Kinetic chemiluminescence as a method for the study of free radical reactions

The review gives concepts of analysis of kinetics of complex reactions with the participation of free-radicals. The concepts are based on the comparison of the kinetic curves of the chemiluminescent reactions in the presence of a physical enhancer coumarin C-525 with the simulated kinetic functions. This method was applied to the investigation of the mechanism of a branched-chain reaction of lipid peroxidation in biological and phospholipid membranes as well as to the effect of antioxidants and determination of their activities. It was also used for studying the reaction of the formation of free radicals in the complex of cytochrome c with cardiolipin. This reaction plays a key role in the initiation of apoptosis.     

Mechanism of copper-catalyzed autoxidation of cysteine

The kinetics of copper-catalyzed autoxidation of cysteine and its derivatives were investigated using oxygen consumption, spectroscopy and hydroxyl radical detection by fluorescence of a coumarin probe. The process has complex two-phase kinetics. During the first phase a stoichiometric amount of oxygen (0.25 moles per mole of thiol) is consumed without production of hydroxyl radicals. In the second reaction phase excess oxygen is consumed in a hydrogen peroxide-mediated process with significant ^·OH production. The reaction rate in the second phase is decreased for cysteine derivatives with a free aminogroup and increased for compounds with a modified aminogroup. The kinetic data suggest the catalytic action of copper in the form of a cysteine complex. The reaction mechanism consists of two simultaneous reactions (superoxide-dependent and peroxide-dependent) in the first phase, and peroxide-dependent in the second phase. The second reaction phase begins after oxidation of free thiol. This consists of a Fenton-type reaction between cuprous-cysteinyl complex and following oxidation of cysteinyl radical to sulfonate with the consumption of excessive oxygen and significant production of hydroxyl radicals.     

Synchrotron radiation-induced formation and reactions of free radicals in human acellular dermal matrix

The present work characterizes the formation of free radicals in an implantable human acellular dermal tissue (Alloderm, LifeCell Corp., Branchburg, NJ) upon irradiation. The tissue was preserved in a vitreous carbohydrate matrix by freeze-drying. Freeze-dried samples were irradiated using a synchrotron light source, and free radicals generated were investigated using the electron paramagnetic resonance (EPR) technique. At least two free radical populations, with g factors of 1.993 (approximately 43%) and 2.002 (approximately 57%), respectively, were identified in the irradiated tissue. The transformation (reaction) kinetics of free radicals produced was investigated in the presence of nitrogen, oxygen and moisture. The reaction kinetics of free radicals was extremely slow in the nitrogen environment. The presence of oxygen and moisture greatly accelerated free radical reactions in the tissue matrix. The reaction of free radicals could not be described by traditional reaction kinetics. A dispersive kinetics model and a diffusion model were developed to analyze the reaction kinetics in the present study. The dispersive model took into consideration molecular mobility and dispersivity of free radicals in the heterogeneous tissue material. The diffusion model described the radical reaction kinetics as two parallel and simultaneous processes: a first-order fast kinetics mainly on tissue surface and a diffusion-limited slow kinetics in deeper layers of the tissue matrix. Both models described quantitative experimental data well. Further investigation is needed to verify whether any of these two models or concepts describes the inherent radical reaction kinetics in the solid tissue matrix.     

Reactions of Low Valent Transition Metal Complexes with Hydrogen Peroxide. Are they “Fenton-Like” or not? 4. The Case of Fe(II)L,L = Edta; Hedta and Tcma

The question whether hydroxyl free radicals are formed in the reactions of divalent iron complexes Fe(II)L; L = edta; hedta; tcma (tcma = l-acetato-l,4,7-triazacyclononane) with hydrogen peroxide in neutral and slightly acidic solutions was studied by using the β elimination reaction as an assay for the formation of hydroxyl free radicals, OH. The results show that at pH<5.5 the iron(II)peroxide intermediate complex decomposes rapidly to yield free hydroxyl radicals for L=edta and hedta. This is in contrast to the mechanism of the corresponding Fe(II)nta peroxide complex, which probably decomposes to form Fe(IV)nta which then reacts with organic substrates to yield aliphatic free radicals. Thus, the non-participating ligand L has an appreciable effect on the mechanism of reaction of the metal center with hydrogen peroxide. Blank experiments using ionizing radiation as the source of CH₂CR(CH₃)OH, R = H or CH₃ radicals indicate that when L = tcma intermediates of the type LFe^III-CH₂CR(CH₃)OH_aq are formed, but their major mode of decomposition is not the β elimination reaction. Thus, the present assay for the formation of hydroxyl free radicals by the Fenton Reaction does not fit the latter system.     

Acyl-CoA-binding protein from cow. Binding characteristics and cellular and tissue distribution.

Hydroxyl radicals (OH.) in free solution react with scavengers at rates predictable from their known second-order rate constants. However, when OH. radicals are produced in biological systems by metal-ion-dependent Fenton-type reactions scavengers do not always appear to conform to these established rate constants. The detector molecules deoxyribose and benzoate were used to study damage by OH. involving a hydrogen-abstraction reaction and an aromatic hydroxylation. In the presence of EDTA the rate constant for the reaction of scavengers with OH. was generally higher than in the absence of EDTA. This radiomimetic effect of EDTA can be explained by the removal of iron from the detector molecule, where it brings about a site-specific reaction, by EDTA allowing more OH. radicals to escape into free solution to react with added scavengers. The deoxyribose assay, although chemically complex, in the presence of EDTA appears to give a simple and cheap method of obtaining rate constants for OH. reactions that compare well with those obtained by using pulse radiolysis.     

On the complex •OH/•O−-induced free radical chemistry of arylalkylamines with special emphasis on the contribution of the alkylamine side chain

A full account of the ^?OH-induced free radical chemistry of an arylalkylamine is given taking all the possible reaction pathways quantitatively into consideration. Such knowledge is indispensable when the alkylamine side chain plays a crucial role in biological activity. The fundamental reactions are investigated on the model compound N-methyl-3-phenypropylamine (MPPA), and extended to its biologically active analog, to the antidepressant fluoxetine (FLX). Pulse radiolysis techniques were applied including redox titration and transient spectral analysis supplemented with DFT calculations. The contribution of the amine moiety to the free radical-induced oxidation mechanism appeared to be appreciable. ^?O^? was used to observe hydrogen atom abstraction events at pH 14 giving rise to the strongly reducing α-aminoalkyl radicals (～38% of the radical yield) and to benzyl (～4%), β-aminoalkyl (～24%), and aminyl radicals (～31%) of MPPA. One-electron transfer was also observed yielding aminium radicals with low efficiency (～3%). In the ^?OH-induced oxidation protonated α-aminoalkyl (～49%), β-aminoalkyl (～27%), benzyl radicals (～4%), and aminium radicals (～5%) are initially generated on the side chain of MPPA at pH 6, whereas hydroxycyclohexadienyl radicals (～15%) were also produced. These initial events are followed by complex protonation–deprotonation reactions establishing acid–base equilibria; however, these processes are limited by the transient nature of the radicals and the kinetics of the ongoing reactions. The contribution of the radicals from the side chain alkylamine substituent of FLX totals up to ～54% of the initially available oxidant yield.     

Kinetics of NADH oxidation of NAD+ reduction by mitochondrial complex I]

The kinetics of the NAD: artificial acceptor-oxidoreductase and delta mu H(+)-dependent succinate: NAD(+)-oxidoreductase reactions (reverse electron transfer) reactions catalyzed by the membrane-bound complex I was studied. The values of apparent rate constants of dissociation of complexes of the oxidized and reduced enzyme with NAD+ and NADH were determined. It was shown that the apparent affinity of NADH for the oxidized complex I is by nearly three orders of magnitude as high as that of the reduced one; a reverse correlation is found for NAD+. A kinetic scheme of complex I functioning in the forward and reverse reactions, according to which the free reduced enzyme is not an intermediate of the forward (NADH-oxidase) reaction and the free oxidized enzyme is not an intermediate of the reverse (NAD(+)-reductase) reaction, is proposed.     

Kinetic analysis-based quantitation of free radical generation in EPR spin trapping

Because short-lived reactive oxygen radicals such as superoxide have been implicated in a variety of disease processes, methods to measure their production quantitatively in biological systems are critical for understanding disease pathophysiology. Electron paramagnetic resonance (EPR) spin trapping is a direct and sensitive technique that has been used to study radical formation in biological systems. Short-lived oxygen free radicals react with the spin trap and produce paramagnetic adducts with much higher stability than that of the free radicals. In many cases, the quantity of the measured adduct is considered to be an adequate measure of the amount of the free radical generated. Although the intensity of the EPR signal reflects the magnitude of free radical generation, the actual quantity of radicals produced may be different due to modulation of the spin adduct kinetics caused by a variety of factors. Because the kinetics of spin trapping in biochemical and cellular systems is a complex process that is altered by the biochemical and cellular environment, it is not always possible to define all of the reactions that occur and the related kinetic parameters of the spin-trapping process. We present a method based on a combination of measured kinetic data for the formation and decay of the spin adduct alone with the parameters that control the kinetics of spin trapping and radical generation. The method is applied to quantitate superoxide trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In principle, this method is broadly applicable to enable spin trapping-based quantitative determination of free radical generation in complex biological systems.     

Kinetic and enzymatic adsorption model in a recirculation hollow-fibre bioreactor

A general procedure has been developed to model the behaviour of enzymatic reactions in a membrane bioreactor. This procedure unifies the kinetics of the reaction and the adsorption of the enzyme or enzymatic complexes on the membrane, enabling the selection of the most appropriate kinetic model. The general procedure proposed has been particularized and applied to experimental results obtained with two enzymatic reactions carried out in a hollow-fibre reactor, enzymatic hydrolysis of lactose by β-galactosidase and glucose–fructose isomerization by glucose isomerase. The application of the general model has allowed us to determine the mechanism of the reaction for both kinetic reactions, assuming the adsorption of the enzymatic complex EGa for lactose hydrolysis and the adsorption of the free enzyme onto the membrane for glucose–fructose isomerization.     

Kinetic modeling of lipase‐catalyzed esterification reaction between oleic acid and trimethylolpropane: A simplified model for multi‐substrate multi‐product ping–pong mechanisms

Kinetic models are among the tools that can be used for optimization of biocatalytic reactions as well as for facilitating process design and upscaling in order to improve productivity and economy of these processes. Mechanism pathways for multi‐substrate multi‐product enzyme‐catalyzed reactions can become very complex and lead to kinetic models comprising several tens of terms. Hence the models comprise too many parameters, which are in general highly correlated and their estimations are often prone to huge errors. In this study, Novozym^®435 catalyzed esterification reaction between oleic acid (OA) and trimethylolpropane (TMP) with continuous removal of side‐product (water) was carried out as an example for reactions that follow multi‐substrate multi‐product ping‐pong mechanisms. A kinetic model was developed based on a simplified ping‐pong mechanism proposed for the reaction. The model considered both enzymatic and spontaneous reactions involved and also the effect of product removal during the reaction. The kinetic model parameters were estimated using nonlinear curve fitting through unconstrained optimization methodology and the model was verified by using empirical data from different experiments and showed good predictability of the reaction under different conditions. This approach can be applied to similar biocatalytic processes to facilitate their optimization and design. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1422–1429, 2013     

One-electron redox reactions of free radicals in solution. Rate of electron transfer processes to quinones

A large number of biologically-important organic and inorganic free radicals have been produced in aqueous solutions, using the fast-reaction technique of pulse radiolysis and kinetic absorption spectrophotometry. The reactions of these free radicals with menaquinone (vitamin K₃, E₀ = 0.42 V) were followed by observing the formation kinetics of the semiquinone radical anion of menaquinone, •MK⁻. The absorption spectrum of •MK⁻ has maxima at 395 nm and 300 nm, with extinction coefficients of 1.1·10⁴ and 1.25·10⁴ M⁻¹·cm⁻¹, respectively. The pK_a of the radical •MK⁻-H⁺ is 4.6±0.1. The free radicals were produced by a one-electron oxidation or reduction of various compounds by hydroxyl radicals and solvated electrons, e_aq⁻. Alcohols, sugars, carboxylic acids, amino acids, peptides, aliphatic amines and amides, aromatic and heterocyclic molecules, pyridine derivatives (nicotinamide, NAD⁺), and transition metal ions have been examined. Significant differences have been observed in both the efficiency (expressed in percentage) and the rate constants of the electron transfer reactions from these free radicals to menaquinone. Absolute rates of electron transfer from approx. 5·10⁸–5·10⁹ M⁻¹·s⁻¹ have been observed for most of the free radicals studied. Information relating to the nature of the radicals and the acid-base properties of these radicals for effective one-electron redox reactions with quinones is indicated.     

Drugs with susceptible sites for free radical induced oxidative transformations: the case of a penicillin

Penicillins, as bactericidal antibiotics, have been widely used to treat infections for several decades. Their structure contains both aromatic and thioether moieties susceptible to free radical oxidation. The ^?OH induced oxidation mechanism of amoxicillin was investigated by pulse radiolysis techniques and by final product analysis performed after steady-state γ-irradiation. The predominant sites of the ^?OH attack are suggested to be the thioether group, initially yielding an ^?OH adduct to the sulfur, and the aromatic ring. This adduct to the sulfur converts to sulfur radical cation, which has three competitive reaction paths: (1) by deprotonation at the adjacent carbon α-(alkylthio)alkyl radicals form, which undergo disproportionation leading presumably to sulfoxide as main product; (2) via the pseudo-Kolbe mechanism it may transform to α-aminoalkyl radicals; (3) the radical cation can be stabilized through intramolecular S.˙.O bond formation. The reaction mechanism suggests the presence of a short-living and a stabilized (via hydrogen bonding) long-living ^?OH adduct to the sulfur. The three-electron bonded dimers of amoxicillin were not formed owing to steric hindrance. Thiyl radicals were also present in equilibrium with α-aminoalkyl radicals. In the presence of dissolved oxygen, aromatic ring hydroxylation occurred along with complex reactions resulting in e.g. oxidation of the methyl groups. The formation of the sulfoxide is especially effective in the presence of dissolved oxygen, under anaerobic condition, however, it is also generated owing to H₂O₂ and α-(alkylthio)alkyl radicals. The thioether moiety appears to be more sensitive to oxidation compared to the aromatic ring in case of amoxicillin.     

Reactivity of semiquinones with ascorbate and the ascorbate radical as studied by pulse radiolysis

Free-radical interactions between hydroquinones (QH2) and ascorbate (AscH-) have a profound impact in many biological situations. Despite the obvious biological significance, not much is known about the kinetics of reactions of QH2 and AscH- with their corresponding free radicals, i.e., semiquinones, Q1.-, and the ascorbate radical, Asc.-. Furthermore, a general approach to reliably measure rate constants for the above reactions is fraught with complications. In this work, the kinetic behavior of Q.- and Asc.-, after pulse radiolytic oxidation of mixtures of a series of alkyl- and methoxysubstituted hydroquinones and ascorbate by azide radicals in aqueous buffer, pH 7.40, was monitored in submillisecond range by time-resolved UV spectroscopy. Rate constants for reactions of Q.- with AscH-(reaction [1]) and Asc.- (reaction [2]) were directly determined by using new kinetic procedures which distinguished between reactions [1] and [2]. The results show that the rate constants for reaction [2] vary only within a narrow range from 1.2 x 10(8) to 2.5 x 10(8) M(-1) s(-1) and do not display any pronounced correlation with Q.- structures. In contrast, the value of k1 for nonsubstituted Q.- was found to be (1.8 +/- 0.2) x 10(5) M(-1) s(-1) and decreases with the number of alkyl and methoxy substituents as well as with the decrease of the one-electron reduction potential E(Q.-/QH2).     

Lipid radicals--possible mediators of charge transfer and energy transformation (a hypothesis)

A number of enzymatic reactions with the participation of lipid radicals is discussed in the article. It is supposed that NADPH- and NADH-dependent formation of the lipid radicals has a functional importance. The uptake of oxygen by free radicals is considered as one of the reactions of radicals utilization. It is proposed that other reactions with participation of lipid radicals can take place in the membranes of microsomes and mitochondria: the reaction of electron transfer from flavoprotein to cytochrome P448 and the reaction of energy transfer which provide the coupling of oxidation and phosphorylation.     

On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation

Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α-d -glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. At a G1P concentration that excludes the substrate-bound E-Glc, the transfer/hydrolysis ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group-transfer reactions by hydrolytic enzymes.     

Free radicals in primary photobiological processes.

Formation of a semiquinone free radical derived from chlorophyll in the reaction of photoreduction has been discovered by A. A. Krasnovsky, Sr. in 1953. This review consider the results obtained in the author's laboratory, concerning the participation of free radicals in photochemical reactions under UV-irradiation of aromatic amino acids, proteins, and lipids, as well as in the reactions of chemiluminescence (CL) in the protein and chlorophyll-containing systems. Free radicals are the very first products of photochemical reactions in all systems studied. The back reactions of radicals are accompanied with photon emission. From the point of view of the molecular energetics, the radiativeless electronic transition in molecules is the most probable event, the transition triplet level is less probable, and the transition to the singlet excited level is virtually impossible. This may explain the low quantum yield of CL, similarity of CL and phosphorescence (rather than fluorescence) spectrum of the reaction products, low quantum yield of CL, and its high temperature coefficient.     

Low-reactive free radicals inhibiting nonbranched chain processes of addition

The kinetics of inhibition of nonbranched chain processes of addition of reactive free radicals at double bonds O=O, C=O, and C=C of molecules in liquid homogeneous binar systems from saturated and unsaturated components by low-reactive free radicals (o-CH3C6H4CH2O*4, HO*4; HC=O; CH2=C(CH3)CH2, and CH2=CHCHOH) was studied. It was assumed that as the concentration of the unsaturated component (as a source of low-reactive free radicals) increases (after the optimum concentration is attained, which corresponds to the maximum rate of the process) the phenomena of increasing inhibition of these processes may act as the elements of self-regulation of similar processes in nature, which returns them into the stationary state. The energetics of the key radical-molecular gas phase reactions was analyzed. The thermal effects of total dissociation reactions of simple alkylperoxyl (exo effect) and alkoxyl (endo effect) reactive free radicals in the gas phase were compared. The kinetic equations (with one to three parameters being directly determined), obtained by the quasi-steady-state treatment, are presented, which make it possible to describe nonmonotonic (with a maximum) dependencies of the rates of formation of molecular 1:1-adducts on the concentration of the unsaturated component.     

Vitamins C and E donate single hydrogen atoms in vivo.

The antioxidant vitamins, C and E, eliminate cytotoxic free radicals by redox cycling. Energetic and kinetic considerations suggest that cycling of vitamin C and vitamin E between their reduced and free radical forms occurs via the transfer of single hydrogen atoms rather than via separate electron transfer and protonation reactions. This may enable these vitamins to reduce many of the damaging free radicals commonly encountered by biological systems while minimizing the reduction of molecular oxygen to superoxide.     

Arguments against the significance of the Fenton reaction contributing to signal pathways under in vivo conditions

One of the common explanations for oxidative stress in the physiological milieu is based on the Fenton reaction, i.e. the assumption that radical chain reactions are initiated by metal-catalyzed electron transfer to hydrogen peroxide yielding hydroxyl radicals. On the other hand — especially in the context of so-called “iron switches” — it is postulated that cellular signaling pathways originate from the interaction of reduced iron with hydrogen peroxide.

Using fluorescence detection and EPR for identification of radical intermediates, we determined the rate of iron complexation by physiological buffer together with the reaction rate of concomitant hydroxylations of aromatic compounds under aerobic and anaerobic conditions. With the obtained overall reaction rate of 1,700 M^-1s^-1 for the buffer-dependent reactions and the known rates for Fenton reactions, we derive estimates for the relative reaction probabilities of both processes.

As a consequence we suggest that under in vivo conditions initiation of chain reactions by hydroxyl radicals generated by the Fenton reaction is of minor importance and hence metal-dependent oxidative stress must be rather independent of the so-called “peroxide tone”. Furthermore, it is proposed that — in the low (subtoxic) concentration range — hydroxylated compounds derived from reactions of “non-free” (crypto) OH radicals are better candidates for iron-dependent sensing of redox-states and for explaining the origin of cellular signals than the generation of “free” hydroxyl radicals.     

Effects of Free Radicals on Partial Reactions of the Na,K-ATPase

The function of the Na,K-ATPase is known to be considerably impaired in the presence of free radicals such as OH^•. While previous experiments were largely based on the loss of enzymatic activity of the protein, this is the first communication dealing with partial reactions of the pump cycle in the presence of free radicals produced by water radiolysis. Three different system states, which are directly involved in ion transfer catalyzed by the enzyme, showed similar sensitivity to free radical action. This is indicated by largely identical D₃₇-doses of the decay of the reaction amplitudes investigated. The decrease in the efficiency of the enzyme functions was largely due to a lethal damage of pump molecules. A kinetic analysis of the ATP-induced conformational transition E₁→ E₂ revealed, however, that a minor component of the inactivation is due to a reduction of the transition rate constant. The decrease of the enzymatic activity could be simulated by the decay of the rate-limiting conformational transition. This finding indicates the conservation of a close coupling between ATP-hydrolysis and sodium translocation process throughout free-radical induced inactivation. As a result of the tight coupling, enzyme modification at different system states leads to similar functional consequences for the protein. Received: 19 July 1996/Revised: 21 October 1996