Radical trapping properties of imidazolyl nitrones

The ability of ten imidazolyl nitrones to directly scavenge free radicals (R(*)) generated in polar ((*)OH, O(*)(2)(-), SO(*)(3)(-) cysteinyl, (*)CH(3)) or in apolar (CH(3)-(*)CH-CH(3)) media has been studied. When oxygen or sulfur-centered radicals are generated in polar media, EPR spectra are not or weakly observed with simple spectral features. Strong line intensities and more complicated spectra are observed with the isopropyl radical generated in an apolar medium. Intermediate results are obtained with (*)CH(3) generated in a polar medium. EPR demonstrates the ability of these nitrones to trap radicals to the nitrone C(alpha) atom (alpha radical adduct) and to the imidazol C(5) atom (5-radical adduct). Beside the nucleophilic addition of the radical to the C(alpha) atom, the EPR studies suggest a two-step mechanism for the overall reaction of R(*) attacking the imidazol core. The two steps seem to occur very fast with the (*)OH radical obtained in a polar medium and slower with the isopropyl radical prepared in benzene. In conclusion, imidazolyl nitrones present a high capacity to trap and stabilize carbon-centered radicals.     

Extending the coverage of spectral libraries: A neighbor‐based approach to predicting intensities of peptide fragmentation spectra

Searching spectral libraries in MS/MS is an important new approach to improving the quality of peptide and protein identification. The idea relies on the observation that ion intensities in an MS/MS spectrum of a given peptide are generally reproducible across experiments, and thus, matching between spectra from an experiment and the spectra of previously identified peptides stored in a spectral library can lead to better peptide identification compared to the traditional database search. However, the use of libraries is greatly limited by their coverage of peptide sequences: even for well‐studied organisms a large fraction of peptides have not been previously identified. To address this issue, we propose to expand spectral libraries by predicting the MS/MS spectra of peptides based on the spectra of peptides with similar sequences. We first demonstrate that the intensity patterns of dominant fragment ions between similar peptides tend to be similar. In accordance with this observation, we develop a neighbor‐based approach that first selects peptides that are likely to have spectra similar to the target peptide and then combines their spectra using a weighted K‐nearest neighbor method to accurately predict fragment ion intensities corresponding to the target peptide. This approach has the potential to predict spectra for every peptide in the proteome. When rigorous quality criteria are applied, we estimate that the method increases the coverage of spectral libraries available from the National Institute of Standards and Technology by 20–60%, although the values vary with peptide length and charge state. We find that the overall best search performance is achieved when spectral libraries are supplemented by the high quality predicted spectra.     

Controlling plasma composition during carbon film deposition in microwave discharges by means of optical-emission spectroscopy

Optical emission spectra from the microwave discharge plasma that is used to activate gas-phase deposition of carbon films are systematically investigated under various deposition conditions. The line emission intensities from CH and C₂ radicals, which are responsible for the growth of the diamond and graphite phases, respectively, are studied as functions of the main macroparameters of the process. To find the relation between the features of the emission spectra and the composition of the films obtained, the films were examined using Raman spectroscopy and electron microscopy. It is shown that monitoring the relative intensities of the spectral lines can be used to obtain the desired type of film, in which case the state of the substrate surface and the presence of a catalyst on it also play an important role. Experiments on the deposition of carbon films in the pulsed regime of plasma excitation show the possibility of changing the phase composition of the film by varying both the pulse repetition rate and the off-duty factor. At the same average microwave power, the rate of film deposition in the pulsed regime of plasma excitation is lower than that in a continuous discharge; however, the growth rate of the graphite phase decreases insignificantly.     

Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects.     

Sugar radicals in DNA: isolation of neutral radicals in gamma-irradiated DNA by hole and electron scavenging

In this investigation of the radical formation and the reaction of radicals in gamma-irradiated DNA, we report the isolation of putative neutral radicals by the scavenging of holes by Fe(CN)6(4-) and of electrons by Fe(CN)6(3-). Experiments are performed under conditions that emphasize direct and quasi-direct effects (collectively called direct-type effects.) Samples containing Fe(CN)6(4-) show effective scavenging of holes and the ESR spectra obtained arise principally from DNA anion radicals and neutral radicals. On the other hand, for samples containing Fe(CN)6(3-), electron scavenging is highly efficient, and the resulting spectra arise principally from guanine cation radicals and neutral radicals. When both Fe(CN)6(4-) and Fe(CN)6(3-) are present, a near complete scavenging of cation radicals and anion radicals is observed at 77 K, and the ESR spectra that result originate predominantly with neutral radicals which are assigned predominantly to radicals on the sugar phosphate backbone. A notable finding is the presence of spectral components that indicate the formation, through the rupture of the C3'-O bond, of a neutral deoxyribose radical; a concurrent strand break must accompany formation of this radical. This radical was previously reported in argon-ion-irradiated DNA and now, for the first time, is reported in DNA irradiated with low-LET radiation.     

Spectrophotometric characteristics of the coccoid forms of actinomycetes]

IR spectra of thw whole cells of the coccoid forms (Mycococcus and Micrococcus) isolated from lithophilous lichen were compared with IR spectra of the collection cultures of Micrococcus and Arthrobacter. Generic spectral characteristics of Mycococcus and Micrococcus are presented. Spectral heterogeneity within the genus Arthrobacter complicates the diagnosis. The cultures of the Mycococcus genus were divided into three groups according to their spectral characteristics. Spectral scans of the studied coccoid forms differ from the scans of the mycelial actinomycetes, and their intensities within the range of 900-1200 cm-1 (lipids) decrease in the series Mycococcus, Arthrobacter, Micrococcus.     

A comparison of mercury arc lamp and laser illumination for flow cytometers.

Optical differences between a mercury arc lamp and a laser-illuminated flow cytometer are compared. The distributions of spectral intensities of the two light sources are shown in relation to the excitation characteristics of the fluorescent dyes acriflavine, chromomycin A3, mithramycin, ethidium bromide, Hoechst 33258, and 4,6-diamidino-2-phenylindole (DAPI). Fluorescence intensities of microspheres and Hoechst 33258-stained mouse sperm are compared in the two cytometers. The optical efficiencies are similar and depend on the match of the excitation characteristics of the stain with the emission spectra of the light source.     

Measurement analysis of two radials with a common‐origin point and its application

In spectral analysis, a chemical component is usually identified by its characteristic spectra, especially the peaks. If two components have overlapping spectral peaks, they are generally considered to be indiscriminate in current analytical chemistry textbooks and related literature. However, if the intensities of the overlapping major spectral peaks are additive, and have different rates of change with respect to variations in the concentration of the individual components, a simple method, named the ‘common‐origin ray’, for the simultaneous determination of two components can be established. Several case studies highlighting its applications are presented.     

Synthesis,structure, and EPR characterization of deuterated derivatives of Finland trityl radical

Substituted trityl radicals are important spin probes for functional electron paramagnetic resonance spectroscopy and imaging including oxygen and pH mapping in vivo. Here we report the synthetic procedure for large scale synthesis of deuterated Finland trityl radical with superior EPR spectral properties and higher sensitivity towards oxygen concentrations in solution. Additionally Finland trityl radicals substituted with linkers suitable for attaching peptide, or other synthetic precursors have been synthesized. The effect of deutero-substitution on EPR spectra of homologous derivatives has been evaluated. The compounds are potential candidates for targeted spin probes in EPR imaging.     

Analysis of multi-component fluorescence emission by phase-sensitive detection using one modulation frequency

We describe the use of phase-sensitive detection of fluorescence to resolve the lifetimes and fractional intensities from multi-component fluorescence samples, using data obtained at a single modulation frequency. Phase-sensitive spectra of the mixture are recorded at arbitrarily chosen detector phase angles. The steady-state spectrum of each component must be known. The phase-sensitive spectra are fitted, using a nonlinear least-squares algorithm, to obtain the lifetimes and fractional intensities of each fluorophore in the mixture. Simulations for two- and three-component mixtures are presented to illustrate how the resolution is affected by spectral overlap and lifetime separation. Experimentally, we resolved two- and three-component mixtures of protein-like fluorophores (N-acetyl-L-tyrosinamide, N-acetyl- L-tryptophanamide, indole and 2,3-dimethylindole) using data collected at 30 MHz. These fluorophores have closely spaced lifetimes of 1.5, 2.9, 4.5 and 4.3 ns, respectively, and display extensive spectral overlap. These results demonstrate that phase-sensitive spectra, recorded at only one modulation frequency with a standard phase fluorometer, can be used to resolve multi-component emissions.     

Absorption spectra of radicals of substrates for p-hydroxybenzoate hydroxylase following electrophilic attack of the .OH radical in the 3 position

The spectra of radicals formed upon the addition of .OH radicals to the 3 position of substrates for p-hydroxybenzoate hydroxylase (4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and 4-aminobenzoic acid) have been determined using pulse radiolysis combined with the results from high performance liquid chromatography measurements. The 3-hydroxy radical forms of the substrates absorb maximally in the 365-410 nm region with extinction coefficients in the range 3700-5250 M-1 cm-1. Upon combining these radical spectra with the known spectrum of enzyme-bound reduced flavin that is substituted in the C(4a) position of the isoalloxazine ring, spectra are found which closely resemble the species long thought to be formed concomitantly with the introduction of an oxygen atom into substrates. On the basis of these spectral results a new radical mechanism is proposed for the functioning of this class of flavoprotein hydroxylases.     

HMMatch: peptide identification by spectral matching of tandem mass spectra using hidden Markov models.

Peptide identification by tandem mass spectrometry is the dominant proteomics workflow for protein characterization in complex samples. The peptide fragmentation spectra generated by these workflows exhibit characteristic fragmentation patterns that can be used to identify the peptide. In other fields, where the compounds of interest do not have the convenient linear structure of peptides, fragmentation spectra are identified by comparing new spectra with libraries of identified spectra, an approach called spectral matching. In contrast to sequence-based tandem mass spectrometry search engines used for peptides, spectral matching can make use of the intensities of fragment peaks in library spectra to assess the quality of a match. We evaluate a hidden Markov model approach (HMMatch) to spectral matching, in which many examples of a peptide's fragmentation spectrum are summarized in a generative probabilistic model that captures the consensus and variation of each peak's intensity. We demonstrate that HMMatch has good specificity and superior sensitivity, compared to sequence database search engines such as X!Tandem. HMMatch achieves good results from relatively few training spectra, is fast to train, and can evaluate many spectra per second. A statistical significance model permits HMMatch scores to be compared with each other, and with other peptide identification tools, on a unified scale. HMMatch shows a similar degree of concordance with X!Tandem, Mascot, and NIST's MS Search, as they do with each other, suggesting that each tool can assign peptides to spectra that the others miss. Finally, we show that it is possible to extrapolate HMMatch models beyond a single peptide's training spectra to the spectra of related peptides, expanding the application of spectral matching techniques beyond the set of peptides previously observed.     

Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN.

The generation of free radicals in rat liver following the acute oral administration of ethanol was studied with the spin-trapping method, using a deuterated derivative of phenyl-N-tert-butylnitrone (PBN-d14) as the spin-trapping agent. After administration of ethanol and PBN-d14 to rats, organic extracts of the liver were prepared and subjected to ESR spectroscopy. In the case of ethanol-treated rats, the ESR spectra indicated that mixtures of radicals had been trapped, while spectra from control rats were essentially negative. The predominant spin adduct detected after ethanol treatment is proposed to be from a carbon-centered, primary alkyl radical, based on gamma-hydrogen hyperfine splitting patterns observed with PBN-d14. Oxygen-centered radicals also contributed to the ESR spectra. Liver extracts also contained low concentrations of the 1-hydroxyethyl radical spin adduct, which was indicated by weak spectral lines corresponding to those of the 1-13C-ethanol adduct. These data confirm previous suggestions that ethanol is metabolized to a free radical metabolite in rat liver. In addition, some information on types of lipid radicals generated during alcohol intoxication has been obtained.     

Absorption spectra of the hydroxycyclohexadienyl radicals of substrates for phenol hydroxylase

The absorption spectra of the hydroxycyclohexadienyl radicals formed upon the addition of OH radicals to six substrates for phenol hydroxylase have been determined using pulse radiolysis. Combining the radical spectra of thiophenol (lambda max, 390 nm; epsilon, 10,500 M-1 cm-1) and resorcinol (lambda max, 340 nm; epsilon, 4,100 M-1 cm-1) with their respective published spectra of enzyme-bound reduced flavin that is substituted in the C(4a) position of the dihydroflavin ring gave composite spectra that closely match the spectra formed concomitantly with the introduction of an oxygen atom into the substrates, the so-called Intermediate II species. A similar procedure for the substrates hydroquinone, 3-aminophenol, 3-chlorophenol, and 3-methylphenol yielded spectra that are also consistent with the known characteristics of their Intermediate II species. These spectral results give further support to the proposed biradical mechanism (Anderson, R.F., Patel, K. B., and Stratford, M. R. L. (1987) J. Biol. Chem. 262, 17475-17479) for the functioning of this class of flavoprotein hydroxylases.     

Ionic base radicals in gamma-irradiated oriented non-deuterated and fully deuterated DNA.

The free radicals induced by gamma-irradiation at 77 K in non-deuterated and fully deuterated oriented samples of moist algal DNA have been studied by electron paramagnetic resonance (e.p.r.). The e.p.r. spectra from the non-deuterated sample were found to be similar to those from calf-thymus DNA studied previously. Numerical spectra simulations were performed based on the previously proposed interpretation of the DNA spectra at 77 K in terms of a mixture of two spectral components arising from anionic and cationic base radicals (probably on thymine and guanine, respectively). The simulations were found to account satisfactorily for the main characteristics of the e.p.r. spectra from both the non-deuterated and the fully deuterated algal DNA sample.     

ENDOR-assisted study of the stable EPR spectrum of X-irradiated alpha-L-sorbose single crystals: MLCFA and simulation decomposition analyses

After X irradiation of single crystals of alpha-L-sorbose at 295 K, previous electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR-induced EPR (EI-EPR) results have indicated the formation of at least 10 different free radicals, and also that conceivably each carbon in the pyranose ring is a possible radical center. The radicals appear to be formed mostly by net H-abstraction reactions followed by standard elimination (e.g. beta-OH elimination) reactions or proton shifts, in turn leading to ring opening and fragmentation. In the present work, EPR spectra were recorded at room temperature with the external magnetic field along each of the three crystallographic axes subsequent to careful annealing at different temperatures using a high-temperature cavity. Each of the three sets of spectra was subjected to a maximum likelihood common factor analysis (MLCFA) that contributed to a better understanding of the spectral decays. Furthermore, the most stable spectra were simulated by optimization of previous ENDOR and EI-EPR results. The optimized EPR parameters resulted in excellent simulations of the experimental stable sorbose spectra and hence provided an improved insight of their spectral compositions.     

ESR spin trapping studies of free radicals generated from nitrofuran derivative analogues of nifurtimox by electrochemical and Trypanosoma cruzi reduction

Electron spin resonance (ESR) spectra of radicals obtained from two analogues of the antiprotozoal drug nifurtimox by electrolytic and Trypanosoma cruzi reduction were analyzed. The electrochemistry of these compounds was studied using cyclic voltammetry. STO 3-21G ab initio and INDO molecular orbital calculations were performed to obtain the optimized geometries and spin distribution, respectively. The antioxidant effect of glutathione on the nitroheterocycle radical was evaluated. DMPO spin trapping was used to investigate the possible formation of free radicals in the trypanosome microsomal system. Nitro1 and Nitro2 nitrofuran analogues showed better antiparasitic activity than nifurtimox. Nitro2 produced oxygen redox cycling in T. cruzi epimastigotes. The ESR signal intensities were consistent with the trapping of either the hydroxyl radical or the Nitro2 analogue radicals. These results are in agreement with the biological observation that Nitro2 showed anti-Chagas activity by an oxidative stress mechanism.     

A model considering light reabsorption processes to correct in vivo chlorophyll fluorescence spectra in apples.

Chlorophyll-a contained in the peel of Granny Smith apples emits fluorescence upon excitation with blue light. The observed emission, collected by an external detector and corrected by its spectral response, is still distorted by light reabsorption processes taking place in the fruit skin and differs appreciably from the true spectral distribution of fluorescence emerging from chlorophyll molecules in the biological tissue. Reabsorption processes particularly affect the ratio of fluorescence intensities at 680 nm and at 730 nm. A model to obtain the correct spectral distribution of the emission, from the experimental fluorescence recorded at a fluorometer detector and corrected for the detector spectral sensitivity, is developed in the present work. Measurements of the whole fruit reflectance, the peel transmittance and the flesh reflectance allow the calculation of the reabsorption-corrected spectra. The model is validated by comparing the corrected emission spectra with that obtained for a thin layer of apple-peel-chloroplasts, where no reabsorption takes place. It is recommended to correct distortions in emission spectra of intact fruits due to light reabsorption effects whenever a correlation between the physiological state of the fruit and its fluorescence spectra is investigated.     

Fourier transform electron paramagnetic resonance studies of photochemical reactions in heterogeneous media

FT-EPR has proved to be an excellent spectroscopic technique for the study of free radicals generated in photochemical reactions. The high spectral and time resolution make it possible to identify transient free radicals. Analysis of the time dependence of the spectra gives data on the kinetics of radical formation and decay. Chemically induced dynamic electron polarization (CIDEP) effects give information on reaction mechanisms and radical pair characteristics. Relaxation and linewidths data provide an insight into molecular motion. This contribution reviews instrumental aspects of the technique and CIDEP mechanisms that can affect the spectra. The utility of the method is illustrated with a discussion of applications in the study of photoinduced electron transfer reactions in homogeneous and heterogeneous media.     

Intensity-based protein identification by machine learning from a library of tandem mass spectra

Tandem mass spectrometry (MS/MS) has emerged as a cornerstone of proteomics owing in part to robust spectral interpretation algorithms. Widely used algorithms do not fully exploit the intensity patterns present in mass spectra. Here, we demonstrate that intensity pattern modeling improves peptide and protein identification from MS/MS spectra. We modeled fragment ion intensities using a machine-learning approach that estimates the likelihood of observed intensities given peptide and fragment attributes. From 1,000,000 spectra, we chose 27,000 with high-quality, nonredundant matches as training data. Using the same 27,000 spectra, intensity was similarly modeled with mismatched peptides. We used these two probabilistic models to compute the relative likelihood of an observed spectrum given that a candidate peptide is matched or mismatched. We used a 'decoy' proteome approach to estimate incorrect match frequency, and demonstrated that an intensity-based method reduces peptide identification error by 50-96% without any loss in sensitivity.