Thiyl radical reaction with amino acid side chains: rate constants for hydrogen transfer and relevance for posttranslational protein modification

Thiyl radicals are prominent intermediates during biological conditions of oxidative stress and have been suggested to be involved in the mutagenic effects of thiols. While several enzymatic processes rely on the formation and selective reactions of protein thiyl radicals with substrates, such reactions may represent a source for biological damage when occurring uncontrolled during oxidative stress. For example, intramolecular hydrogen transfer reactions to protein cysteine thiyl radicals may lead to secondary amino acid oxidation products, which may represent starting points for protein aggregation and/or fragmentation. Here, we have used a kinetic NMR method to determine rate constants, k(sc), for hydrogen transfer reactions between thiyl radicals and amino acid side chain C-H bonds at 37 degrees C. Rate constants cover a range between k(sc) 相似文献   

Kinetic constraints for the thiolysis of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) and related dithiole-3-thiones in aqueous solution

This report summarizes an investigation of the reactions of biological and other thiols with the cancer chemopreventive oltipraz and other dithiolethiones. Analysis of the kinetics of reaction of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) 1 with monothiols and dithiols in the range of 0.75-20 mM in aqueous 15% ethanol, at pH 7.5 (0.1 M Tris buffer) and at 37 degrees C has been undertaken. A plot of k(obsd) against [thiol] shows that reactions of mono- and dithiols are first order in thiol concentration. The dependence on pH of these reactions shows that the active species is the thiolate anion. Specific second-order rate constants, k(2) (M(-1) s(-1)) for reaction of the thiolate anions with oltipraz have been determined to be cysteine, 0.040 +/- 0.001; 2-mercaptoethanol, 2.0 +/- 0.02; glutathione, 0.099 +/- 0.001; mercaptoacetic acid anion, 4.0 +/- 0.01; dithiothreitol, 1.33 +/- 0.02; 1,3-propanedithiol, 10 +/- 0.5; 1-mercaptopropane-3-ol, 6.5 +/- 0.1; 1-mercaptopropane-2,3-diol, 1.26 +/- 0.05. A plot of pK(a) against log k(2) for monothiols shows a linear dependence of k(2) on pK(a), beta(nuc) 1.1 +/- 0.07, which accounts for most of the reportedly enhanced reactivity of dithiols over monothiols. The pseudo-first-order rate constant for the solvolysis of oltipraz has been measured as 2.2 (+/-0.2) x 10(-8) s(-1). The kinetics of reaction of three other dithiole-3-thiones with glutathione has also been studied for comparison with oltipraz. The specific second-order rate constants, k(2) (M(-1) s(-1)) are 5-phenyl-1,2-dithiole-3-thione, 4.7 x 10(-)(4); 5-(4-methoxyphenyl)-1,2-dithiole-3-thione, 4.1 x 10(-4); and 1,2-dithiole-3-thione 0.08. Important implications for the mode of biological action of these compounds and the nature of the putative biological targets of the compounds are discussed.     

Reactions of nitric oxide, peroxynitrite, and carbonate radicals with nitroxides and their corresponding oxoammonium cations

Cyclic nitroxides effectively protect biological systems against radical-induced damage. However, the mechanism of the reactions of nitroxides with nitrogen-derived reactive species and carbonate radicals is far from being elucidated. In the present study, the reactions of several representative piperidine- and pyrrolidine-based nitroxides with *NO, peroxynitrite, and CO3*- were investigated, and the results are as follows: (i) There is no evidence for any direct reaction between the nitroxides and the *NO. In the presence of oxygen, the nitroxides are readily oxidized by *NO2, which is formed as an intermediate during autoxidation of *NO. (ii) *NO reacts with the oxoammonium cations to form nitrite and the corresponding nitroxides with k1 = (9.8 +/- 0.2) x 10(3) and (3.7 +/- 0.1) x 10(5) M(-1) s(-1) for the oxoammonium cations derived from 2,2,6,6-tetramethylpiperidine-1-oxyl (TPO) and 3-carbamoyl-proxyl (3-CP), respectively. (iii) CO3*- oxidizes all nitroxides tested to their oxoammonium cations with similar rate constants of (4.0 +/- 0.5) x 10(8) M(-1) s(-1), which are about 3-4 times higher than those determined for H-abstraction from the corresponding hydroxylamines TPO-H and 4-OH-TPO-H. (iv) Peroxynitrite ion does not react directly with the nitroxides but rather with their oxoammonium cations with k(10) = (6.0 +/- 0.9) x 10(6) and (2.7 +/- 0.9) x 10(6) M(-1) s(-1) for TPO+ and 3-CP+, respectively. These results provide a better insight into the complex mechanism of the reaction of peroxynitrite with nitroxides, which has been a controversial subject. The small effect of relatively low concentrations of nitroxides on the decomposition rate of peroxynitrite is attributed to their ability to scavenge efficiently *NO2 radicals, which are formed during the decomposition of peroxynitrite in the absence and in the presence of CO2. The oxoammonium cations, thus formed, are readily reduced back to the nitroxides by ONOO-, while forming *NO and O2. Hence, nitroxides act as true catalysts in diverting peroxynitrite decomposition from forming nitrating species to producing nitrosating ones.     

Pathways of arachidonic acid peroxyl radical reactions and product formation with guanine radicals

Peroxyl radicals were derived from the one-electron oxidation of polyunsaturated fatty acids by sulfate radicals that were generated by the photodissociation of peroxodisulfate anions in air-equilibrated aqueous solutions. Reactions of these peroxyl and neutral guanine radicals, also generated by oxidation with sulfate radicals, were investigated by laser kinetic spectroscopy, and the guanine oxidation products were identified by HPLC and mass spectrometry methods. Sulfate radicals rapidly oxidize arachidonic (ArAc), linoleic (LnAc), and palmitoleic (PmAc) acids with similar rate constants, (2-4) x 10 (9) M (-1) s (-1). The C-centered radicals derived from the oxidation of ArAc and LnAc include nonconjugated Rn(.) ( approximately 80%) and conjugated bis-allylic Rba(.) ( approximately 20%) radicals. The latter were detectable in the absence of oxygen by their prominent, narrow absorption band at 280 nm. The Rn(.) radicals of ArAc (containing three bis-allylic sites) transform to the Rba(.) radicals via an intramolecular H-atom abstraction [rate constant (7.5 +/- 0.7) x 10 (4) s (-1)]. In contrast, the Rn(.) radicals of LnAc that contain only one bis-allylic site do not transform intramolecularly to the Rba(.) radicals. In the case of PmAc, which contains only one double bond, the Rba(.) radicals are not observed. The Rn(.) radicals of PmAc rapidly combine with oxygen with a rate constant of (3.8 +/- 0.4) x 10(9) M(-1) s(-1). The Rba(.) radicals of ArAc are less reactive and react with oxygen with a rate constant of (2.2 +/- 0.2) x 10 (8) M (-1) s (-1). The ArAc peroxyl radicals formed spontaneously eliminate superoxide radical anions [rate constant = (3.4 +/- 0.3) x 10 (4) M (-1) s (-1)]. The stable oxidative lesions derived from the 2',3',5'-tri- O-acetylguanosine or 2',3',5'-tri- O-acetyl-8-oxo-7,8-dihydroguanosine radicals and their subsequent reactions with ArAc peroxyl radicals were also investigated. The major products found were the 2,5-diamino-4 H-imidazolone (Iz), dehydroguanidinohydantoin (Gh ox), and diastereomeric spiroiminodihydantoin (Sp) nucleosides from 2',3',5'-tri- O-acetylguanosine and the Gh ox and Sp nucleosides from 2',3',5'-tri- O-acetyl-8-oxo-7,8-dihydroguanosine. In air-saturated aqueous solutions, covalent alkylated guanine adducts were not detected.     

DNA lesions derived from the site selective oxidation of Guanine by carbonate radical anions

Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The latter were monitored by their characteristic transient absorption band at 600 nm. The rate constants of reactions of carbonate radicals with oligonucleotides increase in the ascending order: 5'-d(CCATCCTACC) [(5.7 +/- 0.6) x 10(6) M(-)(1) s(-)(1)] < 5'-d(TATAACGTTATA), self-complementary duplex [(1.4 +/- 0.2) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATCGCTACC [(2.4 +/- 0.3) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATC[8-oxo-G]CTACC) [(3.2 +/- 0.4) x 10(8) M(-)(1) s(-)(1)], where 8-oxo-G is 8-oxo-7,8-dihydroguanine, the product of a two electron oxidation of guanine. This remarkable enhancement of the rate constants is correlated with the presence of either G or 8-oxo-G bases in the oligonucleotides. The rate constant for the oxidation of G in a single-stranded oligonuclotide is faster by a factor of approximately 2 than in the double-stranded form. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40-60%), were isolated by reversed phase HPLC and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects and biological implications of these site selective reactions in DNA initiated by carbonate radicals are discussed.     

Nitrogen dioxide as an oxidizing agent of 8-oxo-7,8-dihydro-2'-deoxyguanosine but not of 2'-deoxyguanosine

The redox reactions of guanine and its widely studied oxidation product, the 8-oxo-7,8-dihydro derivative, are of significant importance for understanding the mechanisms of oxidative damage in DNA. Employing 2'-deoxyguanosine 5'-monophosphate (dGMP) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in neutral aqueous solutions as model systems, we have used nanosecond laser flash photolysis to demonstrate that neutral radicals, dGMP(-H)(*), derived by the one-electron oxidation and deprotonation of dGMP, can oxidize nitrite anions (NO2(-)) to the nitrogen dioxide radical (*)NO2. In turn, we show that (*)NO2 can give rise to a one-electron oxidation of 8-oxo-G, but not of dGMP. The one-electron oxidation of dGMP was initiated by a radical cation generated by the laser pulse-induced photoionization of a pyrene derivative with enhanced water solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The dGMP(-H)(*) neutral radicals formed via deprotonation of the dGMP(*)(+) radical cations and identified by their characteristic transient absorption spectrum (lambda(max) approximately 310 nm) oxidize nitrite anions with a rate constant of (2.6 +/- 0.3) x 10(6) M(-1) s(-1). The 8-oxo-dG is oxidized by (*)NO2 with a rate constant of (5.3 +/- 0.5) x 10(6) M(-1) s(-1). The 8-oxo-dG(-H)(*) neutral radicals thus generated are clearly identified by their characteristic transient absorption spectra (lambda(max) approximately 320 nm). The rate constant of 8-oxo-dG oxidation (k(12)) by the (*)NO2 one-electron oxidant (the (*)NO2/NO2(-) redox potential, E degrees approximately 1.04 V vs NHE) is lower than k(12) for a series of oxidizing aromatic radical cations with known redox potentials. The k(12) values for 8-oxo-dG oxidation by different aromatic radical cations derived from the photoionization of their parent compounds depend on the redox potentials of the latter, which were in the range of 0.8-1.6 V versus NHE. The magnitude of k(12) gradually decreases from a value of 2.2 x 10(9) M(-1) s(-1) (E degrees = 1.62 V) to 5.8 x 10(8) M(-1) s(-1) (E degrees = 1.13 V) and eventually to 5 x 10(7) M(-1) s(-1) (E degrees = 0.91 V). The implications of these results, including the possibility that the redox cycling of the (*)NO2/NO2(-) species can be involved in the further oxidative damage of 8-oxo-dG in DNA in cellular environments, are discussed.     

Generation of thymine radicals from 6-alkoxy-5-bromo-5,6-dihydrothymine derivatives by radiolytic reduction and photolytic dehalogenation

The reduction of 6-alkoxy-5-bromo-5,6-dihydrothymine derivatives ( 1a, b) by hydrated electrons (e aq (-)) generated in the radiolysis of deoxygenated aqueous solution was investigated. As the major products, 1-(6'-alkoxy-5',6'-dihydrothymin-5'-yl)thymines ( 6a, b), 5-(hydroxymethyl)uracil ( 8), 6-alkoxy-5,6-dihydrothymines ( 9a, b), and thymine ( 10) were produced in sufficient yields. This product distribution is indicative of the generation of 6-alkoxy-5,6-dihydrothymin-5-yl radicals ( 2a, b) as primary intermediates that undergo elimination of alkoxide ions (RO (-)) into thymine radical cations ( 3) followed by deprotonation at the N1 to form N-centered thymine radicals ( 4). The transient absorption spectra of the 5-yl radicals 2a- c were observed by means of nanosecond laser flash photolysis of 1a, b and 5-bromo-6-ethoxy-5,6-dihydrothymidine ( 1c) in deoxygenated aqueous solution, in which homolytic C5-Br bond dissociation occurred. In contrast to the reaction characteristics in aqueous solutions, the dimeric products were not obtained in acetonitrile, probably because in-cage hydrogen abstraction from the C5 methyl group by bromine atom leads to formation of methide type intermediates 20.     

New insights into the reaction paths of hydroxyl radicals with 2'-deoxyguanosine

The reaction of HO(?) radical with 2'-deoxyguanosine is intensively studied as a model for DNA damage. Several aspects related to the reaction paths responsible for the most relevant lesions are not well understood. We have reinvestigated the reaction of HO(?) with 2'-deoxyguanosine by pulse radiolysis and extended our studies to a variety of substituted derivatives. The main path of hydrogen abstraction was confirmed to be from the exocyclic NH(2) group, followed by a water-assisted tautomerization. The rate constant (k = 2.3 × 10(4) s(-1)) obtained from the spectral changes at 620 nm is influenced by the substituent at the C8 position. When N1-H is replaced by N1-CH(3), the tautomerization does not occur. The spectral changes at 370 nm that correspond to a rate constant of 6.9 × 10(5) s(-1) were assigned to the cyclization of 2'-deoxyguanosin-5'-yl radical with formation of 5',8-cyclo-2'-deoxyguanosine as the product. When NEt(2) replaces the exocyclic NH(2), the spectral changes at all wavelengths follow second-order kinetics, suggesting a "slow" ring-opening of the 8-hydroxyl adduct of 2'-deoxyguanosine.     

Hydrogen exchange equilibria in glutathione radicals: rate constants

The reduction of oxidized glutathione GSSG by hydrated electrons and hydrogen atoms to form GSSG(?-) is quantitative. The radical anion dissociates into GS(?) and GS(-), and the S-centered radical subsequently abstracts a hydrogen intramolecularly. We observe sequential development of UV absorbance signatures that indicate the formation of both α- and β-carbon-centered radicals. From experiments performed at pH 2 and pH 11.8, we determined forward and reverse rate constants for the overall equilibrium between sulfur-centered and carbon-centered radicals: k(forward) = 3·10(5) s(-1), k(reverse) = 7·10(5) s(-1), and K = 0.4. Furthermore, on the basis of the differences between the kinetics traces at 240 and 280 nm, we estimate that α- and β-carbon-centered radicals are formed at a surprising ratio of 1:3. The ratios found at pH 2 also apply to pH 7, with the conclusion that the equilibrium ratio of S-centered:β-centered:α-centered radicals is, very approximately, 8:3:1. The formation of carbon-centered radicals could lead to irreversible damage in proteins via the formation of carbon-carbon bonds or backbone fragmentation.     

Alkaline hydrolysis of oxaliplatin--isolation and identification of the oxalato monodentate intermediate

The alkaline degradation of the chemotherapeutic agent oxaliplatin has been studied using liquid chromatography. The oxalato ligand is lost in two consecutive steps. First, the oxalato ring is opened, forming an oxalato monodentate intermediate, as identified by electrospray ionization mass spectrometry. Subsequently, the oxalato ligand is lost and the dihydrated oxaliplatin complex is formed. The observed rate constants for the first step (k(1)) and the second step (k(2)) follow the equation k(1) or k(2) = k(0) + k(OH(-) )[OH(-)], where k(0) is the rate constant for the degradation catalyzed by water and k(OH(-) ) represents the second-order rate constant for the degradation catalyzed by the hydroxide ion. At 37 degrees C the rate constants for the first step are k(OH(-) ) = 5.5 x 10(-2) min(-1) M(-1) [95% confidence interval (CI), 2.7 x 10(-2) to 8.4 x 10(-2) min(-1) M(-1)] and k(0) = 4.3 x 10(-2) min(-1) (95% CI, 4.0 x 10(-2) to 4.7 x 10(-2) min(-1)). For the second step the rate constants are k(OH(-) ) = 1.1 x 10(-3) min(-1) M(-1) (95% CI, -1.1 x 10(-3) to 3.3 x 10(-3)) min(-1) M(-1) and k(0) = 7.5 x 10(-3) min(-1) (95% CI, 7.2 x 10(-3) to 7.8 x 10(-3) min(-1)). Thus, the ring-opening step is nearly six times faster than the step involving the loss of the oxalato ligand.     

Disproportionation of clozapine radical: a link between one-electron oxidation of clozapine and formation of its nitrenium cation

The primary products of one-electron oxidation of clozapine and olanzapine, very effective atypical antipsychotic drugs, have been spectroscopically characterized. The oxidation process has been studied under glassy matrix conditions and by a pulse radiolysis technique in aqueous solution. The rate constants for the oxidation of clozapine with dibromide radical anion ( k = 2 x 10 (9) M (-1) s (-1)) and azide radical ( k = 2.3 x 10 (9) M (-1) s (-1)) in aqueous solution were measured. The computational DFT results support the identification of the transient species. The mechanistic aspects of reactivity of radical cations, radicals, and nitrenium cations have been investigated. A disproportionation reaction ( k > or = 1 x 10 (8) M (-1) s (-1)) was proposed as a link between the products of one-electron oxidation and formation of the nitrenium cations of clozapine and olanzapine, products likely responsible for the pathogenesis of adverse drug reactions. The rate constants for the reactions of nitrenium cation of clozapine with glutathione ( k = 3.4 x 10 (4) M (-1) s (-1)) and cysteine ( k = 9.8 x 10 (4) M (-1) s (-1)) were determined.     

Scavenging effects of natural phenols on oxidizing intermediates of peroxynitrite

Most of peroxynitrite (ONOO-/ONOOH) is formed via the diffusion-limited reaction between nitric oxide and superoxide. In biological systems, the decomposition of ONOO- yields 30-35% of carbonate radical (CO3*-) and nitrogen dioxide (NO2*), which are strongly oxidizing intermediates and are suggested to take a part of the responsibility for the toxicity of nitric oxide (NO*) or ONOO-. Therefore, the current study focuses on the scavenging activities of phenols toward CO3*- and NO2* to protect biomolecules from damage caused by NO* or ONOO- using the technique of pulse radiolysis. From the build-up kinetic of the phenoxyl radicals and the decay kinetic of CO3*- radical, the rate constants of scavenging reactions were determined to be 1.9-3.4 x 10(8) M(-1) x s(-1) and 0.11-1.9 x 10(8) M(-1) x s(-1) for CO3*- and NO2* respectively. The results indicated that the tested phenols are the efficient scavengers of CO3*- and NO2*.     

Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.     

Properties of the radicals formed by one-electron oxidation of acetaminophen--a pulse radiolysis study

The semi-iminoquinone radical of acetaminophen, which has previously been proposed as a possible hepatotoxic intermediate in the cytochrome P-450 catalysed oxidation of acetaminophen, has been generated and studied by pulse radiolysis. In the absence of other reactive solutes, the radical decays rapidly by second order kinetics with a rate constant (2k2) of (2.2 +/- 0.4) x 10(9) M-1 sec-1. In alkaline solutions the radical deprotonates with a pK of 11.1 +/- 0.1 to form a radical-anion, as confirmed by the effect of ionic strength on the rate of radical decay. The acetaminophen radical-anion reacts with resorcinol at high pH values, leading to the formation of a transient equilibrium from which the one-electron reduction potential of the semi-iminoquinone radical of acetaminophen is estimated to be +0.707 +/- 0.01 V at pH 7. This value predicts that acetaminophen should be oxidised by thiyl radicals. This was confirmed by pulse radiolysis experiments for reaction of the cysteinyl radical, for which rate constants of 7 x 10(6) M-1 sec-1 at pH 7 and 2.7 x 10(8) M-1 sec-1 at pH 11.3 were obtained. The reaction of O2 with the acetaminophen semi-iminoquinone radical could not be detected by pulse radiolysis, and alternative mechanisms for superoxide radical formation are discussed.     

Kinetics and mechanism of the reaction of aminoguanidine with the alpha-oxoaldehydes glyoxal, methylglyoxal, and 3-deoxyglucosone under physiological conditions

Aminoguanidine (AG), a prototype agent for the preventive therapy of diabetic complications, reacts with the physiological alpha-oxoaldehydes glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG) to form 3-amino-1,2,4-triazine derivatives (T) and prevent glycation by these agents in vitro and in vivo. The reaction kinetics of these alpha-oxoaldehydes with AG under physiological conditions pH 7.4 and 37 degrees was investigated. The rate of reaction of AG with glyoxal was first order with respect to both reactants; the rate constant k(AG,G) was 0.892 +/- 0.037 M(-1) sec(-1). The kinetics of the reaction of AG with 3-DG were more complex: the rate equation was d[T](o)/dt (initial rate of T formation) = [3-DG](k(AG,3-DG)[AG] + k(3-DG)), where k(AG,3-DG) = (3. 23 +/- 0.25) x 10(-3) M(-1) sec(-1) and k(3-DG) = (1.73 +/- 0.08) x 10(-5) sec(-1). The kinetics of the reaction of AG with methylglyoxal were consistent with the reaction of both unhydrated (MG) and monohydrate (MG-H(2)O) forms. The rate equation was d[T](o)/dt = ?k(1)k(AG,MG)/(k(-1) + k(AG,MG)[AG]) + k(AG, MG-H(2)O)?[MG-H(2)O][AG], where the rate constant for the reaction of AG with MG, k(AG,MG), was 178 +/- 15 M(-1) sec(-1) and for the reaction of AG with MG-H(2)O, k(AG,MG-H(2)O), was 0.102 +/- 0.001 M(-1) sec(-1); k(1) and k(-1) are the forward and reverse rate constants for methylglyoxal dehydration MG-H(2)O right harpoon over left harpoon MG. The kinetics of these reactions were not influenced by ionic strength, but the reaction of AG with glyoxal and with methylglyoxal under MG-H(2)O dehydration rate-limited conditions increased with increasing phosphate buffer concentration. Kinetic modelling indicated that the rapid reaction of AG with the MG perturbed the MG/MG-H(2)O equilibrium, and the ratio of the isomeric triazine products varied with initial reactant concentration. AG is kinetically competent to scavenge the alpha-oxoaldehydes studied and decrease related advanced glycated endproduct (AGE) formation in vivo. This effect is limited, however, by the rapid renal elimination of AG. Decreased AGE formation is implicated in the prevention of microvascular complications of diabetes by AG.     

Evaluation and interpretation of voltage- and frequency-dependent electrophysiologic effects of a new class I antiarrhythmic agent (nicainoprol) on guinea pig papillary muscle and isolated heart

Frequency- and voltage-dependent electrophysiologic effects of a chemically novel compound, nicainoprol, were evaluated by recording transmembrane action potentials (APs) from papillary muscles and electrograms (EGs) from isolated perfused hearts of guinea pigs. At 0.2 Hz stimulation, nicainoprol (3.3 x 10(-6) M and 10(-5) M) significantly reduces the maximal upstroke velocity (Vmax) of APs without significant change in resting membrane potential (RMP), functional refractory period (FRP), and action potential duration. Nicainoprol prolongs the spread of excitation but has little effect on the duration of the ventricular EG. The Vmax depression is frequency dependent in the range of 0.02-2.5 Hz, showing saturation at higher frequencies. Under resting conditions, nicainoprol (3.3 x 10(-6) M and 10(-5) M) has no effect on Vmax. The onset of frequency-dependent Vmax reduction follows monoexponential time courses with rate constants of 0.053 +/- 0.007 AP-1 (3.3 x 10(-6) M) and of 0.066 +/- 0.005 AP-1 (10(-5) M) at 1 Hz. Vmax recovers from frequency-dependent depression with time constants of 45.4 +/- 3.2 s (3.3 x 10(-6) M) and 48.4 +/- 3.5 s (10(-5) M). Nicainoprol significantly shifts the Vmax-RMP relation in hyperpolarizing direction by 2.6 +/- 1.1 mV (3.3 x 10(-6) M) and 5.4 +/- 1.3 mV (10(-5) M) at membrane potentials where Vmax is half maximal. It is concluded that nicainoprol can be classified as a class 1C drug and does preferentially bind to inactivated sodium channels with a dissociation constant of about 10(-5) M.     

Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid.

1. Carnosol and carnosic acid have been suggested to account for over 90% of the antioxidant properties of rosemary extract. 2. Purified carnosol and carnosic acid are powerful inhibitors of lipid peroxidation in microsomal and liposomal systems, more effective than propyl gallate. 3. Carnosol and carnosic acid are good scavengers of peroxyl radicals (CCl3O2.) generated by pulse radiolysis, with calculated rate constants of 1-3 x 10(6) M-1 s-1 and 2.7 x 10(7) M-1 s-1 respectively. 4. Carnosic acid reacted with HOCl in such a way as to protect the protein alpha 1-antiproteinase against inactivation. 5. Both carnosol and carnosic acid stimulated DNA damage in the bleomycin assay but they scavenged hydroxyl radicals in the deoxyribose assay. The calculated rate constants for reaction with .OH in the deoxyribose system for carnosol and carnosic acid were 8.7 x 10(10) M-1 s-1 and 5.9 x 10(10) M-1 s-1 respectively. 6. Carnosic acid appears to scavenge H2O2, but it could also act as a substrate for the peroxidase system. 7. Carnosic acid and carnosol reduce cytochrome c but with a rate constant significantly lower than that of O2(-.).     

Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro.

Antioxidants play a vital role in the cellular protection against oxidative damage. Quercetin is a well-investigated antioxidant and known to be able to protect against cellular oxidative DNA damage. In this study, we tried to relate the protection by quercetin pre-treatment against oxidative DNA damage in human leucocytes in vitro to the interaction of quercetin in solution with hydroxyl and superoxide anion radicals as measured by electron spin resonance (ESR) spectrometry, using DMPO as a spin trap. Further, scavenging capacity of quercetin-treated leucocytes in vitro was evaluated by ESR spectrometry. Quercetin appears capable of protecting human leucocytes against oxidative DNA damage caused by hydrogen peroxide in a dose-dependent manner. The protection of leucocytes against superoxides is ambiguous. Incubation concentrations of quercetin (1, 10, and 50 microM) reduced levels of superoxide-induced oxidative DNA damage, while at 100 microM the amount of damage was increased. These results are supported by ESR-findings on quercetin in solution, also showing a prooxidant effect at 100 microM. ESR spectroscopy showed rate constant values for the reaction kinetics of quercetin in lowering iron-dependent hydroxyl radical formation and NADH-dependent superoxide anion formation of respectively 3.2 x 10(12)M(-1)s(-1) and 1.1 x 10(4)M(-1)s(-1). This shows that quercetin is a more potent inhibitor of hydroxyl radical formation than a scavenger of superoxide anions.     

The antioxidant activity of caroverine

Caroverine, 1-(2-diethylaminoethyl)-3-(p-methoxy benzyl)-1,2-dihydro-2-quinoxalin-2-on-hydrochloride, is a class B calcium-channel-blocker and antiglutamatergic agent with significant effects on the brain function. Caroverine exhibits competitive AMPA antagonism, and at higher concentrations, noncompetitive NMDA antagonism. In clinical practice caroverine is used as a spasmolytic and otoneuroprotective agent. Since reactive oxygen species are supposed to be involved in the pathogenesis of inner ear diseases in which caroverine shows beneficial effects, the present study aimed to investigate the antioxidant properties of caroverine. Lipid peroxidation of liposomal membranes was suppressed in the presence of caroverine. In order to understand the mechanism of this antioxidant action of caroverine, we determined the rate constants both for a possible reaction with superoxide (O(2)(.-)) radicals from xanthine/xanthine oxidase and for a possible reaction with hydroxyl (.OH) radicals in Fenton system. Using a defined chemical reaction model O(2)(.-) scavenging was found to occur at a rather low rate constant only (3 x 10(2)M(-1)s(-1)). Thus, a reaction of caroverine with O(2)(.-) radicals is of marginal significance. In contrast, the reaction of caroverine with .OH radicals occurs at an extremely high rate constant (k=1.9 x 10(10)M(-1)s(-1)). The strong antioxidant activity of caroverine is therefore based both on the partial prevention and highly active scavenging of hydroxyl radicals.     

Influence of pH, temperature and buffers on cefepime degradation kinetics and stability predictions in aqueous solutions

First-order rate constants (k) were determined for cefepime degradation at 45, 55, 65, and 75 degrees C, pH 0.5 to 8.6, using an HPLC assay. Each pH-rate profile exhibited an inflection between pH 1 and 2. The pH-rate expression was k(pH) = kH1 f1(aH+) + kH2 f2(aH+) + ks + kOH(aOH-), where kH1 and kH2 are the catalytic constants (M(-1) h(-1)) for hydrogen ion activity (aH+), kOH is the catalytic constant for hydroxyl ion activity (aOH-), and ks is the first-order rate constant (h(-1)) for spontaneous degradation. The protonated (f1) and unprotonated (f2) fractions were calculated from the dissociation constant, Ka = (8.32x10(-6))e(5295)/RT where T was absolute temperature (T). Accelerated loss due to formate, acetate, phosphate, and borate buffer catalysis was quantitatively described with the catalytic constant, kGA (M(-1) h(-1)) for the acidic component, [GA], and kGB (M(-1) h(-1) for the basic component, [GB], of each buffer. The temperature dependency for each rate constant was defined with experimentally determined values for A and E and the Arrhenius expression, kT = Ae-E/RT, where kT represented kH1, kH2 , kS, kOH, kGA, or kGB. Degradation rate constants were calculated for all experimental pH, temperature, and buffer conditions by combining the contributions from pH and buffer effects to yield, k = k(pH) + kGA[GA] + kGB[GB]. The calculated k values had <10% error for 103 of the 106 experimentally determined values. Maximum stability was observed in the pH-independent region, 4 to 6. Degradation rate constants were predicted and experimentally verified for cefepime solutions stored at 30 degrees C, pH 4.6 and 5.6. These solutions maintained 90% of their initial concentration (T90) for approximately 2 days.