Quantitative elucidation of the molecular mechanisms of hydroxyl radical quenching reactivity of phenolic compounds

Reported discrepancies have confused the understanding of the molecular mechanisms of antioxidant reactivity somewhat. The consequent problems necessitate systematic investigations on the molecular orbital features of antioxidants and their correlation with antioxidant potentials. In the present work, phenolic compounds as typical antioxidants were selected to investigate their hydroxyl radical-scavenging properties, and the related mechanisms of action were studied theoretically by computational chemistry. A good correlation was observed between antioxidant activity and theoretical parameters, such as O-H bond dissociation energy (BDE), ionization potential (IP), enthalpy of electron transfer (E(a)), chemical hardness (HOMO-LUMO gap), and spin delocalization of the phenoxyl radicals (D(s)(r)). The results demonstrate that the molecular mechanisms regulating the antioxidant action were more complex than hydrogen or electron-transfer processes and explain previous contradictions. Meanwhile, a satisfactory quantitative structure-activity relationship (QSAR) model was established which should be of predictive value in evaluating or screening hydroxyl radical-scavenging antioxidants.     

Neural and hydroxyl radical mechanisms underlying laryngeal airway hyperreactivity induced by laryngeal acid-pepsin insult in anesthetized rats.

Laryngopharyngeal or gastroesophageal reflux is associated with laryngeal airway hyperreactivity (LAH), but neither the cause-effect relationship nor the underlying mechanism has been elucidated. Here we established a rat model with enhanced laryngeal reflex reactivity induced by laryngeal acid-pepsin insult and investigated the neural and hydroxyl radical (*OH) mechanisms involved. The laryngeal segments of 103 anesthetized rats were functionally isolated while animals breathed spontaneously. Ammonia vapor was delivered into the laryngeal segment to measure laryngeal reflex reactivity. We found that the laryngeal pH 5-pepsin treatment doubled the reflex apneic response to ammonia, whereas laryngeal pH 7.4-pepsin, pH 2-pepsin, and pH 5-denatured pepsin treatment had no effect. Histological examination revealed limited laryngeal inflammation and epithelial damage after pH 5-pepsin treatment and more severe damage after pH 2-pepsin treatment. In rats that had received the laryngeal pH 5-pepsin treatment, the apneic response to ammonia was abolished by either denervation or perineural capsaicin treatment (PCT; a procedure that selectively blocks capsaicin-sensitive afferent fibers) of the superior laryngeal nerves, but was unaffected by perineural sham treatment. LAH was prevented by laryngeal application of either dimethylthiourea (DMTU; a *OH scavenger) or deferoxamine (DEF; an antioxidant for *OH), but was unaltered by the DMTU vehicle or iron-saturated DEF (ineffective DEF). LAH reappeared after recovery from PCT, DMTU, or DEF treatment. We conclude that 1) laryngeal insult by pepsin at a weakly acidic pH, but not at acidic pH, can produce LAH; and 2) LAH is probably mediated through sensitization of the capsaicin-sensitive laryngeal afferent fibers by a *OH mechanism.     

Quantitation of the hydroxyl radical by reaction with dimethyl sulfoxide

This investigation was conducted to validate the use of dimethyl sulfoxide (DMSO) as a quantitative molecular probe for the generation of hydroxyl radicals (HO.) in aqueous systems. Reaction of HO. with DMSO produces methane sulfinic acid as a primary product, which can be detected by a simple colorimetric assay. To evaluate this method for estimating total HO. production, we studied three model systems, including the Fenton reaction, gamma irradiation of water, and ultraviolet photolysis of hydrogen peroxide, for which the theoretical maximum yield of HO. could be calculated and compared to measured DMSO oxidation. The results confirm that 0.05 to 1 M DMSO may be used to capture nearly all of the expected HO. radicals formed. Thus, methane sulfinic acid production from DMSO holds promise as an easily measured marker for HO. formation in aqueous systems pretreated with DMSO.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

The mechanisms and process of acephate degradation by hydroxyl radical and hydrated electron

The degradation process of acephate in aqueous solution with OH and ${\text{e}}_{\text{aq}}^{?}$ produced by ⁶⁰Co-γ irradiation and electron pulse radiolysis was studied in the present paper. In the aqueous solution, acephate reacted with ${\text{e}}_{\text{aq}}^{?}$ and transformed to transient species which can absorb weakly in the wavelength range of 300–400?nm and decay very fast. According to the decay of hydrated electron, the reaction rate constant of ${\text{e}}_{\text{aq}}^{?}$ and acephate is (3.51?±?0.076)?×?10⁹?dm³·mol^?1·s^?1. The transient species produced in the reaction of OH and acephate do not distinctly absorb the light in the wavelength range of 300–700?nm, so the decay and kinetics of the transient species cannot determinedirectly. The competing reaction of KSCN oracephate with OH were studied to obtain the reaction rate constant of OH and acephate, which is (9.1?±?0.11)?×?10⁸?dm³·mol^?1·s^?1. Although acetylamide and inorganic ions were determined in the products of the reaction of acephate with OH or ${\text{e}}_{\text{aq}}^{?}$, the concentration of inorganic ions in the products of the reaction of acephate with OH is higher than that in the product of the reaction of acephate with ${\text{e}}_{\text{aq}}^{?}$. Moreover, there were sulfide in the products of the reaction of acephatewith ${\text{e}}_{\text{aq}}^{?}$. The degradation pathways of acephate by OH and ${\text{e}}_{\text{aq}}^{?}$ were also proposed based on the products from GC-MS.     

Formation of TEMPOL-hydroxylamine during reaction between TEMPOL and hydroxyl radical: HPLC/ECD study

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical (^?OH) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with ^?OH does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary ^?OH-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and ^?OH generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary ^?OH-derived radicals in acidic condition. In the presence of NADH, a large proportion of the non-paramagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the ^?OH sources in pathological processes such as Parkinson's disease.     

Formation of TEMPOL-hydroxylamine during reaction between TEMPOL and hydroxyl radical: HPLC/ECD study

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical ((*)OH) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with (*)OH does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary (*)OH-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and (*)OH generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary (*)OH-derived radicals in acidic condition. In the presence of NADH, a large proportion of the non-paramagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the (*)OH sources in pathological processes such as Parkinson's disease.     

Study of the relationship between analgesic activity and structure of synthetic melanocortin analogs

Melanocortins, peptides of the family of adrenocorticotropic (ACTH) and melanocyte-stimulating (MSH) hormones, have a wide range of physiological activity. Heptapeptide semax (MEHFPGP) is an analog of the ACTH_4–10 fragment. Previously, pronounced nootropic and neuroprotective activities were shown for this peptide; in addition, it decreases pain sensitivity in animals. In this work, the relationship between analgesic activity of the peptide and its structure was studied. The following analogs of the N-terminal ACTH fragments were used: rMEHFPGP, where r is glucuronic acid, KEHFPGP, GEHFPGP, EHFPGP, HFPGP, and ERP. The peptides were administered intraperitoneally at doses of 0.015, 0.05 and 0.5 mg/kg. Rat pain sensitivity was assessed using the paw-withdrawal test. Truncations of the N-terminal residues eliminated the analgesic activity. The peptide analgesic effect was observed after the replacement of the N-terminal methionine with lysine or after the attachment of glucuronic acid to the methionine, while the peptide with glycine instead of the methionine had no effect on pain sensitivity. Hence, the amino acid at position 1 of semax analogs plays a key role in mediating the analgesic effects of the peptides.     

Effect of phenolic compounds on abscisic acid-induced stomatal movement: Structure – activity relationship

Application of abscisic acid (ABA) brings about stomatal closure within 30 min in epidermal peels of Vicia faba . A number of phenolic compounds antagonise the effect of ABA. Derivatives of benzoic acid, cinnamic acid, coumarin and flavonoids have been studied in order to establish structure – activity relationship. Derivatives of benzoic acid reverse the ABA effects. Coumarin, esculetin and three hydro derivatives of cinnamic acid fail to show the anti-ABA activity. Thus, the presence of parahydroxyl group and double bond in the side chain is necessary for anti-ABA activity.     

Monitoring equilibrium changes in RNA structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (•OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.¹ When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA¹ and RNA² with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein¹ and RNA-protein complexes. Equilibrium³ and kinetic⁴ transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., ''single-hit kinetics'') results in the uniform sampling of each nucleotide of the polymer.⁵In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H₂O₂ (we call this the ''peroxidative'' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O₂ (we call this the ''oxidative'' protocol). An overview of the data reduction, transformation and analysis procedures is presented.Download video file.^{(82M, mov)}     

Sulfate anion free radical formation by the peroxidation of (Bi)sulfite and its reaction with hydroxyl radical scavengers

The one-electron oxidation of (bi)sulfite is catalyzed by peroxidases to yield the sulfur trioxide radical anion (SO3-), a predominantly sulfur-centered radical as shown by studies with 33S-labeled (bi)sulfite. This radical reacts with molecular oxygen to form a peroxyl radical. The subsequent reaction of this peroxyl radical with (bi)sulfite has been proposed to form the sulfate anion radical, which is nearly as strong an oxidant as the hydroxyl radical. We used the spin trapping electron spin resonance technique to provide for the first time direct evidence for sulfate anion radical formation during (bi)sulfite peroxidation. The sulfate anion radical is known to react with many compounds more commonly thought of as hydroxyl radical scavengers such as formate and ethanol. Free radicals derived from these scavengers are trapped in systems where (bi)sulfite peroxidation has been inhibited by these scavengers.     

The relationship between peptide structure and antibacterial activity

Cationic antimicrobial peptides are a class of small, positively charged peptides known for their broad-spectrum antimicrobial activity. These peptides have also been shown to possess anti-viral and anti-cancer activity and, most recently, the ability to modulate the innate immune response. To date, a large number of antimicrobial peptides have been chemically characterized, however, few high-resolution structures are available. Structure-activity studies of these peptides reveal two main requirements for antimicrobial activity, (1) a cationic charge and (2) an induced amphipathic conformation. In addition to peptide conformation, the role of membrane lipid composition, specifically non-bilayer lipids, on peptide activity will also be discussed.     

The relationship between quaternary structure and enzyme activity

Methods have been developed to study the influence of quaternary structure on enzyme activity. Some enzymes which normally exist as stable oligomers remain catalytically active when the subunits are dissociated by artificial means.     

The relationship between oxidase activity,peroxidase activity,hydrogen peroxide,and phenolic compounds in the degradation of indole-3-acetic acid in vitro

The peroxidase catalyzed degradation of indole-3-acetic acid (IAA) results in the formation of indole-3-methanol (IM) in the presence of phenolic compounds or in 3-hydroxymethyloxindole (HMOx) in their absence. Apparently the phenols compote with a methyleneindolenine intermediate for H₂O₂ which is produced by oxidase action preceding the peroxidase action in the course of IAA degradation. The substitution pattern of various phenolic compounds tested strongly effects the rate of the reaction. However, this substitution pattern does not appear to effect the type of the reaction or the products formed. We suggest that the function of the “oxindole pathway” is to detoxify excess H₂O₂ in the absence of phenolic cosubstrates. The results lead to a number of interesting aspects of IAA biochemistry and to the proposal of a new reaction scheme for the peroxidase catalyzed degradation of IAA.