Cysteine and Crocin Oxidation Catalyzed by Horseradish Peroxidase

The amino acid cysteine is oxidized by horseradish peroxidase, and the water-soluble carotenoid crocin is bleached by cooxidation. The rnonophenol p-hydroxyacetophenone stimulates oxygen uptake, cysteine oxidation and crocin bleaching, whereas its concentration does not change. Superoxide dismutase significantly enhances all these oxidative reactions. Addition of H₂O₂ is not required for these peroxidase-catalyzed oxidations.     

The protective effect of crocin on the amyloid fibril formation of aβ42 peptide in vitro

Aβ is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer’s disease. There are two common isoforms of Aβ: the more common form, Aβ₄₀, and the less common but more amyloidogenic form, Aβ₄₂. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aβ₄₂ amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ₄₂ amyloids. To further investigate the relationship between crocin and Aβ₄₂ structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aβ₄₂. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aβ. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aβ₄₂. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and β-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aβ amyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.     

Crocin Abrogates Carbon Tetrachloride‐Induced Renal Toxicity in Rats via Modulation of Metabolizing Enzymes and Diminution of Oxidative Stress,Apoptosis, and Inflammatory Cytokines

This work aimed at investigating the potential modulatory effects and mechanisms of crocin against CCl₄‐induced nephrotoxicity. Forty male rats were allocated for three weeks treatment with corn oil, CCl₄, crocin, or crocin plus CCl₄. Crocin effectively mitigated CCl₄‐induced kidney injury as evidenced by amelioration of alterations in kidney histopathology, renal weight/100 g body weight ratio and kidney functions. Crocin modulated CCl₄‐induced disturbance of kidney cytochrom‐P450 subfamily 2E1 and glutathione‐S‐transferase. The attenuation of crocin to kidney injury was also associated with suppression of oxidative stress via reduction of lipid peroxides along with induction of renal glutathione content and enhancement of superoxide dismutase, glutathione peroxidase, and catalase activities. Crocin mitigated CCl₄‐induced elevation of the renal levels of tumor necrosis factor‐alpha, interleukin‐6, prostaglandin E2, and active caspases‐3. Collectively, crocin alleviated CCl₄‐induced renal damage via modulation of kidney metabolizing enzymes, suppression of oxidative stress, inhibition of inflammatory cytokines, PGE2, and active caspase3 in kidney.     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

Biochemical basis of sulphenomics: how protein sulphenic acids may be stabilized by the protein microenvironment

Among protein residues, cysteines are one of the prominent candidates to ROS‐mediated and RNS‐mediated post‐translational modifications, and hydrogen peroxide (H₂O₂) is the main ROS candidate for inducing cysteine oxidation. The reaction with H₂O₂ is not common to all cysteine residues, being their reactivity an utmost prerequisite for the sensitivity towards H₂O₂. Indeed, only deprotonated Cys (i.e. thiolate form, ? S^?) can react with H₂O₂ leading to sulphenic acid formation (? SOH), which is considered as a major/central player of ROS sensing pathways. However, cysteine sulphenic acids are generally unstable because they can be further oxidized to irreversible forms (sulphinic and sulphonic acids, ? SO₂H and ? SO₃H, respectively), or alternatively, they can proceed towards further modifications including disulphide bond formation (? SS? ), S‐glutathionylation (? SSG) and sulphenamide formation (? SN?). To understand why and how cysteine residues undergo primary oxidation to sulphenic acid, and to explore the stability of cysteine sulphenic acids, a combination of biochemical, structural and computational studies are required. Here, we will discuss the current knowledge of the structural determinants for cysteine reactivity and sulphenic acid stability within protein microenvironments.     

Effect of hydrogen peroxide on nitrate reductase activity in detached oat leaves in darkness

Nitrate reductase (NR, EC 1.6.6.1) is sensitive to O₂ concentration, and therefore it was of interest to study the action of H₂O₂, a normal substance in plant metabolism, on NR activity in segments of 7-, 14- and 17-day-old leaves of oat (Avena sativa L. ev. Suregrain). After 4 h of treatment in the dark, H₂O₂ decreased NR activity as measured with the in vivo assay. The effect was stronger in 14- and 17- than in 7-day-old leaves. Vacuum infiltration of cysteine did not prevent this decrease. When NR was determined with the in vitro assay, H₂O₂ did not seem to affect the activity after the 4 h treatment. but NR decreased when crude extracts prepared from untreated 14-day-old leaves were incubated directly with H₂O₂. This effect was prevented by addition of cysteine, ascorbate or reduced glutathione to the extracts. In order to study the possibility that low activity of the system for defense against oxidations could account for the age-dependent response of NR to H₂O₂ in the in vivo test, activities of catalase, ascorbate peroxidase and glutathione reductase were measured during leaf development and after a 4-h treatment with H₂O₂ in the dark. No clear correlation was found between the activities of those enzymes and changes in in vivo NR activity caused by H₂O₂. The results suggest that H₂O₂ might affect NR both directly by oxidizing SH-groups and indirectly by decreasing reductant availability as a result of NADH oxidation. The age-dependent response of NR to H₂O₂ treatment could also be explained in terms of decreased NADH availability in the tissues due to decreased NADH synthesis and/or increased degradation.     

Oxidation of proteins in rat heart and lungs by polymorphonuclear leukocyte oxidants

Summary The ability of the polymorphonuclear leukocyte (PMN) oxidants, hypochlorous acid (HOC1) and hydrogen peroxide (H₂O₂), to oxidize proteins in rat heart and lung tissues was investigated. Cardiac myocytes, heart tissue slices, isolated perfused hearts, and lung tissue slices, were treated with HOCI and H₂O₂ and the extent of methionine and cysteine oxidation was determined in the cellular proteins. Cardiac tissues were found to be highly susceptible to oxidation by physiological concentrations of HOCl. For example, in isolated hearts perfused for 60 min with 100 M HOCI, approximately 18010 of the methionine and 2801o of the cysteine residues were oxidized. Lung tissues, unlike those of the heart, were resistant to physiological concentrations of HOCI, showing no oxidation of proteins. HOCI was much more effective than H₂O₂ in oxidizing proteins, suggesting that HOCI may be the most reactive oxidant produced by activated PMN. These studies show that PMN oxidants, in particular HOC I, can cause significant oxidation of proteins in target tissues, and may therefore constitute a primary cause of tissue injury at sites of inflammation. In addition, these studies show that different tissues may have varying susceptibilities to PMN oxidants.     

Differential Effects of Methionine and Cysteine Oxidation on [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Cultured Hippocampal Neurons

Methionine and cysteine residues in proteins are the major targets of reactive oxygen species (ROS). The present work was designed to characterize the impact of methionine and cysteine oxidation upon [Ca²⁺]_i in hippocampal neurons. We investigated the effects of H₂O₂ and chloramine T(Ch-T) agents known to oxidize both cysteine and methionine residues, and 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB)—a cysteine-specific oxidant, on the intracellular calcium in hippocampal neurons. The results showed that these three oxidants, 1 mM H₂O₂, 1 mM Ch-T, and 500 μM DTNB, induced an sustained elevation of [Ca²⁺]_i by 76.1 ± 3.9%, 86.5 ± 5.0%, and 24.4 ± 3.2% over the basal level, respectively. The elevation induced by H₂O₂ and Ch-T was significantly higher than DTNB. Pretreatment with reductant DTT at 1 mM for 10 min completely prevented the action of DTNB on [Ca²⁺]_i, but only partially reduced the effects of H₂O₂ and Ch-T on [Ca²⁺]_i, the reductions were 44.6 ± 4.2% and 29.6 ± 6.1% over baseline, respectively. The elevation of [Ca²⁺]_i induced by H₂O₂ and Ch-T after pretreatment with DTT were statistically higher than that induced by single administration of DTNB. Further investigation showed that the elevation of [Ca²⁺]_i mainly resulted from internal calcium stores. From our data, we propose that methionine oxidation plays an important role in the regulation of intracellular calcium and this regulation may mainly be due to internal calcium stores.     

Role of o-acetylserine in hydrogen sulfide emission from pumpkin leaves in response to sulfate

In the presence of excess sulfate, cysteine synthesis in pumpkin (Cucurbita pepo) leaves is not limited by sulfate reduction, but by the availability of O-acetylserine. Feeding of O-acetylserine or its metabolic precursors S-acetyl-coenzyme-A and coenzyme A to leaf discs enhanced the incorportion of [³⁵S]sulfate into reduced sulfur compounds, mainly into cysteine, at the cost of lowered H₂S emission; the uptake and reduction of sulfate is not affected by these treatments. β-Fluoropyruvate, an inhibitor of the generation of S-acetyl-coenzyme A via pyruvate dehydrogenase, stimulated H₂S emission in response to sulfate. This stimulation is overcompensated by addition of O-acetylserine, S-acetyl-coenzyme A, or coenzyme A. These results indicate that, in the presence of high amounts of sulfate, excess sulfur is reduced and emitted as H₂S into the atmosphere. The H₂S emitted seems to be produced by liberation from a precursor of cysteine rather than by cysteine desulfhydration.     

Regulation of apoptosis by Bcl-2 cysteine oxidation in human lung epithelial cells

Hydrogen peroxide is a key mediator of oxidative stress known to be important in various cellular processes, including apoptosis. B-cell lymphoma-2 (Bcl-2) is an oxidative stress–responsive protein and a key regulator of apoptosis; however, the underlying mechanisms of oxidative regulation of Bcl-2 are not well understood. The present study investigates the direct effect of H₂O₂ on Bcl-2 cysteine oxidation as a potential mechanism of apoptosis regulation. Exposure of human lung epithelial cells to H₂O₂ induces apoptosis concomitant with cysteine oxidation and down-regulation of Bcl-2. Inhibition of Bcl-2 oxidation by antioxidants or by site-directed mutagenesis of Bcl-2 at Cys-158 and Cys-229 abrogates the effects of H₂O₂ on Bcl-2 and apoptosis. Immunoprecipitation and confocal microscopic studies show that Bcl-2 interacts with mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2 [ERK1/2]) to suppress apoptosis and that this interaction is modulated by cysteine oxidation of Bcl-2. The H₂O₂-induced Bcl-2 cysteine oxidation interferes with Bcl-2 and ERK1/2 interaction. Mutation of the cysteine residues inhibits the disruption of Bcl-2–ERK complex, as well as the induction of apoptosis by H₂O₂. Taken together, these results demonstrate the critical role of Bcl-2 cysteine oxidation in the regulation of apoptosis through ERK signaling. This new finding reveals crucial redox regulatory mechanisms that control the antiapoptotic function of Bcl-2.     

Alternatives to the ‘water oxidation pathway’ of biological ozone formation

Recent studies have shown that ozone (O₃) is endogenously generated in living tissues, where it makes both positive and negative physiological contributions. A pathway for the formation of both O₃ and hydrogen peroxide (H₂O₂) was previously proposed, beginning with the antibody or amino acid-catalyzed oxidation of water by singlet oxygen (¹O₂₎ to form hydrogen trioxide (H₂O₃) as a key intermediate. A key pillar of this hypothesis is that some of the H₂O₂ molecules incorporate water-derived oxygen atoms. However, H₂O₃ decomposes extremely readily in water to form ¹O₂ and water, rather than O₃ and H₂O₂. This article highlights key literature indicating that the oxidation of organic molecules such as the amino acids methionine, tryptophan, histidine, and cysteine by ¹O₂ is involved in ozone formation. Based on this, an alternative hypothesis for ozone formation is developed involving a further reaction of singlet oxygen with various oxidized organic intermediates. H₂O₂ having water-derived oxygen atoms is subsequently formed during ozone decomposition in water by known reactions.     

The Effect of Continued Training with Crocin on Apoptosis Markers in Liver Tissue of High Fat Diet Induced Diabetic Rats

Diabetes mellitus (DM) disease can affect process of apoptosis by increasing oxidative stress, nevertheless exercise and crocin can improve apoptosis; therefore present study aimed to investigate the effect of continued training with crocin on apoptosis markers in liver tissue of diabetic rats. In this experimental study 32 diabetic rats based on fasting glucose divided into four groups of eight rats including: 1) sham, 2) training, 3) crocin, and 4) training with crocin also for investigate the effect of DM induction on apoptosis markers, eight healthy rats assigned in healthy control group. During eight weeks groups 2 and 4 ran 60 minutes on treadmill with intensity of 50–55% maximum speed for three sessions per week and groups 3 and 4 received 25 mg/kg/day crocin peritoneally. Shapiro–Wilk, one-way ANOVA with Tukey’s post-hot tests were used for statistical analysis of data (P ≤ 0.05). DM induction significantly increased Bcl-2 as well as decreased Bax and P52 (P ≤ 0.05) nevertheless training and training with crocin significantly decreased Bcl-2 and increased Bax and P53 (P ≤ 0.05); crocin significantly decreased Bcl-2 and increased P53 (P ≤ 0.05) and training with crocin had higher effect on increase of Bax and P53 compare to training (P ≤ 0.05) also increase of Bax compare to crocin. Although training and crocin alone can improve apoptotic markers in diabetic rats, nevertheless training simultaneously with crocin have better effects than training alone.     

Inactivation of Lipoxygenase by Hydrogen Peroxide,Cysteine and Some Other Reagents

The polarographic data show that H₂O₂ is not formed during the course of the coupled oxidation of antioxidants by lipoxygenase from defatted soybean meal. A lower concentration of H₂O₂ or autoxidizing cysteine has been found to induce an irreversible inactivation of the enzyme. Inactivation activity of cysteine is reduced either by the addition of catalase or under anaerobic condition. These facts are indicative of the oxidative function of autoxidizing cysteine for the enzyme. The inactivation by cysteine and H₂O₂ respectively is in additive and is impeded by the addition of competitive inhibitors such as linolelaidic and conjugated linoleic acids, indicating a possible reaction with a certain amino acid residue involved in the enzymic catalysis. The experimental evidences obtained with H₂O₂ and some other modifying reagents have been integrated to furnish a basis of later identification of the residue that is exerting the specific catalytic function.     

Light-dependent incorporation of selenite and sulphite into selenocysteine and cysteine by isolated pea chloroplasts

Illuminated intact pea chloroplasts in the presence of O-acetylserine (OAS) catalysed incorporation of SeO₃^2- and SO₃^2- into selenocysteine and cysteine at rates of ca 0.36 and 6 μmol/mg Chl per hr respectively. Sonicated chloroplasts catalysed SeO₃^2- and SO₃^2- incorporation at ca 3.9 and 32% respectively of the rates of intact chloroplasts. Addition of GSH and NADPH increased the rates to ca 91 and 98% of the intact rates, but SeO₃^2- incorporation under these conditions was essentially light-independent. In the absence of OAS, intact chloroplasts catalysed reduction of SO₃^2- to S^2- at rates of ca 5.8 μmol/mg Chl per hr. In the presence of OAS, S^2- did not accumulate. Glutathione (GSH) reductase was purified from peas and was inhibited by ZnCl₂. This enzyme, in the presence of purified clover cysteine synthase, OAS, GSH and NADPH, catalysed incorporation of SeO₃^2- into selenocysteine (but not SO₃^2- into cysteine). The reaction was inhibited by ZnCl₂. Incorporation of SeO₃^2- into selenocysteine by illuminated intact chloroplasts and sonicated chloroplasts (with NADPH and GSH) was also inhibited by ZnCl₂ but not by KCN. Conversely, incorporation of SO₃^2- into cysteine was inhibited by KCN but not by ZnCl₂. It was concluded that SeO₃^2- and SO₃^2- are reduced in chloroplasts by independent light-requiring mechanisms. It is proposed that SeO₃^2- is reduced by light-coupled GSH reductase and that the Se^2- produced is incorporated into selenocysteine by cysteine synthase.     

Cysteine becomes conditionally essential during hypobaric hypoxia and regulates adaptive neuro-physiological responses through CBS/H2S pathway

Brain is well known for its disproportionate oxygen consumption and high energy-budget for optimal functioning. The decrease in oxygen supply to brain, thus, necessitates rapid activation of adaptive pathways – the absence of which manifest into vivid pathological conditions. Amongst these, oxygen sensing in glio-vascular milieu and H₂S-dependent compensatory increase in cerebral blood flow (CBF) is a major adaptive response. We had recently demonstrated that the levels of H₂S were significantly decreased during chronic hypobaric hypoxia (HH)-induced neuro-pathological effects. The mechanistic basis of this phenomenon, however, remained to be deciphered. We, here, describe experimental evidence for marked limitation of cysteine during HH – both in animal model as well as human volunteers ascending to high altitude. We show that the preservation of brain cysteine level, employing cysteine pro-drug (N-acetyl-_L-cysteine, NAC), markedly curtailed effects of HH – not only on endogenous H₂S levels but also, impairment of spatial reference memory in our animal model. We, further, present multiple lines of experimental evidence that the limitation of cysteine was causally governed by physiological propensity of brain to utilize cysteine, in cystathionine beta synthase (CBS)-dependent manner, past its endogenous replenishment potential. Notably, decrease in the levels of brain cysteine manifested despite positive effect (up-regulation) of HH on endogenous cysteine maintenance pathways and thus, qualifying cysteine as a conditionally essential nutrient (CEN) during HH. In brief, our data supports an adaptive, physiological role of CBS-mediated cysteine-utilization pathway – activated to increase endogenous levels of H₂S – for optimal responses of brain to hypobaric hypoxia.     

The reaction of cysteine with ceruloplasmin copper

The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A₆₁₀/A₂₈₀ ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H₂O₂ by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H₂O₂. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b.     

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN of Rhizobium meliloti involved in symbiotic nitrogen fixation.

Summary TheRhizobium meliloti fdxN gene, which is part of thenifA-nifB fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix^– phenotype of anR. meliloti fdxN: :[Tc] mutant could be rescued by theR. leguminosarum fdxN gene, whereas no complementation was observed withnif-associated genes encoding ferredoxins fromBradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum andRhodobacter capsulatus. In addition to these heterologous genes, severalR. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of theR. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X₂-C-X₂-C-X₃-C,nif-associated ferredoxins, includingR. meliloti FdxN, are characterized by two different cysteine motifs. Six additional amino acids separate the second (Cys₄₂) and the third cysteine (Cys₅₁) in the C-terminal motif (C-X₂-C-X₈-C-X₃-C). By molecular modelling, it was predicted that these amino acid residues form a loop, which does not alter the relative positions of the neighbouring cysteines. Deletion of this loop resulted in anR. meliloti FdxN mutant protein, which exhibited almost 70% wild-type activity, indicating that the predicted loop does not affect Fe-S cluster binding and plays no crucial role in activity of the FdxN protein.     

Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H₂O) of 16.5 kJ mol⁻¹ and 22 kJ mol⁻¹, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H₂O) of 25.2 kJ mol⁻¹ at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC_p), the unfolding enthalpy (H_g) and the temperature at ΔG = 0 (T_g) are 17.9 ± 0.7 kJ mol⁻¹ K⁻¹, 501.2 ± 18.2 kJ mol⁻¹ and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol⁻¹ K⁻¹, 509.3 ± 21.7 kJ mol⁻¹ and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H₂O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.