Reactions of e(-)(aq), CO(2)(*)(-), HO(*), O(2)(*)(-) and O(2)((1)delta(g)) with a dendro[60]fullerene and C(60)[C(COOH)(2)](n) (n = 2-6)

Using pulse radiolysis and laser flash photolysis, we have investigated the reactions of the deleterious species, e(-)(aq), HO&z.rad;, O(2)(*)(-) and O(2)((1)Delta(g)) with 10 water-soluble cyclopropyl-fused C(60) derivatives including a mono-adduct dendro[60]fullerene (d) and C(60) derivatives based on C(60)[C(COOH)(2)](n=2-6), some of which are known to be neuroprotective in vivo. The rate constants for reactions of e(-)(aq) and HO&z.rad; lie in the range 0.5-3.3 x 10(10) M(-1) s(-1). The d and bis-adduct monoanion radicals display sharp absorption peaks around 1000 nm (epsilon = 7 000-11 500 M(-1) cm(-1)); the anions of the tris-, tetra-, and penta-adduct derivatives have broader, weaker absorptions. The monohydroxylated radicals have their most intense absorption maxima around 390-440 nm (epsilon = 1000-3000 M(-1) cm(-1)). The anion and hydroxylated radical absorption spectra display a blue-shift as the number of addends increases. The radical anions react with oxygen (k approximately 10(7)-10(9) M(-1) s(-1)). The reaction of O(2)(*)(-) with the C(60) derivatives does not occur via an electron transfer. The rate constants for singlet oxygen reaction with the dendrofullerene and eee-derivative in D(2)O at pH 7.4 are k approximately 7 x 10(7) and approximately 2 x 10(7) M(-1) s(-1) respectively, in contrast to approximately 1.2 x 10(5) M(-1) s(-1) for the reaction with C(60) in C(6)D(6). The large acceleration of the rates for electron reduction and singlet oxygen reactions in water is due to a solvophobic process.     

One-electron reduction of S-nitrosothiols in aqueous medium

One-electron reduction of S-nitrosothiols (RSNO) has been studied using radiolytically produced reducing entity, the hydrated electron (e(aq)(-)), in aqueous medium. Both kinetics of the reaction and the mechanistic aspects of the decomposition of S-nitroso derivatives of glutathione, L-cysteine, N-acetyl-L-cysteine, N-acetyl-D,L-penicillamine, N-acetylcysteamine, L-cysteine methyl ester, and D,L-penicillamine have been investigated at neutral and acidic pH. The second-order rate constants of the reaction of e(aq)(-) with RSNOs were determined using a pulse radiolysis technique and were found to be diffusion controlled (10(10) dm(3) mol(-1) s(-1)) at neutral pH. The product analysis using HPLC, fluorimetry, and MS revealed the formation of thiol and nitric oxide as the major end products. It is therefore proposed that one-electron reduction of RSNO leads to the liberation of NO. There is no intermediacy of a thiyl radical as in the case of oxidation reactions of RSNOs. The radical anion of RSNO (RSN(*)O(-)) is proposed as a possible intermediate. The overall reaction could be written as RSNO + e(aq)(-) --H+--> RSH + (*)NO.     

Formation of hydroxyl radicals by irradiated 1-nitronaphthalene (1NN): oxidation of hydroxyl ions and water by the 1NN triplet state

The excited triplet state of 1-nitronaphthalene ((3)1NN*) reacts with OH(-) with a second-order reaction rate constant of (1.66 ± 0.08)×10(7) M(-1) s(-1) (μ±σ). The reaction yields the ˙OH radical and the radical anion 1NN(-)˙. In aerated solution, the radical 1NN(-)˙ would react with O(2) to finally produce H(2)O(2) upon hydroperoxide/superoxide disproportionation. The photolysis of H(2)O(2) is another potential source of ˙OH, but such a pathway would be a minor one in circumneutral (pH 6.5) or in basic solution ([OH(-)] = 0.3-0.5 M). The oxidation of H(2)O by (3)1NN*, with rate constant 3.8 ± 0.3 M(-1) s(-1), could be the main ˙OH source at pH 6.5.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6 ± 1 × 10(7) M(-1) s(-1) at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7 ± 3 × 10(7) M(-1) s(-1) at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK(a) of phenolic hydroxyl dissociation in tyrosine is ≈ 10.3, this infers a much lower rate constant, about 3 × 10(5) M(-1) s(-1), for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest

Hydrogen sulfide (H(2)S) is an endogenously generated gas that can also be administered exogenously. It modulates physiological functions and has reported cytoprotective effects. To evaluate a possible antioxidant role, we investigated the reactivity of hydrogen sulfide with several one- and two-electron oxidants. The rate constant of the direct reaction with peroxynitrite was (4.8±1.4)×10(3)M(-1) s(-1) (pH 7.4, 37°C). At low hydrogen sulfide concentrations, oxidation by peroxynitrite led to oxygen consumption, consistent with a one-electron oxidation that initiated a radical chain reaction. Accordingly, pulse radiolysis studies indicated that hydrogen sulfide reacted with nitrogen dioxide at (3.0±0.3)×10(6)M(-1) s(-1) at pH 6 and (1.2±0.1)×10(7)M(-1) s(-1) at pH 7.5 (25°C). The reactions of hydrogen sulfide with hydrogen peroxide, hypochlorite, and taurine chloramine had rate constants of 0.73±0.03, (8±3)×10(7), and 303±27M(-1) s(-1), respectively (pH 7.4, 37°C). The reactivity of hydrogen sulfide was compared to that of low-molecular-weight thiols such as cysteine and glutathione. Considering the low tissue concentrations of endogenous hydrogen sulfide, direct reactions with oxidants probably cannot completely account for its protective effects.     

Oxidation-state-dependent reactions of cytochrome <Emphasis Type="Italic">c</Emphasis> with the trioxidocarbonate(•1−) radical: a pulse radiolysis study

The reaction of the trioxidocarbonate(*1-) radical (CO (3) (*-) , "carbonate radical anion") with cytochrome c was studied by pulse radiolysis at alkaline pH and room temperature. With iron(III) cytochrome c, CO (3) (*-) reacts with the protein moiety with rate constants of (5.1 +/- 0.6) x 10(7) M(-1) s(-1) (pH 8.4, I approximately 0.27 M) and (1.0 +/- 0.2) x 10(8) M(-1) s(-1) (pH 10, I = 0.5 M). The absorption spectrum of the haem moiety was not changed, thus, amino acid radicals produced on the protein do not reduce the haem. The pH-dependent difference in rate constants may be attributed to differences in ionization states of amino acids and to the change in the conformation of the protein. With iron(II) cytochrome c, CO (3) (*-) oxidizes the haem quantitatively, presumably via electrostatic guidance of the radical to the solvent-accessible haem edge, with a different pH dependence: at pH 8.4, the rate constant is (1.1 +/- 0.1) x 10(9) M(-1) s(-1) and, at pH 10, (7.6 +/- 0.6) x 10(8) M(-1) s(-1). We propose that CO (3) (*-) oxidizes the iron center directly, and that the lower rate observed at pH 10 is due to the different charge distribution of iron(II) cytochrome c.     

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.     

Oxidation of cytochrome c peroxidase to compound I by peroxyacids: evidence for rate-limiting diffusion through the protein matrix

The rate of the reaction between p-nitroperoxybenzoic acid and cytochrome c peroxidase (CcP) has been investigated as a function of pH and ionic strength. The pH dependence of the reaction between CcP and peracetic acid has also been determined. The rate of the reactions are influenced by two heme-linked ionizations in the protein. The enzyme is active when His-52 (pK(a) 3.8 +/- 0.1) is unprotonated and an unknown group with a pK(a) of 9.8 +/- 0.1 is protonated. The bimolecular rate constant for the reaction between peracetic acid and CcP and between p-nitroperoxybenzoic acid and CcP are (1.8 +/- 0.1) x 10(7) and (1.6 +/- 0.2) x 10(7) M(-)(1) s(-)(1), respectively. These rates are about 60% slower than the reaction between hydrogen peroxide and CcP. A critical comparison of the pH dependence of the reactions of hydrogen peroxide, peracetic acid, and p-nitroperoxybenzoic acid with CcP provides evidence that both the neutral and anionic forms of the two peroxyacids react directly with the enzyme. The peracetate and p-nitroperoxybenzoate anions react with CcP with rates of (1.5 +/- 0.1) x 10(6) and (1.6 +/- 0.2) x 10(6) M(-)(1) s(-)(1), respectively, about 10 times slower than the neutral peroxyacids. These data indicate that CcP discriminates between the neutral peroxyacids and their negatively charged ions. However, the apparent bimolecular rate constant for reaction between p-nitroperoxybenzoate and CcP is independent of ionic strength in the range of 0.01-1.0 M, suggesting that electrostatic repulsion between the anion and CcP is not the cause of the lower reactivity for the peroxybenzoate anion. The data are consistent with the hypothesis that the rate-limiting step for the oxidation of CcP to compound I by both neutral peroxyacid and the negatively charged peroxide ion is diffusion of the reactants through the protein matrix, from the surface of the protein to the distal heme pocket.     

The carbonate radical is a site-selective oxidizing agent of guanine in double-stranded oligonucleotides

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.     

Cytotoxicity, apoptosis, cellular uptake, cell cycle distribution, and DNA-binding investigation of ruthenium complexes

Three new ruthenium(II) polypyridyl complexes [Ru(bpy)(2)(BHIP)](2+) 1, [Ru(phen)(2)(BHIP)](2+) 2, and [Ru(dip)(2)(BHIP)](2+) 3 were synthesized and characterized. The cytotoxicity of the three complexes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis induced by the complexes was studied by cell morphology and flow cytometry. The results showed that the percentage of apoptotic cells is 7.19%, 75.58%, and 3.51% in the presence of complexes 1, 2, and 3, respectively. The cellular uptakes were also performed and the results indicated that complexes 1, 2, and 3 can enter into the cytoplasm and also into the nucleus. The studies on antiproliferative mechanism showed the induction of S-phase arrest by complexes 1, 2, and 3. DNA-binding constants of these complexes with calf thymus DNA (CT-DNA) were determined to be 1.07 (± 0.47) × 10(5) M(-1) (s = 2.04), 1.21 (± 0.32) × 10(5) M(-1) (s = 1.88), and 2.75 (± 0.27) × 10(5) M(-1) (s = 2.17), respectively. Upon irradiation at 365 nm, complexes 1, 2, and 3 can induce cleavage of pBR322 DNA.     

Interaction of hydrated electron with dietary flavonoids and phenolic acids: rate constants and transient spectra studied by pulse radiolysis.

The reaction rate constants and transient spectra of 11 flavonoids and 4 phenolic acids reacting with e(aq)- at neutral pH were measured. Absorption bands of the transients of e(aq)- reacting with the above compounds all located at a wavelength shorter than 400 nm. The e(aq)- scavenging abilities were divided into three groups: (+)catechin ((1.2 +/-0.1) x 10(8) M(-1)s(-1)) < 4-chromanol ((4.4 +/- 0.4) x 10(8) M(-1)s(-1)) < genistein ((6.2+/-0.4) x 10(9) M (-1) s(-1) approximately genistin ((8 +/- 1) x 10(9) M(-1)s(-1)) approximately rutin ((7.6 +/- 0.4) x M(-1)s(-1) approximately caffeic acid ((8.3 +/- 0.5) x 10(9)M(-1)s(-1)) < transcinnamic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately p-coumaric acid ((1.1 +/- 0.1) x 10(10) M(-1)s(-1) approximately 2,4,6-trihydroxylbenzoic acid((1.1 +/- 0.1) x 10(10) M(-1)s(-1)) approximately baicalein ((1.1 +/- 0.5) x 10(10) M(-1)s(-1)) approximately baicalin((1.3 + 0.1) X 10(10) M(-1)s(-1)) approximately naringenin ((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately naringin ((1.0 +/- 0.1) x 10(10) M(-1)s(-1)) approximately gossypin((1.2 +/- 0.1) x 10(10) M(-1)s(-1)) approximately quercetin((1.3 +/- 0.5) x 10(10) M(-1)s(-1)). These results suggested that C4 keto group is the active site for e(aq)- to attack on flavonoids and phenolic acids, whereas the o-dihydroxy structure in B ring, the C2,3 double bond, the C3-OH group, and glucosylation, which are key structures that influence the antioxidant activities of flavonoids and phenolic acids, have little effects on the e(aq)- scavenging activities.     

The reduction of porphyrin cytochrome c by hydrated electrons and the subsequent electron transfer reaction from reduced porphyrin cytochrome c to ferricytochrome c.

1. Hydrated electrons, produced by pulse radiolysis react with porphyrin cytochrome c with a bimolecular rate constant of 3-10(10) M-1 S-1 at 21 degrees C and pH 7.4. 2. After the reduction step an absorbance change with a half-life of 5 microns is observed with the spectral range of 430-470 nm. A relatively stable intermediate then decays with a half-life of 15 s. 3. The spectrum of the intermediate observed 50 microns after the generation of hydrated electrons shows a broad absorption band between 600 and 700 nm and a peak at 408 nm. The spectrum is attributed to the protonated form of an initially produced porphyrin anion radical. 4. Reduced porphyrin cytochrome c reacts with ferricytochrome c with a bimolecular constant of 2-10(5) M-1- S-1 in 2 mM phosphate pH 7.4, at 21 degrees C and of 2 - 10(6) M-1-S-1 under the same conditions but at 1 M ionic strength. It is proposed that electron transfer in an analogous exchange reaction between ferrocytochrome c and ferricytochrome c occurs via the exposed part of the haem.     

Antioxidant properties of melatonin: a pulse radiolysis study

Various one-electron oxidants such as OH*, tert-BuO*, CCl3OO*, Br2*- and N3*, generated pulse radiolytically in aqueous solutions at pH 7, were scavenged by melatonin to form two main absorption bands with lambda(max) = 335 nm and 500 nm. The assignment of the spectra and determination of extinction coefficients of the transients have been reported. Rate constants for the formation of these species ranged from 0.6-12.5x10(9) dm3 mol(-1) s(-1). These transients decayed by second order, as observed in the case of Br2*- and N3* radical reactions. Both the NO2* and NO* radicals react with the substrate with k = 0.37x10(7) and 3x10(7) dm3 mol(-1) s(-1), respectively. At pH approximately 2.5, the protonated form of the transient is formed due to the reaction of Br2*- radical with melatonin, pKa ( MelH* <=> Mel* + H+) = 4.7+/-0.1. Reduction potential of the couple (Mel*/MelH), determined both by cyclic voltammetric and pulse-radiolytic techniques, gave a value E(1)7 = 0.95+/-0.02 V vs. NHE. Repair of guanosine radical and regeneration of melatonin radicals by ascorbate and urate ions at pH 7 have been reported. Reactions of the reducing radicals e(aq)- and H* atoms with melatonin have been shown to occur at near diffusion rates.     

Fast repair of dAMP radical anions by phenylpropanoid glycosides and their analogs

Repair effect on 2'-deoxyadenosine-5'-monophosphate (dAMP) radical anions by phenylpropanoid glycosides (PPGs) and their analogs, isolated from Chinese folk medicinal herb, was studied using pulse radiolysis technique. The radical anion of dAMP was formed by the reaction of hydrated electron with dAMP. On pulse irradiation of nitrogen-saturated dAMP aqueous solution containing 0.2 M t-BuOH and one of PPGs or their analogs, the transient absorption spectrum of the radical anion of dAMP decayed with the formation of that of the radical anion of PPGs or their analogs within several decades of microseconds after electron pulse irradiation. The results indicated that dAMP radical anions can be repaired by PPGs or their analogs. The rate constants of the repair reactions were deduced to be 1.6-4.5 x 10(8) M(-1) s(-1).     

Transient quinonimines and 1,4-benzothiazines of pheomelanogenesis: new pulse radiolytic and spectrophotometric evidence.

Biosynthetic and model in vitro studies have shown that pheomelanins, the distinctive pigments of red human hair, arise by oxidative cyclization of cysteinyldopas mainly 5-S-cysteinyldopa (1) via a critical o-quinonimine intermediate, which rearranges to unstable 1,4-benzothiazines. To get new evidence for these labile species, fast time resolution pulse radiolytic oxidation by dibromide radical anion of a suitable precursor, the dihydro-1,4-benzothiazine-3-carboxylic acid 7 was performed in comparison with that of 1. In the case of 7, dibromide radical anion oxidation leads over a few microseconds (k = 2.1 x 10(9) M(-1) s(-1)) to a phenoxyl radical (lambda(max) 330 nm, epsilon = 6300 M(-1) cm(-1)) which within tens of milliseconds gives rise with second-order kinetics (2k = 2.7 x 10(7) M(-1) s(-1)) to a species exhibiting an absorption maximum at 540 nm (epsilon = 2200 M(-1) cm(-1)). This was formulated as the o-quinonimine 3 arising from disproportionation of the initial radical. The quinonimine chromophore is converted over hundreds of milliseconds (k = 6.0 s(-1)) to a broad maximum at around 330 nm interpreted as due to a 1,4-benzothiazine or a mixture of 1,4-benzothiazines, which as expected are unstable and subsequently decay over a few seconds (k = 0.5 s(-1)). Interestingly, the quinonimine is observed as a labile intermediate also in the alternative reaction route examined, involving cyclization of the o-quinone (lambda(max) 390 nm, epsilon = 6900 M(-1) cm(-1)) arising by disproportionation (2k = 1.7 x 10(8) M(-1) s(-1)) of an o-semiquinone (lambda(max) 320 nm, epsilon = 4700 M(-1) cm(-1)) directly generated by dibromide radical anion oxidation of 1. Structural formulation of the 540 nm species as an o-quinonimine was further supported by rapid scanning diode array spectrophotometric monitoring of the ferricyanide oxidation of a series of model dihydrobenzothiazines.     

Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: implications for the mechanism of electron and proton transfer in proteins.

A proton-activated electron transfer (PAET) mechanism, involving a protonated semiquinone intermediate state, had been proposed for the electron-transfer reaction k(2)AB [Q(A)(-)(*)Q(B)(-)(*) + H(+) <--> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)] in reaction centers (RCs) from Rhodobacter sphaeroides [Graige, M. S., Paddock, M. L., Bruce, M. L., Feher, G., and Okamura, M. Y. (1996) J. Am. Chem. Soc. 118, 9005-9016]. Confirmation of this mechanism by observing the protonated semiquinone (Q(B)H)(*) had not been possible, presumably because of its low pK(a). By replacing the native Q(10) in the Q(B) site with rhodoquinone (RQ), which has a higher pK(a), we were able to observe the (Q(B)H)(*) state. The pH dependence of the semiquinone optical spectrum gave a pK(a) = 7.3 +/- 0.2. At pH < pK(a), the observed rate for the reaction was constant and attributed to the intrinsic electron-transfer rate from Q(A)(-)(*) to the protonated semiquinone (i.e., k(2)AB = k(ET)(RQ) = 2 x 10(4) s(-)(1)). The rate decreased at pH > pK(a) as predicted by the PAET mechanism in which fast reversible proton transfer precedes rate-limiting electron transfer. Consequently, near pH 7, the proton-transfer rate k(H) > 10(4) s(-)(1). Applying the two step mechanism to RCs containing native Q(10) and taking into account the change in redox potential, we find reasonable values for the fraction of (Q(B)H)(*) congruent with 0.1% (consistent with a pK(a)(Q(10)) of approximately 4.5) and k(ET)(Q(10)) congruent with 10(6) s(-)(1). These results confirm the PAET mechanism in RCs with RQ and give strong support that this mechanism is active in RCs with Q(10) as well.     

Study on the polarographic catalytic wave of vitamin P in the presence of persulfate and its application

The polarographic catalytic wave of vitamin P in the presence of persulfate was studied by linear potential scan polarography and cyclic voltammetry. Vitamin P yielded a single reduction wave in acidic aqueous solution, which was ascribed to a 2e(-), 2H(+) reduction of the carbonyl group in the C-4 position. Actually, the carbonyl group C=O first underwent a 1e(-), 1H(+) reduction to form a neutral free radical, and the further 1e(-), 1H(+) reduction of the free radical was simultaneous with its following chemical reactions. When S(2)O(2-)(8) was present, the free radical of vitamin P was oxidized by both S(2)O(2-)(8) and its reduction intermediate, the sulfate radical anion SO(*-)(4), to regenerate the original, which resulted in the production of a polarographic catalytic wave of vitamin P. Based on this catalytic wave, a novel method for the determination of vitamin P was proposed. In 0.02 M tartaric acid-sodium tartrate (pH 3.3) buffer containing 5.0 x 10(-3) M K(2)S(2)O(8), the peak potential of the catalytic wave was -1.42 V (vs SCE) and the peak current was rectilinear to the vitamin P concentration in the range of 8.0 x 10(-9)-1.0 x 10(-6) M (r = 0.9994, n = 13). The catalytic wave of 2.0 x 10(-7) M vitamin P enhanced the polarographic current 70 times compared with the corresponding reduction wave. The detection limit was 2.0 x 10(-9) M, and the relative standard deviation at the 2.0 x 10(-7) M level was 0.7% (n = 15). The proposed method was used for the determination of vitamin P content in the pharmaceutical preparation of tablets and the medicinal plant Sophora japonica L. without previous separation.     

Electrochemical,UV-Visible and EPR studies on nitrofurantoin: Nitro radical anion generation and its interaction with glutathione

This paper deals with the reactivity of the nitro radical anion electrochemically generated from nitrofurantoin with glutathione. Cyclic voltammetry (CV) and controlled potential electrolysis were used to generate the nitro radical anion in situ and in bulk solution, respectively and cyclic voltammetry, UV-Visible and EPR spectroscopy were used to characterize the electrochemically formed radical and to study its interaction with GSH.

By cyclic voltammetry on a hanging mercury drop electrode, the formation of the nitro radical anion was possible in mixed media (0.015M aqueous citrate/DMF, 40/60, pH 9) and in aprotic media. A second order decay of the radicals was determined, with a k₂ value of 201 and 111M^-1 s^-1, respectively. Controlled potential electrolysis generated the radical and its detection by cyclic voltammetry, UV-Visible and EPR spectroscopy was possible. When glutathione (GSH) was added to the solution, an unambiguous decay in the signals corresponding to a nitro radical anion were observed and using a spin trapping technique, a thiyl radical was detected.

Electrochemical and spectroscopic data indicated that it is possible to generate the nitro radical anion from nitrofurantoin in solution and that GSH scavenged this reactive species, in contrast with other authors, which previously reported no interaction between them.     

Thermodynamics of reactions catalyzed by PABA synthase

Microcalorimetry and high-performance liquid chromatography (HPLC) have been used to conduct a thermodynamic investigation of reactions catalyzed by PABA synthase, the enzyme located at the first step in the shikimic acid metabolic pathway leading from chorismate to 4-aminobenzoate (PABA). The overall biochemical reaction catalyzed by the PabB and PabC components of PABA synthase is: chorismate(aq)+ammonia(aq)=4-aminobenzoate(aq)+pyruvate(aq)+H(2)O(l). This reaction can be divided into two partial reactions involving the intermediate 4-amino-4-deoxychorismate (ADC): chorismate(aq)+ammonia(aq)=ADC(aq)+H(2)O(l) and ADC(aq)=4-aminobenzoate(aq)+pyruvate(aq). Microcalorimetric measurements were performed on all three of these reactions at a temperature of 298.15 K and pH values in the range 8.72-8.77. Equilibrium measurements were performed on the first partial (ADC synthase) reaction at T=298.15 K and at pH=8.78. The saturation molality of 4-aminobenzoate(cr) in water is (0.00382+/-0.0004) mol kg(-1) at T=298.15 K. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate(2-)(aq)+NH(4)(+)(aq)=ADC(-)(aq)+H(2)O(l), K=(10.8+/-4.2) and Delta(r)H(m)(o)=-(35+/-15) kJ mol(-1). For the reaction: ADC(-)(aq)=4-aminobenzoate(-)(aq)+pyruvate(-)(aq)+H(+)(aq), Delta(r)H(m)(o)=-(139+/-23) kJ mol(-1). For the reaction: chorismate(2-)(aq)+NH(4)(+)(aq)=4-aminobenzoate(-)(aq)+pyruvate(-)(aq)+H(2)O(l)+H(+)(aq), Delta(r)H(m)(o)=-(174+/-6) kJ mol(-1). Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that utilize L-glutamine rather than ammonia and that are pertinent to this branch point of the shikimic acid pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes Delta(r)G'(m)(o) under approximately physiological conditions are given.     

Radical reactions in aqueous disulphide-thiol systems

Absolute rate constants have been measured for the reaction of (CH3SSCH3)+. and sulphur centred radical cations of lipoic acid, lip (SS)+., with various thiols including penicillamine, cysteamine and cysteine. Under pulse radiolysis conditions no reactions was observed between the disulphide radical cations and the neutral thiols, RSH, i.e. kappa less than or equal to 10(7) M-1 s-1. Rate constants in the order of 10(9) M-1 s-1, i.e. close to the diffusion controlled limit, were, however, found for the corresponding reactions with the thiolates, RS-. In systems containing lipoate and cysteamine the lip (S therefore S)+. induced oxidation of CyaS- proceeds via CyaS., (CyaS therefore SCya)- and lip (S S)- as intermediates, i.e. results in a cysteamine mediated conversion of an oxidizing lip (S therefore S)+. radical cation to a reducing lip (S S)- radical anion along the reaction route. In other cases the reaction of disulphide radical cations with thiolate anions was found to proceed via an optically absorbing transient (lambda max approximately 380 nm) which is suggested to be an adduct radical. The mechanism of the (RSSR)+. induced oxidation of thiolate appears to depend on the stability of the 3-electron bonded disulphide radical anion.