Probing the redox activity of T-lymphocytes deposited at electrode surfaces with voltammetric methods

Background: Reactive oxygen species and redox signaling play an important role in the regulation of many vital biological processes. However, they are also tightly connected with many pathological conditions. The detection and evaluation of these signaling events are very often accompanied with great difficulties. In this article, we describe the development of a novel electrochemically-based technique for monitoring the cellular redox state. Methods and results: T-cells were attached on the surface of a working electrode, which was modified with 2-palmitoylhydroquinone as a redox mediator. Using cyclic voltammetry, we were able to indirectly (via the redox mediator) monitor an electron transport from the cells towards the working electrode, which enabled us to evaluate the redox activity of the cells. Conclusions: This new technique is rather simple and sensitive and may be used in the future as a valid diagnostic procedure in various branches of biomedical science

Review—Women’s Contribution in the Pulse Voltammetric Theories and Applications: Pulse Voltammetry Stands on the Shoulders of Outstanding Women Electrochemists

It is exactly a century since polarography was developed, which is seen as a predecessor of all voltammetric techniques. As cyclic voltammetry (CV) is the most prominent member in the family of voltammetric techniques for mechanistic studies, the so-called “pulse voltammetric techniques” emerged as simple and viable alternatives to CV for mechanistic characterizations and analytical application, as well as for kinetic and thermodynamic evaluations. The theories and practical application of pulse voltammetric techniques were largely developed by several women electrochemists. In this short overview, we outline some of the major achievements of five women electrochemists who contributed immensely to the theoretical and practical application of pulse voltammetric technique. Since the theory and application of pulse voltammetric techniques largely relies on the works of Janet Osteryoung, Sebojka Komorsky Lovric, Angela Molina, Anna Brainina, and Oliveira Brett, we give in this review a short historical overview of the major accomplishments of these five exceptional women electrochemists

Protein film voltammetry: electrochemical enzymatic spectroscopy. A review on recent progress

This review is focused on the basic principles, the main applications, and the theoretical models developed for various redox mechanisms in protein film voltammetry, with a special emphasis to square-wave voltammetry as a working technique. Special attention is paid to the thermodynamic and kinetic parameters of relevant enzymes studied in the last decade at various modified electrodes, and their use as a platform for the detection of reactive oxygen species is also discussed. A set of recurrent formulas for simulations of different redox mechanisms of lipophilic enzymes is supplied together with representative simulated voltammograms that illustrate the most relevant voltammetric features of proteins studied under conditions of square-wave voltammetry

Redox Chemistry of Ca-Transporter 2-Palmitoylhydroquinone in an Artificial Thin Organic Film Membrane

The redox chemistry of 2-palmitoylhydroquinone (H2Q), a recently introduced synthetic transmembrane Ca2+ transporter, was studied with cyclic and square-wave voltammetry in an artificial thin organic-film membrane sandwiched between a pyrolytic graphite electrode and an aqueous solution. The membrane has a micrometer dimension and consists of the water immiscible organic solvent nitrobenzene, which contains suitable electrolyte and H2Q as a redox active compound. The potential drop at the electrode/membrane interface is controlled by the potentiostat, whereas the potential drop at the membrane/water interface is dependent on the ClO4 - concentration, which is present in a large excess in both liquid phases. The redox transformation of H2Q at the electrode/membrane interface is accompanied by a corresponding ion-transfer reaction at the other side of the membrane. Proton transfer at the membrane/water interface is critical for the redox transformation of H2Q in the interior of the membrane, as a strong dependence of the voltammetric response on the pH of the aqueous medium was observed. H2Q undergoes two oxidation processes due to existence of two distinctive redox forms of H2Q. The electrochemical mechanism can be explained with two tautomer forms of H2Q formed by migration of a proton between the 1-hydroxyl group and the adjacent carbonyl group of the palmitoyl residue. Both tautomers undergo 2e/2H+ distinctive redox transformations to form the quinone form of the studied compound. In the presence of Ca2+ in the aqueous phase, voltammetric experiments confirmed the capability of both tautomers to form 1:1 complexes with Ca2+ and to extract it into the organic membrane. Upon the oxidation of the complexes, Ca2+ is expelled back to the aqueous phase. The studied compound exhibits very similar complexing affinity toward Mg2+, implying that it is not highly selective for transmembrane Ca2+ transport

Inhibition of protein tyrosine phosphatase 1B by reactive oxygen species leads to maintenance of Ca2+ influx following store depletion in HEK 293 cells

Depletion of inositol 1,4,5 trisphosphate-sensitive Ca2+ stores generates a yet unknown signal, which leads to increase in Ca2+ influx in different cell types [J.W. Putney Jr., A model for receptor-regulated calcium entry, Cell Calcium 7 (1986) 1–12]. Here, we describe a mechanism that modulates this store-operated Ca2+ entry (SOC). Ca2+ influx leads to inhibition of protein tyrosine phosphatase 1B (PTP1B) activity in HEK 293 cells [L. Sternfeld, et al., Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells, Cell Signal 17 (2005) 951–960]. Since Ca2+ does not directly inhibit PTP1B, we assumed an intermediate signal, which links the rise in cytosolic Ca2+ concentration and PTP1B inhibition.We now show that Ca2+ influx is followed by generation of reactive oxygen species (ROS) and that it is reduced in cells preincubated with catalase. Furthermore, Ca2+-dependent inhibition of PTP1B can be abolished in the presence of catalase. H2O2 (100�M) directly added to cells inhibits PTP1B and leads to increase in Ca2+ influx after store depletion. PP1, an inhibitor of the Src family tyrosine kinases, prevents H2O2-induced Ca2+ influx. Our results show that ROS act as fine tuning modulators of Ca2+ entry. We assume that the Ca2+ influx channel or a protein involved in its regulation remains tyrosine phosphorylated as a consequence of PTP1B inhibition by ROS. This leads to maintained Ca2+ influx in the manner of a positive feedback loop

Redox regulation of calcium ion channels: Chemical and physiological aspects

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease. Changes in the intracellular free Ca2+ concentration are key triggers for diverse cellular functions. Ca2+ homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca2+ permeable ion channels such as voltage-gated (CaV) Ca2+ channels, transient receptor potential (TRP) channels and Orai channels

Calcium Binding and Transport by Coenzyme Q

Coenzyme Q10 (CoQ10) is one of the essential components of the mitochondrial electron-transport chain (ETC) with the primary function to transfer electrons along and protons across the inner mitochondrial membrane (IMM). The concomitant proton gradient across the IMM is essential for the process of oxidative phosphorylation and consequently ATP production. Cytochrome P450 (CYP450) monoxygenase enzymes are known to induce structural changes in a variety of compounds and are expressed in the IMM. However, it is unknown if CYP450 interacts with CoQ10 and how such an interaction would affect mitochondrial function. Using voltammetry, UV�vis spectrometry, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), fluorescence microscopy and high performance liquid chromatography�mass spectrometry (HPLC�MS), we show that both CoQ10 and its analogue CoQ1, when exposed to CYP450 or alkaline media, undergo structural changes through a complex reaction pathway and form quinone structures with distinct properties. Hereby, one or both methoxy groups at positions 2 and 3 on the quinone ring are replaced by hydroxyl groups in a time-dependent manner. In comparison with the native forms, the electrochemically reduced forms of the new hydroxylated CoQs have higher antioxidative potential and are also now able to bind and transport Ca2þ across artificial biomimetic membranes. Our results open new perspectives on the physiological importance of CoQ10 and its analogues, not only as electron and proton transporters, but also as potential regulators of mitochondrial Ca2þ and redox homeostasis

Experiments towards understanding binding potential of hydroxilated CoQ's lipophilic forms to earth-alkaline cations

This is a set of experimental data of Coenzyme Q1 and Coenzyme Q10 related to the redox chemistry and the ability of native and hydroxylated CoQ’s forms to bind earth-alkaline cations. While there is an evident potential of hydroxylated CoQ1 and CoQ10 to bind strongly calcium ions, a bit less complexing potential exists towards binding Ba2+ and Mg2

A Peptoid Delivers CoQ-derivative to Plant Mitochondria via Endocytosis

Controlled delivery of molecules interfering specifically with target activities in a cell of interest can be a powerful tool for experimental manipulation, because it can be administered at a defined time point and does not require genetic transformation, which in some systems is difficult and time consuming. Peptides as versatile tools that can be tailored for binding numerous binding partners, are of special interest. However, their passage through membranes, their intracellular targeting, and their sensitivity to proteases is limiting. The use of peptoids, where cationic amino-acid side chains are linked to nitrogen (rather than to carbon) of the peptide bond, can circumvent these limitations, because they are not cleavable by proteases. In the current work, we provide a proof-of-concept that such Trojan Peptoids, the plant PeptoQ, can be used to target a functional cargo (i.e. a rhodamine-labelled peptoid and a coenzyme Q10 derivative) into mitochondria of tobacco BY-2 cells as experimental model. We show that the uptake is specific for mitochondria, rapid, dose-dependent, and requires clathrin-mediated endocytosis, as well as actin filaments, while microtubules seem to be dispensable. Viability of the treated cells is not affected, and they show better survival under salt stress, a condition that perturbs oxidative homeostasis in mitochondria. In congruence with improved homeostasis, we observe that the salt induced accumulation of superoxide is mitigated and even inverted by pretreatment with PeptoQ. Using double labelling with appropriate fluorescent markers, we show that targeting of this Trojan Peptoid to the mitochondria is not based on a passage through the plasma membrane (as thought hitherto), but on import via endocytotic vesicles and subsequent accumulation in the mitochondrial intermembrane space, from where it can enter the matrix, e.g. when the permeability of the inner membrane is increased under salt stress