A. V. Bychkova

Ozone-induced oxidative modification of plasma fibrin-stabilizing factor.

The plasma fibrin-stabilizing factor (pFXIII) function is to maintain a hemostasis by the fibrin clot stabilization. The conversion of pFXIII to the active form of the enzyme (FXIIIа) is a multistage process. Ozone-induced oxidation of pFXIII has been investigated at different stages of its enzyme activation. The biochemical results point to a decrease of an enzymatic activity of FXIIIа depending largely on the stage of the pFXIII conversion into FXIIIа at which oxidation was carried out. UV-, FTIR- and Raman spectroscopy demonstrated that chemical transformation of cyclic, NH, SH and S-S groups mainly determines the oxidation of amino acid residues of pFXIII polypeptide chains. Conversion of pFXIII to FXIIIa proved to increase protein sensitivity to oxidation in the order: pFXIII<pFXIII activated by thrombin<pFXIII in the presence of calcium ions<FXIIIa. The dynamic light scattering data indicate that the three-dimensional structure of pFXIII becomes loosened due to oxidative modification. ESR spectroscopy data also point to conformational changes of the fibrin-stabilizing factor under oxidation. Taking into account these new findings it seems reasonable to assume that the inhibitory/carrier FXIII-B subunits can serve as scavengers of ROS. Hypothetically, this mechanism could help to protect the key amino acid residues of the FXIII-A subunits responsible for the enzymatic function of FXIIIa.

Oxidized modification of fragments D and E from fibrinogen induced by ozone

Ozone-induced free-radical oxidation of fragments D and E from fibrinogen has been studied. The methods of elastic and dynamic light scattering in combination with electrophoresis of unreduced samples have shown the acceleration of enzymatic covalent crosslinking of molecules of oxidation-modified fragment D under the action of factor XIIIa. UV and IR spectroscopy shows that free-radical oxidation of amino acid residues of polypeptide chains catalyzed by ozone affects the cyclic and amino groups, giving rise to generation of mainly oxygen-containing products. Comparison of the IR spectra obtained for the oxidation-modified D and E fragments revealed more significant transformation of functional groups for the D fragment. EPR spectroscopy showed that the rotational correlation time of spin labels bound to the ozonized proteins decreased in comparison with the non-ozonized proteins. The rotation correlation time of the radicals covalently bound to the ozonized D and E fragments suggests that D fragment of fibrinogen is more sensitive to free-radical oxidation followed by local structural changes. Possible causes of different degrees of oxidation for fragments D and E are discussed.

Covalent structure of single-stranded fibrin oligomers cross-linked by FXIIIa

FXIIIa-mediated isopeptide γ-γ bonds are produced between γ polypeptide chains of adjacent monomeric fibrin. Despite the use of the different methodological approaches there are apparently conflicting ideas regarding the orientation of γ-γ bonds. To identify the orientation of these bonds a novel approach has been applied. It was based on self-assembly of soluble cross-linked fibrin protofibrils ongoing in the urea solution of moderate concentrations followed by dissociation of protofibrils in the conditions of increasing urea concentration. The oligomers were composed of monomeric desA fibrin molecules created by cleavage of the fibrinopeptides A from fibrinogen molecules with thrombin-like enzyme, reptilase. The results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated an emergence of the double-stranded rod-like fibrin protofibrils. For the first time, the protofibrils are proved to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures. Since no accumulation of α polymers has been found the covalent structure of soluble single-stranded fibrin oligomers is entirely brought about by γ-γ bonds. The results of this study provide an extra evidence to support the model of the longitudinal γ-γ bonds that form between the γ chains end-to-end within the same strand of a protofibril.

Free-radical cross-linking of serum albumin molecules on the surface of magnetite nanoparticles in aqueous dispersion

A novel universal approach to cross-linking of protein macromolecules on the surface of magnetite nanoparticles has been developed. The approach is based on protein liability to free-radical modification, leading to the formation of intermolecular covalent cross-links. Free radicals are locally generated on the surface of nanoparticles. Stable coatings of serum albumin 3 nm thick are formed on the surface of magnetite nanoparticles. Using a set of physicochemical methods, it has been proven that stable coatings composed of protein macromolecules are formed around individual nanoparticles. The presence of reactive groups in the protein structure makes it possible to perform subsequent modification of the surface layers-in particular, to graft nonprotein drugs. The approach developed can be used to create superfine systems with desired surface properties for targeted delivery of drugs and biologically active substances.

Free-radical oxidation of plasma fibrin-stabilizing factor.

213 Fibrinnstabilizing factor (FXIII) belongs to the family of transglutaminases (endooγglutamine:ε lysine transferase, EC. 2.3.2.13) and is one of the key proteins of the blood coagulation system. Similarly to other blood clotting factors, FXIII circulates in plasma as an inactive precursor. This is a heterotett ramer (FXIIIIA 2 B 2) with a molecular weight of 320 kDa, consisting of two identical singleechain catt alytic subunits А (FXIIIIA 2 ; molecular weight, ~83 kDa) and two identical singleechain regulatory subunits B (FXIIIIB 2 ; molecular weight, ~78 kDa), which are held together by weak noncovalent bonds [1]. Factor XIII is activated in several stages. The first stage, catalyzed by thrombin, is the hydrolytic cleavv age of the peptide bond between Arg37 and Gly38 in the NH 2 terminal region of subunit А, causing the removal of the activation peptide АP from each sub unit A. As a result, the FXIIIIA 2 B 2 fibrinnstabilizing factor becomes the FXIIII B 2 factor, which still has no enzymatic activity [2]. The second activation stage requires the presence of calcium ions, which can cause dissociation of heterosubunits to form FXIIII and FXIIIIB 2. At the last stage, which also proceeds in the presence of Ca 2+ , FXIIII undergoes conformaa tional transformations, resulting in the exposure of the active site Cys314 with the formation of enzyme FXIIII (FXIIIa) [3]. The main function of factor XIII is to maintain hemostasis by covalent stabilization of the fibrin clot, which is accompanied by an increase in its mechanical strength and resistance to plasmin degradation. In the presence of the active form of fibrinnstabilizing factor, fibrin polymers undergo covalent crossslinking due to formation of ε/γglutamyl–lysine isopeptide bonds [4]. A 2 * To date, the catalytic function of FXIIII has been studied sufficiently well, whereas the role of the noncatalytic FXIIIIВ 2 subunits is less obvious. It is believed that, in the bloodstream, these subunits funcc tion as carriers of the catalytic subunits FXIIIIA 2 , proo tecting them from possible proteolytic degradation and thereby maintaining the required level of zymogen in the bloodstream [5, 6]. In addition, they fulfill a regg ulatory function by controlling the activation of FXIIIIA 2 B 2 by thrombin [7]. It is known that fibrinnstabilizing factor, similarly to many other proteins circulating in the blood plasma, may be a target for reactive oxygen species (ROS), which disturb its functional properties [8]. Previously, by …

Study of protein coatings cross-linked via the free-radical mechanism on magnetic nanoparticles by the method of spectral and fluorescent probes

Magnetite nanoparticles have been obtained with stable coatings formed from bovine and human serum albumins. The coatings are fixed by free-radical cross-linking of the proteins with the use of their ability to form interchain covalent bonds under the action of free radicals, which are generated with participation of transition metals present on nanoparticle surface. The method of spectral and fluorescent probes has been employed for the first time to describe the properties of coatings with the use of various dyes. It has been shown that, when studying the adsorption and free-radical cross-linking of proteins on nanoparticles, it is reasonable to use polymethine and squarylium dyes for estimating the functional properties of the proteins forming the coatings. It has been found that as many as 50% of molecules forming an albumin coating crosslinked via the free-radical mechanism retain their capability of bonding to a fluorescent dye. It has been concluded that the proteins occurring in the structure of the coatings retain their functional properties.

Ozone-induced oxidative modification of fibrinogen molecules

Ozone-induced oxidation of fibrinogen has been investigated. The conversion of oxidized fibrinogen to fibrin catalyzed either by thrombin or by a reptilase-like enzyme, ancistron, in both cases is accompanied by production of gels characterized by a higher weight/length ratio of fibrils in comparison with the native fibrin gels. IR spectra of the D and E fragments isolated from unoxidized and oxidized fibrinogen suggest a noticeable transformation of functional groups by oxidation. A decrease in content of N-H groups in the peptide backbone and in the number of C-H bonds in aromatic structures, as well as a decrease in the intensity of the C-H valence vibrations in aliphatic fragments CH2 and CH3 were found. The appearance in the differential spectra of the D fragments of rather intense peaks in the interval of 1200–800 cm−1 clearly indicates the interaction of ozone with amino acid residues of methionine, tryptophan, histidine, and phenylalanine. Comparison of the differential spectra for the D and E fragments suggests that fibrinogen fragment D is more sensitive to the oxidant action than fragment E. Using EPR spectroscopy, differences are found in the spectra of spin labels bound with degradation products of oxidized and unoxidized fibrinogen, the D and E fragments, caused by structural and dynamical modifications of the protein molecules in the areas of localization of the spin labels. The relationship between the molecular mechanism of oxidation of fibrinogen and its three-dimensional structure is discussed.

Interaction of fibrinogen with magnetite nanoparticles

The interaction between fibrinogen and magnetite nanoparticles in solution has been studied by the methods of spin labeling, ferromagnetic resonance, dynamic and Rayleigh light scattering. It is shown that protein molecules adsorb on the surface of nanoparticles to form multilayer protein covers. The number of molecules adsorbed on one nanoparticle amounts to ∼65 and the thickness of the adsorption layer amounts to ∼27 nm. Separate nanoparticles with fibrinogen covers (clusters) form aggregates due to interactions of the end D domains of fibrinogen. Under the influence of direct magnetic field, nanoparticles with adsorbed proteins form linear aggregates parallel to the force lines. It is shown that the rate of protein coagulation during the formation of fibrin gel under the action of thrombin on fibrinogen decreases ∼2 times in the presence of magnetite nanoparticles, and the magnitude of the average fiber mass/length ratio grows.

Modification of human serum albumin under induced oxidation

For the first time, by using the complex of physicochemical methods (mass-spectrometry, differential scanning calorimetry, spectrofluorimetry, method of spectral and fluorescent probes, dynamic light scattering, and UV spectrophotometry), the oxidation-mediated modification of chemical and spatial structure of albumin has been studied. All albumin structural regions are subjected to oxidation, methionine and aromatic amino acids primarily involved in oxidation. The albumin melting shows a decrease in thermal stabilization of the structure and changing of aggregation upon oxidation. The change in physical and chemical properties of albumin under different quantity of the oxidizer has been analyzed.

Nature of active intermediate particles formed during ozone-induced oxidation

139 Ozone as a representative of reactive oxygen spee cies (ROS) is one of the most toxic components of the atmosphere. A number of studies have shown that ozone is able to generate other ROS, including HO • , , H 2 O 2 , etc. [1, 2]. In some cases, these secondary ROS can be even more deleterious than the ozone molecules themselves. It is now generally recognized that proteins are among the main targets of ROS. Under the action of ROS, proteins undergo oxidative modifications, which disturb their structures and functions. Oxidationnmodified proteins accumulate in the course of aging, oxidative stress, and various diss eases [3]. It was shown that fibrinogen is 20 times more sensitive to oxidative modification than other major plasma proteins (albumin, immunoglobulins, transs ferrin, and ceruloplasmin) [4]. Therefore, fibrinogen, which accounts for approximately 4% of the total plasma proteins, is an easily vulnerable target for oxii dants. It was previously shown that a number of proteins (fibrinogen [5, 6], fibrinnstabilizing factor [7], bovine serum albumin [8], etc.) are involved in oxidative proo cesses under the influence of ozone. In studying the ozonization of fibrinogen, it was found that moderate oxidation leads to a decrease in the content of not only reactive groups (NH x , > SH, etc.), but also to a marked reduction in the content of СН 2 groups, whose reactivity compared to that of NH x and SHHgroups is smaller by 7 and 4 orders of magnitude, respectively [9]. This interesting feature of the reaction could not be attributed to the molecular reaction of ozone but, rather, was due to the action of an as yet unidentified secondary ROS and required further explanation. Since ozone in the liquid phase can form highly toxic secondary ROS, the discriminaa O 2 • − tion between the effects of the molecular oxidation and the free radical oxidation caused by the generation of free radicals under the influence of ozone during ozonation is one of the key problems in understanding the mechanism of oxidation of different biological tarr gets with ozone. In this study, we investigated this problem using fibrinogen as an example. The oxidation of fibrinogen under the action of ozone was performed as described previously [6]. The amount of ozone in the reactor varied from 5 × 10 –8 to 2 × 10 ⎯7 moles. The generation of hydroxyl radicals generated under the …