Regularity of changes in magnetic characteristics of Saccharomyces cerevisiae yeast cells at various stages of culture growth

Correlation between cell cycle of the synchronous yeast culture and ESR signal intensity at g 2.2 and 77 K was studied. It was shown that the maximal intensity of ESR signal was reached 10-15 min before the beginning of intensive cell division. The ESR signal with g 2.2 (77 K) is caused by the spin-glass like structure. The "freezing" temperature of these spin-glasses was measured.     

Functions of pheophytin, plastoquinone, iron and carotenoids in plant photosystem 2 reaction centers

Photoreduction of the intermediary electron acceptor, pheophytin (Ph), in photosystem-2 (PS-2) reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and Eh approximately -450 mV produces a narrow ESR signal of Ph. (g = 2.0033; delta H approximately 13 G) and a "doublet" centered at g = 2.00 with a splitting of 52 G at 7 K. The doublet (but not the narrow signal) is eliminated after extraction of lyophylized TSF-II with hexane, containing 0.1-0.2% methanol, or after extraction of Fe with LiClO4 and o-phenantroline, and the signal is restored by reconstitution with plastoquinone-A (PQ) or Fe++, respectively. The Fe removal results also in the development of a photoinduced ESR signal of PQ. (g approximately 2.0044; delta H approximately 9.2 G). The conclusion is made that the primary electron acceptor, Q, is in fact a complex PQ-Fe++ and that the exchange interaction of Ph. with PQ. -Fe++ is responsible for the ESR doublet. Photoreduction of Ph in TSF-IIa is accompanied by the 3-fold decrease in the formation of carotenoid triplet state (measured by the characteristic flash-induced absorbance changes) which is suggested to be a result of charge recombination in the pair [P680+ .PH.].     

Complex formation of spin-labeled 9-aminoacridine with DNA and polynucleotides

Complex formation between spin-labeled 9-aminoacridine and DNA or polynucleotides has been studied by differential spectrophotometry and ESR. The differential spectra of the strong type 9-aminoacridine-DNA complex showed characteristic absorption bands at 270 and 290 nm, and the intensity ratio of these bands varied according to the degree of DNA denaturation. The ESR spectra of this complex were characterized by slow rotation of the radical; as the macromolecule became increasingly denatured and in the polynucleotide complex, a rapid signal appeared in the ESR spectrum. The temperature at which DNA undergoes a structural transition in the premelting region could be determined from the temperature dependence of the ESR spectral form of the dye-DNA complex. The spectral characteristics of the complexes give additional information about structural disturbances in DNA.     

Electron spin resonance (ESR) spectrum of paraffin embedded human liver tissue. A possible method for the estimation of copper content.

Human liver tissues embedded in paraffin wax for histological examination have been studied by Electron Spin Resonance (ESR) spectroscopy. A signal was detected at g approximately 2.05 section of the spectrum. The amplitude of this signal was correlated with the copper content of the embedded specimens measured by flame atomic absorption technique. The positive correlation which has been found can make ESR spectroscopy suitable for estimating the copper content of tissues without damaging the sample. The limits and errors of this method have also been analysed.     

Participation of Free Radicals in Photoreduction of Protochlorophyllide to Chlorophyllide in an Artificial Pigment–Protein Complex

The primary stages of protochlorophyllide phototransformation in an artificially formed complex containing heterologously expressed photoenzyme protochlorophyllide-oxidoreductase (POR), protochlorophyllide, and NADPH were investigated by optical and ESR spectroscopy. An ESR signal (g = 2.002; H = 1 mT) appeared after illumination of the complex with intense white light at 77 K. The ESR signal appeared with simultaneous quenching of the initial protochlorophyllide fluorescence, this being due to the formation of a primary non-fluorescent intermediate. The ESR signal disappeared on raising the temperature to 253 K, and a new fluorescence maximum at 695 nm belonging to chlorophyllide simultaneously appeared. The data show that the mechanism of protochlorophyllide photoreduction in the complex is practically identical to the in vivo mechanism: this includes the formation of a short-lived non-fluorescent free radical that is transformed into chlorophyllide in a dark reaction.     

Computer modeling of ESR spectra of the plasma in patients with Down's syndrome

ESR technique at 77 degrees K was used for studying the blood plasma ESR signals of patients with Down syndrome and of healthy people. It was observed that the first exhibited a tendency towards a decrease of ESR signal with g = 4.3 and increase of the ratio of ceruplasmin (g = 2.05) and transferrin (g = 4.3) ESR signal amplitude. A computer simulation has been carried out by means of special mathematical program of experimental ESR spectra of the blood plasma.     

菠菜光系统I反应中心的电子自旋共振研究

利用温和的SDS-PAGE法分离纯化了菠菜光系统Ⅰ的反应中心复合物(CP1),并进行了低温(77K)和室温下的电子自旋共振谱(ESR)测定,获得了自由基P700~+的ESR吸收信号.其一级微分谱的g值为2.0024,谱线近以于高斯型,与从菠菜叶绿体中发现的P700~+的ESR信号相符.根据这些结果,我们认为以温和SDS-PAGE法从菠菜得到的CP1组份保存了大部分P700的光氧化活性.     

Computer simulation of ESR signals of liver paramagnetic centers

Changes of paramagnetic centres concentration characterized by g-factors values of 1.94, 2.2, and 2.03 in the rat liver were studied by ESR method under acute intoxication by diethylnitrosamine (DENA) and at preliminary threefold treatment of animals with butylhydroxytoluene (BHT). A protective effect of BHT can be explained by its stabilizing action of the membrane structures. A comparison has been carried out with a similar study of paramagnetic centres in the experiment of chronic intoxication by DENA. A simulation was performed of the liver tissue ESR spectra by means of special computer program. The parameters of simulated ESR spectra of the liver tissue with due regard for ESR signal g 2.03 corresponded to the parameters of the experimental spectra. Confirmations were obtained for the nature and number of paramagnetic centres in the liver tissue.     

Photosystem I charge separation in the absence of centers A and B. II. ESR spectral characterization of center 'X' and correlation with optical signal 'A2'

The Photosystem I electron acceptor complex was characterized by optical flash photolysis and electron spin resonance (ESR) spectroscopy after treatment of a subchloroplast particle with lithium dodecyl sulfate (LDS). The following properties were observed after 60 s of incubation with 1% LDS followed by rapid freezing. (i) ESR centers A and B were not observed during or after illumination of the sample at 19 K, although the P-700+ radical at g = 2.0026 showed a large, reversible light-minus-dark difference signal. (ii) Center 'X', characterized by g factors of 2.08, 1.88 and 1.78, exhibited reversible photoreduction at 8 K in the absence of reduced centers A and B. (iii) The backreaction kinetics at 8 K between P-700, observed at g = 2.0026, and center X, observed at g = 1.78, was 0.30 s. (iv) The amplitudes of the reversible g = 2.0026 radical observed at 19 K and the 1.2 ms optical 698 nm transient observed at 298 K were diminished to the same extent when treated with 1% LDS at room temperature for periods of 1 and 45 min. We interpret the strict correlation between the properties and lifetimes of the optical P-700+ A2 reaction pair and the ESR P-700+ center X- reaction pair to indicate that signal A2 and center X represent the same iron-sulfur center in Photosystem I.     

Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe-4S]cluster

Redox titrations of the iron-sulphur clusters in fumarate reductase purified from Escherichia coli, monitored by ESR spectroscopy, identified three redox events, similar to those observed in other fumarate reductases and succinate dehydrogenases: Centre 1, a [2Fe-2S] cluster, at g = 2.03, 1.93, appeared on reduction with Em = -20 mV. Centre 3, probably a [3Fe-xS] cluster, at g = 2.02 appeared in the oxidized state with Em = -70 mV. Centre 2 has been observed as an increase in the electron-spin relaxation of Centre 1. It titrates as an n = 1 species with Em = -320 mV, but in our hands did not appear to contribute significant intensity to the g = 2.03, 1.93 signal. It therefore appears to be an additional centre which undergoes spin-spin interaction with Centre 1. The reduction of Centre 2 coincided with the appearance of an extremely broad ESR spectrum, observed at temperatures below 20 K, with features at g = 2.17, 1.9, 1.68. The broad signal was observed in both soluble and membrane-bound preparations. Its midpoint potential was -320 mV. Its integrated intensity was approximately equal to that of Centre 1, if its broad outer wings were taken into account. Consideration of the ESR properties of this signal, together with the amino acid sequence of the frdB subunit of the enzyme, indicates that Centre 2 is a [4Fe-4S] cluster which, in its reduced state, enhances the spin relaxation of the [2Fe-2S] Centre 1.     

Assignment of ESR signals of Escherichia coli terminal oxidase complexes

The ESR signals of all the major components of the aerobic respiratory chain of Escherichia coli were measured and assigned at liquid helium temperature. Cytochrome b-556 gives a weak high-spin signal at g = 6.0. The terminal oxidase cytochrome b-562 . o complex gives signals at g = 6.0, 3.0 and 2.26, and the terminal oxidase cytochrome b-558 . d complex gives signals at g = 6.0, 2.5 and 2.3. A signal derived from cupric ions in the purified cytochrome b-562 . o complex was observed near g = 2.0. It was shown by the effects of KCN or NaN3 on cytochromes under the air-oxidized conditions that cytochrome o has a high-spin heme and cytochrome d has a low-spin heme. The E'm values for cytochromes b-558 and d, respectively, determined by potentiometric titration of the ESR signals were 140 and 240 mV in the membrane preparation, and 30 and 240 mV in the purified preparation. The oxidized cytochrome d gave intense low-spin signals at g = 2.5 and 2.3, while cytochrome d under the air-oxidized conditions gave corresponding signals of only very low intensity. These results suggested that most of the cytochrome d under the air-oxidized conditions contains a diamagnetic iron atom with a bound dioxygen.     

A flash photolysis ESR study of photosystem II signal IIvf, the physiological donor to P-680+.

In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II (g approximately 2.0045, deltaHpp approximately 19G). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with "red" illumination (lambda less than 680 nm) and occurs stoichiometrically in a 1:1 ratio with P-700+. The Photosystem II transient is formed in less than 100 mus and decays via first-order kinetics with a halftime of 400-900 mus. Additionally, the t1/2 for radical decay is temperature independent between 20 and 4 degrees C; however, below 4 degrees C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal-mol-1. Inhibition of electron transport through Photosystem II by o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (1-5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this "quinoidal" type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, P-680+.     

Free radicals and oxidative phosphorylation in chemical carcinogenesis

Rat liver mitochondria were investigated at DEN induced chemical cancerogenesis. The intensity of ESR signal (g = 2.00) at 77 K was measured in parallel with respiratory control indicating the relationship between oxidation and ATP synthesis levels. Correlation between these values was obtained. Consequently the change of ESR signal (g = 2.00) intensity at cancerogenesis may be to a great extent associated with the state of the coupling system in mitochondria.     

Study of steroid-proteininteractions by electron spin resonance spectroscopy. Binding of a spin-labelled dihydrotestosterone to bovine serum albumin.

The interaction of bovine serum albumin with dihydrotestosterone bearing a spin label at C-3 was studied using electron spin resonance (ESR) spectroscopy. Quantitative binding parameters (Ka approximately 10(5) M-1; maximum binding capacity; two sites/mol albumin) obtained by ESR were in good agreement with those given by equilibrium dialysis. ESR study at various temperatures allowed the calculation of the thermodynamic parameters of the steroid-protein interaction: deltaG=-6.8 kcal/mol; deltaH=-7.9 kcal/mol; deltaS=-3.2 cal/mol per degree and confirmed a transition temperature of about 65 degrees C for albumin. Na, Liland Ca salts had a generally favorable effect on the interaction whereas other ions (e.g. Hg, Cu) impaired the binding process. Study of the width of the ESR spectra of the protein-bound spin-labelled steroid and extrapolation of a 2 T value to infinite viscosity (Azz coupling constant) indicated a non-polar binding site, which became increasingly hydrophobic as the temperature was raised. Since this methodology can give both pertinent quantitative and qualitative data, ESR spectroscopy should be of value in the study of steroid-protein interactions of biological significance.     

Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase

Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase.     

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.     

The source of the iron binding with nitric oxide in activated macrophages]

No decrease in iron-sulphur centers was found in cultured macrophage cells (J774) after the treatment with nitric oxide (10(-7) M NO/10(7) cells) during 5 min. The center content was controlled by the electron spin resonance (ESR) method. The macrophages pretreated with dithionite + methyl viologen showed the formation of dinitrosyl iron complexes (DNIC) with a characteristic ESR signal at g approximately 2.03. The data suggest that loosely bound nonheme iron (free iron) mostly contributes to the formation of these complexes. Iron from iron-containing proteins does not release from these centers under the direct action of nitric oxide. The iron-sulphur centers can be destroyed by the products of nitric oxide oxidation (NO2, N2O3, etc.) as oxidizing and acid agents.     

Evidence that electron-dense bodies in Cyanidium caldarium have an iron-storage role

The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs. Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm. Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0. The electron spin resonance (ESR) spectrum of the intact C. caldarium cells showed an isotropic signal at a g value of 2.00. Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal. EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA. The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal. Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.     

pH and microwave power effects on the electron spin resonance spectra of Rhus vernicifera laccase and Cucumis sativus ascorbate oxidase

The present study shows that the electron spin resonance (ESR) spectral features of Rhus laccase depend considerably on the pH value of the enzyme solution and the irradiated microwave power. Because of the local protein structure change, the type 1 copper is appreciably autoreduced at alkaline pH as monitored both by the ESR and absorption spectroscopies. In addition, the ESR signal of the type 2 copper, especially its g perpendicular region, becomes prominent at alkaline pH. Protein dissociation from a water or an imidazole group coordinated to the type 2 copper is supposed to be responsible for this behavior. Besides above pH effects, the g perpendicular component of the type 2 copper ESR signal is obscured with rising microwave power level. The power saturation behavior of native laccase and its derivatives reveals that the type 2 copper is more easily saturated than the type 1 copper. Cucumis ascorbate oxidase also exhibits similar behavior upon pH variation and microwave power saturation.     

Free Radicals and Sod Activity of Jaw Cyst. Direct Measurement and Spin Trapping Studies by Esr

Free radicals produced in the fluid of jaw cysts were directly measured at room temperature using ESR. With these samples, SOD activity of the cyst fluid was measured by the ESR spin trapping method with DMPO as a trapping agent. Freeze-dried samples of cyst fluid showed a broad ESR signal at g = 2.005. Relative signal intensity of samples from jaw cysts with inflammation was higher than jaw cysts without inflammation. SOD activity of cyst fluid with high viscosity showed higher values than that of cyst fluid with low viscosity. We suggest that free radicals produced in jaw cyst damage tissues while higher SOD activity of cyst fluid play a role in a self-defense mechanism against free radicals.