Detection and imaging of superoxide in roots by an electron spin resonance spin-probe method

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.     

Quantitative determination and reversible modification of sulfhydryl groups in low and high molecular weight compounds using a biradical spin marker]

A new method for quantitation of sulfhydryl groups of low and high molecular weight compounds is proposed. The method is based on the use of a biradical spin label carrying a disulfide bond, RS-SR, where R is the imidazoline radical. It was found that this biradical is involved in the reaction of thiol-disulfide exchange with thiols; the EPR spectra of the original biradical and monoradical products differ essentially. This circumstance made it possible to determine the bimolecular rate constant for the biradical interaction with cysteamine, cysteine, glutathione and human serum albumin. The method was used for measuring glutathione and cysteine levels in murine and rat blood and for assaying the insect acetylcholine esterase activity and reversible inhibition of NADPH-cytochrome P-450 reductase. The method is marked for a high sensitivity (10(-6)-10(-7) M) towards sulfhydryl groups and allows the determination of thiol groups in coloured and nontransparent solutions.     

High-field electron spin resonance of spin labels in membranes

High-field electron spin resonance (ESR) spectroscopy is currently undergoing rapid development. This considerably increases the versatility of spin labelling which, at conventional field strengths, is already well established as a powerful physical technique in membrane biology. Among the unique advantages offered by high-field spectroscopy, particularly for spin-labelled lipids, are sensitivity to non-axial rotation and lateral ordering, a better orientational selection, an extended application to rotational dynamics, and an enhanced sensitivity to environmental polarity. These areas are treated in some depth, along with a detailed consideration of recent developments in the investigation of transmembrane polarity profiles.     

Electron spin relaxation of synthetic melanin and melanin-containing human tissues as studied by electron spin echo and electron spin resonance

Electron spin lattice relaxation times (T1) and the phase memory times (Tm) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 degrees K. Saturation behavior of the ESR spectra of melanins in melanin-containing tissue and of the synthetic melanin was also determined at the same temperature. The spin lattice relaxation time and the spectral diffusion time of the synthetic melanin are very long (4.3 ms and 101 microseconds, respectively, in the solid state), and the ESR signal saturates readily at low microwave powers. On the other hand, ESR spectra of natural melanins from the tissues chosen for this study, as well as those of synthetic melanins which contain Fe3+ of g = 4.3 and Mn2+ of g = 2, are relatively difficult to saturate compared with samples without such metal ions. These results show clearly that a large part of those two metal ions in sites responsible for the ESR spectral components with these particular g values are coordinated to melanin in melanin-containing tissue, and modify the magnetic relaxation behavior of the melanin. Accumulations of these metal ions in melanins are different from system to system, and they increase in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin.     

Electron spin resonance and electron nuclear double resonance studies of cation radicals derived from tocopherol model compounds

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) measurements were performed for the cation radicals obtained from the model compounds of α-, β-, γ- and δ-tocopherol (vitamin E) by oxidizing the tocopherol precursors in an AlCl₃-CH₂Cl₂ solution. The proton hyperfine coupling constants g-values were precisely determined. The ENDOR spectra of the cation radicals of α-, β-, γ- and δ-tocopherol models in CH₂Cl₂ at ?100°C clearly show 10, 6, 6 and 12 different proton hyperfine couplings, respectively. By varying the temperature, the ESR spectra of the α- and δ-tocopherol model cations exhibit line-width alternation phenomena characteristic of the hindered rotation of the OH group. However, neither the β- nor the γ-tocopherol model cation radical ESR spectra show any sign of an alternative line-width effect. These results are interpreted by assuming that the β- and γ-tocopherol model cations are stabilized in the trans and cis conformations, respectively. On tocopherol model cations are stabilized in the trans and cis conformations, respectively. On the other hand, both the α- and δ-tocopherol model cations exist as cis and trans isomers.     

Translational diffusion of lipid monomers determined by spin label electron spin resonance

The collision rates between spin-labelled valeric acid in water, and between the corresponding mixed-chain, spin-labelled phosphatidylcholine in water-methanol mixtures, and also between spin-labelled phosphatidylcholine monomers and micelles in water have been determined from the spin-spin broadening of the electron spin resonance spectrum. In each case the second order rate constants are consistent with a diffusion-controlled process. For spin-labelled valeric acid in water the translational diffusion coefficient at 20°C is 3.4 · 10⁻⁶ cm² · s⁻¹, and for spin-labelled phosphatidylcholine varies between 2.3 · 10⁻⁶ and 3.8 · 10⁻⁶ cm² · s⁻¹ within the range 44 to 88 wt% methanol. The spin-labelled phosphatidylcholine monomer diffusion coefficient in water at 20°C is 2.4 · 10⁻⁶ cm² · s⁻¹, deduced from the monomer-micelle association rate, with an activation energy of 4.0 kcal · mol⁻¹. The much slower on-rates for association of lipid monomers with phospholipid bilayer vesicles reported in the literature, therefore indicate that incorporation into bilayers is not a diffusion-controlled process.     

Radiolysis of spin-labeled DNA: an electron spin resonance investigation

The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.     

Iron and copper binding by fungal phenolic polymers: an electron spin resonance study

In an electron spin resonance (ESR) survey of fungal phenolic polymers and melanins, it was found that the phenolic polymers fromEpicoccum purpurascens andPenicillium funiculosum displayed hyperfine splitting, indicating the presence of transition metal bonding. The ESR parameters agree well with those of iron and copper organic complexes. Further, the copper-complex ESR parameters were similar to those reported for mixed complexes of copper, bipyridyl and dicarboxylic acids; this suggests that nitrogen-containing groups from the proteinaceous moiety are involved in the process. Acid hydrolysis removed the proteins and metals, but the hydrolyzed polymers were still able to remove 72% of the copper present in a 0.5× 10^–4 M solution. It was concluded that fungal phenolic polymers, which constitute a part of the soil humus, may play an important role in the translocation of metal ions and their availability to biological systems.Deceased     

Quantitative determination of the rate of superoxide radical formation in mitochondrial membranes by electron paramagnetic resonance

The reaction of hydroxylamine (1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidine) with O2-. resulting in the stable nitroxyl radical formation recorded by ESR-technique was applied to estimate quantitatively the rate of O2-. superoxide radical generation (VO2.-) by submitochondrial particles (SMP) of liver (of mice and rats). The VO2.- dependence on concentrations of NADH, succinate and protein of SMP was established. The method allows detecting VO2.- greater than 0.05 nmol.min-1.ml-1. It has been shown that in the NADH-dependent site of the chain VO2.- is 3-4 times that in the succinate-dependent site. In the presence of rhotenone and antimycin A VO2.- increases by 30-35%, while cyanide retards VO2.- by about 30%. The data comparison with regard to VO2.- and O2 absorption rate polarographically determined has indicated that about 2% of the absorbed O2 is consumed to form O2-.     

Quantitative electron probe X-ray microanalysis of thin objects using an independent mass determination method

Elemental mass fraction determinations on thin biological specimens by quantitative electron probe X-ray microanalysis commonly use a total mass estimation based on registration of X-ray continuum. We have tested whether the accurary of such determinations on individual biological objects can be improved when a densitometric method for mass determination is used. Air-dried femtoliter droplets with dry masses in the order of 10⁻¹⁶ kg were used as model objects. An improvement of over two-fold upon the continuum method could be achieved.     

Superoxide formation in spinach chloroplasts: electron spin resonance detection by spin trapping.

The new technique of spin trapping has been applied to a biological system for the first time. The light induced generation of O₂^? by chloroplasts in the presence of oxygen has been shown by the production of the O₂^? adduct of the spin trap 5,5-dimethyl-1-pyrroline-1-oxide. The O₂^? adduct was detected by electron spin resonance spectroscopy. Methyl viologen enhanced the production of the O₂^? adduct thus providing support for the hypothes is that methyl viologen accepts electrons from the primary acceptor of photosystem I and subsequently reduces O₂ to O₂^?.     

Heparin modulates conformational states of plasma fibronectin: an electron spin resonance spin label approach

We have examined the interaction between heparin and human plasma fibronectin using electron spin resonance (ESR) spin label methods. The titratable sulfhydryl groups of plasma fibronectin were modified with a maleimide spin label [Lai and Tooney (1984) Arch. Biochem. Biophys. 228, 465-473]. Addition of heparin resulted in a decrease in the maximum splitting value of the ESR spectrum of spin-labeled fibronectin from 66.8 to 64.3 G, suggesting that heparin induces a conformational alteration of plasma fibronectin. This heparin effect was noticeable at a heparin-to-fibronectin ratio of 20 to 1 and reached a plateau at about 100 to 1. Other sulfated carbohydrates were tested; dextran sulfate was found to be as effective as heparin but chondroitin sulfates were ineffective. The results presented suggest that the binding of heparin changes the molecular conformation of plasma fibronectin to a more relaxed or flexible state.     

Hyperthermic modification of bleomycin--DNA interaction detected by electron spin resonance

Electron spin resonance spectra of DNA labeled with each of four spin-labeling compounds have been studied to detect interaction between the antibiotic bleomycin and DNA. Only one of these labels, compound IV, resulted in a modified spectrum when bound to DNA and the latter was subjected to bleomycin. This property has been used to monitor DNA-bleomycin interactions under physiological and hyperthermic conditions. Bleomycin produced an increase in rotational correlation time of the residue bound to DNA at 37 degrees C and a significantly higher increase at 43 degrees C. Some effect was still detected with bleomycin at 37 degrees C after preheating at 43 degrees C. Parallel studies have revealed enhanced binding of 59Fe-bleomycin to DNA during and after hyperthermic treatment.     

Oxidation of low-density lipoprotein by Cu2+ and lipoxygenase: an electron spin resonance study

The aim of this work was to obtain spectroscopic evidence for free radicals formed during copper ion- and lipoxygenase-catalyzed oxidation of the low-density lipoprotein. During the initial oxidation phase, a free-radical metabolite derived from the endogenous alpha-tocopherol present in the low-density lipoprotein was detected by the electron spin resonance technique. The divalent copper ions were bound to the residual EDTA present in the low-density lipoprotein and to the protein. Production of the alpha-tocopherol radical was suppressed in the presence of spin traps. Evidence for the low-density lipoprotein-lipid derived radicals was obtained by ESR-spin trapping methods. Implications of these findings in the oxidative modification of the low-density lipoprotein are discussed.