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While the Fe(2+)-dithiocarbamate complexes have been commonly used as NO traps to estimate NO production in biological systems, these complexes can undergo complex redox chemistry. Characterization of this redox chemistry is of critical importance for the use of this method as a quantitative assay of NO generation. We observe that the commonly used Fe(2+) complexes of N-methyl-D-glucamine dithiocarbamate (MGD) or diethyldithiocarbamate (DETC) are rapidly oxidized under aerobic conditions to form Fe(3+) complexes. Following exposure to NO, diamagnetic NO-Fe(3+) complexes are formed as demonstrated by the optical, electron paramagnetic resonance and gamma-resonance spectroscopy, chemiluminescence and electrochemical methods. Under anaerobic conditions the aqueous NO-Fe(3+)-MGD and lipid soluble NO-Fe(2+)-DETC complexes gradually self transform by reductive nitrosylation into paramagnetic NO-Fe(2+)-MGD complexes with yield of up to 50% and the balance is converted to Fe(3+)-MGD and nitrite. In dimethylsulfoxide this process is greatly accelerated. More efficient transformation of NO-Fe(3+)-MGD into NO-Fe(2+)-MGD (60-90% levels) was observed after addition of reducing equivalents such as ascorbate, hydroquinone or cysteine or with addition of excess Fe(2+)-MGD. With isotope labeling of the NO-Fe(3+)-MGD with (57)Fe, it was shown that these complexes donate NO to Fe(2+)-MGD. NO-Fe(3+)-MGD complexes were also formed by reversible oxidation of NO-Fe(2+)-MGD in air. The stability of NO-Fe(3+)-MGD and NO-Fe(2+)-MGD complexes increased with increasing the ratio of MGD to Fe. Thus, the iron-dithiocarbamate complexes and their NO derivatives exhibit complex redox chemistry that should be considered in their application for detection of NO in biological systems.

In EPR experiments on mice it was demonstrated that a hydrophobic complex Fe2+ with diethyldithiocarbamate (DETC) is a more efficient selective NO trap than a hydrophilic complex Fe2+ with N-methyl-D-glutamine dithiocarbamate (MGD). This difference can be due to the higher stability of paramagnetic nitrosyl iron complex with DETC (MNIC-DETC) formed by NO binding to Fe2+-DETC in animal tissues in vivo. The complex analogue MNIC-MGD is reversibly oxidized in animal blood to transform into the diamagnetic EPR-silent form. The latter is detectable also in urine of animals, especially of those treated with bacterial lipopolysaccharide which initiates the enhanced NO production in the organism. We suggest that NO2 or peroxynitrite formed from endogenous NO can serve as an agent reversibly oxidizing MNIC-MGD in these animals.

It has been shown by using EPR method that hydrophobic complexes Fe(2+)-diethyldithiocarbamate (DETC) act more efficiently as a selective traps of nitric oxide (NO) in mice organisms than hydrophilic complexes Fe(2+)-N-methyl-D-glutamyldithiocarbamate (MGD). This difference seemed to be due to higher stability of paramagnetic mononitrosyl iron complexes with DETC (MNIC-DETC) formed in vivo in animal tissues in a result of NO binding with Fe(2+)-DETC complexes. Analogous complexes MNIC-MGD appeared a blood were oxidized to diamagnetic, EPR silent form. The latter was also detected in mouse urine especially for animals which were pretreated with bacterial lipopolysaccharide inducing increased NO generation in mice organisms. Nitrogen dioxide or peroxynitrite formed endogenous NO were suggested to be the agents which oxidized MNIC-MGD by reversible way in mice organisms.

When studying the vasodilator activity of dinitrosyl iron complexes (DNIC) with thiol-containing ligands as NO donors, it should be taken into consideration that these complexes depending on the content of thiols in the environment can occur in either form that differ by their EPR, gamma-resonance and optical characteristics and also by their vasodilator effect on isolated blood vessels. The more stable diamagnetic form appears at the ratio Fe2+:RS(-)-1:2. It reversibly dissociates to the monomeric paramagnetic form [(RS-)2Fe+(NO+)2] on increasing the thiol content to the level 20 times and more exceeding the quantity of iron. It is suggested that stabilization of the dimeric form is provided by formation of RS(-)-NO+ bonds between monomeric components of the dimer. This process is favored by a corresponding orientation of the monomers relative to each other. A high stability of the DNIC dimeric form correlates with more prolonged vasodilator effect of this complex as an NO source. Replacement of cysteine by reduced glutathione in dimeric and monomeric forms of DNIC increases both stability of the complexes and the duration of their vasodilator effect.

When studying the vasodilator activity of dinitrosyl iron complexes (DNIC) with thiol-containing ligands as NO donors, it should be taken into consideration that these complexes depending on the content of thiols in the environment can occur in either of two forms that differ by their EPR, gamma-resonance and optical characteristics and also by their vasodilator effect on isolated blood vessels. The more stable diamagnetic form appears at the ratio Fe2+:RS- = 1:2. It reversibly dissociates to the monomeric paramagnetic form [(RS-)2Fe+(NO+)2] on increasing the thiol content to the level 20-times and more exceeding the quantity of iron. It is suggested that stabilization of the dimeric form is provided by formation of RS(-)-NO+ bonds between monomeric components of the dimer. This process is favored by a corresponding orientation of the monomers relative to each other. A high stability of the DNIC dimeric form correlates with more prolonged vasodilator effect of this complex as an NO source. Replacement of cysteine by reduced glutathione in dimeric and monomeric forms of DNIC increases both stability of the complexes and the duration of their vasodilator effect.

Murine tetrameric oxyhaemoglobin and insect monomeric erythrocruorin were studied. A doublet with the Lorentz form of lines (delta EQ = 2.22 mm s-1; delta alpha-Fe = 0.27 mm s-1; gamma 1/2 = 0.29 mm s-1) was observed in the oxyhaemoglobin spectrum at 4.2 K. In the 80-170 K interval the doublet components are distorted, but at T greater than 175 K, the lines again become symmetrical. In the 175-210 K region the value of gamma 1/2 is approximately 0.42 mm s-1. The profiles of the oxyhaemoglobin spectra are not dependent on the nature of the samples (blood; whole, or in aqueous or water-glycerol solutions at different pH values), or on the rate at which the latter are frozen. The oxyerythrocruorin spectra in aqueous solution and in a water-glycerol solution at 80 K and 170 K were doublets with Lorentz lines (the delta EQ values are equal to 2.20 mm s-1 and 2.12 mm s-1, respectively). It is concluded that the characteristics of the oxyhaemoglobin spectra reflect the specific electronic and structural properties of the oxy-complex in this protein. It was found that the oxyhaemoglobin spectra are very adequately described by two doublets with equal delta and gamma 1/2 values, but with different delta EQ values and relative intensities. A model is described in which these doublets correspond to two types of hydrogen bond associated with the distal histidine (E7), involving the terminal atom of molecular oxygen and the oxygen atom bound with the haem iron, respectively.

Reduction of frozen water-glycerol solutions of methemoglobin by thermolysed electrons at 77 degrees K has been studied by Mossbauer spectroscopy. The formation of nonequilibrium hemoglobin states with two slightly different low-spin ferroforms was observed. The latter is explained by the presence of two ferroforms in initial methemoglobin. There was observed relaxation of the conformation - nonequilibrium states up to equilibrium hemoglobin with high-spin Fe(II) at the sample disfreezing.

When mice were given water diet with 57Fe a practically complete exchange in the blood 56Fe and 57Fe was observed during 2 months. 57Fe isotope was accumulated in iron storage of the liver, kidney and spleen. Partial isotope exchange for free iron occurred in these tissues too. The free iron content was not changed. This isotope exchange for free iron did not occur in tissues which were unable to accumulate 57Fe in iron storage.

Nonequilibrium hemoglobin states formed at low-temperature (T = 77K) reduction of its derivatives (MetHb and HbO2) by thermolysed electrons have been studied by Mössbauer spectroscopy. Relaxation of nonequilibrium states at the samples heating was observed. Correlation between the relaxation temperatures and the changes of protein dynamic structure determined from Mössbauer data were stated.

Interaction between Fe2+ ions and melanoproteid granules (MPG) of bovine eye pigment epithelium was studied by gammaresonance spectroscopy. MPG was found to form complexes with bi- and three-valent ferrum ions. MPG can both directly bind Fe2+ ions and oxidize them Fe3+ inactive in prooxidant state and bind the latter. The activity of ferrum ion binding increases when suspension is illuminated with visible light and pH of the incubation solution increases. The protein part of MPG does not participate in the complex formation with ferrum ions. The complex formation proceeds mainly by carboxyl, amino- and imino-groups of melanin polymer.

The possibility of using gamma resonance spectroscopy (GRS) for studying the dynamics of water-protein systems is shown in the present work. The experiments were carried out on an albumin-water system. The results obtained are compared with those obtained by the spin label method under the same conditions. The different behaviour of Fe ions which are firmly- and weakly-related with the protein matrix allows to separate the mobility of the whole protein macromolecule and also the mobility of its subunits.