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- ItemOn the liquid chemistry of the reactive nitrogen species peroxynitrite and nitrogen dioxide generated by physical plasmas(Basel : MDPI, 2020) Bruno, Giuliana; Wenske, Sebastian; Lackmann, Jan-Wilm; Lalk, Michael; Von Woedtke, Thomas; Wende, KristianCold physical plasmas modulate cellular redox signaling processes, leading to the evolution of a number of clinical applications in recent years. They are a source of small reactive species, including reactive nitrogen species (RNS). Wound healing is a major application and, as its physiology involves RNS signaling, a correlation between clinical effectiveness and the activity of plasma‐derived RNS seems evident. To investigate the type and reactivity of plasma‐derived RNS in aqueous systems, a model with tyrosine as a tracer was utilized. By high‐resolution mass spectrometry, 26 different tyrosine derivatives including the physiologic nitrotyrosine were identified. The product pattern was distinctive in terms of plasma parameters, especially gas phase composition. By scavenger experiments and isotopic labelling, gaseous nitric dioxide radicals and liquid phase peroxynitrite ions were determined as dominant RNS. The presence of water molecules in the active plasma favored the generation of peroxynitrite. A pilot study, identifying RNS driven post‐translational modifications of proteins in healing human wounds after the treatment with cold plasma (kINPen), demonstrated the presence of in vitro determined chemical pathways. The plasma‐driven nitration and nitrosylation of tyrosine allows the conclusion that covalent modification of biomolecules by RNS contributes to the clinically observed impact of cold plasmas. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemNonenzymatic post-translational modifications in peptides by cold plasma-derived reactive oxygen and nitrogen species(Melville, NY : AIP, 2020) Wenske, Sebastian; Lackmann, Jan-Wilm; Bekeschus, Sander; Weltmann, Klaus-Dieter; Von Woedtke, Thomas; Wende, KristianCold physical plasmas are emerging tools for wound care and cancer control that deliver reactive oxygen species (ROS) and nitrogen species (RNS). Alongside direct effects on cellular signaling processes, covalent modification of biomolecules may contribute to the observed physiological consequences. The potential of ROS/RNS generated by two different plasma sources (kINPen and COST-Jet) to introduce post-translational modifications (PTMs) in the peptides angiotensin and bradykinin was explored. While the peptide backbone was kept intact, a significant introduction of oxidative PTMs was observed. The modifications cluster at aromatic (tyrosine, histidine, and phenylalanine) and neutral amino acids (isoleucine and proline) with the introduction of one, two, or three oxygen atoms, ring cleavages of histidine and tryptophan, and nitration/nitrosylation predominantly observed. Alkaline and acidic amino acid (arginine and aspartic acid) residues showed a high resilience, indicating that local charges and the chemical environment at large modulate the attack of the electron-rich ROS/RNS. Previously published simulations, which include only OH radicals as ROS, do not match the experimental results in full, suggesting the contribution of other short-lived species, i.e., atomic oxygen, singlet oxygen, and peroxynitrite. The observed PTMs are relevant for the biological activity of peptides and proteins, changing polarity, folding, and function. In conclusion, it can be assumed that an introduction of covalent oxidative modifications at the amino acid chain level occurs during a plasma treatment. The introduced changes, in part, mimic naturally occurring patterns that can be interpreted by the cell, and subsequently, these PTMs allow for prolonged secondary effects on cell physiology. © 2020 Author(s).
- ItemSinglet-Oxygen-Induced Phospholipase A2 Inhibition: A Major Role for Interfacial Tryptophan Dioxidation(Weinheim : Wiley-VCH, 2021) Nasri, Zahra; Memari, Seyedali; Wenske, Sebastian; Clemen, Ramona; Martens, Ulrike; Delcea, Mihaela; Bekeschus, Sander; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Wende, KristianSeveral studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A2 (PLA2) activity. Therefore, the regulation of PLA2 catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA2 due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA2 inhibition. A more detailed analysis of the plasma-treated PLA2 identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N-formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation. © 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH
- ItemReactive species driven oxidative modifications of peptides—Tracing physical plasma liquid chemistry(Melville, NY : American Inst. of Physics, 2021) Wenske, Sebastian; Lackmann, Jan-Wilm; Busch, Larissa Milena; Bekeschus, Sander; von Woedtke, Thomas; Wende, KristianThe effluence of physical plasma consists of a significant share of reactive species, which may interact with biomolecules and yield chemical modifications comparable to those of physiological processes, e.g., post-translational protein modifications (oxPTMs). Consequentially, the aim of this work is to understand the role of physical plasma-derived reactive species in the introduction of oxPTM-like modifications in proteins. An artificial peptide library consisting of ten peptides was screened against the impact of two plasma sources, the argon-driven MHz-jet kINPen and the helium-driven RF-jet COST-Jet. Changes in the peptide molecular structure were analyzed by liquid chromatography–mass spectrometry. The amino acids cysteine, methionine, tyrosine, and tryptophan were identified as major targets. The introduction of one, two, or three oxygen atoms was the most common modification observed. Distinct modification patterns were observed for nitration (+N + 2O–H), which occurred in kINPen only (peroxynitrite), and chlorination (+Cl–H) that was exclusive for the COST-Jet in the presence of chloride ions (atomic oxygen/hypochlorite). Predominantly for the kINPen, singlet oxygen-related modifications, e.g., cleavage of tryptophan, were observed. Oxidation, carbonylation, and double oxidations were attributed to the impact of hydroxyl radicals and atomic oxygen. Leading to a significant change in the peptide side chain, most of these oxPTM-like modifications affect the secondary structure of amino acid chains, and amino acid polarity/functionality, ultimately modifying the performance and stability of cellular proteins.
- ItemAssessment of Phycocyanin Extraction from Cyanidium caldarium by Spark Discharges, Compared to Freeze-Thaw Cycles, Sonication, and Pulsed Electric Fields(Basel : MDPI, 2021) Sommer, Marie-Christine; Balazinski, Martina; Rataj, Raphael; Wenske, Sebastian; Kolb, Juergen F.; Zocher, KatjaPhycocyanin is a blue colored pigment, synthesized by several species of cyanobacteria and red algae. Besides the application as a food-colorant, the pigmented protein is of high interest as a pharmaceutically and nutritionally valuable compound. Since cyanobacteria-derived phycocyanin is thermolabile, red algae that are adapted to high temperatures are an interesting source for phycocyanin extraction. Still, the extraction of high quality phycocyanin from red algae is challenging due to the strong and rigid cell wall. Since standard techniques show low yields, alternative methods are needed. Recently, spark discharges have been shown to gently disintegrate microalgae and thereby enable the efficient extraction of susceptible proteins. In this study, the applicability of spark discharges for phycocyanin extraction from the red alga Cyanidium caldarium was investigated. The efficiency of 30 min spark discharges was compared with standard treatment protocols, such as three times repeated freeze-thaw cycles, sonication, and pulsed electric fields. Input energy for all physical methods were kept constant at 11,880 J to ensure comparability. The obtained extracts were evaluated by photometric and fluorescent spectroscopy. Highest extraction yields were achieved with sonication (53 mg/g dry weight (dw)) and disintegration by spark discharges (4 mg/g dw) while neither freeze-thawing nor pulsed electric field disintegration proved effective. The protein analysis via LC-MS of the former two extracts revealed a comparable composition of phycobiliproteins. Despite the lower total concentration of phycocyanin after application of spark discharges, the purity in the raw extract was higher in comparison to the extract attained by sonication.