Hydroxyl Radical-Induced Reactions in Polyadenylic Acid as Studied by Pulse Radiolysis: Part I. Transformation Reactions of Two Isomeric OH-Adducts

The absorption spectra of polyadenylic acid (polyA) radicals in N₂0 saturated aqueous solution have been measured as a function of time (up to 15 s) following an 0.4μS electron pulse. The spectra and their changes were analysed by comparison with those from monomeric adenine derivatives (nucleosides and nucleotides) which had been studied by Steenken.¹

The reaction of OH^· radicals with the adenine moiety in poly A results in the formation of two hvdroxvl adducts at the positions C-4 [polyA40H^·] and C-8 [polyA80H^·]. Each OH-adduct undergoes a unimol-ecular transformation reaction before any bimolecular or other unimolecular decay occurs. These reactions are characterized by different rate constants and _pH dependencies. The polyA40H^· adduct undergoes a dehydration reaction to yield a neutral N⁶ centered radical (rate constant K_deh= 1.4 × 10⁴s^-1 at ^pH7.3). This reaction is strongly inhibited by H⁺. In comparison with the analogous reactions in adenosine phosphates, the kinetic _pK value for its inhibition is two ^pH units higher. This shift is the result of the counter ion condensation or double-strand formation. The polyA80H^· adduct undergoes an imidazole ring opening reaction to yield an enol type of formamidopyrimidine radical with the resulting base damage (k^r.o. = 3.5 × 10⁴ s ^-1 at pH7.3). This reaction in contrast is strongly catalysed by H⁺and OH^-, similar as for adenosine but different compared to the nucleotides.     

On the generation of OH<Superscript>·</Superscript> radical species from H<Subscript>2</Subscript>O<Subscript>2</Subscript> by Cu(I) amyloid beta peptide model complexes: a DFT investigation

According to different studies, the interaction between amyloid β-peptide (Aβ) and copper ions could yield radical oxygen species production, in particular the highly toxic hydroxyl radical OH^· that is suspected to contribute to Alzheimer’s disease pathogenesis. Despite intensive experimental and computational studies, the nature of the interaction between copper and Aβ peptide, as well as the redox reactivity of the system, are still matter of debate. It was proposed that in Cu(II) → Cu(I) reduction the complex Cu(II)–Aβ could follow a multi-step conformational change with redox active intermediates that may be responsible for OH^· radical production from H₂O₂ through a Fenton-like process. The purpose of this work is to evaluate, using ab initio Density Functional Theory computations, the reactivity of different Cu(I)–Aβ coordination modes proposed in the literature, in terms of OH^· production. For each coordination model, we considered the corresponding H₂O₂ adduct and performed a potential energy surface scan along the reaction coordinate of O–O bond dissociation of the peroxide, resulting in the production of OH^· radical, obtaining reaction profiles for the evaluation of the energetic of the process. This procedure allowed us to confirm the hypothesis according to which the most populated Cu(I)–Aβ two-histidine coordination is not able to perform efficiently H₂O₂ reduction, while a less populated three-coordinated form would be responsible for the OH^· production. We show that coordination modes featuring a third nitrogen containing electron-donor ligand (an imidazole ring of an histidine residue is slightly favored over the N-terminal amine group) are more active towards H₂O₂ reduction.     

Nitric oxide and peroxynitrite. The ugly,the uglier and the not so good

Nitric oxide, a gaseous free radical, is poorly reactive with most biomolecules but highly reactive with other free radicals. Its ability to scavenge peroxyl and other damaging radicals may make it an important antioxidant in vivo, particular in the cardiovascular system, although this ability has been somewhat eclipsed in the literature by a focus on the toxicity of peroxynitrite, generated by reaction of O^·-₂ with NO^· (or of NO^- with O₂). On balance, experimental and theoretical data support the view that ONOO^- can lead to hydroxyl radical (OH^·) generation at pH 7.4, but it seems unlikely that OH^· contributes much to the cytotoxicity of ONOO^-. The cytotoxicity of ONOO^- may have been over-emphasized: its formation and rapid reaction with antioxidants may provide a mechanism of using NO^· to dispose of excess O^·-₂, or even of using O^·-₂ to dispose of excess NO^·, in order to maintain the correct balance between these radicals in vivo. Injection or instillation of “bolus” ONOO^- into animals has produced tissue injury, however, although more experiments generating ONOO^- at steady rates in vivo are required. The presence of 3-nitrotyrosine in tissues is still frequently taken as evidence of ONOO^- generation in vivo, but abundant evidence now exists to support the view that it is a biomarker of several “reactive nitrogen species”. Another under-addressed problem is the reliability of assays used to detect and measure 3-nitrotyrosine in tissues and body fluids: immunostaining results vary between laboratories and simple HPLC methods are susceptible to artefacts. Exposure of biological material to low pH (e.g. during acidic hydrolysis to liberate nitrotyrosine from proteins) or to H₂O₂ might cause artefactual generation of nitrotyrosine from NO^-₂ in the samples. This may be the origin of some of the very large values for tissue nitrotyrosine levels quoted in the literature. Nitrous acid causes not only tyrosine nitration but also DNA base deamination at low pH: these events are relevant to the human stomach since saliva and many foods are rich in nitrite. Several plant phenolics inhibit nitration and deamination in vitro, an effect that could conceivably contribute to their protective effects against gastric cancer development.     

Modeling the scavenging activity of ellagic acid and its methyl derivatives towards hydroxyl, methoxy, and nitrogen dioxide radicals

The reaction mechanisms involved in the scavenging of hydroxyl (OH^·), methoxy (OCH₃ ^·), and nitrogen dioxide (NO₂ ^·) radicals by ellagic acid and its monomethyl and dimethyl derivatives were investigated using the transition state theory and density functional theory. The calculated Gibbs barrier energies associated with the abstraction of hydrogen from the hydroxyl groups of ellagic acid and its monomethyl and dimethyl derivatives by an OH· radical in aqueous media were all found to be negative. When NO₂ ^· was the radical involved in hydrogen abstraction, the Gibbs barrier energies were much larger than those calculated when the OH^· radical was involved. When OCH₃ ^· was the hydrogen-abstracting radical, the Gibbs barrier energies lay between those obtained with OH^· and NO₂ ^· radicals. Therefore, the scavenging efficiencies of ellagic acid and its monomethyl and dimethyl derivatives towards the three radicals decrease in the order OH^· >> OCH₃ ^· > NO₂ ^·. Our calculated rate constants are broadly in agreement with those obtained experimentally for hydrogen abstraction reactions of ellagic acid with OH· and NO₂· radicals.

Solvent Effects in the Spin Trapping Of Lipid Oxyl Radicals

Studies documenting spin trapping of lipid radicals in defined model systems have shown some surprising solvent effects with the spin trap DMPO. In aqueous reactions comparing the reduction of H₂O₂ and methyl linoleate hydroperoxide (MLOOH) by Fe^z+, hydroxyl (HO·) and lipid alkoxyl (LO·) radicals produce identical four-line spectra with line intensities 1:2:2:1. Both types of radicals react with commonly-used HO· scavengers, e.g. with ethanol to produce ·C(CH₃)HOH and with dirnethylsulfoxide (DMSO)togive ·CH₃. However, DMSO radicals (either ·CH₃or ·OOCH₃) react further with lipids, and when radicals are trapped in these MLOOH systems, multiple adducts are evident. When acetonitrile is added to the aqueous reaction systems in increasing concentrations, ·CH₂CN radicals resulting from HO· attack on acetonitrile are evident, even with trace quantities of that solvent. In contrast, little, if any, reaction of LO· with acetonitrile occurs, even in 100% acetonitrile. A single four-line signal persists in the lipid systems as long as any water is present, although the relative intensity of the two center lines decreases as solvent-induced changes gradually dissociate the nitrogen and β-hydrogen splitting constants. Extraction of the aqueous-phase adducts into ethyl acetate shows clearly that the identical four-line spectra in the H₂0₂ and MLOOH systems arise from different radical species in this study, but the lack of stability of the adducts to phase transfer may limit the use of this technique for routine adduct identification in more complex systems. These results indicate that the four-line 1:2:2:1. a_N = a_H = 14.9G spectrum from DMPO cannot automatically be assigned to the HO· adduct in reaction systems where lipid is present, even when the expected spin adducts from ethanol or DMSO appear confirmatory for HO-. Conclusive distinction between HO· and LO· ultimately will require use of ¹³C-labelled DMPO or HPLC-MS separation and specific identification of adducts when DMPO is used as the spin trap.     

Stereochemistry of Decarboxylation Reactions Catalyzed by a Constitutive Aromatic l-Amino Acid Decarboxylase

The stereochemistry of the decarboxylation reaction catalyzed by an aromatic l-amino acid decarboxylase, purified from Micrococcus percitreus, was studied using stereospecifically deuterium labelled phenylalanine (Phe). The ¹H NMR spectrum of [1,2-²H₂]-β-phenethylamine enzymatically derived from (2S, 3R)-[3-²H]-Phe in ²H₂O was compared with that of [1-²H]-β-phenethylamine from unlabelled Phe in ²H₂O. The results clearly indicate that the decarboxylation reaction of this enzyme proceeds exclusively through a course in which the configuration at C-2 of Phe is retained.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Pulse-radiolytic investigations of catechols and catecholamines II. Reactions of Tiron with oxygen radical species

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (°OH), superoxide anions (O₂^?) or a combination of both oxygen radicals. The rate constant with °OH radicals was determined at 1.0·10⁹ M^?1·s^?1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11–16), the rate constant with O₂^? of 1.0·10⁷ M^?1·s^?1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with °OH and O₂^? of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of °OH radicals, leading to the same products with catechol compounds.     

The effect of antioxidants on the formation of free radicals and primary products of the peroxidase reaction

Antioxidants suppress the formation of radicals in peroxidase processes. The chemiluminescence kinetic curves in the horseradish peroxidase/luminol/H₂O₂ system have been compared with those obtained from the mathematical model of the reaction. It was shown that the effect of trolox, ascorbate, and mexidol is a result of the reaction of the luminol radical with the inhibitor molecule (rate constants, 1.0·10¹⁰, 9.0·10⁹, and 2.3·10⁵ mol^–1 min^–1, respectively). The antiradical action of quercetin has been described by eight reactions that were based on the assumption of two reaction centers in the molecule, each reacting with two radicals. The hypothesis that the antioxidant molecule captures the enzyme intermediate radicals in peroxidase cycle, rather than the radical of the reaction product was not confirmed because the calculated curves differed from the experimental point positions. Apparently, the formation of radicals in the presence of peroxidases in living cells and the subsequent events, such as apoptosis, may be prevented not only by the inhibition of an enzyme but also by antioxidants that capture free-radical reaction products     

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N⁶-ethano[¹⁴C]adenosine nor 1,N⁶-ethanol[¹⁴C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-³H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N⁶-Benzyl[¹⁴C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N⁶-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8-¹⁴C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

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.     

In Vitro Effect of H2O2, Some Transition Metals and Hydroxyl Radical Produced Via Fenton and Fenton-Like Reactions,on the Catalytic Activity of AChE and the Hydrolysis of ACh

It is well known that the principal biomolecules involved in Alzheimer’s disease (AD) are acetylcholinesterase (AChE), acetylcholine (ACh) and the amyloid beta peptide of 42 amino acid residues (Aβ42). ACh plays an important role in human memory and learning, but it is susceptible to hydrolysis by AChE, while the aggregation of Aβ42 forms oligomers and fibrils, which form senile plaques in the brain. The Aβ42 oligomers are able to produce hydrogen peroxide (H₂O₂), which reacts with metals (Fe²⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺) present at high concentrations in the brain of AD patients, generating the hydroxyl radical (^·OH) via Fenton (FR) and Fenton-like (FLR) reactions. This mechanism generates high levels of free radicals and, hence, oxidative stress, which has been correlated with the generation and progression of AD. Therefore, we have studied in vitro how AChE catalytic activity and ACh levels are affected by the presence of metals (Fe³⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Cd²⁺), H₂O₂ (without Aβ42), and ^· OH radicals produced from FR and FLR. The results showed that the H₂O₂ and the metals do not modify the AChE catalytic activity, but the ^·OH radical causes a decrease in it. On the other hand, metals, H₂O₂ and ^·OH radicals, increase the ACh hydrolysis. This finding suggests that when H₂O₂, the metals and the ^·OH radicals are present, both, the AChE catalytic activity and ACh levels diminish. Furthermore, in the future it may be interesting to study whether these effects are observed when H₂O₂ is produced directly from Aβ42.     

Direct demonstration that ferrous ion complexes of di- and triphosphate nucleotides catalyze hydroxyl free radical formation from hydrogen peroxide

Utilizing an electron paramagnetic resonance (EPR) spin-trapping technique it was demonstrated that the di- and triphosphate nucleotides of adenosine, cytidine, thymidine, and guanosine in the presence of Fe(II) catalyze hydroxyl free radical formation from H₂O₂. The triphosphate nucleotides in general were about 20% more effective than the diphosphate nucleotides. The amount of ?H produced from H₂O₂ as a function of nucleotide level tended to increase in a sigmoidal fashion beginning at a nucleotide/Fe(II) ratio of 2 but then rose rapidly up to a ratio of 5 at which point the increase became more gradual. The monophosphate nucleotides did not cause an increase in the amount of hydroxyl free radical produced from H₂O₂ over the low level obtained in the buffer system only. The cations, Mg²⁺ and Ca²⁺, even at much higher than physiological levels and much higher than the level of added Fe(II), did not cause a substantial diminution of the Fe(II)-nucleotide-catalyzed breakdown of H₂O₂ to yield ?H. A study of the time course of the effectiveness of Fe(II)-nucleotide-mediated ?H formation from H₂O₂ demonstrated that Fe(II) in the presence of nucleotides remained in an effective catalytic state with a halftime of about 160 s whereas in the absence of the nucleotides the halftime was 7.5 s. All observations indicate that Fe(II) ligates with di- and triphosphate nucleotides and remains in the ferrous state which is then capable of catalyzing ?H formation from H₂O₂; but with time, oxidation of the metal ion to the ferric state occurs, which either ligated to the nucleotide or to buffer ions, is ineffective in H₂O₂ catalysis to yield ?H. Iron-nucleotide complexes may be of importance in mediating oxygen free radical damage to biological systems. The observations presented here indicate that hydroxyl free radicals will be produced when H₂O₂ is present with ferrous-nucleotide complexes.     

Spin polymerization of DNA/RNA nucleotides

The Car-Parrinello Molecular Dynamics (CPMD) has been used to study the ion-radical (IR) polymerization (triplet (T) and singlet (S/T₀) states) of adenine mononucleotides upon interaction with Mg²⁺(H₂O)₂-ATP⁴⁻. It has been found that the IR polymerization occurs only upon Mg²⁺(H₂O)₂-ATP⁴⁻ excitation into a T state (the Franck-Condon or femtosecond laser excitation); it naturally occurs in the dark with DNA polymerase or another Mg-holoenzyme upon interaction of Mg with two Asp residues. The IR path affects only the HO-C_3′ group of ribose, leaving the HO-C_2′ group inactive. The IR polymerization starts with the homolytic removal of the hydrogen atom from the HO-C_3′ group and its transfer onto the hydroxyl radical ·OH, a product of the ATP cleavage, which yields a water molecule. A further progress of the reaction involves interaction between two ion-radicals ·ATP. The reaction is sensitive to the recombination of ·OH and ·ATP. It is mostly suppressed by the appearance of identically directed electron spins on both radicals (the radical pair in the T₀ state) in the vicinity of the HO-C_3′ group and not suppressed in the vicinity of the HO-C_2′ group (the spins in the radical pair are oppositely directed, the radical pair in the T₀ state), making the latter inert on the IR polymerization, but allowing it to be active in the ionic (hydrolytic) polymerization.     

C-11 H-abstraction from linoleic acid, the rate-limiting step in lipoxygenase catalysis

[11,11-²H₂]-, [9,10,12,13-²H₄]-, [9,10,11,11,12,13-²H₆]- and unlabelled linoleic acids were incubated with pure lipoxygenase-1 from soya beans. The apparent rate constants of the overall reactions and the apparent Michaelis constants in air-equilibrated solutions at 25°C and pH 9.0 were obtained from Lineweaver-Burk plots. The apparent K_m-values were hardly affected by the type of substrate used. Substrates bearing ²H instead of ¹H at C-11 gave rise to considerable isotope effects, k_H/k_2H values being 8.7 and 9.3 for dideutero- and hexadeutero linoleate, respectively. From the observed isotope effects it was concluded, that H-abstraction from C-11 is the rate-determining step in the overall reaction. All substrates used gave identical product distributions. No measurable exchange of deuterium with solvent hydrogen occurred during oxygenation.     

The hemoglobin of the crossopterygian fish, Latimeria chalumnae (Smith). Subunit structure and oxygen equilibria

There are five oxidation-reduction states of horseradish peroxidase which are interconvertible. These states are ferrous, ferric, Compound II (ferryl), Compound I (primary compound of peroxidase and H₂O₂), and Compound III (oxy-ferrous). The presence of heme-linked ionization groups was confirmed in the ferrous enzyme by spectrophotometric and pH stat titration experiments. The values of pK were 5.87 for isoenzyme A and 7.17 for isoenzymes (B + C). The proton was released when the ferrous enzyme was oxidized to the ferric enzyme while the uptake of the proton occurred when the ferrous enzyme reacted with oxygen to form Compound III. The results could be explained by assuming that the heme-linked ionization group is in the vicinity of the sixth ligand and forms a stable hydrogen bond with the ligand.The measurements of uptake and release of protons in various reactions also yielded the following stoichiometries: Ferric peroxidase + H₂O₂ → Compound I, Compound I + e^? + H⁺ → Compound II, Compound II + e^? + H⁺ → ferric peroxidase, Compound II + H₂O₂ → Compound III, Compound III + 3e^? + 3H⁺ → ferric peroxidase.Based on the above stoichiometries and assuming the interaction between the sixth ligand and heme-linked ionization group of the protein, it was possible to picture simple models showing structural relations between five oxidation-reduction states of peroxidase. Tentative formulae are as follows: [Pr·Po·Fe-(II) $\text{\$}\text{?}$PrH⁺·Po·Fe(II)] is for the ferrous enzyme, Pr·Po·Fe(III)OH₂ for the ferric one, Pr·Po·Fe(IV)OH^? for Compound II, Pr(OH^?)·Po⁺·Fe(IV)OH^? for Compound I, and PrH⁺·Po·Fe(III)O₂^? for Compound III, in which Pr stands for protein and Po for porphyrin. And by Fe(IV)OH^?, for instance, is meant that OH^? is coordinated at the sixth position of the heme iron and the formal oxidation state of the iron is four.     

Magnesium cation initiates nucleoside triphosphate to monophosphate conversion by a radical mechanism: Quantum molecular dynamics simulation

A DFT:B3LYP (6-311G** basis set) quantum molecular dynamics simulation was used to study the mechanism of Mg²⁺-induced conversion of guanosine triphosphate (GTP) to guanosine monophosphate (GMP). The computations were performed at 310 K in a bath of 178 water molecules surrounding the Mg(H₂O)₂-GTP complex and mimicking the hydration shells. Dissociation of the Mg²⁺-GTP complex (process duration, 5 ps) produces two phosphate anions (P_i), a hydrated Mg²⁺ cation, atomic oxygen, and a highly reactive GMP radical. This radical appears as a result of the action of Mg²⁺, which initiates the radical mechanism of GTP cleavage. At the initial stage of the interaction with GTP, Mg²⁺ is reduced to Mg⁺, producing an ion-radical pair ⁺Mg^·-^·GTP^3?. In the absence of Mg²⁺, an inert GMP molecule forms instead of the GMP radical as a result of hydrolytic cleavage of GTP via an ionic mechanism. Presumably, formation of the GMP radical and analogous radicals with adenosine, cytidine, thymidine, and uridine is a key point in the syntheses of deoxyribonucleic and ribonucleic acids.     

Spin Trapping by Use of N-Tert-Butylhydroxylamine. Involvement of Fenton Reactions

Spin trapping of short-lived R. radicals is done by use of N-tert-butylhydroxylamine (1) and H²O². The hydroxylamine is oxidized to the radical t-BuN(O)H (2) which is converted into the spin trap 2-methyl-2-nitrosopropane (3). Simultaneously, hydroxyl radicals. OH are formed from H²O². The latter radical species abstracts hydrogen atoms from suitable molecules HR to give R. radicals, which are trapped with the formation of aminooxyl radicals, i. e., t-BuN(O)R (4) detectable by EPR spectroscopy. The reaction is enhanced by the presence of iron ions. The cleavage of H²O² into. OH radicals is considered to involve both a radical-driven (t-BuN(O)H 2) and an iron-driven Fenton reaction.     

The role of interactions between O2, H2O2, .OH,e- and O2- in free radical damage to biological systems

The occurrence of the Haber-Weiss reaction and other interactions between free radicals has been investigated in the effects of mixtures of free radicals on the permeability of resealed erythrocyte ghosts and on the activity of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. The following mixtures were found to induce damage greater than that which could be accounted for by the independent actions of the constituent free radicals: (i) · OH + H₂O₂, and (ii) · OH + H₂O₂ + O₂^?. In contrast, the following mixtures were found to induce less damage than that predicted on the basis of independent actions of constituent free radicals: (i) H₂O₂ + O₂^?, and (ii) oxidizing radicals ( · OH, H₂O₂) + reducing radicals (e^?, H · ). These results suggest a Haber-Weiss-like interaction between H₂O₂ and O₂^?and an interaction between H₂O₂ and · OH to produce a species more potent than either in causing increased permeability. The decrease in damage observed in the simultaneous presence of oxidizing and reducing radicals suggests an antagonistic effect by which each tends to moderate damage by the other. Inactivation of glyceraldehyde-3-phosphate dehydrogenase was found to be more sensitive to radiation than permeability by an order of magnitude, while permeability was more sensitive to the enhancement of damage by oxygen. Comparison of the effectiveness of free radical scavengers in inhibiting the increase in permeability caused by free radicals showed the following order of effectiveness, expressed in terms of percentage protection: formate (90%) > nitrogen (65%) > catalase (60%) > dismutase (32%), and with respect to enzymatic inactivation, nitrogen (100%) > formate (77%) > dismutase (48%) > catalase (44%). The relative rates observed anaerobically and aerobically in the presence and absence of the above scavengers suggest that (at least in the case of radiation damage to the membranes of erythrocyte ghost cells) the “oxygen effect” is due to the interaction of oxygen with e^? and H., producing O₂^? which aggravates damage under conditions which allow consequent Haber-Weiss-like reactions. The further increase in damage when oxygen concentration is raised yet higher is due to the interaction of oxygen with the sites of initial damage.     

Non-enzymatic hydrolysis of creatine ethyl ester

The rate of the non-enzymatic hydrolysis of creatine ethyl ester (CEE) was studied at 37 °C over the pH range of 1.6-7.0 using ¹H NMR. The ester can be present in solution in three forms: the unprotonated form (CEE), the monoprotonated form (HCEE⁺), and the diprotonated form (H₂CEE²⁺). The values of pK_a1 and pK_a2 of H₂CEE²⁺ were found to be 2.30 and 5.25, respectively. The rate law is found to be

Rate=-dC_CEE/dt=k₊₊[H₂CEE²⁺][OH^-]+k₊[HCEE⁺][OH^-]+k₀[CEE][OH^-]