halogen substituted nucleic acid constituents, the free radicals produced in iodo-, bromo-, chloro- and fluoro-derivatives of uracil, uridine and deoxyuridine by reaction with hydrated electrons and with hydroxyl radicals and by direct U.V. photolysis have been studied by e.s.r. and spin-trapping. t-Nitrosobutane was used as the spin-trap. From 5-halogenated bases (except 5-fluorouracil) U.V. photolysis and reactions with hydrated electrons produced the uracilyl radical which was subsequently spin-trapped. When hydroxyl radical reactions were studied, the free radical at the N(1) position of the base was identified. From 5-fluorouracil U.V. photolysis generated the alpha-halo radical at the C(5) position of the base. For 5-halogenated ribonucleosides and deoxyribonucleosides, free radicals located on the sugar moiety were observed for reactions with hydrated electrons, hydroxyl radicals and for U.V. photolysis. The implications of these results for understanding the mechanism of radio- and photosensitization by 5-halogenated nucleic acids are discussed.     

E.s.r. of free radicals in irradiated uracil-beta-D-arabinofuranoside.

Electron-spin-resonance measurements have been made on single crystals of uracil-beta-D-arabinofuranoside, which were irradiated by 4-0 MeV electrons at 77 K. At low temperatures, two radicals have been identified, one attributed to a hydrogen abstraction from 05' in the sugar moiety and the other to a radical anion located on the pyrimidine ring. The former is very unstable and seems to act as a precursor to other unidentified radical species stable at 77K. At room temperature, the main resonance is due to hydrogen addition to C5 and is probably produced by protonation of the anion. This same radical is also produced by X-irradiation at room temperature.     

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids. Chromatographic separation of radicals

The free radicals produced by gamma-radiolysis of polycrystalline amino acids (L-valine, L-leucine, L-isoleucine and L-proline) at room temperature in the absence of air were investigated by spin trapping with 2-methyl-2-nitrosopropane (MNP). The spin adducts produced by dissolving the irradiated solids in aqueous MNP solutions were separated by high-performance liquid chromatography and then identified by e.s.r. Deamination (ring-opening reaction for L-proline) was observed for all amino acid. For L-valine and L-leucine, H-abstraction from the tertiary carbon in the side chains occurred. For isoleucine, H-abstractions from the alpha-carbon of the amino acid and from a non-terminal carbon in the side chain were found.     

OH radical formation by photolysis of aqueous porphyrin solutions. A spin trapping and e.s.r. study

Radical production during the photolysis of deaerated aqueous alkaline solutions (pH 11) of some water-soluble porphyrins was investigated. Metal-free and metallo complexes of tetrakis (4-N-methylpyridyl)porphyrin (TMPyP) and tetra (4-sulphonatophenyl)porphyrin (TPPS4) were studied. Evidence for the formation of OH radicals during photolysis at 615, 545, 435, 408 and 335 nm of Fe(III) TPPS4 is presented. Fe(III) TMPyP, Mn(III) TPPS4 and Mn(III) TMPyP also gave OH radicals but only during photolysis at 335 nm. The method of spin trapping with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 4-pyridyl-1-oxide-N-tert-butylnitrone (POBN) combined with e.s.r. was used for the detection of OH, H and hydrated electrons. With the spin trap DMPO, photolysis generated DMPO-OH adducts under certain conditions but no DMPO-H adducts could be observed. With POBN, no POBN-H adducts were found. The formation of OH was confirmed by studying competition reactions for OH between the spin traps and OH scavengers (formate, isopropanol) and the concomitant formation of the CO-2 adduct and the (CH3)2COH adduct with both DMPO and POBN. The photochemical generation of OH radicals was pH dependent; at pH 7.5 no OH radicals could be detected. Photolysis (615-335 nm) of dicyanocomplexes of the Fe(III) porphyrins did not produce OH radicals. When corresponding Cu(II), Ni(II), Zn(II) and metal-free porphyrins were photolysed at 615 and 335 nm, no OH radicals could be spin trapped. These results tend to associate the well-known phenomenon of photoreduction of Fe(III) and Mn(III) porphyrins with the formation of OH radicals. This process is described mainly as the photoreduction of the metal ion by the ligand-bound hydroxyl ion via an intramolecular process.     

The oxidation of some polysaccharides by the hydroxyl radical: an e.s.r. investigation

E.s.r. Experiments employing a flow system in conjunction with the TiIII-H2O2 couple show that dextrans react with the hydroxyl radical (HO.) via indiscriminate attack (except that abstraction of hydrogen atoms from carbons which are both linked by glycosidic bonds and included in the pyranose ring may be inhibited, possibly for steric reasons). Acid- and base-catalysed transformations of first-formed radicals have been demonstrated; the suggestion that such reactions can lead to glycosidic cleavage is supported by viscosity studies which confirm the pH-dependence of radical-initiated degradation. For galacturonan and related compounds, e.s.r. results indicate that reaction with HO. proceeds preferentially via abstraction of the hydrogen on the carbon adjacent to the carboxyl group. The crucial step in the subsequent degradation pathway probably involves a pH-independent rearrangement.     

Free radicals in U.V.-irradiated aqueous solutions of substituted amides: an e.s.r. and spin-trapping study.

The radicals produced by reactions of hydroxyl radicals with alkyl substituted ureas and amides in aqueous solutions have been investigated. Hydroxyl radicals were produced by U.V. photolysis of H2O2 and the short-lived amide and urea radicals were spin-trapped by t-nitrosobutane and identified by e.s.r. For all N-alkyl derivatives of urea and acetamide, and for N,N-dimethyl propionamide and N,N-diethyl formamide, only radicals centred on N-alkyl groups were detected. Radicals situated only on alkyl groups attached to the carbonyl carbon were observed for dimethyl acetamide, trimethyl acetamide and butyramide. However, for N,N-dimethyl butyramide, N, N-diethyl butyramide, N-methyl propionamide and N, N-diethyl propionamide, free radicals were formed which were localized on the alkyl group attached to the amide carbon as well as those attached to nitrogen. The hydrogen atom bound to the carbonyl carbon was abstracted in N-ethyl formamide. Acyl radicals formed by C-N scission due to direct U.V. photolysis of N, N-dimethyl butyramide and N,N-dimethyl propionamide were also detected.