Electron spin resonance study of peroxidase activity and kinetics.

An electron spin resonance (ESR) assay has been developed for peroxidase activity. The assay measures the formation of the paramagnetic nitroxide Tempol from the oxidation of its hydroxylamine derivative (TOLH) by short-lived radicals produced by peroxidase cycle intermediates, Compounds I and II. Using phenol as a peroxidase electron donor, the ESR approach is suitable for measurements of peroxidase activity ( > or = 0.003 U/ml) and micromolar quantities of H2O2 in sample sizes as small as 2 microliters. In addition, the ESR method can be used to continuously monitor activity in cell suspensions and other media that are susceptible to optical artifacts. The high membrane permeability of TOLH also makes it possible to estimate peroxidase activity in membrane-enclosed compartments, provided that TOLH oxidation rates can be stimulated with exogenous peroxidase reductants, e.g., phenol. Analysis of TOLH oxidation rates under conditions of low electron donor concentrations and high concentrations of H2O2 also shows clear indications of substrate-dependent inhibition and increased catalytic activity. Computer simulations indicate that the results obtained are consistent with the peroxidase reaction scheme proposed by Kohler et al. (1988, Arch. Biochem. Biophys. 264, 438-449) modified to correct for a nitroxide dependent stimulation of peroxidase catalytic activity.     

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.     

Electron spin resonance study of melanin treated with reducing agents.

The electron spin resonances (ESR) of several native and modified melanins have been determined. Melanins isolated from black wool and synthesized from 3,4-dihydroxyl-L-phenylalanine (L-DOPA) and tyrosine all show similar ESR signals. Modification of the isolated melanins by treatment with reducing agents causes some lightening in color and slight changes in the ESR spectra. Lithium and liquid ammonia (Birch) reduction applied to melanins from wool and L-DOPA gave very different results, as reflected by ESR spectra, but in both cases the changes were much greater than those produced by other treatments. In general, reductive treatments in nonaqueous media in the presence of metals increase the free radical content and line width, whereas treatment in aqueous media resulted in decreased free radical content. These observations are consistent with a melanin pigment which is an irregular polymer and has unpaired electrons localized on different but similar monomer units.     

An electron spin resonance study of interactions between phosphatidylcholine and phosphatidylserine in oriented membranes.

A detailed electron spin resonance (ESR) study of mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and phosphatidylserine (POPS) in oriented multilayers in the liquid crystalline phase is reported with the purpose of characterizing the effects of headgroup mixing on the structural and dynamical properties of the acyl chains. These studies were performed over a range of blends of POPC and POPS and temperatures, utilizing the spin-labeled lipids 16-phosphatidylcholine and 5-phosphatidylcholine as well as cholestane (CSL). The ESR spectra were analyzed by nonlinear least-squares fitting using detailed spectral simulations. Whereas CSL shows almost no variation in ordering and rotational dynamics versus mole fraction POPS, (i.e. XPS), and 5-PC shows small effects, the weakly ordered end-chain labeled 16-PC shows large relative effects, such that the orientational order parameter, S is at a minimum for XPS = 0.5 where it is about one-third the value observed for XPS = 0 and 1. This is directly reflected in the ESR spectrum as a substantial variation in the hyperfine splitting with XPS. The least-squares analysis also shows a reduction in rotational diffusion coefficient, R perpendicular by a fractor of 2 for XPS = 0.5 and permits the estimation of S2, the ordering parameter representing deviations from cylindrically symmetric alignment. These results are contrasted with 2H NMR studies which were insensitive to effects of mixing headgroups on the acyl chains. The ESR results are consistent with a somewhat increased disorder in the end-chain region as well as a small amount of chain tilting upon mixing POPC and POPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron spin resonance of calmodulin-vanadyl complexes

X-band (9.2 GHz) electron spin resonance spectroscopy was used to investigate the binding of vanadyl to calmodulin. Solution spectra, obtained at ambient temperature with various VO2+:calmodulin molar ratios, suggested a binding stoichioimetry of 4 mol of VO2+/mol of protein and the possibility of two classes of binding sites. The latter was confirmed by using frozen solutions of calmodulin-VO2+ complexes that gave splitting of the spectral bands corresponding to the parallel components, which was particularly pronounced with the three high-field peaks. Competition of Ca2+ for the VO2+ binding sites was investigated, and the results indicated that two of the VO2+ sites corresponded to two of the Ca2+ sites; the other two VO2+ binding sites may have a higher affinity for VO2+ than for Ca2+ or they may correspond to Ca2+-independent sites. These results demonstrate that electron spin resonance spectroscopy can be used advantageously to probe subtle differences in the microenvironments of metal-binding sites in calmodulin.     

An electron spin resonance spin-label study of lipophilin in oriented phospholipid bilayers

Model membranes consisting of dimyristoyl phosphatidylcholine and a hydrophobic protein from bovine myelin, lipophilin, were studied using the cholesterol-resembling cholestane ESR spin label. Orientation of the membranes made it possible to deconvolute the spectra into two fractions, one of oriented spin labels reflecting phospholipid bilayer of high order, and one of isotropically tumbling spin labels ascribed to the lipid fraction surrounding the protein molecule (boundary lipid). This isotropic tumbling is different from the behavior of phospholipid molecules near the protein, which retain some degree of order, and indicates that the boundary lipid fraction in our model system forms a rather fluid environment for the protein. A nonlinear relation was found between protein concentration and amount of boundary spin labels. Addition of cholesterol decreases the amount of boundary spin labels. Both findings form evidence for a preferential binding of cholesterol by the membrane protein.     

Electron spin resonance study on peroxidase- and oxidase-reactions of horse radish peroxidase and methemoglobin.

The intermediate free radicals generated from phenols, naphthols and benzoate, in the peroxidase- and oxidase-reactions of horse radish peroxidase and in the peroxidase-reaction of methemoglobin, were studied by electron spin resonance spectroscopy.The difference between the peroxidase- and oxidase-reactions of HRP are demonstrated, i.e., the ferro horse radish peroxidase-O₂ system attacks both phenols and benzoate yielding unidentified radicals, which may be hydroxy-cyclohexadienyl radicals, while the horse radish peroxidase-H₂O₂ system reacts only with phenols and naphthols producing the phenoxy-and naphthoxy-radicals.Phenoxy-radical formation from phenols, in the reactions horse radish peroxidase-H₂O₂ and methemoglobin-H₂O₂, occurs independently of the molecular sizes of phenols but dependently on their redox-potentials.On the basis of kinetic studies on methemoglobin-H₂O₂ system, the existence of a reactive intermediate complex between methemoglobin and H₂O₂ is proposed, which may be similar to compound-I or -II of horse radish peroxidase and which further degenerates to MetHb radical. The oxidation of phenols and naphthols takes place outside of the hemepocket of methemoglobin.     

Electron spin resonance studies on anthocyanins

The ESR characteristics of anthocyanins and their congeners have been determined using both crystalline and non-crystalline preparations. For the latter, a micro-technique has been proposed. The results show that red and violet pigments specifically exhibit a distinct signal at g=2·057 being different from blue anthocyanins. It has been suggested that the g=2·057 signal arises from the unpaired electron of pyrylium ring. Comparison of ESR spectra of isolated anthocyanins with those in fresh petals has further shown that there is no difference in the g=2 region of ESR spectra and that the appearance of the g=2·003 signal is thus not due to anthocyanins alone.     

Electron spin resonance study of free radicals generated from retinyl- and ionyl-derivatives

Free radicals generated from alpha- and beta-ionyl bromides gave well resolved ESR spectra, but retinyl bromide and chloride gave only broad signals. Delocalised radicals were also spectroscopically observed on hydrogen abstraction from alpha-ionane, alpha-ionyltrimethylsilylether and buten-3-ynyl-2,6,6-trimethyl-2-cyclohexene. Retinyl and beta-ionyl radicals, derived from the corresponding xanthates, were successfully spin trapped with nitrosodurene. The results suggested that the secondary sites C(7) and C(9) were the most reactive in the beta-ionyl radical and that the secondary sites C(7) and C(11) and probably the primary site C(15) were the most reactive in the retinyl radical.     

Electron spin resonance dating in paleoanthropology

Many archeological and paleoanthropological sites cannot be dated by well established and common dating techniques such as uranium series (U-series) or argon-argon (⁴⁰Ar/³⁹Ar) because of the lack of materials that are suitable for these techniques. Most sites, however, contain bones and teeth, and the latter can be used to obtain electron spin resonance (ESR) age estimates. The theoretical age range of ESR dating accuracy lies between a few thousand and more than a million years. In practice, continuing uranium accumulation increases the uncertainty of ESR age assessments in such a way that most age assignments beyond 300,000 years are very uncertain.     

Electron spin resonance spin label studies of intercalation of nitrobenzene in DNA.

Nitrobenzene-DNA intercalation mechanisms have been studied by means of electron spin resonance spin label techniques. Of the seven derivatives prepared and examined, 2,4-dinitrobenzene analogs with amine linkage to the nitroxide reporter demonstrate the strongest binding with DNA by intercalation, and the reporter nitroxide is oriented 45 ° to the plane of the benzene ring and is due primarily to the steric hindrance of the 2-nitro substituent. This binding is found to be largely dependent upon the number of nitrosubstituents, their relative position on the benzene ring, and the type of linkage between the ligand and the nitroxide reporter, suggesting that polarization bonding is a major driving force in their complex formation with DNA.     

Electron spin resonance study of human transcortin binding properties. Interaction of monomeric and polymeric forms with spin labeled corticosteroids.

Corticosteroids bearing a nitroxide radical on the side chain were shown to bind with a high affinity to purified human serum transcortin. ESR spectroscopy data allowed calculation of the thermodynamic parameters (ΔH, ΔS) of the interaction and characterization of a hydrophobic spin label binding site. Transcortin spontaneously associated upon storage into reversible polymeric forms which partly retained the steroid binding properties. Apparent rotational correlation times of 34 and 70 n sec were obtained by ESR analysis at 20°C for the transcortin monomer and dimer respectively.