HOBr Research Articles

Electroreduction of the bromate anion on a microlectrode in excess acid: solution of the inverse kinetic problem

Acidic aqueous solutions of bromic acid salts (bromates) are promising electrolytes for redox flow batteries due to their record high power capacity and the rate of electrode reactions proceeding in the autocatalytic regime. The paper gives a comparison of the results of mathematical modeling and experimental measurements of steady state currents of bromate anion electroreduction in sulfuric acid medium on microelectrodes of various radii. An algorithm of solving the inverse problem suitable for determination of the key transport and kinetic process parameters was proposed and tested.

Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis

Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 μM, cross-linked calprotectin (5 μM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 μM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9)2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.

Bromate Oxidation of Ammonium Salts: In Situ Acid Formation for Reservoir Stimulation.

A redox chemistry approach has been employed to synthesize an assortment of acids in the subterranean environment for the purpose of enhancing productivity from hydrocarbon-bearing rock formations. Experimental studies revealed that bromate selectively oxidizes a series of ammonium salts NH4X where X = F-, Cl-, Br-, SO42-, and CF3CO2- to produce 5-17 wt % HX. Importantly, the in situ method allows strategic placement of the acid in the zone of interest where the fluid is heated, and the reaction is triggered. Ammonium counteranions are shown to influence the kinetics of the bromate-ammonium reaction, and the conditions are tailored to promote oxidation of ammonium at reservoir temperatures. The reaction is observed to be acid-catalyzed, where the formation of bromous acid (HBrO2) is involved in the rate-limiting step. As a result, an induction period that scales with the p Ka of the acid being formed is followed by rapid formation of the reaction products.

Competitive hydrogen-bonding and halogen-bonding interactions in the dimerization of hypobromous acid (HOBr) molecules

The dimerization of hypobromous acid molecules was investigated using MP2 method with aug-cc-pVTZ basis set. Six different configurations were found as the local minima, corresponding to different dimers with one hydrogen bond or one halogen bond, or cyclic structures formed via simultaneously two hydrogen bonds or two halogen bonds, or one hydrogen bond along with one halogen-bonding-like interaction. These dimers exhibit different binding energies. The dimers with one O–H···O hydrogen bond and the cyclic one with an O–H···O HB and an O–Br···O interaction as well as another one with two O–H···Br hydrogen bonds are much stronger than the dimer with one O–Br···O halogen bond, while the dimer with one O–Br···Br halogen bond is even weaker. The topological property of the hydrogen bond or halogen bond in each dimer was assessed by AIM analysis. NBO analysis was employed to reveal the important roles of the intermolecular donor-acceptor orbital interactions and the charge transfer in each dimer. EDA analyses suggest that the electrostatic, exchange, polarization, and dispersion interactions are the attractive forces in stabilizing the complex, while the Pauli term is the repulsive force.

Electrochemical Oxidation of Bromides on DSA/RuO2 Anode in the Semi-Industrial Batch Reactor for On-Site Water Disinfection

The electrochemical oxidation of bromides for on-site water disinfection, on dimensionally stable DSA titanium coated RuO2 anode is investigated by the polarization measurements and in the semi-industrial batch reactor under constant current conditions. During electrolysis of 0.4 M bromide with the current of 5 A, the concentration of bromine, Br2, tribromide ions, Br3−, hypobromous acid, HOBr, and hypobromite, BrO−, so-called equivalent bromine, Breq, is determined using arsenometric titration at pH∼9 and pH∼3.5. At pH∼9, the concentration of 0.1 M is obtained, but solutions undergo to fast decomposition. In the acidified electrolyte, the stable bromine solution with the concentration of 0.15 M after 1.75 h of electrolysis is obtained, corresponding to 14 g dm−3 of “active bromine”, a mixture of HOBr and OBr−. With typical dosing rate of active bromine for disinfection of ∼0.5 mg per liter of water, it is suggested that investigated batch semi-industrial reactor can be used for the treatment of ∼28.000 liters of water. Basic electrolysis parameters like, reactor voltage, temperature, pH, current efficiency, energy consumption is also determined. The reaction products are determined using thermodynamic equilibrium consideration and UV-vis spectroscopy measurements. The main loss reaction during electrolysis is considered, and appropriate analytical expressions are suggested.

Lysosome-targeted two-photon fluorescent probe for detection of hypobromous acid in vitro and in vivo.

It is found that hypobromous acid (HOBr) can affect the activity of type IV collagen. Herein, we synthesized a lysosome-targeted fluorescence probe NA-lyso based on Suzuki coupling reaction with naphthalimide as a fluorescent group. HOBr can oxidize the amino group and methylthio group, which increased the degree of conjugation of the probe, thereby affecting its optical properties. Accordingly, it can establish a method for the specific detection of HOBr. NA-lyso has the properties including fast response, high fluorescence quantum yield (Φ = 59.17%), high selectivity, low cytotoxicity and good membrane-permeability. The probe can locate to lysosome of cells. The potential of the probe as biosensor for HOBr was demonstrated by imaging of exogenous and endogenous HOBr in living cells and in mice. In consequence, NA-lyso is expected to be a powerful tool to detect HOBr in complex biosystem and provides a means of exploring physiological functions associated with HOBr in living organisms.

32 - Hypobromous acid and loss of cardio-protective paraoxonase-1 activity from human plasma

Paraoxonase-1 (PON1) is a cardio-protective enzyme associated with plasma high-density lipoprotein (HDL). In this study we investigated effects of HOBr, a pro-inflammatory mediator, on plasma PON1 activity. In initial studies, HOBr or HOCl was added to 5-fold diluted human plasma and PON1 phosphotriesterase activity was assayed. Consistent with prior reports, addition of HOCl (2000 uM) to plasma caused loss of PON1 activity, which declined to 71 ± 3 % normal (P 0.05). In conclusion, our results demonstrate HOBr and brominated amines, such as taurine may be important mediators of PON1 activity during inflammation. Supported by Midwestern University.

235 - Influence of pH on the reactions of heme peroxidase-derived oxidants with R19S

Oxidant production by heme peroxidases (such as myeloperoxidase [MPO], lactoperoxidase, eosinophil peroxidase and peroxidasin) impacts host defense and macromolecular structure and has the potential to cause tissue damage. Marked increases in phagocyte MPO, capable of producing highly reactive hypochlorous acid (HOCl), have been noted in multiple chronic inflammatory diseases. However, measuring MPO-derived oxidants at the site of production in vivo is technically challenging. The rhodamine-based probe R19S, first described by X. Chen et al (Chem Commun 2011; 47:4373-4375), becomes fluorescent R19 after reaction with HOCl but does not react with superoxide, hydrogen peroxide or several other reactive species. Thus, R19S may be useful to study MPO activity in situ. We sought to characterize the potential responses of R19S to other heme peroxidase-derived oxidants to establish whether a fluorescent response is solely indicative of HOCl or may include others. We analyzed responses of R19S to HOCl (positive control), hydrogen peroxide (negative control), hypobromous acid (HOBr), hypothiocyanous acid (HOSCN), hypoiodous acid (HOI) and taurine chloramine (TauCl) at pH 5.5 and 7.5. A similar study was recently published by A.M. Albrett et al (J Biol Chem 2018; ahead of print). Our results show strong agreement with their study at neutral pH (with a response hierarchy of HOI > HOCl > HOBr > HOSCN), although the overall responses of HOBr and HOSCN relative to HOCl were greater. At pH 5.5, we observed an altered hierarchy, with HOBr and HOI responses diminished (mean of 14.0±0.5% and 65.1±1.0% of HOCl, respectively, vs. 83.2±2.3% and 165.8±6.4% at pH 7.5), while HOSCN response was increased (93.3±5.1% of HOCl, vs. 37.6±0.2% at pH 7.5). Although TauCl did not cause fluorescence at neutral pH, modest response was detectible at acidic pH (9.8±0.2% of HOCl). These data illustrate that the environment of R19S determines its responsiveness to peroxidase-derived oxidants and should be considered when interpreting results.

Inhibitory Anti-Peroxidasin Antibodies in Pulmonary-Renal Syndromes.

Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP. We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score. We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro. The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease. Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.

Heterogeneity of hypochlorous acid production in individual neutrophil phagosomes revealed by a rhodamine-based probe

The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or Staphylococcus aureus by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to >30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in ∼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.

Selective Monitoring and Imaging of Eosinophil Peroxidase Activity with a J-Aggregating Probe.

The specific detection of eosinophil peroxidase (EPO) activity requires the difficult distinction between hypobromous acid generated by EPO and hypochlorous acid generated by other haloperoxidases. Here we report a fluorogenic probe that is halogenated with high kinetic selectivity (≥1200:1) for HOBr over HOCl. Heavy-atom effects do not quench the dibrominated product because of its self-assembly into emissive J-aggregates that provide a turn-on signal. Applications of this fluorogen to EPO activity assays, dipstick sensors, fluorescence imaging of EPO activity, assays of oxidative stress in cancer cells, and immune response detection in live mice are reported.

Bromate formation during the oxidation of bromide-containing water by ozone/peroxymonosulfate process: Influencing factors and mechanisms

Previously, we proposed a novel advanced oxidation process combining ozone (O3) with peroxymonosulfate (PMS), which simultaneously produces sulfate radical (SO4−) and hydroxyl radical (OH) and significantly enhances the removal of organic matter. However, bromate formation is poorly understood during the oxidative treatment of bromide-containing water by O3/PMS process. In this study, factors influencing bromate formation were explored during the O3/PMS process, including the influences of solution pH, PMS dosage, and bromide concentration in distilled water and in actual water samples. Furthermore, the bromate formation mechanism was also clarified by monitoring the intermediate hypobromous acid (HOBr/OBr−), calculating the change of bromide species at different pH levels, and was further confirmed by adding radical scavengers. Generally, the O3/PMS process significantly promotes bromate formation compared with ozone treatment alone. During the O3/PMS process, increasing the PMS dosage and bromide concentrations promoted bromate formation, which was also significantly improved by raising the pH from 4.0 to 10.0. Bromate formation was a result of the combined effect of molecular ozone, OH, and SO4−, and the HOBr/OBr− was the essential intermediate species. Bromate formation was inhibited in the actual water sample due to the competitive effect of natural organic matter, bicarbonate, and reaction of HOBr/OBr− with ammonia. Therefore, the bromate formation should be evaluated as applying the O3/PMS process for organic matters abatement in bromide-containing actual water.

A novel hypobromous acid scavenging activity assay using p-cresol as a spectrofluorometric probe

In this study, a novel spectrofluorometric assay based on $p$-cresol (4-methyl phenol) probe is developed for the measurement of HOBr scavenging activity. It is the first study involving the use of a $p$-cresol probe for the determination of the HOBr scavenging activity of biothiols. While the $p$-cresol probe ($\lambda_{ex}$ = 260 nm, $\lambda_{em}$ = 305 nm) has fluorescence characteristics, its brominated derivatives emerging at the end of the oxidation reaction with HOBr do not show fluorescence. The initial fluorescence intensity of the $p$-cresol probe is decreased in the presence of the brominating agent, HOBr, and this decrease is lower in the presence of HOBr scavenging antioxidants. The scavenging activities of biothiols tested with respect to the developed method decrease in the following order: penicillamine $>$ N-acetyl cysteine $>$ L-glutathione (reduced) $>$ cysteamine $>$ homocysteine $>$ glutathione ethyl ester $>$ cysteine $>$ 1,4-dithiothreitol $>$ lipoic acid $>$ methionine. Penicillamine (IC$_{50}$ = 10.12 $\mu $M) was the most effective HOBr-scavenger among the tested biothiols. The results obtained with the developed method for biothiols and some pharmaceutical samples were statistically compared (using ANOVA) to those found by the reference methods (KI/taurine and UPLC). The advantage of the proposed method over the KI/taurine assay was demonstrated

X-ray Diffraction and Density Functional Theory Provide Insight into Vanadate Binding to Homohexameric Bromoperoxidase II and the Mechanism of Bromide Oxidation.

X-ray diffraction of native bromoperoxidase II (EC 1.11.1.18) from the brown alga Ascophyllum nodosum reveals at a resolution of 2.26 Å details of orthovanadate binding and homohexameric protein organization. Three dimers interwoven in contact regions and tightened by hydrogen-bond-clamped guanidinium stacks along with regularly aligned water molecules form the basic structure of the enyzme. Intra- and intermolecular disulfide bridges further stabilize the enzyme preventing altogether the protein from denaturing up to a temperature of 90 °C, as evident from dynamic light scattering and the on-gel ortho-dianisidine assay. Every monomer binds one equivalent of orthovanadate in a cavity formed from side chains of three histidines, two arginines, one lysine, serine, and tryptophan. Protein binding occurs primarily through hydrogen bridges and superimposed by Coulomb attraction according to thermochemical model on density functional level of theory (B3LYP/6-311++G**). The strongest attractor is the arginine side chain mimic N-methylguanidinium, enhancing in positive cooperative manner hydrogen bridges toward weaker acceptors, such as residues from lysine and serine. Activating hydrogen peroxide occurs in the thermochemical model by side-on binding in orthovanadium peroxoic acid, oxidizing bromide with virtually no activation energy to hydrogen bonded hypobromous acid.

Characterisation of peroxidasin activity in isolated extracellular matrix and direct detection of hypobromous acid formation

Peroxidasin is a heme peroxidase that catalyses the oxidation of bromide by hydrogen peroxide to form an essential sulfilimine cross-link between methionine and hydroxylysine residues in collagen IV. We investigated cross-linking by peroxidasin embedded in extracellular matrix isolated from cultured epithelial cells and its sensitivity to alternative substrates and peroxidase inhibitors. Peroxidasin showed peroxidase activity as measured with hydrogen peroxide and Amplex red. Using a specific mass spectrometry assay that measures NADH bromohydrin, we showed definitively that the enzyme releases hypobromous acid (HOBr). Less than 1 μM of the added hydrogen peroxide was used by peroxidasin. The remainder was consumed by catalase activity that was associated with the matrix. Results from NADH bromohydrin measurements indicates that low micromolar HOBr generated by peroxidasin was sufficient for maximum sulfilimine cross-linking, whereas 100 μM reagent HOBr or taurine bromamine was less efficient. This implies selectivity for the enzymatic process. Physiological concentrations of thiocyanate and urate partially inhibited cross-link formation. 4-Aminobenzoic acid hydrazide, a commonly used myeloperoxidase inhibitor, also inhibited peroxidasin, whereas acetaminophen and a 2-thioxanthine were much less effective. In conclusion, HOBr is produced by peroxidasin in the extracellular matrix. It appears to be directed at the site of collagen IV sulfilimine formation but the released HOBr may also undergo other reactions.

Bromofatty aldehyde derived from bromine exposure and myeloperoxidase and eosinophil peroxidase modify GSH and protein

α-Chlorofatty aldehydes (α-ClFALDs) and α-bromofatty aldehydes (α-BrFALDs) are produced in activated neutrophils and eosinophils. This study investigated the ability of α-BrFALD and α-ClFALD to react with the thiols of GSH and protein cysteinyl residues. Initial studies showed that 2-bromohexadecanal (2-BrHDA) and 2-chlorohexadecanal (2-ClHDA) react with GSH producing the same fatty aldehyde-GSH adduct (FALD-GSH). In both synthetic and cellular reactions, FALD-GSH production was more robust with 2-BrHDA compared with 2-ClHDA as precursor. NaBr-supplemented phorbol myristate acetate (PMA)-activated neutrophils formed more α-BrFALD and FALD-GSH compared with non-NaBr-supplemented neutrophils. Primary human eosinophils, which preferentially produce hypobromous acid and α-BrFALD, accumulated FALD-GSH following PMA stimulation. Mice exposed to Br2 gas had increased levels of both α-BrFALD and FALD-GSH in the lungs, as well as elevated systemic plasma levels of FALD-GSH in comparison to mice exposed to air. Similar relative reactivity of α-ClFALD and α-BrFALD with protein thiols was shown using click analogs of these aldehydes. Collectively, these data demonstrate that GSH and protein adduct formation are much greater as a result of nucleophilic attack of cysteinyl residues on α-BrFALD compared with α-ClFALD, which was observed in both primary leukocytes and in mice exposed to bromine gas.

Thermal Decomposition of Printed Circuit Board in the Presence of Zinc Oxide under Inert and Oxidative Atmosphere: Emission Behavior of Inorganic Brominated Compounds

Thermal degradation of a FR-4 type printed circuit board, PCB, containing brominated flame retardant has been studied both in inert and oxidative atmosphere for the emission control of harmful brominated compounds. The presence of oxygen in atmosphere resulted in the reduction of the yield of hydrogen bromide, one of the major brominated compounds in thermal treatment, and in the enhancement of the formation of bromine and hypobromous acid. The intentional addition of zinc oxide to the PCB powder sample gave rise to the fixation of Br as zinc bromide. It also resulted in the promotion of the release of brominated compounds in comparison to the case of pure PCB. Thus, the addition of the oxide can be a benefit with respect to the bromine fixation and the kinetics of thermal treatment of PCB as well as metal recovery.

Investigation of reaction conditions on synthesis of steroidal bromohydrin and structural analysis of novel 6α-brom-5β-hydroxy derivative

Reaction conditions variation and its influence on the reaction of 3?- acetoxy-17-oxa-17a-homoandrost-5-en-16-one with in situ generated hypobromous acid was investigated. Hypobromous acid was generated from N-bromoacetamide or Nbromosuccinimide and perchloric acid, and as solvent dioxane, dimethoxyethane or tetrahydrofuran were used. After a series of experiments, it was determined that the number of the reaction products depends on the reagent used, solvents, perchloric acid concentration and the presence/absence of daylight. It has also been found that the yields of certain compounds depend also on the reaction time and temperature. 6?-Bromo-5?- hydroxy derivate is obtained by usage of NBA and 0.28 M perchloric acid in dioxane on daylight. Its structure was confirmed by NMR and X-ray crystal structure analysis.

Mechanistic Study of Ruthenium(III) Catalyzed Oxidation of Cyclohexanone by Acidic Bromate

In this work, kinetics and mechanism of Ru(III) catalyzed oxidation of cyclohexanone by acidified solution of potassium bromate has been studied. Present study employ mercuric acetate Hg (OAc)2 as a scavenger for Br¯ ion to prevent parallel oxidation by bromine. The kinetics and mechanism have also been studied in the temperature range of 30°C - 45°C. The reaction exhibits first order kinetics with respect to Ru (III), while zero order kinetics with respect to KBrO3 and HClO4. The influence of Hg(OAc)2 and ionic strength on the rate of reaction was found to be insignificant. Positive effect in the reaction mixture was also observed upon addition of chloride ion; while as the negative effect was revealed with acetic acid. A suitable mechanism in conformity with the kinetic observations has been proposed and the rate law is derived on the basis of obtained data. The various activation parameters such as energy of activation (ΔE*), Arrhenius factor (A), entropy of activation (ΔS*) were calculated from the rate measurements at 30°C, 35°C - 40°C and 45°C.

Bromate electroreduction from acidic solution at rotating disc electrode. Theoretical study of the steady-state convective-diffusion transport for excess of bromate ions compared to protons

Theory of the convective-diffusion transport for the steady-state electroreduction of bromate-anion at rotating disk electrode has been developed for the first time for the compositions of the bulk solution where the bromate concentration is much higher than the proton one. Because of the non-electroactivity of the bromate anions and protons the process proceeds via a redox cycle composed of the electrochemical reduction of bromine (always present in acidic bromate solutions) to bromide and of the comproportionation reaction between bromate, bromide and protons regenerating bromine species. The autocatalytic feature of the cycle can lead under certain conditions on the convection intensity to very strong accumulation of the redox-couple components, bromine and bromide, in the kinetic layer of the solution near the electrode surface, resulting in a very high rate of the overall process, which is controlled by transport of principal components of the bulk solution, namely by that of protons for systems with bromate excess. Theoretical description of such a situation is based on transport equations for all components of the system taking into account of both the convective and diffusional contributions to their fluxes, the bromate concentration being approximately considered as constant across the diffusion layer. Analytical formulas for the concentration distributions have been derived for all current densities that can pass through the RDE system for any rotation frequency. Principal results of this analysis for the bromate excess in the bulk solution are similar to those obtained within the framework of the previous approaches, in particular existence of an “anomalous frequency range” where the maximal current (for a fixed rotation frequency) rises drastically if the rotation frequency (i.e. the solution agitation intensity) decreases, as well as approach of the maximal current for the lower frequency range to the one limited by the convective-diffusion transport of protons. At the same time quantitative predictions of this novel theory and of conventional Nernst model of a stagnant layer has shown significant differences among them that cannot be avoided for any choice of the diffusion layer thickness. On the contrary, the recently proposed theoretical approach of the “Generalized Nernst Layer” model has turned out to be able to reproduce adequately both the concentration distributions inside the kinetic layer (in particular, the values of the concentrations at the electrode surface) for any passing current, as well as the dependence of the maximal current density on the disk rotation frequency (including the frequency ranges of the anomalous behavior and of very strong currents). As it has been expected intuitively, no model replacing the convective-diffusion transport outside the (thin) kinetic layer by a convection-free layer is able to describe properly the concentration profiles there.