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The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO • ) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO • , a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO • . Enzyme mixtures (1:1:1 ratio) were exposed to ROO • produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP + generated ∼175 μM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (∼100 μM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The development of diverse types of biomaterials has significantly contributed to bringing new biomedical strategies to treat clinical conditions. Applications of these biomaterials can range from mechanical support and protection of injured tissues to joint replacement, tissue implants, and drug delivery systems. Among the strategies commonly used to prepare biomaterials, the use of electromagnetic radiation to initiate crosslinking stands out. The predominance of photo-induced polymerization methods relies on a fast, efficient, and straightforward process that can be easily adjusted to clinical needs. This strategy consists of irradiating the components that form the material with photons in the near ultraviolet-visible wavelength range ( i.e. , ∼310 to 750 nm) in the presence of a photoactive molecule. Upon photon absorption, photosensitive molecules can generate excited species that initiate photopolymerization through different reaction mechanisms. However, this process could promote undesired side reactions depending on the target zone or treatment type ( e.g. , oxidative stress and modification of biomolecules such as proteins and lipids). This review explores the basic concepts behind the photopolymerization process of ex situ and in situ biomaterials. Particular emphasis was put on the photosensitization initiated by the most employed photosensitizers and the photoreactions that they mediate in aqueous media. Finally, the undesired oxidation reactions at the bio-interface and potential solutions are presented.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and 'moonlighting' activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H 2 O 2 ) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H 2 O 2 - and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The pentose phosphate pathway (PPP) is a key metabolic pathway. The oxidative phase of this process involves three reactions catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydrogenase (6PGDH) enzymes. The first and third steps (catalyzed by G6PDH and 6PGDH, respectively) are responsible for generating reduced nicotinamide adenine dinucleotide phosphate (NAPDH), a key cofactor for maintaining the reducing power of cells and detoxification of both endogenous and exogenous oxidants and electrophiles. Despite the importance of these enzymes, little attention has been paid to the fact that these proteins are targets of oxidants. In response to oxidative stimuli metabolic pathways are modulated, with the PPP often up-regulated in order to enhance or maintain the reductive capacity of cells. Under such circumstances, oxidation and inactivation of the PPP enzymes could be detrimental. Damage to the PPP enzymes may result in a downward spiral, as depending on the extent and sites of modification, these alterations may result in a loss of enzymatic activity and therefore increased oxidative damage due to NADPH depletion. In recent years, it has become evident that the three enzymes of the oxidative phase of the PPP have different susceptibilities to inactivation on exposure to different oxidants. In this review, we discuss existing knowledge on the role that these enzymes play in the metabolism of cells, and their susceptibility to oxidation and inactivation with special emphasis on NADPH production. Perspectives on achieving a better understanding of the molecular basis of the oxidation these enzymes within cellular environments are given., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Skeletal muscle is crucial for maintaining human health and overall quality of life. Acute exercise introduces a multifaceted intracellular stress, with numerous post-translational modifications believed to underpin the health benefits of sustained exercise training. Reactive oxygen species (ROS) are posited to serve as second messengers, triggering cytoprotective adaptations such as the upregulation of enzymatic scavenger systems. However, a significant knowledge gap exists between the generation of oxidants in muscle and the exact mechanisms driving muscle adaptations. This review delves into the current research on subcellular redox biochemistry and its role in the physiological adaptations to exercise. We propose that the subcellular regulation of specific redox modifications is key to ensuring specificity in the intracellular response., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cutibacterium acnes is one of the most abundant bacteria on the skin. Being exposed to oxygen and oxic stress, the secretion of the bacterial antioxidant protein RoxP ensures an endogenous antioxidant system for the preservation of skin health. To investigate the impact of the antioxidant RoxP on oxidation of the bacteria, wildtype and an isogenic roxp mutant were cultured in anaerobic and oxic conditions. The carbonylated status of proteins were recorded, as were the most significant modifications in a relative intensity of free fatty acids (FFA) and lipids containing fatty acids (FA), such as di- (DG) and triglycerides (TG), di- (DGDG) and sulfoquinozyldiacylglycerol (SQDG) and ceramides. Concerning the fatty acid types, it was observed that the free fatty acids contained mainly C12:0-C26:0 in hydroxy and acylated forms, the DG contained mainly C29:0-C37:0, the TG contained mainly C19:0-C33:0, and the DGDG/SQDGs contained very long fatty acids (C29:0-C37:0) demonstrating the interdependence of de novo synthesis of lipids and RoxP. The area of DGDG peaks (924.52, 929.56 and 930.58) were affected by bacterial growth conditions, with the exception of m / z 910.61. Moreover, the FFA unsaturation is wider in the SQDG species (C30:0 to C36:6) than in DG, TG or free FFA species. It could be concluded that both environmental oxidative statuses, as well as the prevalence of bacterial antioxidant systems, significantly shape the lipidome of C. acnes .

Diabetes mellitus currently affects ∼10% of the population worldwide, with Type 2 predominating, and this incidence is increasing steadily. Both Type 1 and 2 are complex diseases, involving β-cell death and chronic inflammation, but the pathways involved are unresolved. Chronic inflammation is characterized by increased oxidant formation, with this inducing protein modification, altered function and immunogenicity. Amylin, a peptide hormone co-secreted with insulin by β-cells, has attracted considerable interest for its amyloidogenic properties, however, the effects that oxidants have on amylin aggregation and function are poorly understood. Amylin was exposed in vitro to hypochlorous acid, hydrogen peroxide and peroxynitrous acid/peroxynitrite to investigate the formation of post-translational oxidative modifications (oxPTMs, via mass spectrometry) and fibril formation (via transmission electron microscopy). Amylin free acid (AFA) was also examined to investigate the role of the C-terminal amide in amylin. Oxidant exposure led to changes in aggregate morphology and abundance of oxPTMs in a concentration-dependent manner. The toxicity and immunogenic potential of oxidant-modified amylin or AFA on pancreatic islet cells (INS-1E), human monocyte cell line (THP-1) and monocyte-derived dendritic cells (moDCs) were examined using metabolic activity and cytokine assays, and flow cytometry. No significant changes in vitality or viability were detected, but exposure to oxidant-modified amylin or AFA resulted in altered immunogenicity when compared to the native proteins. THP-1 and moDCs show altered expression of activation markers and changes in cytokine secretion. Furthermore, oxidant-treated amylin and AFA promoted maturation of THP-1 and pre-mature moDCs, as determined by changes in size, and maturation markers., Competing Interests: Declaration of competing interest MJD declares consultancy contracts with Novo Nordisk A/S. This funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The other authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

6-phosphogluconolactonase (6PGL) catalyzes the second reaction of the pentose phosphate pathway (PPP) converting 6-phosphogluconolactone to 6-phosphogluconate. The PPP is critical to the generation of NADPH and metabolic intermediates, but some of its components are susceptible to oxidative inactivation. Previous studies have characterized damage to the first (glucose-6-phosphate dehydrogenase) and third (6-phosphogluconate dehydrogenase) enzymes of the pathway, but no data are available for 6PGL. This knowledge gap is addressed here. Oxidation of Escherichia coli 6PGL by peroxyl radicals (ROO • , from AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was examined using SDS-PAGE, amino acid consumption, liquid chromatography with mass detection (LC-MS), protein carbonyl formation and computational methods. NADPH generation was assessed using mixtures all three enzymes of the oxidative phase of the PPP. Incubation of 6PGL with 10 or 100 mM AAPH resulted in protein aggregation mostly due to reducible (disulfide) bonds. High fluxes of ROO • induced consumption of Cys, Met and Trp, with the Cys oxidation rationalizing the aggregate formation. Low levels of carbonyls were detected, while LC-MS analyses provided evidence for oxidation of selected Trp and Met residues (Met1, Trp18, Met41, Trp203, Met220 and Met221). ROO • elicited little loss of enzymatic activity of monomeric 6PGL, but the aggregates showed diminished NADPH generation. This is consistent with in silico analyses that indicate that the modified Trp and Met are far from the 6-phosphogluconolactone binding site and the catalytic dyad (His130 and Arg179). Together these data indicate that monomeric 6PGL is a robust enzyme towards oxidative inactivation by ROO • and when compared to other PPP enzymes., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Biological milieus are highly crowded and heterogeneous systems where organization of macromolecules within nanodomains (e.g. membraneless compartments) is vital to the regulation of metabolic processes. There is an increasing interest in understanding the effects that such packed environments have on different biochemical and biological processes. In this context, the redox biochemistry and redox signaling fields are moving towards investigating oxidative processes under conditions that exhibit these key features of biological systems in order to solve existing paradigms including those related to the generation and transmission of specific redox signals within and between cells in both normal physiology and under conditions of oxidative stress. This review outlines the effects that crowding, nanodomain formation and altered local viscosities can have on biochemical processes involving proteins, and then discusses some of the reactions and pathways involving proteins and oxidants that may, or are known to, be modulated by these factors. We postulate that knowledge of protein modification processes (e.g. kinetics, pathways and product formation) under conditions that mimic biological milieus, will provide a better understanding of the response of cells to endogenous and exogenous stressors, and their role in ageing, signaling, health and disease., Competing Interests: Declaration of competing interest E.F-L. declares no conflicts of interest. M.J.D. declares commercial consultancy contracts with Novo Nordisk A/S. This funder had no role in the conceptualization and writing of the manuscript, or in the decision to publish this., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteins are modified during milk processing and storage, with sidechain oxidation and crosslinking being major consequences. Despite the prevalence and importance of proteins in milk, and particularly caseins (∼80% of total content), the nature of the cross-links formed by oxidation, and their mechanisms of formation, are poorly characterized. In this study, we investigated the formation and stability of cross-links generated by the nucleophilic addition of Cys residues to quinones generated on oxidation of Tyr residues. The mechanisms and stability of these adducts was explored using ubiquitin as a model protein, and β-casein. Ubiquitin and β-casein were oxidized using a rose Bengal/visible light/O 2 system, or by the enzyme tyrosinase. The oxidized proteins were incubated with glutathione or β-lactoglobulin (non-oxidized, but unfolded by treatment at 70 °C), before analysis by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that Cys-quinone adducts are readily-formed, and are stable for >48 h. Thus, oxidized β-casein reacts efficiently with the thermally unfolded β-lactoglobulin, likely via Michael addition of the exposed Cys to a Tyr-derived quinone. These data provide a novel, and possibly general, mechanism of protein cross-link formation, and provides information of the stability of these species that have potential as markers of protein quality., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Escherichia coli glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are key enzymes of the pentose phosphate pathway, responsible for the NADPH production in cells. We investigated modification of both enzymes mediated by peroxyl radicals (ROO · ) to determine their respective susceptibilities to and mechanisms of oxidation. G6PDH and 6PGDH were incubated with AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride), which was employed as ROO · source. The enzymatic activities of both enzymes were determined by NADPH release, with oxidative modifications examined by electrophoresis and liquid chromatography (LC) with fluorescence and mass (MS) detection. The activity of G6PDH decreased up to 62.0 ± 15.0% after 180 min incubation with 100 mM AAPH, whilst almost total inactivation of 6PGDH was determined under the same conditions. Although both proteins contain abundant Tyr (particularly 6PGDH), these residues were minimally affected by ROO · , with Trp and Met being major targets. LC-MS and in silico analysis showed that the modification sites of G6PDH are distant to the active site, consistent with a dispersed distribution of modifications, and inactivation resulting from oxidation of multiple Trp and Met residues. In contrast, the sites of oxidation detected on 6PGDH are located close to its catalytic site indicating a more localized oxidation, and a consequent high susceptibility to ROO · -mediated inactivation., (© 2022. The Author(s).)

Protein modification occurs in biological milieus that are characterized by high concentrations of (macro)molecules (i.e. heterogeneous and packed environments). Recent data indicate that crowding can modulate the extent and rate of protein oxidation, however its effect on other post-translational modifications remains to be explored. In this work we hypothesized that crowding would affect the glycation of plasma proteins. Physiologically-relevant concentrations of albumin (35 mg mL -1 ) and transferrin (2 mg mL -1 ) were incubated with methylglyoxal and glyoxal (5 μM-5 mM), two α-oxoaldehyde metabolites that are elevated in the plasma of people with diabetes. Crowding was induced by adding dextran or ficoll polymers. Electrophoresis, electron microscopy, fluorescence spectroscopy and mass spectrometry were employed to investigate the structural consequences of glycation under crowded conditions. Our data demonstrate that crowding modulates the extent of formation of transferrin cross-links, and also the modification pathways in both albumin and transferrin. Arginine was the most susceptible residue to modification, with lysine and cysteine also affected. Loss of 0.48 and 7.28 arginine residues per protein molecule were determined on incubation with 500 μM methylglyoxal for albumin and transferrin, respectively. Crowding did not influence the extent of loss of arginine and lysine for either protein, but the sites of modification, detected by LC-MS, were different between dilute and crowded conditions. These data confirm the relevance of studying modification processes under conditions that closely mimic biological milieus. These data unveil additional factors that influence the pattern and extent of protein modification, and their structural consequences, in biological systems., Competing Interests: Declaration of competing interest M.J.D. declares commercial consultancy contracts with Novo Nordisk A/S. This funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish these results. The other authors declare no conflicts of interest with regard to the data presented., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO ● ) and peroxynitrite (ONOO - ), were explored. G6PDH was incubated with AAPH (2,2' -azobis(2-methylpropionamidine)dihydrochloride), used as ROO ● source, and ONOO - . Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4 μM) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to ∼59% of the initial level after 90 min. ONOO - induced a significant and concentration-dependent loss of G6PDH activity with ∼46% of the initial activity lost on treatment with 1.5 mM ONOO - . CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO ● , but not ONOO - . LC-MS analysis provided evidence for ONOO - -mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO ● . These two oxidant systems therefore have markedly different effects on G6PDH structure and activity., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Photo-oxidation of casein proteins is commonplace during milk processing and storage. A major consequence of such light exposure is protein cross-linking and aggregation. Although caseins are key milk components, the nature of the cross-links and the mechanisms involved are poorly characterized, with most previous work having been focused on detecting and quantifying di-tyrosine formed on dimerization of two tyrosine-derived phenoxyl radicals. However, this is only one of a large number of possible cross-links that might be formed. In this study, we have investigated the potential involvement of secondary reactions between oxidized protein side-chains and the thiol group of cysteine (Cys) residues in casein cross-linking. Casein proteins were subjected to photo-oxidation using visible light in the presence of a sensitizer (riboflavin or rose Bengal) and O 2 , then incubated with a Cys-containing peptide (glutathione, GSH) or protein (κ-casein), and subsequently analyzed by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that that photo-oxidized (but not parent) caseins react efficiently with the Cys-containing species, likely via Michael addition to quinones formed from tyrosine residues to give glutathionylated species or protein adducts. Thus, oxidized α-casein reacts with native κ-casein to give high molecular mass aggregates. This adduct formation was prevented by alkylation of the Cys thiol group. The cross-link site and the residues involved have been confirmed by liquid chromatography-mass spectrometry (LC-MS) proteomic analysis. Together, these data extend our knowledge of the mechanisms involved in casein oxidation and aggregation., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Beta-2-microglobulin (B2M) is synthesized by all nucleated cells and forms part of the major histocompatibility complex (MHC) class-1 present on cell surfaces, which presents peptide fragments to cytotoxic CD8 + T-lymphocytes, or by association with CD1, antigenic lipids to natural killer T-cells. Knockout of B2M results in loss of these functions and severe combined immunodeficiency. Plasma levels of this protein are low in healthy serum, but are elevated up to 50-fold in some pathologies including chronic kidney disease and multiple myeloma, where it has both diagnostic and prognostic value. High levels of the protein are associated with amyloid formation, with such deposits containing significant levels of modified or truncated protein. In the current study we examine the chemical and structural changes induced of B2M generated by both inflammatory oxidants (HOCl and ONOOH), and photo-oxidation ( 1 O 2 ) which is linked with immunosuppression. Oxidation results in oligomer formation, with this occurring most readily with HOCl and 1 O 2 , and a loss of native protein conformation. LC-MS analysis provided evidence for nitrated (from ONOOH), chlorinated (from HOCl) and oxidized residues (all oxidants) with damage detected at Tyr, Trp, and Met residues, together with cleavage of the disulfide (cystine) bond. An intermolecular di-tyrosine crosslink is also formed between Tyr10 and Tyr63. The pattern of these modifications is oxidant specific, with ONOOH inducing a greater range of modifications than HOCl. Comparison of the sites of modification with regions identified as amyloidogenic indicate significant co-localization, consistent with the hypothesis that oxidation may contribute, and predispose B2M, to amyloid formation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Covalent crosslinks within or between proteins play a key role in determining the structure and function of proteins. Some of these are formed intentionally by either enzymatic or molecular reactions and are critical to normal physiological function. Others are generated as a consequence of exposure to oxidants (radicals, excited states or two-electron species) and other endogenous or external stimuli, or as a result of the actions of a number of enzymes (e.g., oxidases and peroxidases). Increasing evidence indicates that the accumulation of unwanted crosslinks, as is seen in ageing and multiple pathologies, has adverse effects on biological function. In this article, we review the spectrum of crosslinks, both reducible and non-reducible, currently known to be formed on proteins; the mechanisms of their formation; and experimental approaches to the detection, identification and characterization of these species.

Biological systems are heterogeneous and crowded environments. Such packed milieus are expected to modulate reactions both inside and outside the cell, including protein oxidation. In this work, we explored the effect of macromolecular crowding on the rate and extent of oxidation of Trp and Tyr, in free amino acids, peptides and proteins. These species were chosen as they are readily oxidized and contribute to damage propagation. Dextran was employed as an inert crowding agent, as this polymer decreases the fraction of volume available to other (macro)molecules. Kinetic analysis demonstrated that dextran enhanced the rate of oxidation of free Trp, and peptide Trp, elicited by AAPH-derived peroxyl radicals. For free Trp, the rates of oxidation were 15.0 ± 2.1 and 30.5 ± 3.4 μM min -1 without and with dextran (60 mg mL -1 ) respectively. Significant increases were also detected for peptide-incorporated Trp. Dextran increased the extent of Trp consumption (up to 2-fold) and induced short chain reactions. In contrast, Tyr oxidation was not affected by the presence of dextran. Studies on proteins, using SDS-PAGE and LC-MS, indicated that oxidation was also affected by crowding, with enhanced amino acid loss (45% for casein), chain reactions and altered extents of oligomer formation. The overall effects of dextran-mediated crowding were however dependent on the protein structure. Overall, these data indicate that molecular crowding, as commonly encountered in biological systems affect the rates, and extents of oxidation, and particularly of Trp residues, illustrating the importance of appropriate choice of in vitro systems to study biological oxidations., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The present work examined the oxidation and crosslinking of the anti-bacterial enzyme lysozyme (Lyso), which is present in multiple biological fluids, and released from the cytoplasmic granules of macrophages and neutrophils at sites of infection and inflammation. It is therefore widely exposed to oxidants including peroxyl radicals (ROO•). We hypothesized that exposure to ROO• would generate specific modifications and inter- and intra-protein crosslinks via radical-radical reactions. Lyso was incubated with AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) as a ROO• source. Enzymatic activity was assessed, while oxidative modifications were detected and quantified using electrophoresis and liquid chromatography (UPLC) with fluorescence or mass detection (MS). Computational models of AAPH-Lyso interactions were developed. Exposure of Lyso to AAPH (10 and 100 mM for 3 h, and 20 mM for 1 h), at 37 °C, decreased enzymatic activity. 20 mM AAPH showed the highest efficiency of Lyso inactivation (1.78 mol of Lyso inactivated per ROO•). Conversion of Met to its sulfoxide, and to a lesser extent, Tyr oxidation to 3,4-dihydroxyphenylalanine and diTyr, were detected by UPLC-MS. Extensive transformation of Trp, involving short chain reactions, to kynurenine, oxindole, hydroxytryptophan, hydroperoxides or di-alcohols, and N-formyl-kynurenine was detected, with Trp62, Trp63 and Trp108 the most affected residues. Interactions of AAPH inside the negatively-charged catalytic pocket of Lyso, with Trp108, Asp52, and Glu35, suggest that Trp108 oxidation mediates, at least partly, Lyso inactivation. Crosslinks between Tyr20-Tyr23 (intra-molecular), and Trp62-Tyr23 (inter-molecular), were detected with both proximity (Tyr20-Tyr23), and chain flexibility (Trp62) appearing to favor the formation of covalent crosslinks., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oxidation and inactivation of FtsZ is of interest due to the key role of this protein in bacterial cell division. In the present work, we studied peroxyl radical (from AAPH, 2,2'-azobis(2-methylpropionamidine)dihydrochloride) mediated oxidation of the highly stable FtsZ protein (MjFtsZ) from M. jannaschii, a thermophilic microorganism. MjFtsZ contains eleven Met, and single Tyr and Trp residues which would be expected to be susceptible to oxidation. We hypothesized that exposure of MjFtsZ to AAPH-derived radicals would induce Met oxidation, and cross-linking (via di-Tyr and di-Trp formation), with concomitant loss of its functional polymerization and depolymerization (GTPase) activities. Solutions containing MjFtsZ and AAPH (10 or 100 mM) were incubated at 37 °C for 3 h. Polymerization/depolymerization were assessed by light scattering, while changes in mass were analyzed by SDS-PAGE. Amino acid consumption was quantified by HPLC with fluorescence detection, or direct fluorescence (Trp). Oxidation products and modifications at individual Met residues were quantified by UPLC with mass detection. Oxidation inhibited polymerization-depolymerization activity, and yielded low levels of irreversible protein dimers. With 10 mM AAPH only Trp and Met were consumed giving di-alcohols, kynurenine and di-Trp (from Trp) and the sulfoxide (from Met). With 100 mM AAPH low levels of Tyr oxidation (but not di-Tyr formation) were also observed. Correlation with the functional analyses indicates that Met oxidation, and particularly Met164 is the key driver of MjFtsZ inactivation, probably as a result of the position of this residue at the protein-protein interface of longitudinal interactions and in close proximity to the GTP binding site., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Peroxyl radicals participate in multiple processes involved in critical changes to cells, tissues, pharmacueticals and foods. Some of these reactions explain their association with degenerative pathologies, including cardiovascular and neurological diseases, as well as cancer development. Azocompounds, and particularly AAPH (2,2'-Azobis(2-methylpropionamidine) dihydrochloride), a cationic water-soluble derivative, have been employed extensively as sources of model peroxyl radicals. A considerable number of studies have reported mechanistic data on the oxidation of biologically-relevant targets, the scavenging activity of foods and natural products, and the reactions with, and responses of, cultured cells. However, despite the (supposed) experimental simplicity of using azocompounds, the chemistry of peroxyl radical production and subsequent reactions is complicated, and not always considered in sufficient depth when analyzing experimental data. The present work discusses the chemical aspects of azocompounds as generators of peroxyl (and other) radicals, together with their contribution to our understanding of biochemistry, pharmaceutical and food chemistry research. The evidence supporting a role for the formation of alkoxyl (RO•) and other radicals during thermal and photochemical decomposition of azocompounds is assessed, together with the potential influence of such species on the reactions under study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Amongst the available methodologies for protein determination, the bicinchoninic acid (BCA) assay highlights for its simplicity, sensitivity, repeatability and reproducibility. Nevertheless, in spite that the general principle behind this methodology is known, there are still unanswered questions regarding the chemistry behind the assay and the experimental conditions commonly employed. The present work explored the kinetics, and the analytical response of the assay to free amino acids, peptides (containing tryptophan and tyrosine), and proteins. Results revealed kinetic profiles characterized by the absence of plateaus, with behaviors depending on the type of the sample. The latter, along with contribution to the BCA index elicited by oxidation products generated at the side chain of tryptophan and tyrosine, as well as pre-oxidized β-casein, evidenced the presence of complex reaction mechanisms. In spite of such complexity, our results showed that the BCA index is not modulated by the incubation time. This applies for responses producing absorbance intensities (at 562 nm) higher than 0.1. Therefore, we propose that the assay can be applied at shorter incubation times (15 min) than those indicated in manufactures specifications, and usually used by researches and industry (30 min at 37 °C)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dityrosine and ditryptophan bonds have been implied in protein crosslinking. This is associated with oxidative stress conditions including those involved in neurodegenerative pathologies and age-related processes. Formation of dityrosine and ditryptophan derives from radical-radical reactions involving Tyr˙ and Trp˙ radicals. However, cross reactions of Tyr˙ and Trp˙ leading to Tyr-Trp crosslinks and their biological consequences have been less explored. In the present work we hypothesized that exposure of free Tyr and Trp to a high concentration of carbonate anion radicals (CO 3 ˙ - ), under anaerobic conditions, would result in the formation of Tyr-Trp species, as well as dityrosine and ditryptophan crosslinks. Here we report a simple experimental procedure, employing CO 3 ˙ - generated photochemically by illumination of a Co(iii) complex at 254 nm, that produces micromolar concentrations of Tyr-Trp crosslinks. Analysis by mass spectrometry of solutions containing only the individual amino acids, and the Co(iii) complex, provided evidence for the formation of o , o '-dityrosine and isodityrosine from Tyr, and three ditryptophan dimers from Trp. When mixtures of Tyr and Trp were illuminated in an identical manner, Tyr-Trp crosslinks were detected together with dityrosine and ditryptophan dimers. These results indicate that there is a balance between the formation of these three classes of crosslinks, which is dependent on the Tyr and Trp concentrations. The methods reported here allow the generation of significant yields of isolated Tyr-Trp adducts and their characterization. This technology should facilitate the detection, and examination of the biological consequences of Tyr-Trp crosslink formation in complex systems in future investigations., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Photosensitized protein oxidation is a promising tool for medical procedures such as photochemical tissue bonding (PTB). We have recently reported that the binding of rose Bengal, a sensitizer employed in PTB, to lysozyme modulates the photooxidation and crosslinking of this protein. In this work we examined the photooxidation and crosslinking of lysozyme mediated by riboflavin (RF) an endogenous sensitizer also employed in PTB. We hypothesized that since RF does not bind strongly to proteins, the mechanism(s) and extent of enzymatic inactivation, amino acid modification and protein crosslinking would be dependent on the presence of O 2 , and differ to that induced by rose Bengal. This hypothesis was tested using UV-visible spectrophotometry, isothermal titration calorimetry (ITC), SDS-PAGE gels, quantification of amino acid consumption, and LC-MS analysis of sites of modification and crosslinks. Under N 2 , limited damage was detected arising from type 1 (radical) chemistry with formation of specific intra- (Tyr20-Tyr23) and inter- (Tyr23-Trp108) molecular crosslinks. In contrast, the presence of O 2 triggered extensive protein damage through mixed type 1 and type 2 ( 1 O 2 ) mechanisms leading to Trp, Met, Tyr and His oxidation, loss of enzymatic activity and protein dimerization. LC-MS analysis provided evidence for crosslinking via radical-radical recombination reactions (Trp28-Tyr53), and secondary reactions involving nucleophilic attack of the side-chain amine of Lys116 on carbonyl groups. Overall, this behavior is in marked contrast to that detected with rose Bengal indicating that the mechanisms and sites of photo-oxidative damage, and consequences for protein function, can be modulated by the choice of sensitizing dye., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The enhanced green fluorescent protein (eGFP) is one of the most employed variants of fluorescent proteins. Nonetheless little is known about the oxidative modifications that this protein can undergo in the cellular milieu. The present work explored the consequences of the exposure of eGFP to free radicals derived from γ-radiolysis of water, and AAPH thermolysis. Results demonstrated that protein crosslinking was the major pathway of modification of eGFP towards these oxidants. As evidenced by HPLC-FLD and UPLC-MS, eGFP crosslinking would occur as consequence of a mixture of pathways including the recombination of two protein radicals, as well as secondary reactions between nucleophilic residues (e.g. lysine, Lys) with protein carbonyls. The first mechanism was supported by detection of dityrosine and cysteine-tyrosine bonds, whilst evidence of formation of protein carbonyls, along with Lys consumption, would suggest the formation and participation of Schiff bases in the crosslinking process. Despite of the degree of oxidative modifications elicited by peroxyl radicals (ROO • ) generated from the thermolysis of AAPH, and free radicals generated from γ-radiolysis of water, that were evidenced at amino acidic level, only the highest dose of γ-irradiation (10 kGy) triggered significant changes in the secondary structure of eGFP. These results were accompanied by the complete loss of fluorescence arising from the chromophore unit of eGFP in γ-irradiation-treated samples, whereas it was conserved in ROO • -treated samples. These data have potential biological significance, as this fluorescent protein is widely employed to study interactions between cytosolic proteins; consequently, the formation of fluorescent eGFP dimers could act as artifacts in such experiments., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Irradiation from the sun has played a crucial role in the origin and evolution of life on the earth. Due to the presence of ozone in the stratosphere most of the hazardous irradiation is absorbed, nonetheless UVB, UVA, and visible light reach the earth's surface. The high abundance of proteins in most living organisms, and the presence of chromophores in the side chains of certain amino acids, explain why these macromolecules are principal targets when biological systems are illuminated. Light absorption triggers the formation of excited species that can initiate photo-modification of proteins. The major pathways involve modifications derived from direct irradiation and photo-sensitized reactions. In this review we explored the basic concepts behind these photochemical pathways, with special emphasis on the photosensitized mechanisms (type 1 and type 2) leading to protein oxidation, and how this affects protein structure and functions. Finally, a description of the photochemical reactions involved in some human diseases, and medical applications of protein oxidation are presented., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

This work examined the hypothesis that interactions of Rose Bengal (RB 2- ) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring 1 O 2 formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB 2- -Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H 2 18 O and H 2 O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB 2- giving a K d of 4.8 μM, while data from ITC studies, yielded a K d of 0.68 μM as an average of the interactions with stoichiometry of 3.3 RB 2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 ( 1 O 2 ) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Carbonate radicals (CO 3 - ) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO 3 - formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared to previously reported methods using electron paramagnetic resonance spin trapping with DMPO, and the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid). The kinetics of PGR consumption elicited by CO 3 - was followed by visible spectrophotometry. Solutions containing PGR (5-200 μM), SOD-1 (0.3-3 μM), H 2 O 2 (2 mM) in bicarbonate buffer (200 mM, pH 7.4) showed a rapid loss of the PGR absorption band centered at 540 nm. The initial consumption rate (R i ) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO 3 - release of 24.6 ± 4.3 μM min -1 for 3 μM bSOD-1. Both bSOD-1 and hSOD-1 showed a similar peroxidase activity, with enzymatic inactivation occurring over a period of 20 min. The single Trp residue (Trp 32 ) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 ± 0.1 μM min -1 ) with this occurring more rapidly than hSOD-1 inactivation, suggesting that these processes are not directly related. Added free Trp was rapidly oxidized in competition with PGR. These data indicate that PGR reacts rapidly and efficiently with CO 3 - resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO 3 - release from bSOD-1 and hSOD-1 peroxidase activity., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The mechanism of photo-oxidation of tryptophan (Trp) sensitized by riboflavin (RF) was examined employing high concentrations of Trp and RF, with a high intensity 365 nm light emitting diode (LED) source under N 2 , 20% and 100% O 2 atmospheres. Dimerization of Trp was a major pathway under the N 2 atmosphere, though this occurred with a low yield ( D φ Trp = 5.9 × 10 -3 ), probably as a result of extensive back electron transfer reactions between RF •- and Trp(H) •+ . The presence of O 2 decreased the extent of this back electron transfer reaction, and the extent of Trp dimerization. This difference is attributed to the formation of O 2 •- (generated via electron transfer from RF •- to O 2 ) which reacts rapidly with Trp • leading to extensive consumption of the parent amino acid and formation of peroxides and multiple other oxygenated products (N-formylkynurenine, alcohols, diols) of Trp, as detected by LC-MS. Thus, it appears that the first step of the Type 1 mechanism of Trp photo-oxidation, induced by this high intensity 365 nm light source, is an electron transfer reaction between the amino acid and 3 RF, with the presence of O 2 modulating the subsequent reactions and the products formed, as a result of O 2 •- formation. These data have potential biological significance as LED systems and RF-based treatments have been proposed for the treatment of pathological myopia and keratitis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present work examined the role of Tyr and Trp in oxidative modifications of caseins, the most abundant milk proteins, induced by peroxyl radicals (ROO • ). We hypothesized that the selectivity of ROO • and the high flexibility of caseins (implying a high exposure of Tyr and Trp residues) would favor radical-radical reactions, and di-tyrosine (di-Tyr) and di-tryptophan (di-Trp) formation. Solutions of α- and β-caseins were exposed to ROO • from thermolysis and photolysis of AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride). Oxidative modifications were examined using electrophoresis, western blotting, fluorescence, and chromatographic methodologies with diode array, fluorescence and mass detection. Exposure of caseins to AAPH at 37 °C gave fragmentation, cross-linking and protein aggregation. Amino acid analysis showed consumption of Trp, Tyr, Met, His and Lys residues. Quantification of Trp and Tyr products, showed low levels of di-Tyr and di-Trp, together with an accumulation of carbonyls indicating that casein aggregation is, at least partly, associated with secondary reactions between carbonyls and Lys and His residues. AAPH photolysis, which generates a high flux of free radicals increased the extent of formation of di-Tyr in both model peptides and α- and β- caseins; di-Trp was only detected in peptides and α-casein. Thus, in spite of the high flexibility of caseins, which would be expected to favor radical-radical reactions, the low flux of ROO • generated during AAPH thermolysis disfavours the formation of dimeric radical-radical cross-links such as di-Tyr and di-Trp, instead favoring other O 2 -dependent crosslinking pathways such as those involving secondary reactions of initial carbonyl products., (Copyright © 2018 Elsevier Inc. All rights reserved.)