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The influence of oxidized fibrinogen on the intensity of luminol-dependent chemilumin escence of blood leukocytes, stimulated by opsonized zymosan was studied. It was shown that the introduction of fibrinogen modified by UV-irradiation in to a suspension of cells resulted in a significant increase in the intensity of the luminol-dependent chemiluminescence of leukocytes. It was suggested that oxidized fibrinogen can influence blood leukocytes, enhancing their functional activity.

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.

The effect of metal cations on copper-catalyzed lipid peroxidation (LPO) of low density lipoproteins (LDL) was examined. The presence of metal cations in the incubation media containing LDL (0.8 mg protein/ml) and CuSO4 (0-80 microM) influenced on LPO of LDL as evident by the measurement of TBARS. With the concentrations of CuSO4 less than 10 microM, the metal cations caused an increase in LDL peroxidation. Zn2+ appeared to be the most effective inductor, Mn2+ was less effective, and the influence of Ca2+ and Mg2+ was insignificant. With greater CuSO4 concentrations Mg2+ showed no effect on TBARS formation in LDL while the addition of other nontransition metal cations to the incubation mixture led to the inhibition of LDL peroxidation. The capacity for inhibition decreased in the row Mn2+ > Zn2+ > Ca2+ > Mg2+. The possible mechanism explaining these results may be in the competition of metal ions for copper binding sites on LDL. Our results allow to suggest the existence of two types of copper binding sites on LDL, tight-binding sites which are non-effective in LPO and effective weak-binding sites.

Copper-catalyzed lipid peroxidation (LPO) in low density lipoprotein (LDL) and two subfractions of high density lipoprotein (HDL2 and HDL3) isolated from human serum was studied. The levels of LPO were estimated as thiobarbituric acid-reactive substances (TBARS). The amount of TBARS increased in a time-dependent manner when either LDL, HDL2 or HDL3 (0.4-1.2 mg protein/ml) was incubated at 37 degrees C during 4 h with phosphate-buffered saline containing CuSO4 (2-80 microM). The increase in CuSO4 concentration to some value therewith caused the enhancement of the rate of TBARS formation but the following increase in CuSO4 concentration beyond that value didn't already affect the kinetics of LPO in lipoproteins. It was also shown that the lesser was the concentration of lipoprotein the lesser CuSO4 concentration was needed to reach maximal rate of TBARS formation. The data obtained point to the versatility of the mechanism of copper-catalyzed oxidation of low and high density lipoproteins and suggest that the decisive role in the process belongs to copper ions bound to lipoprotein particle.

Rapid dissociated Fe(III)-ascorbic acid complexes are formed after the mixing of Fe(II) (100 mkM) with ascorbic acid (1 mM) at pH-7,4. As a result of these complexes formation Fe(II) concentration was decreased in solution, but o-phenantrolin addition or pH decreased up to 6.1 lead to the destruction of these complexes and reduction of iron up to Fe(II). Iron-ascorbic acid paramagnetic complexes were not observed in the perfused rat liver homogenate. Probably, these facts were due to the presence of other stronger reductants in the tissue.

Using ESR spectroscopy of spin probes and spin labelling, the effects of lipid peroxidation (LPO) on the molecular organization of Ca-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membranes were studied. Accumulation of LPO products caused an increase of the hydrophobicity and a decrease of the mobility of the Ca-ATPase polypeptide chain fragment immediately proximal to the enzyme active site. Induction of LPO in the membranes resulted in a decrease of the thermal stability of the lipoprotein Ca-ATPase complex which occurred as a biphasic process. At temperatures of 36-38 degrees C this thermodenaturation consisted in a shift of the Ca-transport ability towards a lower temperature region corresponding to the shift in the low temperature break on the Arrhenius plots for parameter 2A'zz of the maleimide spin label. Within the temperature range of 49-52 degrees C an earlier LPO-induced denaturation determines the low temperature shift of the point for a sharp decrease of the Ca-ATPase activity and of the corresponding break for parameter 2A'zz as well as the dissociation point for the lipoprotein Ca-ATPase complex and the corresponding breaks on the Arrhenius plots for the solubilization parameter, alpha, and the order parameter of spin probes, S.

The dependence of the ESR spectral parameters of spin labelled sarcoplasmic reticulum membranes on the amount of bound maleimide spin label is found which is shown to result from non-equivalence of the different SH groups rather than from enzyme activity changes on modification. Some structural characteristics (local polarity, the accessibility to water soluble reagents, the distance from ATP binding site) of the fast and slowly reacting SH groups were studied using selective labelling technique. The main contribution to the ESR absorption for both types of thiol groups gives the spin labelled Ca-ATPase. The slow rotational mobility of both types of strongly immobilized spin labels is measured using microwave saturation technique. This mobility is shown to be due essentially to intramolecular motion in different regions of the Ca-ATPase. The frequency of this motion correlates with the enzyme activity and it is sensitive also to the interaction of the enzyme with the substrates (ATP, acetylphosphate) and to the changes of lipid-protein interaction by temperature variation.

The mechanism of free radical generation in the reaction of ferrous ion with t-butyl and linolenic acid hydroperoxide was investigated by spin trapping method. The t-butoxyl, methyl, linolenic acid alkoxyl and alkyl radical spin adducts EPR spectra were observed and identified.

Formation of free radicals under UV-irradiation of egg phosphatidyl choline, and synthetic phosphatidyl cholines and their analogs, as well as of their model compounds was studied. When saturated fat lipids are UV-irradiated radicals--CH2CH2 are accumulated at the expense of the breakage of--CH2CH2--COOH-bond. Radicals CH3--OCH2, HCO are formed in aminoalcohols due to the breakage of --OCH2--CH2N+(CH3)3- and--CH2--N+(CH3)3-bonds. It has been found that in natural and synthetic phospholipids radicals are formed in the course of photochemical reactions in complex ether and amine groups. The mechanism of radical formation is discussed.

The interaction between 3-acyl-2-(6-doxylpalmitoyl) phosphatidyl choline used as a hydrophobic spin probe and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes of rabbit and carp white skeletal muscles was studied. The spin label incorporation into ATPase preparations was performed at initial steps of ATPase isolation by incubating reticulum membranes with the spin probe in the presence of cholic acid. A comparison of the molecular mobility of the probe incorporated into ATPase preparations and into liposomes formed from ATPase phospholipids demonstrated that the presence of the protein in the membrane produces the same effect on the probe mobility as does the decrease of temperature by 10-15 degrees C. The molecular mobility of the probe in the ATPase preparation is increased during protein thermal denaturation. The breaks on the Arrhenius plots for the probe molecular mobility are revealed at the same temperatures (25 degrees for rabbit reticulum and 16 degrees for carp reticulum) as those for the ATPase activity.

When incubating SOD for two hours in the buffer with pH 6.0 practically complete loss of the activity of enzyme was observed at insignificant changes of the absorption spectra in the region of 450 and 680 nm. Irradiation of SOD solution with helium-neon laser resulted in the dose-dependent reduction of spectral and enzymic properties. Similar effect of the red light was also observed when SOD was inactivated with hydrogen peroxide: laser irradiation brought about the reduction of initial absorption spectrum of the enzyme. One can believe that the therapeutic effect of helium-neon laser radiation is conditioned, at least partially, by photochemical reactivation of SOD inactivated under pathological conditions (inflammation, hypoxia) in the tissues.

The interaction between free fatty acids and Ca2+-dependent ATPase, an intrinsic protein of sarcoplasmic reticulum membranes, was studied with relevance to the changes in membrane permeability induced by free fatty acids. It was found that only unsaturated fatty acids increase the permeability of reticulum membranes for Ca2+, this effect being completely reversible. The increase in the membrane permeability by fatty acids is coupled to a generation of a channel for Ca2+ efflux under effect of Ca2+-dependent ATPase. The interaction between fatty acids and Ca2+-dependent ATPase was demonstrated by the protein fluorescence and electron paramagnetic resonance methods, using spin-labelled fatty acid derivatives. A model demonstrating the increase of sarcoplasmic reticulum membrane permeability for Ca2+ in the presence of the fatty acid-Ca2+-dependent ATPase complex is proposed.

Interaction of nucleotides with histones F-1, F-2b, F-2a1, F-3 have been studied by NMP method. It has been found that the interaction of nucleotides with histones increase in the line F-1. F-2b, F-3, F-2a1. It is suggested that histone F-1 is bound with the nucleotides mainly by electrostatic forces. Bounding of histones F-2a1 and F-3 with nucleotides is due not only to electrostatic sinteractions but also to more specific bonds of the base ring with protein.

It is shown by ESR method that 8-methoxypsoralen (8-MOP) under low temperature UV-irradiation has no influence on free radical formation of saturated fatty acids (FA). Under the same conditions the quantum yield of free radical photogeneration for unsaturated FA (oleate and limolenate) after addition of 8-MOP increased 7.5-15 times. The ESR spectrum of free radicals generated after interaction of 8-MOP with polyunsaturated FA, may be presented as superposition of signals-CH-(CH = CH)2-, -CH2CHCH2- and -CH2CH2. The molecular mechanism of photosensitized reactions is discussed.

Experimental hypothyreosis in animals was obtained by means of extirpation of thyroid gland; hyperthyreosis was obtained by thyroxine injections intraperitoneally for 10 days. Content of Fe2+ ions of the free pool (not bound with ferritine) was estimated with the ESR method, after treatment with sodium nitrite. A decrease in Fe2+ ions content is observed in liver homogenate of hypothyroid rabbits, and an increase is observed in that of the hyperthyroid animals. Content of Fe2+ ions in cells of liver tissues correlates with the oxidation capacity of the mitochondrial lipids. The changes in the rate of lipid peroxidation under thyroid pathology is supposed to result from changes in free ferrum content.

Changes of structural organization of liposomal phospholipid membranes, after their Fe2+-induced peroxidation were studied at different depth using nitroxyl derivatives of stearic acid. It was established that during Fe2+-induced lipid peroxidation a strong rise in lipid polarity accompanied with immobilization of acyl chains of phospholipids at the depth of 0.6-0.8 nm from the surface was measured. At the same time no significant changes in the structural organization were seen at the depth of 20-22 nm.

Structural and functional modifications of sarcoplasmic reticulum membranes from skeletal muscles by molecular oxygen was studied. Lipid peroxidation (accumulation of 5-10 nmoles hydroperoxides/mg lipids) results in membrane permeability increase for Ca2+-ions whereas the activity of Ca2+-dependant ATPase preserved unchanged. In the temperature range 5-30 degrees C decrease of solubilization parameter alpha (for nitroxide radical 2,2,6,6-tetramethylpiperidin-1-oxyl) was registered while alpha reached the control values at temperatures higher than 30 degrees C. Further increase in lipoperoxide content (more than 20 nmoles/mg lipids) lead to inhibition of Ca2+dependant ATPase. In this case alpha was lower than in intack membranes in the whole temperature interval investigated. The loss of Ca2+-accumulating capacity is explained on the basis of peroxide clusters formation in lipid bilayer regions of sarcoplasmic membrane. One of the mechanisms of Ca2+-dependant ATPase inhibition after lipid peroxidation is the deficiency of polyunsaturated fatty acyls in microenvironment of the enzyme.

Surface potential of human plasma lipoproteins was studied by the use of positively charged spin probe. The calculated values of surface potential of high and low density lipoproteins appeared to be -29 +/- 1 and -16 +/- 1 respectively. It was shown that lipid peroxidation process induces an increase of surface potential of both high and low density lipoproteins. Probably, it is connected with the increase of the negative charge density on their surface. This fact can play an important role in pathogenesis of diseases with lipid metabolism and lipid peroxidation level disorders in plasma (atherosclerosis, ischemic heart disease etc.).

Purified preparations of Ca2+-dependent ATPase were lipid-deleted and incorporated into egg lecithin (EL) and dipalmitoyl lecithin (DPL) liposomes. The temperature dependences of the catalytic activity and of molecular mobility of the spin label (N-1-hydroxyl-2,2,6,6-tetramethyl-4-piperidyl) maleimide linked to a highly reactive SH-group in the vicinity of the active center (15-16 A) and of the fatty acid spin probe (6-doxylpalmitate) located in the protein-lipid moiety were compared. The molecular mobility of the spin label was measured by the saturation transfer method; that of the spin probe was estimated from the maximal splitting value. It was found that the catalytic activity of DPL is correlated with the molecular mobility of the hydrophobic part of ATPase, while that of EL with the segment flexibility in the vicinity of the active center.

On the irradiation of oxidized linolic acid an assymetric ESR signal with g-factor 2.0039 and the linewidth 30--33 Oe has been registered. The signal could be the result of photolysis of hydroperoxides. During prolonged irradiation of all the samples accumulation of alkyl radicals characterized by a symmetric signal with a well-defined ultrafine structure has been observed. An increase of the sample temperature up to 87 K in the presence of oxygen was preceded by transformation of alkyl radical R into RO2. All the radicals disappeared at the temperature of 160 K. All these processes seem to be the first phases of photoperoxidation of unsaturated fat acids in lipid-containing systems, including biological membranes.

The thermo-induced structural changes in lipoproteins (LP) of human plasma were investigated by the electron paramagnetic resonance method, using 5-doxylstearate as a spin probe. LP were shown to undergo thermotropic transformations at temperatures specific for each LP type. Differences were found in the nature of temperature dependences of the order parameter for the probe localized in LP and for isolated lipids. The denser the LP, the more pronounced the differences. 5-Doxylstearate was shown to be localized not only in the lipid phase of LP, but also in the area of the protein--lipid contact. This indicates that 5-doxylstearate is a useful tool for the study of protein--lipid interactions in pathological processes coupled with their modifications.

Using spin trapping method there were discovered and identified the radicals of linolenic acid formed when initiating its peroxidation by the system Fe2+-ascorbate. Mechanism of formation of linolenic acid radicals and their role in initiation of peroxidation were studied. A scheme of reactions of peroxidation initiation in the system Fe2+-ascorbate. linolenic acid is proposed.