Post-Translational Oxidative Modifications of Hemostasis Proteins: Structure, Function, and Regulation.

Reactive oxygen species (ROS) are constantly generated in a living organism. An imbalance between the amount of generated reactive species in the body and their destruction leads to the development of oxidative stress. Proteins are extremely vulnerable targets for ROS molecules, which can cause oxidative modifications of amino acid residues, thus altering structure and function of intra- and extracellular proteins. The current review considers the effect of oxidation on the structural rearrangements and functional activity of hemostasis proteins: coagulation system proteins such as fibrinogen, prothrombin/thrombin, factor VII/VIIa; anticoagulant proteins - thrombomodulin and protein C; proteins of the fibrinolytic system such as plasminogen, tissue plasminogen activator and plasminogen activator inhibitor-1. Structure and function of the proteins, oxidative modifications, and their detrimental consequences resulting from the induced oxidation or oxidative stress in vivo are described. Possible effects of oxidative modifications of proteins in vitro and in vivo leading to disruption of the coagulation and fibrinolysis processes are summarized and systematized, and the possibility of a compensatory mechanism in maintaining hemostasis under oxidative stress is analyzed.

Life Cycle of Functional All-Green Biocompatible Fibrous Materials Based on Biodegradable Polyhydroxybutyrate and Hemin: Synthesis, Service Life, and the End-of-Life via Biodegradation.

This article addresses the entire life cycle of the all-green fibrous materials based on poly(3-hydroxybutyrate) (PHB) containing a natural biocompatible additive Hemin (Hmi): from preparation, service life, and the end of life upon in-soil biodegradation. Fibrous PHB/Hmi materials with a highly developed surface and interconnected porosity were prepared by electrospinning (ES) from Hmi-containing feed solutions. Structural organization of the PHB/Hmi materials (porosity, uniform structure, diameter of fibers, surface area, distribution of Hmi within the PHB matrix, phase composition, etc.) is shown to be governed by the ES conditions: the presence of even minor amounts of Hmi in the PHB/Hmi (below 5 wt %) serves as a powerful tool for the control over their structure, performance, and biodegradation. Service characteristics of the PHB/Hmi materials (wettability, prolonged release of Hmi, antibacterial activity, breathability, and mechanical properties) were studied by different physicochemical methods (scanning electron microscopy, Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, differential scanning calorimetry, contact angle measurements, antibacterial tests, etc.). The effect of the structural organization of the PHB/Hmi materials on their in-soil biodegradation at the end of life was analyzed, and key factors providing efficient biodegradation of the PHB/Hmi materials at all stages (from adaptation to mineralization) are highlighted (high surface area and porosity, thin fibers, release of Hmi, etc.). The proposed approach allows for target-oriented preparation and structural design of the functional PHB/Hmi nonwovens when their structural supramolecular organization with a highly developed surface area controls both their service properties as efficient antibacterial materials and in-soil biodegradation upon the end of life.

Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Electrospinning of biomimetic materials with fibrinogen for effective early-stage wound healing.

Electrospun biomimetic materials based on polyester of natural origin poly-3-hudroxybutyrate (PHB) modified with hemin (Hmi) and fibrinogen (Fbg) represent a great interest and are potentially applicable in various fields. Here, we describe formulation of the new fibrous PHB-Fbg and PHB-Hmi-Fbg materials with complex structure for biomedical application. The average diameter of the fibers was 3.5 µm and 1.8 µm respectively. Hmi presence increased porosity from 80 % to 94 %, significantly reduced the number of defects, ensured the formation of a larger number of open pores, and improved mechanical properties. Hmi presence significantly improved the molding properties of the material. Hmi facilitated effective Fbg adsorption on the of the PHB wound-healing material, ensuring uniform localization of the protein on the surface of the fibers. Next, we evaluated cytocompatibility, cell behavior, and open wound healing in mice. The results demonstrated that PHB-Fbg and PHB-Hmi-Fbg electrospun materials had pronounced properties and may be promising for early-stage wound healing - the PHB-Hmi-Fbg sample accelerated wound closure by 35 % on the 3rd day, and PHB-Hmi showed 45 % more effective wound closure on the 15th day.

Antioxidant role of methionine-containing intra- and extracellular proteins.

Significant evidence suggests that reversible oxidation of methionine residues provides a mechanism capable of scavenging reactive species, thus creating a cycle with catalytic efficiency to counteract or mitigate deleterious effects of ROS on other functionally important amino acid residues. Because of the absence of MSRs in the blood plasma, oxidation of methionines in extracellular proteins is effectively irreversible and, therefore, the ability of methionines to serve as interceptors of oxidant molecules without impairment of the structure and function of plasma proteins is still debatable. This review presents data on the oxidative modification of both intracellular and extracellular proteins that differ drastically in their spatial structures and functions indicating that the proteins contain antioxidant methionines/the oxidation of which does not affect (or has a minor effect) on their functional properties. The functional consequences of methionine oxidation in proteins have been mainly identified from studies in vitro and, to a very limited extent, in vivo. Hence, much of the functioning of plasma proteins constantly subjected to oxidative stress remains unclear and requires further research to understand the evolutionary role of methionine oxidation in proteins for the maintenance of homeostasis and risk factors affecting the development of ROS-related pathologies. Data presented in this review contribute to increased evidence of antioxidant role of surface-exposed methionines and can be useful for understanding a possible mechanism that supports or impairs structure-function relationships of proteins subjected to oxidative stress.

Hypochlorite-induced oxidation of fibrinogen: Effects on its thermal denaturation and fibrin structure.

BACKGROUND: Human fibrinogen, which plays a key role in plasma haemostasis, is a highly vulnerable target for oxidants. Fibrinogen undergoes posttranslational modifications that can potentially disrupt protein structure and function. METHODS: For the first time, by differential scanning calorimetry, dynamic and elastic light scattering and confocal laser scanning microscopy, the consequences of HOCl/-OCl-induced oxidation of fibrinogen on its thermal denaturation, molecular size distribution and fibrin clot network have been explored. RESULTS: Within a wide range of HOCl/-OCl concentrations (50-300 µM), the molecular size distribution remained unimodal; however, the average size of the hydrated molecules decreased. HOCl/-OCl-induced oxidation of fibrinogen resulted in the diminished thermal stability of regions D and E. As evidenced by elastic light scattering and confocal laser scanning microscopy, HOCl/-OCl caused the formation of abnormal fibrin with a decreased diameter of individual fibres. CONCLUSIONS: The current results along with data from previous studies enable one to conclude that the effect of HOCl/-OCl-mediated oxidation on the thermal stability of region D is influenced directly by oxidative damage to the D region structure. Since the E region is not subjected to oxidative modification, its structural damage is likely to be mediated by the oxidation of other protein structures, in particular α-helical coiled-coils. GENERAL SIGNIFICANCE: The experimental findings acquired in the current study could help to elucidate the consequences of oxidative stress in vivo on damage to the structure of fibrinogen/fibrin under the action of different ROS species.

Simultaneous express immunoassay of multiple cardiac biomarkers with an automatic platform in human plasma.

C-reactive protein, cystatin C, myoglobin, and D-dimer represent the inflammatory or thromboembolic status of the patient and play important roles in early diagnostics of acute myocardial infarction. Each protein can indicate some health problems, but their simultaneous detection can be crucial for differential diagnostics. The express analysis of these proteins in a small drop of plasma was developed using magnetic beads. The suggested method is based on immunomagnetic extraction of the target analyte from plasma samples and its simultaneous labelling by fluorescent dye. Reaction time was optimized for quantification of cardiac biomarkers in the spike solutions and human plasma samples. In this paper, we developed a one-protein detection technique for each cardiac biomarker and united it to a four-protein facility using an automatic platform. The proposed technique requires only 17 µL of the human plasma and takes 14 min for four-protein measuring. The suggested technique covers concentration difference by more than two orders of magnitude and demonstrates analytical applicability by measurements of human plasma samples of 16 volunteers.

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.

Oxidation of proteins: is it a programmed process?

Proteins represent extremely susceptible targets for oxidants. Oxidative modifications of proteins may bring about violation of their structure and functionality. It implies that the structures of proteins are not infallible in terms of their antioxidant defence. The protection mechanisms in preventing oxidative damages for proteins within cells are mainly related to a large variety of antioxidant enzymatic systems. In contrast, plasma proteins are scarcely protected by these systems, so the mechanism that provides their functioning in the conditions of generating reactive oxygen species (ROS) seems to be much more complicated. Oxidation of many proteins was long considered as a random process. However, the highly site-specific oxidation processes was convincingly demonstrated for some proteins, indicating that protein structure could be adapted to oxidation. According to our hypothesis, some of the structural elements present in proteins are capable of scavenging ROS to protect other protein structures against ROS toxicity. Various antioxidant elements (distinct subdomains, domains, regions, and polypeptide chains) may act as ROS interceptors, thus mitigating the ROS action on functionally crucial amino acid residues of proteins. In the review, the oxidative modifications of certain plasma proteins, such as α2-macroglobulin, serum human albumin, fibrinogen, and fibrin-stabilising factor, which differ drastically in their spatial structures and functions, are analysed. The arguments that demonstrate the possibility of existing hypothetical antioxidant structures are presented. For the first time, the emphasis is being placed on the programmed mechanism of protein oxidation.