Formation Of Thiyl Radicals Research Articles

Internal spin trapping of thiyl radical during the complexation and reduction of cobalamin with glutathione and dithiothrietol

The activation of cobalamin requires the reduction of Cbl(III) to Cbl(II). The reduction by glutathione and dithiothreitol was followed using visible spectroscopy and electron paramagnetic resonance. In addition the oxidation of glutathione was monitored. Glutathione first reacts with oxidized Cbl(III). The binding of a second glutathione required for the reduction to Cbl(II) is presumably located in the dimethyl benzimidazole ribonucleotide ligand cavity. The reduction of Cbl(III) by dithiothreitol, which contains two thiols, is much faster even though no stable Cbl(III) complex is formed. The reduction, by both thiol reagents, results in the formation of thiyl radicals, some of which are released to form oxidized thiol products and some of which remain associated with the reduced cobalamin. In the reduced state the intrinsic lower affinity for the benzimidazole base, coupled with a trans effect from the initial GSH bound to the β-axial site and a possible lowering of the pH results in an equilibrium between base-on and base-off complexes. The dissociation of the base facilitates a closer approach of the thiyl radical to the Co(II) α-axial site resulting in a complex with ferromagnetic exchange coupling between the metal ion and the thiyl radical. This is a unique example of 'internal spin trapping' of a thiyl radical formed during reduction. The finding that the reduction involves a peripheral site and that thiyl radicals produced during the reduction remain associated with the reduced cobalamin provide important new insights into our understanding of the formation and function of cobalamin enzymes.

Superoxide Induces Endothelial Nitric-oxide Synthase Protein Thiyl Radical Formation, a Novel Mechanism Regulating eNOS Function and Coupling

An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.

Reaction of hydroxyl radicals with S-nitrosothiols: Formation of thiyl radical (RS•) as the intermediate

The decomposition studies of S-nitrosothiols (RSNO) are important due to their potential role in vivo in connection with the storage and transport of nitric oxide (•NO) within the body. Reactions of hydroxyl radicals (•OH) with a number of RSNOs (S-nitroso derivatives of N-acetyl-dl-penicillamine, l-cysteinemethylester, N-acetylcysteamine, and dl-penicillamine) in aqueous medium at neutral and acidic pH have been reported in the present study. Radiation chemical technique (steady state and pulse radiolysis) has been utilized for the determination of the reaction rate constants, the end product analyses, and the transient intermediate species. The rate constants for the reaction of •OH with the selected RSNOs were determined using a competition kinetic method with 2′-deoxy-d-ribose as the competitor. All the rate constants were found to be of the order of diffusion controlled (1010 M−1 s−1). The degradation yield of RSNOs was found to be quantitative (i.e., G(–RSNO) ≈ G(•OH)) at neutral and acidic pH. The major products of decomposition were the respective disulfide (RSSR) and nitrite (NO2 −). A good material balance is also obtained between the degradation yield and the formation of the products (i.e., G(–RSNO) ≈ G(RSSR) + G(NO2 −)). The major transient intermediate was the thiyl radical (RS•). Its intermediacy was confirmed by making use of the electron transfer reaction of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS2−) to RS•, which results in the formation of ABTS•− having a transient absorption spectrum with λmax at 410 nm. Based on these results, a generalized reaction mechanism is deduced for the reaction of •OH with RSNO.

ChemInform Abstract: Perthiyl Radicals, Trisulfide Radical Ions, and Sulfate Formation. A Combined Photolysis and Radiolysis Study on Redox Processes with Organic Di- and Trisulfides.

The formation of perthiyl and thiyl radicals has been studied in a combined photochemical and radiation chemical investigation of the di- and trisulfides of penicillamine and cysteine, together with glutathione disulfide, cystamine dihydrochloride, and dithiodiglycolic acid in aqueous solution.

Abstract 5209: Oxidative Stress Uncouples eNOS in a BH4-independent Manner via the Formation of Protein Thiol-radicals

Imbalance of nitric oxide (NO) and superoxide (·O 2 − ) production in the endothelium contributes to many cardiovascular diseases, including hypertension, atherosclerosis, and heart failure. Under conditions of oxidative stress, NO synthase (NOS) can switch from NO to ·O 2 − generation. We have demonstrated that endothelial NOS (eNOS) is reversibly S-glutathiolated in vivo and in vitro , in response to oxidative stress. This redox modification of eNOS leads to uncoupling, with decreased eNOS-derived NO and increased ·O 2 − . There are several proposed mechanisms that can lead to protein S-glutathiolation: thiol-disulfide exchange with GSSG; formation of thiyl radicals, sulphenic acid, or protein-S-nitrosothiols, which in turn react with GSH. Here we demonstrate the oxidant induced formation of an eNOS protein thiyl radical using immunoblotting, immunostaining, and mass spectrometry. BH 4 -free eNOS was used to self generate ·O 2 − , which in turn can modify sulfhydryl groups to form protein radicals. We trapped these short lived protein radicals with the spin trap DMPO. Immunoblotting using an anti-DMPO antibody, demonstrated the formation of eNOS protein radicals, which were abolished by SOD and L-NAME, and were Ca 2+ /CaM sensitive, indicating that protein radical formation is due to ·O 2 − generation from the eNOS heme domain. With BH 4 reconstituted eNOS, which will only generate NO, formation of the eNOS protein radical is completely inhibited. Furthermore, in endothelial cells treated with menadione to trigger cellular ·O 2 − generation, formation of eNOS protein radical as visualized with confocal microscopy was increased compared to control, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Using mass spectrometric analysis, we identified Cys908 as the only residue involved in protein radical formation. Of note, we have also demonstrated the oxidant-induced S-glutathiolation of Cys908. Thus, eNOS protein radical formation provides the groundwork for a mechanism of ·O 2 − -directed regulation of eNOS, involving S-glutathiolation; defining a unique pathway for the redox regulation of cardiovascular function. This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

Light-induced aggregation of type I soluble tumor necrosis factor receptor

We evaluated the effect of UV-B light at 302 nm on a model therapeutic protein, 2.6 D type I soluble tumor necrosis factor receptor (sTNF-RI). This protein contains a single Trp at position 97 and seven native disulfide bonds along its interior from the N to the C-terminus. At a protein concentration of 0.1 mg/mL photoirradiation was found to induce the formation of soluble disulfide cross-linked dimers with greater levels of these species formed at pH 8 than at pH 5. Intermolecular disulfide formation was also directly correlated with the photoinduced unfolding of the protein as measured by changes in secondary structure by CD spectroscopy. Trp was implicated as the initiator of the observed photoreactions by the detection of the Trp oxidation products and the absence of dimer formation when Trp97 was replaced with Gln. Reactive oxygen species or triplet state species of Trp were not involved in the reaction suggesting that disulfides were cleaved through one-electron reduction by either hydrated or peptide bound electrons produced by the photoirradiated Trp resulting in thiyl radical formation with disruption of the protein structure and intermolecular cross-linking. Photodegradation was not prevented by deoxygenation, methionine or sucrose commonly used for formulation of biopharmaceuticals. To our knowledge this is the first report directly documenting disulfide mediated aggregation through thiyl radical formation initiated by photoirradiation of Trp. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3182–3199, 2009

Decomposition of S‐Nitrosothiols Induced by UV and Sunlight

Photochemical release of nitric oxide (NO) from the S‐nitroso derivatives of glutathione, L‐cysteine, N‐acetyl‐L‐cysteine, L‐cysteinemethylester, D,L‐penicillamine, N‐acetyl‐D,L‐penicillamine, and N‐acetylcysteamine has been investigated at neutral and acidic pH. The release of NO from RSNO is one of the key reactions that could be utilized in photodynamic therapy. The UV‐VIS and HPLC analyses have shown that under argon saturated conditions, disulfide (RSSR) is the major product of UV as well as sunlight induced decomposition. While in aerated conditions, nitirite—the end product of the oxidation of NO—was also observed along with disulfide. The formation of thiyl radical as the intermediate was reconfirmed by laser flash photolysis. The initial rate of formation of NO was on the order of 10−5 dm3 mol−1 s−1. The quantum yields of these reactions were in the range of 0.2–0.8. The high quantum yields observed in the photo induced release of NO from RSNO using both UV and sunlight demonstrate the potential application of these reactions in photodynamic therapy.

Mechanisms of Protein Damage Induced by Cysteine Thiyl Radical Formation

This perspective covers the reactions and underlying kinetics of peptide and protein thiyl radicals specifically with respect to reversible hydrogen atom transfer processes, which may ultimately lead to the oxidation and/or epimerization of amino acids adjacent to the thiyl radicals.

The reactivity of ortho-methoxy-substituted catechol radicals with sulfhydryl groups: Contribution for the comprehension of the mechanism of inhibition of NADPH oxidase by apocynin

Redox processes are involved in the mechanism of action of NADPH oxidase inhibitors such as diphenyleneiodonium and apocynin. Here, we studied the structure-activity relationship for apocynin and analogous ortho-methoxy-substituted catechols as inhibitors of the NADPH oxidase in neutrophils and their reactivity with peroxidase. Aiming to alter the reduction potential, the ortho-methoxy-catechol moiety was kept constant and the substituents at para position related to the hydroxyl group were varied. Two series of compounds were employed: methoxy-catechols bearing electron-withdrawing groups (MC-W) such as apocynin, vanillin, 4-nitroguaiacol, 4-cyanoguaiacol, and methoxy-catechol bearing electron-donating groups (MC-D) such as 4-methylguaiacol and 4-ethylguaiacol. We found that MC-D were weaker inhibitors compared to MD-W. Furthermore, the radicals generated by oxidation of MC-W via MPO/H2O2, but not for MC-D, were able to oxidize glutathione (GSH) as verified by the formation of thiyl radicals, depletion of GSH, and recycling of the ortho-methoxy-catechols during their oxidations. The capacity of oxidizing sulfhydryl (SH) groups was also verified when ovalbumin was incubated with MC-W, but not for MC-D. Since the effect of apocynin has been correlated with inactivation of the cytosolic fractions of the NADPH oxidase complex and its oxidation during the inhibitory process develops a special role in this process, we suggest that the close relationship between the reactivity of the radicals of MC-W compounds with thiol groups and their efficacy as NADPH oxidase inhibitor could be the chemical pathway behind the mechanism of action of apocynin and should be taken into account in the design of new and specific NADPH oxidase inhibitors.

Sulfur-alkylation-initiated Cp ∗Ru thiyl radicals

The formation of thiyl radicals from [Cp ∗Ru III{κ 3 SSS′-tpdt}] ( 1A) and [Cp ∗Ru III{κ 3 SSN-apdt}] ( 1B) {Cp ∗ = η 5-C 5Me 5; tpdt = S(CH 2CH 2S −) 2; apdt = HN(CH 2CH 2S −) 2} has been initiated by thiolate alkylation or oxidation with iodine. Subsequent electron transfer processes yielded disulfide-bridged dinuclear complexes. The mechanistic pathways of these processes will be discussed.

Glutathione-induced radical formation on lactoperoxidase does not correlate with the enzyme's peroxidase activity

Lactoperoxidase (LPO) is believed to serve as a mediator of host defense against invading pathogens. The protein is more abundant in body fluids such as milk, saliva, and tears. Lactoperoxidase is known to mediate the oxidation of halides and (pseudo)halides in the presence of hydrogen peroxide to reactive intermediates presumably involved in pathogen killing. More recently, LPO has been shown to oxidize a wide diversity of thiol compounds to thiyl free radicals, which ultimately lead to the formation of a protein radical characterized by DMPO-immunospin trapping. In the same study by our group the authors claimed that a consequence of this protein radical formation was the inactivation of LPO (Guo et al., J. Biol. Chem. 279:13272–13283; 2004). Here we demonstrate that although thiyl radical formation does lead to LPO radical production, the formation of this radical is unrelated to the enzyme's activity. We suggest the source of this misleading interpretation to be the binding of GSH to ELISA plates, which interferes with ABTS and guaiacol oxidation. In addition, DMPO-GSH-nitrone adducts bind to ELISA plates, leading to ambiguities of interpretation since we have demonstrated that DMPO-GSH nitrone does not bind to LPO, and only LPO-protein-DMPO-nitrone adducts can be detected by Western blot.

The Reaction of Hydrogen Atoms with Methionine Residues: A Model of Reductive Radical Stress Causing Tandem Protein–Lipid Damage

The occurrence of tandem damage, due to reductive radical stress involving proteins and lipids, is shown by using a biomimetic model. It is made of unsaturated lipid vesicle suspensions in phosphate buffer in the presence of methionine, either as a single amino acid or as part of a protein such as RNase A, which contains four methionine residues. The radical process starts with the formation of H(.) atoms by reaction of solvated electrons with dihydrogen phosphate anions, which selectively attack the thioether function of methionine. The modification of methionine to alpha-aminobutyric acid is accompanied by the formation of thiyl radicals, which in turn cause the isomerization of the cis fatty acid residues to the trans isomers. The relationship between methionine modification and lipid damage and some details of the reductive radical stress obtained by proteomic analysis of irradiated RNase A are presented.

Experimental and field evidence for thiyl radical-induced stereomutation of alkenones and other lipids in sediments and seawater

Possible origins were investigated for unusual cis/trans alkenones previously identified after incubation of a marine haptophyte Emiliania huxleyi strain with bacterial consortia isolated from cyanobacterial microbial mats. Diunsaturated alkenones and alkenoates isolated from the strain were incubated in the presence of ethanethiol to examine whether double bond stereomutation (i.e., cis/trans equilibration without double bond migration) could be induced by thiyl radicals. Significant stereomutation was observed after 4–5 days of incubation. The reaction was strongly attenuated when incubation was carried out in the presence of a free radical quencher (2,4,6-tri- tert-butylphenol), confirming the involvement of a thiyl radical-dependent mechanism. Room temperature incubation of E. huxleyi in natural seawater amended with ethanethiol also resulted in stereomutation of the alkenones and a significant increase in the measured value for the palaeotemperature proxy U 37 K ′ . In this case, production of thiyl radicals is likely initiated by peroxyl and hydroxyl radicals arising from the homolytic decomposition of photochemically or autoxidatively produced hydroperoxides often found in senescent algal cells. Thiyl radical-induced stereomutation of alkenones also acted in the presence of sulfides and transition metals (e.g., Fe 3+). Based on these results, alkenone cis/trans isomerization previously observed during bacterial incubations of E. huxleyi cells under sulfate-reducing conditions is attributable to formation of thiyl radicals either from methanethiol produced by bacterial degradation of DMSP (β-dimethylsulfoniopropionate) or from oxidation of thiolate ions by transition metals. Analysis of a sediment interval from the Unit II deposit in a Black Sea core revealed the presence of stereomutated isomers, confirming that such a reaction can be geochemically significant. Stereomutation could explain previous observations of a surprising increase in U 37 K ′ values with depth in the anoxic water column of the Black Sea. Since this stereomutation process is induced under sulfate-reducing conditions, it may occur widely in anoxic environments, a prospect that should be checked as part of palaeotemperature studies. Other commonly found lipids such as phytol and unsaturated fatty acids can also be affected by these reactions, thereby providing additional means for recognizing the occurrence of this phenomenon.

The Role of Thiols in Liver Ischemia-Reperfusion Injury

Thiol-containing compounds have an essential role in many biochemical reactions due to their ability to be easily oxidised and then quickly regenerated. Main representatives are glutathione, lipoic acid and thioredoxin which are synthesised de novo in mammalian cells. N-acetylcysteine and Bucillamine are synthetic thiols which have been administered in experimental and clinical studies for treatment of conditions associated with oxidative stress. Ischemia and reperfusion (I/R) injury is characterised by significant oxidative stress, characteristic changes in the antioxidant system and organ injury leading to significant morbidity and mortality. I/R occurs in a variety of clinical settings such as liver resection, organ transplantation, haemorrhagic shock with fluid resuscitation, heart surgery, myocardial infarction followed by reperfusion and laparoscopic surgery. In these circumstances, the administration of antioxidant agents such as thiols, could provide protection from the harmful effects of I/R injury. However, the ability of thiol compounds to reduce free radicals is associated with the formation of thiyl radicals and the rate and efficiency of removal of thiyl radicals has a critical effect on antioxidant or prooxidant actions of thiols in the cells. The aim of this review is to present the mechanisms by which thiols act as antioxidants and signalling molecules and the experimental and clinical evidence regarding their role in I/R injury with a particular emphasis on liver I/R. The current evidence suggests that thiols ameliorate I/R injury and that their clinical significance should be further evaluated in large scale randomised clinical trials.

Heteroaromatic Thiols as Co-initiators for Type II Photoinitiating Systems Based on Camphorquinone and Isopropylthioxanthone

The heteroaromatic thiols (imidazole, oxazole, and thiazole derivatives) were investigated in regard to their abilities to function as co-initiators in free-radical photopolymerizations induced by camphorquinone and isopropylthioxanthone. As shown by the kinetic data, these heteroaromatic thiols are efficient co-initiators with activities comparable to aromatic amines. They quenched the triplet states of isopropylthioxanthone and camphorquinone with rate constants determined to be on the order of 109 and 108 M-1 s-1, respectively. The results suggested that rates of polymerization of a low-viscosity monomer (triethylene glycol dimethacrylate) in an inert atmosphere are dependent on the quantum yields of formation of primary thiyl radicals Φ. However, this effect was also moderated by the reactivities of the thiyl radicals that were generated in the photosensitization stage. The observed activities of the initiator/thiol systems also depended on the possibility of initiator photoreduction by the monomer and, in air, on the ability of the co-initiators to reduce oxygen inhibition.

Intramolecular Electron Transfer between Tyrosyl Radical and Cysteine Residue Inhibits Tyrosine Nitration and Induces Thiyl Radical Formation in Model Peptides Treated with Myeloperoxidase, H2O2, and NO2-: EPR SPIN TRAPPING STUDIES

We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H(2)O(2), and nitrite anion (NO(2)(-)) and with horseradish peroxidase and H(2)O(2). Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H(2)O(2) and MPO/H(2)O(2)/NO(2)(-). The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH(3)) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H(2)O(2)/NO(2)(-). However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H(2)O(2)/NO(2)(-)/YCSSCH(3) system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H(2)O(2)/(*)NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed.

Voltammetric and Spectroscopic Studies on the Interaction of Pentoxifylline with Cysteine in the Presence and Absence of UV Irradiation

Abstract The interaction of pentoxifylline (PTX), a methylxanthine derivative drug, with cysteine on the hanging mercury drop electrode studied by square-wave voltammetry, cyclic voltammetry and UV-Vis spectroscopy at phosphate buffer (pH 7.4) in the presence and absence of UV irradiation. The addition of PTX to cysteine solution results in the decrease of peak currents of mercurous cysteine thiolate and the shift of its voltammetric reversible peak towards positive potentials. Electrochemical data indicated a 1:1 molecular complex formation of PTX with cysteine by means of electrostatic interaction and intermolecular forces. For the long UV irradiation times (25–85 min), voltammogram of the cysteine solution gave the reduction peaks of sulphonyl radical (−0.056 V), disulfidic anion (−0.684 V) and free cystine (−0.800 V) due to probably the formation of thiyl radical. With adding of PTX to the UV irradiated cysteine solution, a new peak at −0.292 V was observed. The peak at −0.292 V was also obtained for the cysteine and PTX mixture under UV irradiation, but the peaks of sulphonyl radical and disulphidic anion were no seen. As a result, this irreversible peak at −0.292 V may be assigned to the reduction of an electroactive species which is formed from PTX–cysteine complex under UV irradiation. PTX prevents the formation of thiyl radical and its further oxidation.

Thiyl radical reaction with amino acid side chains: rate constants for hydrogen transfer and relevance for posttranslational protein modification.

Thiyl radicals are prominent intermediates during biological conditions of oxidative stress and have been suggested to be involved in the mutagenic effects of thiols. While several enzymatic processes rely on the formation and selective reactions of protein thiyl radicals with substrates, such reactions may represent a source for biological damage when occurring uncontrolled during oxidative stress. For example, intramolecular hydrogen transfer reactions to protein cysteine thiyl radicals may lead to secondary amino acid oxidation products, which may represent starting points for protein aggregation and/or fragmentation. Here, we have used a kinetic NMR method to determine rate constants, k(sc), for hydrogen transfer reactions between thiyl radicals and amino acid side chain C-H bonds at 37 degrees C. Rate constants cover a range between k(sc) <or= 1 x 10(3) M(-1) s(-1) (Val) and k(sc) = 1.6 x 10(5) M(-1) s(-1) (Ser). On the basis of these values and earlier data, model calculations are performed, which will demonstrate that protein thiyl radicals may attack protein C-H bonds via intramolecular hydrogen transfer at physiological conditions, potentially resulting in irreversible protein damage.

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS•+ radical cation: 65% and 89% inhibition for compound [3] at 10–4 M and 5 × 10–5 M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models.

Calorimetric and spectroscopic properties of small globular proteins (bovine serum albumin, hemoglobin) after free radical generation

Mild oxidation of SH-containing proteins (serum albumin, hemoglobin) by Ce(IV)-ions in the presence of the spin trap phenyl- tert-butylnitrone (PBN) resulted in the appearance of strongly immobilized nitroxide free radicals which evidences the formation of thiyl radicals on the thiol site of the proteins. In hydroxyl free radical generating system a fraction of strongly immobilized nitroxide radicals was also detected in these proteins, which implies that the oxidation of a fraction of the thiol groups was also involved in the free radical reaction. According to the differential scanning calorimetry (DSC) experiments the melting processes of the proteins were calorimetrically irreversible, therefore the two-state kinetic model was used to evaluate the experiments. The results support the view that site-specific interaction of SH-containing proteins with hydroxyl and thiyl free radicals is able to modify the internal dynamics of proteins and affect the conformation of large molecules.