Intra and intermolecular charge effects on the reaction of the superoxide radical anion with semi-oxidized tryptophan in peptides and N-acetyl tryptophan

The reaction of the superoxide radical anion (O^-·₂), with the semi-oxidized tryptophan neutral radical (Trp^·) generated from tryptophan (Trp) by pulse radiolysis has been observed in a variety of functionalized Trp derivatives including peptides. It is found that the reaction proceeds 4-5 times faster in positively charged peptides, such as Lys-Trp-Lys, Lys-Gly-Trp-Lys and Lys-Gly-Trp-Lys-O-tert-butyl, than in solutions of the negatively charged N-acetyl tryptophan (NAT). However, the reactivity of O^-·₂ with the Trp^· radical is totally inhibited upon binding of these peptides to micelles of negatively charged SDS and is reduced upon binding to native DNA. By contrast, no change in reactivity is observed in a medium containing CTAB, where the peptides cannot bind to the positively charged micelles. On the other hand, the reactivity of the Trp^· radical formed from NAT with O^-·₂ is reduced to half that of the free Trp^· in buffer but is markedly increased in CTAB micelles. The models studied here incorporate elements of the complex environment in which Trp^· and O^-·₂ may be concomitantly formed in biological system and demonstrate the magnitude of the influence such elements may have on the kinetics of reactions involving these two species.     

Interactions of superoxide anion with enzyme radicals: kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer

The kinetics of O^·-₂ reaction with semi-oxidized tryptophan radicals in lysozyme, Trp^·(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp^·(Lyz) radicals were formed by Br^·-₂ oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 × 10⁸M^-1s^-1; but drops to 8 × 10⁷M^-1s^-1 or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp^·(Lyz) + Trp^·(Lyz) recombination reaction is about (4-7 × 10⁶M^-1s^-1) at/or below pH 6.2 then drops to 1.3-1.6 × 10⁶M^-1s^-1 at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semioxidized Trp^· residues upon Br^·-₂ reaction. The kinetics of Trp^·(Lyz) with ascorbate, a reducing species rather larger than O^·-₂ have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals-leading to the repair of the semi-oxidized Trp^·(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp^·(Lyz)+O^·-₂ reaction, as is most of the Trp^·(Lyz)+Trp^·(Lyz) reaction. However, the kinetic behavior of Trp^·(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O^·-₂. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O^·-₂ reactivity. To our knowledge, this is the first report on the kinetics of a protein-O^·-₂ reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.     

Interaction of melanin with carbon- and oxygen-centered radicals from methanol and ethanol

The interaction of dopa-melanin (DM) and cysteinyldopa-melanin (CDM) with carbon- and oxygen-centered radicals generated by benzophenone-photosensitized hydrogen abstraction from ethanol, or by pulse radiolysis of aqueous solutions of methanol and ethanol, is reported. Photosensitized formation of carbon-centered radicals and their interaction with melanin was monitored by electron paramagnetic resonance (EPR) spin trapping using DMPO, and via the melanin free radical signal itself. In the pulse radiolysis experiments, the interaction of DM or CDM with hydroxymethyl, hydroxyethyl, and the corresponding methanol peroxyl radical was monitored by recording time-dependent changes of the melanin absorbance at selected wavelengths. The data indicate that both melanins are good scavengers of carbon-centered radicals, with corresponding rate constants in the range of 10⁷ to 10⁸ M⁻¹ s⁻¹. Significantly, compared to DM, CDM is also an exceptionally efficient scavenger of oxygen-centered radicals derived from methanol with corresponding rate constants of 2.7 × 10⁴ and 2 × 10⁶, M⁻¹ s⁻¹ for DM and CDM, respectively. The results are discussed with reference to the potential role of melanin in protecting the integrity of melanosomes by inhibiting peroxidation of lipid components of the organelle membrane.     

Kinetics of the reactions of nitrogen dioxide with glutathione,cysteine, and uric acid at physiological pH

Nitrogen dioxide (NO₂^•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO₂^• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO₂^• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO₂^• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO₂^• with glutathione, cysteine, and urate were estimated as 2 × 10⁷, 5 × 10⁷, and 2 × 10⁷ M⁻¹ s⁻¹, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO₂^• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10⁷ M⁻¹ s⁻¹. The implications of these values are: (i) the lifetime of NO₂^• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO₂^• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO₂^• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO₂^•; and (v) reactions between NO₂^• and thiols/urate severely limit the likelihood of reaction of NO₂^• with NO• to form N₂O₃ in the cytoplasm.     

Pulse radiolysis kinetics of the reaction of the hydrated electron and the carboxyl anion radical with Pseudomonas aeruginosa cytochrome c-551

The kinetics of the reaction of hydrated electron (e⁻_aq) and carboxyl anion radical (CO₂) with Pseudomonas aeruginosa ferricytochrome c-551 were studied by pulse radiolysis. The rate of reaction of e⁻_aq with the negatively charged ferricytochrome c-551 (17 nM⁻¹ · s⁻¹) is significantly slower than the larger positively charged horse heart ferricytochrome c (70 nM⁻ · s⁻). This difference cannot be explained solely by electrostatic effects on the diffusion-controlled reactions. After the initial encounter of e⁻_aq with the protein, ferricytochrome c-551 is less effective in transferring an electron to the heme which may be due to the negative charge on the protein. The charge on ferricytochrome c-551 is estimated to be −5 at pH 7 from the effect of ionic strength on the reaction rate. A slower relaxation (2 · 10⁴ s⁻¹) observed after fast e⁻_aq reduction is attributed to a small conformational change. The rate of reaction of CO₂ with ferricytochrome c-551 (0.7 nM⁻¹ · s⁻) is, after electrostatic correction, the same as ferricytochrome c, indicating that the steric requirements for reaction are similar. This reaction probably takes place through the exposed heme edge.     

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

The Kinetics of the Reaction of Ferritin with Superoxide

Using pulse radiolysis and competition kinetics with cytochrome c, the reaction of superoxide with horse spleen ferritin was investigated. The second-order rate constant is estimated to be 2 ± 1 × 10⁶dm³mol^-1s^-1     

水环境中活性氧的来源、种类及测定方法

各种环境介质和生命体中许多微观化学过程都与活性氧密切相关.本文介绍了水环境中活性氧的来源、种类和测定.它们主要包括:¹O₂(单线态氧)、O₂^-(超氧自由基)/HO₂·(氢过氧自由基)、·OH(羟基自由基)、H₂O₂、RO·(烷氧基)、ROO·(烷过氧基)和R·OH(氢过氧化物)等.其主要来源于辐射分解、热解和氧化还原法等.测定采用分子探针法、图谱法和酶法.     

Mechanism of reaction of nitrogen dioxide radical with hydroxycinnamic acid derivatives: A pulse radiolysis study

Nitrogen dioxide radical (NO^·₂) is known as a toxic agent produced in the metabolism of nitrates and nitrites. By the use of the pulse radiolysis technique, the mechanism of the reaction of NO^·₂ radical with hydroxycinnamic acid derivatives (HCA) was studied and the rate constants have been measured. The rate constants were found to be 7.4 × 10⁸, 7.2 × 10⁸, 8.6 × 10⁸ dm³ mol^-1s^-1 for ferulic acid, sinapic acid and caffeic acid, respectively. The reactions produce the corresponding phenoxyl radical.     

Kinetics of some electron-transfer reactions in biological Photosystems II. Study of intramolecular electron-transfer rates between ferredoxin-NADP reductase, NADP radical and oxidized ferredoxin

It has been shown by the pulse radiolysis technique that radiation-generated NADP free radicals (NADP·) first combine with ferredoxin-NADP reductase and then transfer the odd electron by a fast intramolecular process to the enzyme flavin moiety yielding the semiquinone (ferredoxin-NADP reductase, FNR-FADH·). The corresponding first-order rate constant k₁₅ varies with ionic strength from 2.6·10³ s⁻¹ at I = 0.66 M to 2.3·10⁴ s⁻¹ at I = 0.005 M In the presence of ferredoxin-NADP reductase-bound oxidized ferredoxin, the electron cascades, thus further reducing the ferredoxin. The transfer of the electron from the flavin semiquinone (ferredoxin-NADP reductase, FNR-FADH·) to the bound oxidized ferredoxin proceeds at a rate of k₁₈ = 2.36 s⁻¹. This process approaches an equilibrium condition which is in favor of the reverse reaction suggesting that k₋₁₈ > k₁₈.     

Interaction of hydroxycinnamic acid derivatives with the Cl3COO radical: a pulse radiolysis study

The electron transfer reactions between the trichloromethylperoxyl radical (Cl₃COO^·) and hydroxycinnamic acid derivatives, including chlorogenic acid, sinapic acid, caffeic acid, ferulic acid and 3,4-(methylenedioxy)cinnamic acid, have been studied by pulse radiolysis. The hydroxycinnamic acid derivatives, especially sinapic acid, are identified as good antioxidants for reduction of Cl₃COO^· via electron transfer reactions. From buildup kinetic analysis of phenoxyl radical, the rate constant for reaction of Cl₃COO^· with sinapic acid has been determined to be 8.2 × 10⁷ dm³ mol^-1 s^-1, while the rate constants of electron transfer from other hydroxycinnamic acid derivatives to Cl₃COO^· were obtained to be about 2 × 10⁷dm³ mol^-1 s^-1. The reaction of 3,4-(methylenedioxy)cinnamic acid with Cl₃COO^· was investigated as an evidence for the electron transfer mechanism.     

Oxidation-Reduction Reactions of Iron Bleomycin in the Absence and Presence of DNA

Using the pulse radiolysis technique, we have demonstrated that bleomycin-Fe(III) is stoichiometrically reduced by CO₂^- to bleomycin-Fe(II) with a rate of (1.9 ± 0.2) × 10⁸M^-1s^-1. In the presence of calf thymus DNA, the reduction proceeds through free bleomycin-Fe(III) and the binding constant of bleomycin-Fe(III) to DNA has been determined to be (3.8 ± 0.5) x 10⁴ M^-1. It has also been demonstrated that in the absence of DNA O₂^-1 reacts with bleomycin-Fe(III) to yield bleomycin-Fe(II)O₂, which is in rapid equilibrium with molecular oxygen, and decomposes at room temperature with a rate of (700 ± 200) s^-1. The resulting product of the decomposition reaction is Fe(III) which is bound to a modified bleomycin molecule. We have demonstrated that during the reaction of bleomycin-Fe(II) with O₂, modification or self-destruction of the drug occurs, while in the presence of DNA no destruction occurs, possibly because the reaction causes degradation of DNA.     

Electron Transfer Reactions in RB90745, A Bioreductive Drug Having Both Aromatic N-Oxide and Nitroarene Moieties

The bifunctional hypoxia-specific cytotoxin RB90745, has a nitroimidazole moiety attached to an imidazo[1,2,-a]quinoxaline mono-N-oxide with a spacer/linking group. The reduction chemistry of the drug was studied by pulse radiolysis using the one electron reductant CO₂^˙-. As N-oxides and nitro compounds react with CO₂^˙- at diffusion controlled rates, initial reaction produced a mixture of the nitro radical (λ_max 410 nm) and the N-oxide radical (λ_max 550 nm) in a few microseconds. Subsequently an intramolecular electron transfer (IET) was observed (k = 1.0 ± 0.25 × 10³ s^-1 at pH 5-9), from the N-oxide to the more electron-affinic nitro group. This was confirmed by the first order decay rate of the radical at 550 nm and formation at 410 nm, which was independent of both the concentration of the parent compound and the radicals. The rates of electron transfer and the decay kinetics of the nitro anion radicals were pH dependent and three different pK_aS could be estimated for the one electron reduced species: 5.6 (nitroimidazole group) and 4.3, and 7.6 (N-oxide function). The radicals react with oxygen with rate constants of 3.1 × 10⁷ and 2.8 × 10⁶ dm³ mol^-1 s^-1 observed at 575 nm and 410 nm respectively. Steady state radiolysis studies indicated four electron stoichiometry for the reduction of the compound.     

Reaction rate constants of superoxide scavenging by plant antioxidants

Plant phenols may exert protective effects by scavenging superoxide, which is implicated in tissue damage and accelerated inactivation of vasorelaxing nitric oxide. Preventing the interaction of superoxide with tissue biomolecules depends not only on the extent of superoxide scavenging but also on scavenging velocity. However, information on superoxide scavenging kinetics of plant phenols is scarce. We describe an improved lucigenin-based chemiluminescence assay for kinetic analysis. The use of potassium superoxide (KO₂) as a nonenzymatic superoxide source allowed simple and reliable determination of the second-order reaction rate constants between superoxide and plant antioxidants at physiologically relevant conditions, avoiding unspecific effects of other reactive oxygen species or superoxide-generating enzymes. We calculated the rate constants for phenols of different structures, ranging from 2.9 × 10³ mol⁻¹ l s⁻¹ for morin to 2.9 × 10⁷ mol⁻¹ l s⁻¹ for proanthocyanidins. Compounds with pyrogallol or catechol moieties were revealed as the most rapid superoxide scavengers, and the gallate moiety was found to be the minimal essential structure for maximal reaction rate constants with superoxide.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

Oxygen-copper (II) interplay in the repair of semi-oxidized urate by quercetin bound to human serum albumin

The 1:1 complex of copper (II) and human serum albumin (HSA) slowly reacts with radiolytically generated ^•O₂^- radical-anion at a rate constant of 6.1×10₆ M_-1 s_-1. Absorbance and fluorescence spectroscopies demonstrate that addition of an equimolar portion of quercetin (QH₂) to the solution of the copper (II)-HSA complex induces a relocalization of the copper resulting in a ternary copper (II)-QH₂-HSA complex. This form of quercetin slowly oxidizes in air-saturated solutions. A 10-fold excess urate, a plasma antioxidant, cannot displace copper (II) bound to HSA. In N₂O-saturated solutions the ternary complex form of QH₂ can repair the urate radical with a rate constant of 2.7×10₆ M_-1 s_-1 by an electron transfer reaction similar to that observed in the absence of copper (II). In O₂-saturated solutions and in the absence of copper, HSA-bound QH₂ fails to repair the urate radical because of the fast competitive reaction of ^•O₂^- with urate radicals. However, addition of equimolar copper (II) restores the electron transfer from QH₂ to the urate radical. These contrasting results are tentatively explained either by an enhanced reactivity of copper (II) with ^•O₂^- in the ternary complex or by direct production of quercetin radicals via a copper-catalyzed reduction of the ^•O₂^- radicals by QH₂.     

Antioxidant action of sodium diethyldithiocarbamate: Reaction with hydrogen peroxide and superoxide radical

The oxidation of sodium diethyldithiocarbamate (DDC) by hydrogen peroxide or superoxide radicals has been investigated. Hydrogen peroxide oxidizes DDC, leading to the formation of a hydrated form of disulfiram, a dimer of DDC having a disulfide group. In equimolar conditions, the overall process appears as a first-order reaction (k = 0.025±0.005 s⁻¹), the first step being a second-order reaction (k = 5.0±0.1mol⁻¹.1. s⁻¹). No radical intermediate was observed in this process. In the presence of an excess of any of the reagents, the hydrated form of disulfiram transforms into different products corresponding to the fixation of oxygen by sulfur atoms or replacement of C = S group by ketone function, in the presence of an excess of hydrogen peroxide. Superoxide anions (produced by steady-state ⁶⁰Co γ-radiolysis) oxidize DDC, yielding similar products to those obtained with hydrogen peroxide with a maximum oxidation G-value of 0.3 μmol.J⁻¹. The rate constant k(O₂^·− + DDC) is equal to 900 mol⁻¹. 1. s⁻¹.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Degradation of Hyaluronic Acid, Poly- and Mono-Saccharides, and Model Compounds by Hypochlorite: Evidence for Radical Intermediates and Fragmentation

Degradation of hyaluronic acid by oxidants such as HO^· and HOCl/ClO⁻ is believed to be important in the progression of rheumatoid arthritis. While reaction of hyaluronic acid with HO^· has been investigated extensively, reaction with HOCl/ClO⁻ is less well defined. Thus, little is known about the site(s) of HOCl/ClO⁻ attack, the intermediates formed, or the mechanism(s) of polymer degradation. In this study reaction of HOCl/ClO⁻ with amides, sugars, polysaccharides, and hyaluronic acid has been monitored by UV-visible (220–340 nm) and EPR spectroscopy. UV-visible experiments have shown that HOCl/ClO⁻ reacts preferentially with N-acetyl groups. This reaction is believed to give rise to transient chloramide (R—NCl—C(O)—R′) species, which decompose rapidly to give radicals via either homolysis (to produce N· and Cl·) or heterolysis (one-electron reduction, to give N· and Cl⁻) of the N—Cl bond. The nature of the radicals formed has been investigated by EPR spin trapping. Reaction of HOCl/ClO⁻ with hyaluronic acid, chondroitin sulphates A and C, N-acetyl sugars, and amides gave novel, carbon-centered, spin adducts, the formation of which is consistent with selective initial attack at the N-acetyl group. Thus, reaction with hyaluronic acid and chondroitin sulphate A, appears to be localized at the N-acetylglucosamine sugar rings. These carbon-centered radicals are suggested to arise from rapid rearrangement of initial nitrogen-centered radicals, formed from the N-acetyl chloramide, by reactions analogous to those observed with alkoxyl radicals. The detection of increasing yields of low-molecular-weight radical adducts from hyaluronic acid and chondroitin sulphate A with increasing HOCl/ClO⁻ concentrations suggests that formation of the initial nitrogen-centered species on the N-acetylglucosamine rings, and the carbon-centered radicals derived from them, brings about polymer fragmentation.