Comparative study of three magnetic nano-particles (FeSO4, FeSO4/SiO2, FeSO4/SiO2/TiO2) in plasmid DNA extraction

Recent updates on Magnetic Nano-Particles (MNPs) based separation of nucleic acids have received more attention due to their easy manipulation, simplicity, ease of automation and cost-effectiveness. It has been indicated that DNA molecules absorb on solid surfaces via hydrogen-bonding, and hydrophobic and electrostatic interactions. These properties highly depend on the surface condition of the solid support. Therefore, surface modification of MNPs may enhance their functionality and specification. In the present study, we functionalized Fe₃O₄ nano-particle surface utilizing SiO₂ and TiO₂ layer as Fe₃O₄/SiO₂ and Fe₃O₄/SiO₂/TiO₂ and then compare their functionality in the adsorption of plasmid DNA molecules with the naked Fe₃O₄ nano-particles. The result obtained showed that the purity and amount of DNA extracted by Fe₃O₄ coated by SiO₂ or SiO₂/TiO₂ were higher than the naked Fe₃O₄ nano-particles. Furthermore, we obtained pH 8 and 1.5 M NaCl as an optimal condition for desorption of DNA from MNPs. The result further showed that, 0.2 mg nano-particle and 10 min at 55 °C are the optimal conditions for DNA desorption from nano-particles. In conclusion, we recommended Fe₃O₄/SiO₂/TiO₂ as a new MNP for separation of DNA molecules from biological sources.     

Multilayered Magnetic Nanoparticles as a Support in Solid-Phase Peptide Synthesis

The synthesis of multilayered magnetic nanoparticles (MNPs) for use as a support in solid-phase peptide synthesis (SPPS) is described. Silanization of magnetite (Fe₃O₄) nanoparticles with 3-(trimethoxysilyl)propyl methacrylate introduced polymerizable groups on the surface. Polymerization with allylamine, trimethylolpropane trimethacrylate, and trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate provided a polymeric coating and amino groups to serve as starting points for the synthesis. After coupling of an internal reference amino acid and a cleavable linker, the coated MNPs were applied as the solid phase during synthesis of Leu-enkephalinamide and acyl carrier protein (65-74) by Fmoc chemistry. A “high-load” version of the MNP support (0.32 mmol/g) was prepared by four consecutive cycles of Fmoc-Lys(Fmoc)-OH coupling and Fmoc deprotection. Successful synthesis of Leu-enkephalin was demonstrated on the “high-load” MNPs. Chemical stability studies proved the particles to be stable under SPPS conditions and magnetization measurements showed that the magnetic properties of the particles were maintained throughout derivatizations and SPPS. The MNPs were further characterized by high-resolution transmission electron microscopy, inductively coupled plasma atomic emission spectrometry, elemental analysis, and nitrogen gas adsorption measurements.     

Double-Layer Magnetic Nanoparticle-Embedded Silica Particles for Efficient Bio-Separation

Superparamagnetic Fe₃O₄ nanoparticles (NPs) based nanomaterials have been exploited in various biotechnology fields including biomolecule separation. However, slow accumulation of Fe₃O₄ NPs by magnets may limit broad applications of Fe₃O₄ NP-based nanomaterials. In this study, we report fabrication of Fe₃O₄ NPs double-layered silica nanoparticles (DL MNPs) with a silica core and highly packed Fe₃O₄ NPs layers. The DL MNPs had a superparamagnetic property and efficient accumulation kinetics under an external magnetic field. Moreover, the magnetic field-exposed DL MNPs show quantitative accumulation, whereas Fe₃O₄ NPs single-layered silica nanoparticles (SL MNPs) and silica-coated Fe₃O₄ NPs produced a saturated plateau under full recovery of the NPs. DL MNPs are promising nanomaterials with great potential to separate and analyze biomolecules.     

A quantitating reagent for methanethiolation of protein sulfhydryl groups

p-Nitrophenoxycarbonyl methyl disulfide has been synthesized for use as a quantitating agent for methanethiolation of protein sulfhydryl groups. This reagent reacts specifically and quantitatively with cysteine residues of proteins to yield an unsymmetrical disulfide containing a CH₃S group and concomitantly releases the chromophore, p-nitrophenol. Titration of the sulfhydryl groups of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with this reagent has been studied. Incorporation of CH₃S as measured by the release of p-nitrophenol paralleled the loss of sulfhydryl group dependent activity of the enzyme. The enzyme was found inactive on modification of four of the eight sulfhydryl groups present in the enzyme. Stability of p-nitrophenoxycarbonyl methyl disulfide has also been studied in different buffer systems. The rate of decomposition of the p-nitrophenyl ester due to hydrolysis was found negligible below a pH of 8.0 compared to its rate of reaction with free sulfhydryl groups.     

Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H₂O₂-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H₂O₂ to form Cys sulfinic acid (Cys-SO₂H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H₂O₂ and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.     

Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP–HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β₄ can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI–MS (electrospray ionization–mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β₄ did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β₄ as well as of other proteins and peptides.     

Syntheses of Isoquinolines by Condensation of Dopamine with Carbonyl Compounds

A cleavable photoactivable heterobifunctional reagent, the N-hydroxysuccinimide ester of 1- azido-5-naphthalene sulfonyl S-carboxymethylthiocysteamine (NHS-ANS-CTC), was synthesized and characterized. The reagent was applicable to the group-directed modification of protein ligands, such as an invertebrate lectin and a trypsin inhibitor. The modified ligands bound to rabbit erythrocyte ghosts and trypsin, respectively. Upon exposure to ultraviolet light, the modified ligands reacted with their binding proteins to form cross-linked fluorescent products. The cross-linked fluorescent complexes were readily cleaved by reducing the disulfide bond of the reagent, leaving the fluorescent probe on the binding proteins. The photolabeled binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The fluorescence intensity of the fluorescent probe was enhanced by 4~8 times to improve sensitivity when the SDS-gel was dehydrated with methanol. This phenomenon was also observed with the proteins labeled with 1-dimethylamino-5-naphthalene sulfonyl chloride.     

Oxidative protein folding: Selective pressure for prolamin evolution in rice

During seed development, endosperm cells of highly productive cereals, including rice, synthesize disulfide-rich proteins in large amounts and deposit them into storage organelles. Disulfide bond formation involves electron transfer and generates H₂O₂ as a by-product. To ensure proper development and maturation of seeds, the endosperm cells must supply large amounts of oxidizing equivalents to dithiols in nascent proteins in a controlled manner. This review compares multiple oxidative protein folding systems in yeast, cultured human cells, and rice endosperm. We discuss possible roles of ERO1, other sulfhydryl oxidases, and the protein disulfide isomerase family in the formation of disulfide bonds in storage proteins and the development of protein bodies. Rice prolamins, encoded by a multigene family, are divided into Cys-rich and Cys-depleted subgroups. We discuss the potential importance of disulfide bond formation in the evolution of the prolamin family in japonica rice.     

Bridging Small Molecules to Modified Bacterial Microparticles Using a Disulphide Linkage: MIS416 as a Cargo Delivery System

MIS416 is an intact minimal cell wall skeleton derived from Proprionibacterium acnes that is phagocytosed by antigen presenting cells, including dendritic cells (DCs). This property allows MIS416 to be exploited as a vehicle for the delivery of peptide antigens or other molecules (for example, nucleic acids) to DCs. We previously showed that covalent (non-cleavable) conjugation of OVA, a model antigen derived from ovalbumin, to MIS416 enhanced immune responses in DCs in vivo, compared to unconjugated MIS416 and OVA. Intracellular trafficking promotes the lysosomal degradation of MIS416, leading to the destruction of MIS416 plus the associated cargos conjugated to MIS416. However, lysosomal degradation of cargo may not be desired for some MIS416 conjugates. Here we have investigated whether a cleavable linkage could facilitate release of the cargo in the cytoplasm of DCs to avoid lysosomal degradation. DCs were treated in vitro with disulfide-containing conjugates, and as hypothesised faster release of SIINFEKL peptide in the cytoplasm of DCs was observed with the inclusion of a disulfide bond between MIS416 and cargo. The inclusion of a cleavable disulfide bond in the conjugates did not significantly alter the amount of SIINFEKL antigens presented on MHC I molecules on DCs as compared with conjugates without a disulfide bond. However, the conjugates containing disulfide-linkages performed either slightly better (p<0.05) than, or the same as conjugates without a disulfide bond with respect to in vitro OT-1 T-cell proliferation induced by the presentation of SIINFEKL antigens on DCs, or DC activation studies, respectively. However, disulfide-containing conjugates were less effective than conjugates without a disulfide bond in in vivo cytotoxicity assays. In conclusion, inclusion of a disulfide bond in MIS416-peptide conjugates was associated with efficient release of peptides in the cytoplasm of DCs, an important consideration for MIS416-mediated delivery of degradation-sensitive cargoes. However, treatment of DCs with disulfide-containing conjugates did not significantly alter the presentation of peptide antigens on MHC class I molecules to T-cells, or greatly enhance antigen-associated T-cell proliferation in vitro.     

The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k_F^H2O = 72,600 s^− 1), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.     

Magnetic nano-Fe3O4 particles targeted gathering and bio-effects on nude mice loading human hepatoma Bel-7402 cell lines model under external magnetic field exposure in vivo

Abstract

Magnetic nano-Fe₃O₄ particles (MNPs), static magnetic field (SMF) and extremely low-frequency altering electric magnetic field (ELFF) were utilized to treat nude mice loading hepatoma Bel-7402 cell lines to investigate the therapeutic values of MNPs combined with ELFF in vivo. Magnetic resonance image (MRI) figures showed that about 98.9% MNPs injected into mice body through tail vein were gathered in tumor focal by SMF directing exposure. Single ELFF and MNPs treatments did not influence mice physiological function obviously. However, gathered MNPs combined with ELFF treatment prolonged mice survival time and inhibited loading tumor cells proliferation significantly compared to other mice groups (p?<?0.05); furthermore, the tumor cells early apoptosis ratio of mice group was significantly higher than other groups (p?<?0.05), and ELFF combined with gathered MNPs treatment improved tumor cells early apoptosis associated with Bcl group protein expression: Bax protein expression was higher than Bcl-2 and the combined treatment improved cells Heat shock protein-27 (Hsp-27) expression which could protect cells avoiding early apoptosis. The possible mechanism that this kind of combination inducing more cells into early apoptosis could be due to ELFF exposure influencing cells ion metabolism, MNPs strengthening the effects, and the ELFF vibrating MNPs to generate extra heat and activate cellular heat shock signal channel.     

Going through the Barrier: COUPLED DISULFIDE EXCHANGE REACTIONS PROMOTE EFFICIENT CATALYSIS IN QUIESCIN SULFHYDRYL OXIDASE*

The quiescin sulfhydryl oxidase (QSOX) family of enzymes generates disulfide bonds in peptides and proteins with the reduction of oxygen to hydrogen peroxide. Determination of the potentials of the redox centers in Trypanosoma brucei QSOX provides a context for understanding catalysis by this facile oxidant of protein thiols. The CXXC motif of the thioredoxin domain is comparatively oxidizing (E′₀ of −144 mV), consistent with an ability to transfer disulfide bonds to a broad range of thiol substrates. In contrast, the proximal CXXC disulfide in the ERV (essential for respiration and vegetative growth) domain of TbQSOX is strongly reducing (E′₀ of −273 mV), representing a major apparent thermodynamic barrier to overall catalysis. Reduction of the oxidizing FAD cofactor (E′₀ of −153 mV) is followed by the strongly favorable reduction of molecular oxygen. The role of a mixed disulfide intermediate between thioredoxin and ERV domains was highlighted by rapid reaction studies in which the wild-type CGAC motif in the thioredoxin domain of TbQSOX was replaced by the more oxidizing CPHC or more reducing CGPC sequence. Mixed disulfide bond formation is accompanied by the generation of a charge transfer complex with the flavin cofactor. This provides thermodynamic coupling among the three redox centers of QSOX and avoids the strongly uphill mismatch between the formal potentials of the thioredoxin and ERV disulfides. This work identifies intriguing mechanistic parallels between the eukaryotic QSOX enzymes and the DsbA/B system catalyzing disulfide bond generation in the bacterial periplasm and suggests that the strategy of linked disulfide exchanges may be exploited in other catalysts of oxidative protein folding.     

Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-coupled Transporter Extracellular Topography

The extracellular loop 3 (EL-3) of SLC4 Na⁺-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na⁺-HCO₃⁻ cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys⁵⁸³-Cys⁵⁸⁵ and Cys⁶¹⁷-Cys⁶⁴², respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.     

Activation of sulfonate ester based matrix metalloproteinase proinhibitors by hydrogen peroxide

This study details the development of matrix metalloproteinase inhibitor prodrugs (proMMPi) that are activated in the presence of reactive-oxygen species (ROS). Conventional matrix metalloproteinase inhibitors (MMPi) utilize a zinc-binding group (ZBG) that chelates to the catalytic zinc(II) ion of matrix metalloproteinases (MMPs) to inhibit their activity. To create ROS-sensitive prodrugs, sulfonate esters were used as a protecting group for the ZBG to block their metal binding ability. Surprisingly, these sulfonate esters were found to be cleaved by H₂O₂ only when the ZBG contained an N-oxide donor atom moiety. Sulfonate ester derivatives of full-length MMPi based on these ROS-triggerable systems were synthesized. It was found that proMMPi with sulfonate ester protecting groups showed relatively high rates of cleavage in the presence of H₂O₂ to release the active MMPi. In vitro MMP inhibition studies confirmed a significant increase in inhibitory activity of proMMPi upon addition of H₂O₂, demonstrating the use of sulfonate esters to act as cleavable triggers for ROS-activated prodrugs.     

Toxicity Assessment of Silica Coated Iron Oxide Nanoparticles and Biocompatibility Improvement by Surface Engineering

We have studied in vitro toxicity of iron oxide nanoparticles (NPs) coated with a thin silica shell (Fe₃O₄/SiO₂ NPs) on A549 and HeLa cells. We compared bare and surface passivated Fe₃O₄/SiO₂ NPs to evaluate the effects of the coating on the particle stability and toxicity. NPs cytotoxicity was investigated by cell viability, membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assays, and their genotoxicity by comet assay. Our results show that NPs surface passivation reduces the oxidative stress and alteration of iron homeostasis and, consequently, the overall toxicity, despite bare and passivated NPs show similar cell internalization efficiency. We found that the higher toxicity of bare NPs is due to their stronger in-situ degradation, with larger intracellular release of iron ions, as compared to surface passivated NPs. Our results indicate that surface engineering of Fe₃O₄/SiO₂ NPs plays a key role in improving particles stability in biological environments reducing both cytotoxic and genotoxic effects.     

Effect of the concentration of protein and nanoparticles on the structure of biohybrid nanocomposites

We present colloidal nanocomposites formed by incorporating magnetite Fe₃O₄ nanoparticles (MNPs) with lysozyme amyloid fibrils (LAFs). Preparation of two types of solutions, with and without addition of salt, was carried out to elucidate the structure of MNPs-incorporated fibrillary nanocomposites and to study the effect of the presence of salt on the stability of the nanocomposites. The structural morphology of the LAFs and their interaction with MNPs were analyzed by atomic force microscopy and small-angle x-ray scattering measurements. The results indicate that conformational properties of the fibrils are dependent on the concentration of protein, and the precise ratio of the concentration of the protein and MNPs is crucially important for the stability of the fibrillary nanocomposites. Our results confirm that despite the change in fibrillary morphology induced by the varying concentration of the protein, the adsorption of MNPs on the surface of LAF is morphologically independent. Moreover, most importantly, the samples containing salt have excellent stability for up to 1 year of shelf-life.     

Aminooxy adsorbents derived from sephareose and toyopearl

Convenient synthetic schemes being suitable for the preparation of aminooxy-Sepharose and hydroxylamine-containing Toyopearl with capacity of 0.35–0.015 mmols of H₂NO-groups per mL gel, including the adsorbent with easily cleavable disulfide bond in the linker are developed.     

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.     

Is the heme pocket region modulated by disulfide-bridge formation in fish and amphibian neuroglobins as in humans?

Neuroglobin, a globin characterized by a bis-histidine ligation of the heme iron, has been identified in mammalian and non-mammalian vertebrates, including fish, amphibians and reptiles. In human neuroglobin, the presence of an internal disulfide bond in the CD loop (CD7–D5) is found to modulate the ligand binding through a change in the heme pocket structure. Although the neuroglobin sequences mostly display conserved Cys at positions CD7, D5 and G18/19, a number of exceptions are known. In this study, neuroglobins from amphibian (Xenopus tropicalis) and fish (Chaenocephalus aceratus, Dissostichus mawsoni and Danio rerio) are investigated using electron paramagnetic resonance and optical absorption spectroscopy. All these neuroglobins differ from human neuroglobin in their Cys-positions. It is demonstrated that if disulfide bonds are formed in fish and amphibian neuroglobins, the reduction of these bonds does not result in alteration of the heme pocket in these globins. Furthermore, it is shown that mutagenesis of the Cys residues of X. tropicalis neuroglobin influences the protein structure. The amphibian neuroglobin is also found to be more resistant to H₂O₂-induced denaturation than the other neuroglobins under study, although all show an overall large stability in high concentrations of this oxidant. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.