Stable Organic Free Radical Research Articles

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.

Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).

Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

Contrast agents for nuclear magnetic resonance imaging

Nuclear magnetic resonance imaging relies upon differences in relaxation times for much of its ability to resolve anatomical structures and to detect changes in tissue. The natural differences can be changed by the administration of paramagnetic substances, such as metal complexes and stable organic free radicals, and ferromagnetic materials, such as small particles of magnetite. Detailed studies of the chemistry and biophysics of such substances in the body are required if they are to become safe and effective contrast agents for use in medical NMR imaging.

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.

Application of the convolution difference resolution method in reconstruction techniques in EPR imaging

Nitroxides are versatile, stable organic free radicals which are important as spin labels or spin probes in biological EPR studies (1). Recently, they have been used as contrast agents in NMR imaging (2, 3) and as “magic bullets” in EPR imaging (4). While we have previously demonstrated the feasibility of making two-dimensional images by EPR, there were several unsolved resolution problems, which may plague NMR image reconstruction as well. The hyper6ne or chemical-shift problem (5) is a general resolution problem caused by superposition of peaks from (1) hyperfine structure: for nitroxides, the low-field peak is superimposed on the central peak of the triplet in a strong magnetic field gradient, ultimately contributing to image distortion and overlapping; and (2) intrinsic linewidth: this may not be small compared with the linewidth in a strong field gradient, again causing image distortion and image overlap. Furthermore, the Lorentzian-like lineshape obtained in EPR imaging experiments makes the spectral analysis very complex. When the same half-width is assumed for both Lorentzian and Gaussian lineshapes, the Lorentzian falls off in the wings more slowly than does the Gaussian (6). In practice, this is a common problem with large field gradients and/or very large samples. While some resolution enhancement methods have been reported for organic radical EPR spectra (7), no reports address the problems above for image reconstruction with nitroxides. Hoch et al. (8) reported deconvolution methods applicable to the single line spectrum of DPPH, where it was relatively trivial to subtract the intrinsic linewidth contribution. Ohno (9) attempted deconvolution on simulated spectra only. We have applied image reconstruction for nitroxides by the convolution difference method. Several examples are compared with and without this treatment. The convolution difference method, which has been applied extensively to NMR resolution enhancement, is a straightforward mathematical manipulation which sacrifices signalto-noise in order to achieve increased resolution (10, II). When the lineshape is Lorentzian, the Fourier transform is multiplied by eVar (where a is a constant and t is time), and the resulting lineshape will also be Lorentzian (L,). In a similar manner, convolution by e -bt leads to another Lorentzian (Lb). By subtracting the two Loren-

Immunospecific NMR contrast agents

Most NMR contrast agents suggested to date have been paramagnetic. These agents, which include the transition and lanthanide metal ions as well as stable organic free radicals, do not provide effective contrast at concentrations much below 1 mM. However, the use of macromolecular ferromagnetic and superparamagnetic particles provides, for the first time, an NMR relaxation agent that is effective at subnanomolar concentrations. Two different sized superparamagnetic particles have been coupled to monoclonal antibodies with high affinity for a neuroblastoma-specific cell surface antigen. The specific binding of these particles, both in vivo and in vitro is demonstrated and the consequences for immunospecific NMR contrast are discussed.

Investigations on Normal and Cataractous Human Lenses by EPR

Normal and senile cataractous human lenses were studied by spin probe electron paramagnetic resonance (EPR) technique analyzing the reduction of added stable organic-free radicals. It has been concluded that the radical reduction kinetics were different at the different states of the senile cataract, and the conformational changes in lenticular proteins induced by the urea had no influence on the kinetics of the radical reduction. It is supposed that the decrease of the reduced glutathione is an exceedingly sensitive indicator of the change in the lens metabolism in the course of cataractous progression.

Effect of protein denaturants on the structure of water: Electron spin resonance spin probe study

The effect of the urea-class of protein denaturants on the structure of liquid water was studied. The method chosen was to monitor the microviscosity of the medium by estimating the reorientational correlation time, τ8 and proton hyperfine linewidth, WH of an inert stable organic free radical (2, 2, 6, 4-tetramethyl-piperid 4-one-l-oxyl) by measuring its electron spin resonance spectra in aqueous denaturant solutions. Urea, thiourea and guanidinium chloride were found to disrupt water structure efficiently and continuously, with the effect significant at low molarities. Dimethyl urea was found to be somewhat less efficient. The relation between the structure-breaking tendency of the denaturants and their ability to weaken hydrophobic interactions is rationalised.

ESR spin probe studies on the alteration of water structure

The electron spin resonance (ESR) spectra of the stable organic free radical 2,2,6,6-tetramethyl piperid-4-one N-oxide (TEMPO) were measured in aqueous solutions of urea and sodium butyrate. From an analysis of the ESR spectral line-widths and tumbling correlation times, inferences have been made about the alteration in the structure of solvent water induced by these solutes.

The Chlorine Nuclear Quadrupole Resonance in an Organic Free Radical, p-Cl-EDPA

Abstract The 35Cl nuclear quadrupole resonance in a stable organic free radical, 1,3-bisdiphenylene-2-p-chlorophenyl-allyl, has been measured from 4.2 to 1.6 K. On lowering the temperature, the resonance line became weaker and broader, and it disappeared near the Néel temperature. This behavior has been interpreted in terms of the presence of a hyperfine interaction.

EPR and ENDOR studies of hyperfine interactions in solutions of stable organic free radicals

ENDOR has been used to measure hyperfine couplings for all protons in solutions of the stable free radicals picryl-N-aminocarbazyl (PAC) and α, γ-bisdiphenylene-β-phenyl allyl (BDPA). The assignment of the measured couplings was aided through computer stimulation of the ENDOR spectra. The intensities of the ENDOR lines were not found to be proportional to the number of contributing protons, and the correction factor suggested by Allendoerfer and Maki was found to be necessary. The values of T2 thus obtained are ∼0.1 μsec and ∼0.05 μsec for PAC and BDPA in mineral oil at room temperature. A further check in the assignments was obtained through comparison of the experimentally observed high resolution EPR spectra with those stimulated on a computer using the proton couplings determined through ENDOR. This also yielded the nitrogen hyperfine couplings in PAC. These results complement our earlier assignment of hyperfine couplings in DPPH and confirm our earlier observations that large ENDOR enhancements can be obtained in nitrogen-centered, strongly exchanging radicals by using solvents with high viscosity and low dielectric loss and by optimizing radical concentration for large EPR signal but lower exchange rates. Well degassed mineral oil at about room temperature appears to be a good solvent. Temperature dependence of the ENDOR spectra shows dynamical changes in line splittings in DPPH and PAC. A model involving the presence of hindered rotations about the Nα–Nβ and the Nβ–Cpicryl bonds helps explain these observations. These results complement the available results on hindered rotations of picryl groups in the related nitroaromatic amines. The present work shows that for paramagnetic systems where NMR might not be successful, ENDOR could be a valuable technique for obtaining information on molecular dynamics. The ENDOR results on BDPA also resolve a controversy between earlier EPR and Knight shift studies.

Iron and Free Radical in Ribonucleotide Reductase: EXCHANGE OF IRON AND MÖSSBAUER SPECTROSCOPY OF THE PROTEIN B2 SUBUNIT OF THE ESCHERICHIA COLI ENZYME

Abstract Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits (Protein B1 and Protein B2), both required for activity. Protein B2 contains 2 iron atoms and a lesser, variable amount of a paramagnetic species, characterized by g = 2.0047 EPR and 410 nm electronic absorptions. Except for the 410 nm band, Protein B2 much resembles oxy- and methemerythrins in electronic spectra. The paramagnetic species is probably organic free radical rather than metal, is dependent upon the presence of iron in Protein B2, is directly correlated with enzymatic activity, and is destroyed by NH2OH and hydroxyurea (Ehrenberg, A., and Reichard, P. (1972) J. Biol. Chem. 247, 3485–3488). We here describe complete removal of iron from Protein B2, reconstitution to active metalloenzyme, and Mossbauer spectroscopy of [57Fe]Protein B2. Addition of excess Fe2+ to metal-free protein yielded reconstituted/reactivated protein with 2:1 iron:subunit stoichiometry and a hemerythrinlike electronic spectrum, but with 120 to 390% of the original specific enzymatic activity and correspondingly increased 410 nm and EPR absorptions. NH2OH- or hydroxyurea-inactivated protein was also converted to highly active enzyme subunit by the same iron substitution procedures. Mossbauer and electronic spectra suggested that Protein B2 contains 2 nonidentical high spin Fe(III) ions in an antiferromagnetically coupled binuclear complex that resembles both methydroxohemerythrin and oxyhemerythrin. Below 195°K the iron in Protein B2 was completely diamagnetic, in confirmation of the previous assignment of the 410 nm- and EPR-absorbing species as stable organic free radical of unknown structure. We propose that the function of iron in Protein B2 is the initial generation of radical from a protein-bound group, and that stability of the radical depends upon some continuing interaction with the iron center. The radical is apparently less stable than is metalloprotein structure: this would account for the variability of radical content in native protein and for the g 100% yield of radical obtainable by substitution of the iron. We further propose that the free radical participates in the reduction of ribonucleotides by the enzymatically active Protein B1-Protein B2 complex.

Vapor deposition of thin films of DPPH and BDPA

A technique for vapor deposition of thin films of the stable organic free radicals DPPH and BDPA is described. The magnetic properties and chemical composition of the resultant films were studied by EPR, X-ray and optical absorption methods and found to be similar to those of the bulk materials. Free radical concentrations as high as 80% were obtained in these films.

Radicals derived from 1,2-dithiolylium ions, a new class of stable organic free radicals

Cathodic reduction of 3,5-diaryl-1,2-dithiolylium ions produces the corresponding stable neutral free radical which is in equilibrium with its dimer at lower temperature.

Spin-quenching by oxygen of a radiosensitizing nitroxide free radical in aqueous solution.

Stable organic nitroxide free radicals have recently become of considerable interest in radiobiology, since they have been found to increase the sensitivity of anoxic cells to radiation-induced reproductive death without affecting the aerobic sensitivity (Emmerson and Howard-Flanders 1964, 1965, Emmerson 1967 and Brustad 1968). Such characteristics make these compounds of potential interest in radiotherapy. Among the nitroxide free radicals studied to date, 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (often referred to as triacetoneamine-N-oxyl or TAN) appears to combine the most desirable qualities of relatively low toxicity with efficient radiosensitization. Emmerson and Howard-Flanders (1965) and Emmerson (1967) have suggested that TAN exerts its sensitizing effect by reacting with radiationinduced radicals in vital cellular constituents in the same way as does oxygen. Pulse radiolysis experiments have revealed that TAN interacts rapidly with OH-induced DNA transients (Willson and Emmerson 1970). We have recently investigated the nature of this radiation-induced interaction in dilute neutral aqueous solution. Our results show that TAN becomes covalently bonded to OH-induced DNA transients under anoxic conditions, whereas no binding occurs when irradiation is performed in the presence of oxygen. This lack of binding, as well as the absence of a radiosensitizing effect upon oxygenated cells, might most obviously be explained by competition between TAN and 02 for reaction with radiation-induced DNA transients. An alternative explanation, which so far has not been considered, could involve an interaction of oxygen with TAN to render the latter ineffective. However, it has been pointed out that the nitroxide free radicals do not react with oxygen, at least to form peroxides (Emmerson and Howard-Flanders 1964, Hoffmann and Henderson 1961). Nevertheless, since TAN is a free radical, its ability to react with radiation-induced DNA transients may conceivably be related to the unpaired electron localized on the NO group. We therefore decided to conduct an electron spin resonance (ESR) study to determine whether oxygen alters the free radical properties of TAN. TAN was synthesized from 2,2,6,6-tetramethyl-4-piperidone-HCl (Aldrich Chemical Company) according to published procedures (Briere, Lemarie and Rassat 1965), with some minor modifications. Our X-band ESR spectrometer with transmission H102 mode cavity has been described previously (Henriksen 1962). All E.S.R. measurements described here were carried out at 77°K. All

Voltammetry without background electrolyte: Redox pattern of DPPH in non-aqueous media

The voltammetric behaviour of diphenylpicrylhydrazyl (DPPH), a stable organic free radical, which has been found to give reversible reduction waves at platinum in 0·1 M NaClO4 solution in acetonitrile, was investigated at platinum and graphite electrodes in several media under a variety of conditions. The reported acetonitrile data were confirmed; comparison with data obtained in acetonitrile in the absence of background electrolyte has shown that voltammetry in such absence is quite feasible when using a three-electrode IR compensation circuit. Variation of polarization rate, polarization direction, electrode rotation rate, background electrolyte (0·1 M NAClO4; 0·1 M (C2H5)4NClO4), indicating electrode (platinum; pyrolytic graphite) and solvent (acetonitrile; pyridine; sulphur dioxide) revealed, ia, semi-reversible behaviour of DPPH dependent on the presence of background electrolyte, adsorption of the neutral organic free radical on pyrolytic graphite, and a lack of stability for the compound in normally available reagent grade pyridine and in liquid sulphur dioxide (one product in-both cases was diphenylpicrylhyrazine). The stability of DPPH in pyridine increased with purity of the solvent, which is of significance in connexion with the postulation of free radical intermediates in organic electrode processes.

Free radical studies of lignin, lignin degradation products and soil humic acids

Native lignin, various lignin preparations and a variety of humic acids isolated from soil and lignite have been shown to contain stable organic free radicals by means of electron paramagnetic resonance spectrometry. The radical species are not contaminants, but an integral part of the natural polymers. Chemical and EPR spectral evidence suggests that the radical species is an ortho- or para-semiquinone, which is coexistent with quinhydrone and quinone moieties. Although the nature of the stable free radical appears to be the same in lignins and humic acids, the concentration of these species varies considerably. Thus, the unpaired electron spin concentration increases from low values in native lignin to intermediate values in lignin preparations and finally to relatively high values in soil humic acids and leonardite. These results are significant because 1. (1) they lend experimental support to present theories regarding the free radical mechanism of the biological degradation of lignin; 2. (2) they illustrate a technique (EPR spectrometry) which may provide valuable information on the structures of humic acids; and 3. (3) they provide a basis for future studies of subtle structural changes in lignin and humic acids.

Stable Organic Free Radicals. I. Synthesis of Substituted α,γ-Bisdiphenylene-β-phenylallyls

Stable Organic Free Radicals. I. Synthesis of Substituted α,γ-Bisdiphenylene-β-phenylallyls

Oxygen-17 Hyperfine Structure in Electron Paramagnetic Resonance

The hyperfine structure for O17 and N14 splittings in an organic radical, which is a model for nitric oxide, were compared. The relatively stable and simple organic free radical di-sec-butyl nitric oxide was enriched to 3.94% O17, and the O17 hyperfine structure observed in solution electron paramagnetic resonance. The O17 isotropic hfs in nitric oxide was calculated. It is given by the same mechanism involved in the nitrogen hfs; predominantly from cross terms, in the configuration interaction wave function, between the ground state (2 pi ) and various excited states having one (2 pi ) and two ( delta ) unpaired electrons. Agreement between the calculated splitting for O17 and that observed in the model compound was quite good.

Stable free radicals in soil humic acid

Solid soil humic acid contains stable organic free radicals, on the order of 10 18 radicals/g. Solutions in organic solvents also exhibit paramagnetism. Fractionation studies reveal that these radicals are not caused by impurities or by unstable species trapped in the solid humic acid polymer but rather are an integral part of the molecule. Analysis of the EPR spectrum suggests that two stable free radicals co-exist in humic acid. One of these could be a semiquinone of a catechol-resorcinol-type copolymer; the other could be a quinhydrone-type radical.