The effect of hypochlorite- and peroxide-induced oxidation of plasminogen on damage to the structure and biological activity.

In this study, we examined for the first time the effect of the HOCl/OCl-- and H2O2-induced oxidation of Glu-plasminogen on damage to its primary structure and the biological activity of plasmin. The consolidated results obtained with the aid of MS/MS, electrophoresis, and colourimetry, demonstrated that none of the oxidised amino acid residues found in the proenzyme treated with 25 µM HOCl/OCl- or 100 µM H2O2 were functionally significant for plasminogen. However, the treatment of plasminogen with increasing concentrations of HOCl/OCl- from 25 µM to 100 µM or H2O2 from 100 µM to 300 µM promoted a partial loss in the activity of oxidised plasmin. Several methionine residues (Met57, Met182, Met385, Met404, Met585, and Met788) localized in different protein domains have been shown to serve as ROS traps, thus providing an efficient defense mechanism against oxidative stress. Oxidised Trp235, Trp417, Trp427, Trp761, and Tyr672 are most likely responsible for the reduced biological activity of Glu-plasminogen subjected to strong oxidation. The results of the present study, along with those of previous studies, indicate that the structure of Glu-plasminogen is adapted to oxidation to withstand oxidative stress induced by ROS.

Interaction between immunoglobulin G and peroxidase-like iron oxide nanoparticles: Physicochemical and structural features of the protein.

The study is devoted to the oxidative modification of immunoglobulin G (IgG) on the surface of peroxidase-like iron oxide magnetic nanoparticles (MNPs) under conditions of induced reactive oxygen species (ROS) generation and without them. A pronounced change of thermodynamic parameters of denaturation has been detected for IgG in solutions containing MNPs under hydrogen peroxide action during 24 h of incubation. Dynamic light scattering measurements and UV-Visible spectrophotometry have been used to show aggregation in these solutions. Ferromagnetic resonance (FMR) was used to compare IgG coating thickness on individual MNPs under conditions of induced ROS generation and without them. The similarity between IgG adsorption on MNPs under these conditions after 24 h of incubation has been confirmed by the fluorescence measurements. The sites of IgG oxidative modifications that take place on MNPs surface and some evidences of the influence of oxidative modification and adsorption on the chemical structure of IgG were revealed by HPLC MS/MS analysis.

Ozone-induced damage of fibrinogen molecules: identification of oxidation sites by high-resolution mass spectrometry.

Fibrinogen is highly susceptible to oxidation compared to other plasma proteins. Fibrinogen oxidation damages its structure and affects the protein function. Ozone-induced oxidative modifications of the fibrinogen Aα, Bß, and γ polypeptide chains upon addition of various amounts of the oxidiser were studied by mass spectrometry. Amino acid residues located on all three chains and main structural parts of the protein were revealed to be involved in oxidation. The αC-connector was shown to be most vulnerable to oxidation as compared to other structural parts while the E region turned out to be the most protected area of the protein. For the first time, it was established that numerous amino acid residues responsible for the conversion of fibrinogen to fibrin remain unaffected upon fibrinogen oxidation. The data obtained in this study indicate that none of the identified residues, which are considered crucial for the binding of both hole "a" and hole "b" to knob "A" and knob "B", respectively, as well as those responsible for the thrombin binding to fibrinogen E region, have been subjected to chemical alterations under moderate oxidation. The data on fibrinogen oxidation acquired in the current study enable one to assume that some of the structural fibrinogen parts and easily oxidisable residues could be endowed with antioxidant properties. New findings presented here could be essential for the detection of adaptive molecular mechanisms capable of mitigating the detrimental action of reactive oxygen species (ROS) on the functioning of oxidatively damaged fibrinogen. Data are available via ProteomeXchange with identifier PXD012046. Highlights Various oxidative modifications were detected in fibrinogen by mass spectrometry αC-connector has been shown to be most susceptible to oxidation E region proved to be least vulnerable to the action of the oxidising agent Some of the Met residues in the fibrinogen structure could operate as ROS scavengers.

Oxidation-induced modifications of the catalytic subunits of plasma fibrin-stabilizing factor at the different stages of its activation identified by mass spectrometry.

Plasma fibrin-stabilizing factor (pFXIII) is a heterotetrameric proenzyme composed of two catalytic A subunits (FXIII-A2) and two inhibitory/carrier B subunits (FXIII-B2). The main function of the protein is the formation of cross-links between the polypeptide chains of the fibrin clot. The conversion of pFXIII into the enzymatic form FXIII-A2* is a multistage process. Like many other blood plasma proteins, pFXIII is an oxidant-susceptible target. The influence of distinct sites susceptible to oxidation-mediated modifications on the changes in the structural-functional characteristics of the protein remains fully unexplored. For the first time, a set of the oxidation sites within FXIII-A2 under ozone-induced oxidation of pFXIII at different stages of its activation have been identified by mass spectrometry, and the extent as well as the chemical nature of these modifications have been explored. It was shown that the set of amino acid residues susceptible to oxidative attack and the degree of oxidation of these residues in FXIII-A2 of non-activated pFXIII, pFXIII activated by Ca2+ and fully activated pFXIII treated with thrombin and Ca2+ significantly differ. The obtained data enable one to postulate that in the process of the proenzyme conversion into FXIII-A2*, new earlier-unexposed amino acid residues become available for the oxidizer while some of the initially surface-exhibited residues are buried within the protein globule.

From Supercomputer Modeling to Highest Mass Resolution in FT-ICR.

Understanding of behavior of ion ensembles inside FT-ICR cell based on the computer simulation of ion motion gives rise to the new ideas of cell designs. The recently introduced novel FT-ICR cell based on a Penning ion trap with specially shaped excitation and detection electrodes prevents distortion of ion cyclotron motion phases (normally caused by non-ideal electric trapping fields) by averaging the trapping DC electric field during the ion motion in the ICR cell. Detection times of 5 min resulting in resolving power close to 40,000,000 have been reached for reserpine at m/z 609 at a magnetic field of only 7 Tesla. Fine structures of resolved 13Cn isotopic cluster groups could be measured for molecular masses up to 5.7 kDa (insulin) with resolving power of 4,000,000 at 7 Tesla. Based on resolved fine structure patterns atomic compositions can be directly determined using a new developed algorithm for fine structure processing. Mass spectra of proteins and multimers of proteins reaching masses up to 186 kDa (enolase tetramer) could be measured with isotopic resolution. For instance, at 7 Tesla resolving power of 800,000 was achieved for enolase dimer (96 kDa) and 500,000 for molecular masses above 100 kDa. Experimental data indicate that there is practically no limit for the resolving power of this ICR cell except by collisional damping in the ultrahigh vacuum chamber.