scholarly journals Protein Formulations Containing Polysorbates: Are Metal Chelators Needed at All?

Antioxidants ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 441 ◽  
Author(s):  
Ema Valentina Brovč ◽  
Stane Pajk ◽  
Roman Šink ◽  
Janez Mravljak
Keyword(s):  
Reactive Oxygen Species ◽  
Hydroxyl Radicals ◽  
Ethylenediaminetetraacetic Acid ◽  
Peptide Mapping ◽  
Reactive Oxygen ◽  
Redox System ◽  
Oxygen Species ◽  
Post Translational Modifications ◽  
Catalysed Oxidation ◽  
Reduced Rate

Proteins are prone to post-translational modifications at specific sites, which can affect their physicochemical properties, and consequently also their safety and efficacy. Sources of post-translational modifications include oxygen and reactive oxygen species. Additionally, catalytic amounts of Fe(II) or Cu(I) can promote increased activities of reactive oxygen species, and thus catalyse the production of particularly reactive hydroxyl radicals. When oxidative post-translational modifications are detected in the biopharmaceutical industry, it is common practice to add chelators to the formulation. However, the resultant complexes with metals can be even more damaging. Indeed, this is supported here using an ascorbate redox system assay and peptide mapping. Ethylenediaminetetraacetic acid (EDTA) addition strongly accelerated the formation of hydroxyl radicals in an iron-ascorbate system, while diethylenetriaminepentaacetic acid (DTPA) addition did not. When Fe(III) was substituted with Cu(II), EDTA addition almost stopped hydroxyl radical production, whereas DTPA addition showed continued production, but at a reduced rate. Further, EDTA accelerated metal-catalysed oxidation of proteins, and thus did not protect them from Fe-mediated oxidative damage. As every formulation is unique, justification for EDTA or DTPA addition should be based on experimental data and not common practice.

scholarly journals Role of mitochondrial superoxide dismutase in contraction-induced generation of reactive oxygen species in skeletal muscle extracellular space

2004 ◽  
Vol 286 (5) ◽  
pp. C1152-C1158 ◽  
Author(s):  
A. McArdle ◽  
J. van der Meulen ◽  
G. L. Close ◽  
D. Pattwell ◽  
H. Van Remmen ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Extracellular Space ◽  
Superoxide Anion ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Superoxide Anions ◽  
Wild Type ◽  
Mnsod Activity

Contractions of skeletal muscles produce increases in concentrations of superoxide anions and activity of hydroxyl radicals in the extracellular space. The sources of these reactive oxygen species are not clear. We tested the hypothesis that, after a demanding isometric contraction protocol, the major source of superoxide and hydroxyl radical activity in the extracellular space of muscles is mitochondrial generation of superoxide anions and that, with a reduction in MnSOD activity, concentration of superoxide anions in the extracellular space is unchanged but concentration of hydroxyl radicals is decreased. For gastrocnemius muscles from adult (6–8 mo old) wild-type ( Sod2+/+) mice and knockout mice heterozygous for the MnSOD gene ( Sod2+/-), concentrations of superoxide anions and hydroxyl radical activity were measured in the extracellular space by microdialysis. A 15-min protocol of 180 isometric contractions induced a rapid, equivalent increase in reduction of cytochrome c as an index of superoxide anion concentrations in the extracellular space of Sod2+/+ and Sod2+/- mice, whereas hydroxyl radical activity measured by formation of 2,3-dihydroxybenzoate from salicylate increased only in the extracellular space of muscles of Sod2+/+ mice. The lack of a difference in increase in superoxide anion concentration in the extracellular space of Sod2+/+ and Sod2+/- mice after the contraction protocol supported the hypothesis that superoxide anions were not directly derived from mitochondria. In contrast, the data obtained suggest that the increase in hydroxyl radical concentration in the extracellular space of muscles from wild-type mice after the contraction protocol most likely results from degradation of hydrogen peroxide generated by MnSOD activity.

Reactive Oxygen Species Influence Biofilm Formation of Acidophilic Mineral-Oxidizing Bacteria on Pyrite

2015 ◽  
Vol 1130 ◽  
pp. 118-122 ◽  
Author(s):  
Sören Bellenberg ◽  
Dieu Huynh ◽  
Laura Castro ◽  
Maria Boretska ◽  
Wolfgang Sand ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Hydroxyl Radicals ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Reactive Oxygen ◽  
Laser Scanning Microscopy ◽  
Metal Sulfides ◽  
Confocal Laser ◽  
Water Molecules ◽  
Oxygen Species

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide (O2-) and hydroxyl radicals (OH.) are known to be formed on the surface of metal sulfides in aqueous solution under oxic and anoxic conditions. Consequently bacteria which have not been adapted to their presence are metabolically inhibited [1], presumably due to the presence of these ROS. Pyrite-grown cells ofAcidithiobacillus ferrooxidansT, in contrast to iron (II)-grown cells, were able to oxidize iron (II)-ions or pyrite after 24 h starvation and contact with 1 mM externally added H2O2. In this study, similar results were obtained withAcidiferrobactersp. SPIII/3. However,Acidithiobacillus ferrivoransSS3 showed the highest tolerance towards contact with H2O2, whileLeptospirillum ferrooxidansDSM 2391 was most sensitive. Similar results were obtained after exposure to defined doses of gamma radiation, which cleaves water molecules and generates ROS. In this study members of the three aforementioned genera of mineral-oxidizing bacteria were compared regarding their ability to survive, colonize pyrite and to oxidize iron (II)-ions after exposure to different concentrations of H2O2. Pyrite colonization was studied after exposure to endogenous ROS formed on pyrite or after external addition of H2O2using confocal laser scanning microscopy (CLSM).

scholarly journals Reactive Oxygen Species and Induction of Lignin Peroxidase in Phanerochaete chrysosporium

2003 ◽  
Vol 69 (11) ◽  
pp. 6500-6506 ◽  
Author(s):  
Paula A. Belinky ◽  
Nufar Flikshtein ◽  
Sergey Lechenko ◽  
Shimon Gepstein ◽  
Carlos G. Dosoretz
Keyword(s):  
Reactive Oxygen Species ◽  
Oxidative Damage ◽  
Phanerochaete Chrysosporium ◽  
Hydroxyl Radicals ◽  
Lignin Peroxidase ◽  
Manganese Superoxide Dismutase ◽  
Reactive Oxygen ◽  
Radical Scavenger ◽  
Oxygen Species ◽  
Atmospheric Air

ABSTRACT We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O2) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O2 gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O2 (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O2 concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O2 is at least partially mediated by the intracellular ROS.

scholarly journals Ebsulfur Is a Benzisothiazolone Cytocidal Inhibitor Targeting the Trypanothione Reductase of Trypanosoma brucei

2013 ◽  
Vol 288 (38) ◽  
pp. 27456-27468 ◽  
Author(s):  
Jun Lu ◽  
Suman K. Vodnala ◽  
Anna-Lena Gustavsson ◽  
Tomas N. Gustafsson ◽  
Birger Sjöberg ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Reactive Oxygen ◽  
Redox System ◽  
Trypanothione Reductase ◽  
Oxygen Species ◽  
African Trypanosomiasis ◽  
Thiol Redox

Trypanosoma brucei is the causing agent of African trypanosomiasis. These parasites possess a unique thiol redox system required for DNA synthesis and defense against oxidative stress. It includes trypanothione and trypanothione reductase (TryR) instead of the thioredoxin and glutaredoxin systems of mammalian hosts. Here, we show that the benzisothiazolone compound ebsulfur (EbS), a sulfur analogue of ebselen, is a potent inhibitor of T. brucei growth with a favorable selectivity index over mammalian cells. EbS inhibited the TryR activity and decreased non-protein thiol levels in cultured parasites. The inhibition of TryR by EbS was irreversible and NADPH-dependent. EbS formed a complex with TryR and caused oxidation and inactivation of the enzyme. EbS was more toxic for T. brucei than for Trypanosoma cruzi, probably due to lower levels of TryR and trypanothione in T. brucei. Furthermore, inhibition of TryR produced high intracellular reactive oxygen species. Hydrogen peroxide, known to be constitutively high in T. brucei, enhanced the EbS inhibition of TryR. The elevation of reactive oxygen species production in parasites caused by EbS induced a programmed cell death. Soluble EbS analogues were synthesized and cured T. brucei brucei infection in mice when used together with nifurtimox. Altogether, EbS and EbS analogues disrupt the trypanothione system, hampering the defense against oxidative stress. Thus, EbS is a promising lead for development of drugs against African trypanosomiasis.

scholarly journals The oxidation of dehydroascorbic acid and 2,3-diketogulonate by distinct reactive oxygen species

2018 ◽  
Vol 475 (21) ◽  
pp. 3451-3470 ◽  
Author(s):  
Rebecca A. Dewhirst ◽  
Stephen C. Fry
Keyword(s):  
Reactive Oxygen Species ◽  
Ascorbic Acid ◽  
Hydroxyl Radicals ◽  
Reactive Oxygen ◽  
Dehydroascorbic Acid ◽  
Oxidation Products ◽  
Oxygen Species ◽  
H2o2 Treatment ◽  
Fold Excess

l-Ascorbate, dehydro-l-ascorbic acid (DHA), and 2,3-diketo-l-gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H2O2), ‘superoxide’ (a ∼50 : 50 mixture of and ), hydroxyl radicals (•OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1O2). Products were monitored electrophoretically. DHA quenched H2O2 far more effectively than superoxide, but the main products in both cases were 4-O-oxalyl-l-threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H2O2, but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical •OH yield; it yielded oxalate, threonate, and OxT. 1O2 had no effect on DHA. DKG was oxidatively decarboxylated by H2O2, Fenton mixture, and 1O2, forming a newly characterised product, 2-oxo-l-threo-pentonate (OTP; ‘2-keto-l-xylonate’). Superoxide yielded negligible OTP. Prolonged H2O2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H2O2, Fenton mixture, or 1O2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H2O2 and superoxide; DKG more readily by 1O2. The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product ‘fingerprints’ are analytically useful, indicating which ROS are acting in vivo.

scholarly journals Reactive Oxygen Species Regulate Oxygen-Sensitive Potassium Flux in Rainbow Trout Erythrocytes

2001 ◽  
Vol 117 (2) ◽  
pp. 181-190 ◽  
Author(s):  
Anna Yu Bogdanova ◽  
Mikko Nikinmaa
Keyword(s):  
Reactive Oxygen Species ◽  
Hydroxyl Radicals ◽  
Fenton Reaction ◽  
Copper Ions ◽  
Reactive Oxygen ◽  
Potassium Transport ◽  
Oxygen Species ◽  
Hypoxic Conditions ◽  
Potassium Flux ◽  
Oxygen Sensitive

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K+-Cl− cotransport). 5 mM hydrogen peroxide caused an increase in K+-Cl− cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K+-Cl− cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K+-Cl− cotransport significantly at hypoxic conditions (1% O2). These data suggest that hydroxyl radicals, formed from O2 in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K+-Cl− cotransporter.

scholarly journals Yap1-Regulated Glutathione Redox System Curtails Accumulation of Formaldehyde and Reactive Oxygen Species in Methanol Metabolism of Pichia pastoris

2009 ◽  
Vol 8 (4) ◽  
pp. 540-549 ◽  
Author(s):  
Taisuke Yano ◽  
Emiko Takigami ◽  
Hiroya Yurimoto ◽  
Yasuyoshi Sakai
Keyword(s):  
Reactive Oxygen Species ◽  
Pichia Pastoris ◽  
Critical Role ◽  
Reactive Oxygen ◽  
Redox System ◽  
Reduced Form ◽  
Growth Defect ◽  
Oxygen Species ◽  
Glutathione Redox System ◽  
Glutathione Redox

ABSTRACT The glutathione redox system, including the glutathione biosynthesis and glutathione regeneration reaction, has been found to play a critical role in the yeast Pichia pastoris during growth on methanol, and this regulation was at least partly executed by the transcription factor PpYap1. During adaptation to methanol medium, PpYap1 transiently localized to the nucleus and activated the expression of the glutathione redox system and upregulated glutathione reductase 1 (Glr1). Glr1 activates the regeneration of the reduced form of glutathione (GSH). Depletion of Glr1 caused a severe growth defect on methanol and hypersensitivity to formaldehyde (HCHO), which could be complemented by addition of GSH to the medium. Disruption of the genes for the HCHO-oxidizing enzymes PpFld1 and PpFgh1 caused a comparable phenotype, but disruption of the downstream gene PpFDH1 did not, demonstrating the importance of maintaining intracellular GSH levels. Absence of the peroxisomal glutathione peroxidase Pmp20 also triggered nuclear localization of PpYap1, and although cells were not sensitive to HCHO, growth on methanol was again severely impaired due to oxidative stress. Thus, the PpYap1-regulated glutathione redox system has two important roles, i.e., HCHO metabolism and detoxification of reactive oxygen species.

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