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Background: Excess 5-aminolevulinic acid (ALA) and α-aminoacetone (AA) are implicated in ketosis, porphyrinpathies and diabetes. Pathologic manifestations involve O₂⁻, H₂O₂, OH, enoyl radicals (ALA and AA) and their oxidation end products., Methods: To characterize enoyl radicals resulting from reaction of OH radicals with ALA and AA, micromolar OH concentrations were produced by pulse radiolysis of ALA and AA in aqueous solutions., Results: ALA and AA react with OH at k=1.5 × 10⁹ M⁻¹s⁻¹. At pH7.4, the ALA absorbance spectrum has a maximum at 330 nm (ε=750 M⁻¹cm⁻¹). This band appears as a shoulder at pH8.3 where two ALA species are present: (NH3)⁺-CH₂-CO-CH₂-CH₂-COO⁻ and NH₂-CH₂-CO-CH₂-CH₂-COO⁻ (pKa=8.3). At pH8.3, ALA reacts with oxygen (k=1.4 × 10⁸ M⁻¹s⁻¹) but not with O₂⁻. At pH8.3, AA oxidation produces two AA species characterized by an absorbance spectrum with maxima at 330 and 450 nm. ALA and AA are repaired by antioxidants (quercetin (QH), catechin, trolox, ascorbate) which are semi-oxidized (k>10(8)M⁻¹s⁻¹). QH bound to HSA or to apoferritin and ferritin repairs ALA and AA. In O₂-saturated apoferritin solutions, Q, O₂⁻, AA and reaction product(s) react with QH., Conclusions: The optical absorption properties and the time evolution of ALA and AA were established for the first time. These radicals and their reaction products may be neutralized by antioxidants free in solution or bound to proteins., General Significance: Adjuvant antioxidant administration may be of interest in pathologies related to excess ALA or AA production., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Ferritin (Ft) impairment through (•)O2(-), H2O2, and (•)OH production occurs in the cases of ketoses, diabetes mellitus, acute intermittent porphyria and tyrosinemia. In addition to (•)Trp and TyrO(•) radical production, ferrous iron liberation and Ft synthesis stimulation, site-specific oxidation reactions are induced leading to toxic iron accumulation in organs with high Ft content, for example, liver and brain. To elucidate the potential pathways to Ft recovery, repair of oxidative damage to horse spleen apoferritin (apoFt) and Ft by quercetin (QH) or rutin (RH) was studied in the presence and absence of oxygen. (•)Trp and TyrO(•) radicals were produced in pulse radiolysis through apoFt oxidation by (•)Br2(-) radicals. QH and RH bind to apoFt on eight sites with binding constants of ˜80,000 and ˜32,000 M(-1), respectively. In deaerated solutions, a repair of apoFt radicals is observed involving both bound and free flavonoids. This repair occurs by a fast intra- and a slow inter-molecular electron transfer from bound and free flavonoids, respectively. With QH, the rate constants are 10(4) s(-1) and 3.5 × 10(7) M(-1) s(-1) for the intra- and intermolecular repair reactions, respectively. Oxygen does not interfere with repair of apoFt or Ft by bound QH but inhibits 90% of Ft repair by RH. These results taken together indicate that flavonoid antioxidants may help alleviate Ft impairment in diseases involving an oxidative stress.

There are numerous studies concerning the effect of UVB light on skin cells but fewer on other skin components such as the interstitial fluid. This review highlights high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as important targets of UVB in interstitial fluid. Tryptophan residues are the sole apolipoprotein residues absorbing solar UVB. The UVB-induced one-electron oxidation of Trp produces (•)Trp and (•)O2 (-) radicals which trigger lipid peroxidation. Immunoblots from buffered solutions or suction blister fluid reveal that propagation of photooxidative damage to other residues such as Tyr or disulfide bonds produces intra- and intermolecular bonds in apolipoproteins A-I, A-II, and B100. Partial repair of phenoxyl tyrosyl radicals (TyrO(•)) by α -tocopherol is observed with LDL and HDL on millisecond or second time scales, whereas limited repair of α -tocopherol by carotenoids occurs in only HDL. More effective repair of Tyr and α -tocopherol is observed with the flavonoid, quercetin, bound to serum albumin, but quercetin is less potent than new synthetic polyphenols in inhibiting LDL lipid peroxidation or restoring α -tocopherol. The systemic consequences of HDL and LDL oxidation and the activation and/or inhibition of signalling pathways by oxidized LDL and their ability to enhance transcription factor DNA binding activity are also reviewed.

A slow, long range electron transfer (SLRET) in human serum albumin (HSA) is observed from an intact tyrosine (Tyr) residue to the neutral tryptophan (Trp) radical (Trp·) generated in pulse radiolysis. This radical is formed, at neutral pH, through oxidation with Br (2) (·-) radical anions of the single Trp 214 present. The SLRET rate constant of ~0.2 s(-1) determined is independent of HSA concentration and radiation dose, consistent with an intra-molecular process. This is the slowest rate constant so far reported for an intra-molecular LRET. In sharp contrast with the LRET reported for other proteins, the SLRET observed here is insensitive to oxygen, suggesting that the oxidized Trp is inaccessible to-or do not react with radiolytically generated O (2) (·-) . In N(2)O-saturated solutions, the SLRET is inhibited by Cu(2+) ions bound to the His 3 residue of the N-terminal group of HSA but it is partially restored in O(2)-saturated solutions.

The flavonoid quercetin is known to reduce the α-tocopheroxyl radical (˙TocO) and reconstitute α-tocopherol (TocOH). Structurally related polyphenolic compounds, hydroxy-2,3-diarylxanthones (XH), exhibit antioxidant activity which exceeds that of quercetin in biological systems. In the present study repair of ˙TocO by a series of these XH has been evaluated using pulse radiolysis. It has been shown that, among the studied XH, only 2,3-bis(3,4-dihydroxyphenyl)-9H-xanthen-9-one (XH9) reduces ˙TocO, though repair depends strongly on the micro-environment. In cationic cetyltrimethylammonium bromide (CTAB) micelles, 30% of ˙TocO radicals are repaired at a rate constant of ~7.4 × 10(6) M(-1) s(-1) by XH9 compared to 1.7 × 10(7) M(-1) s(-1) by ascorbate. Water-soluble Trolox (TrOH) radicals (˙TrO) are restored by XH9 in CTAB (rate constant ~3 × 10(4) M(-1) s(-1)) but not in neutral TX100 micelles where only 15% of ˙TocO are repaired (rate constant ~4.5 × 10(5) M(-1) s(-1)). In basic aqueous solutions ˙TrO is readily reduced by deprotonated XH9 species leading to ionized XH9 radical species (radical pK(a) ~10). An equilibrium is observed (K = 130) yielding an estimate of 130 mV for the reduction potential of the [˙X9,H(+)/XH9] couple at pH 11, lower than the 250 mV for the [˙TrO,H(+)/TrOH] couple. A comparable value (100 mV) has been determined by cyclic voltammetry measurements.

A structure-activity relationship has been established for eight hydroxy-2,3-diarylxanthones (XH) bearing hydroxy groups on the two aryl rings. One-electron oxidation by superoxide radical-anions (˙O(2)(-)) and ˙Trp radicals as well as reaction with ˙CCl(3)O(2) and ˙CHCl(2)O(2) radicals demonstrates that two OH groups are required for efficient antioxidant reactivity in cetyltrimethylammonium bromide micelles. Hydroxy groups at the meta and para positions on either of the two phenyl rings confer enhanced reactivity, but XH bearing an OH at the para position of either phenyl ring is unreactive. While oxidation is favoured by OH in both meta and para positions of 2-aryl xanthone substituents, addition of a third and/or fourth OH enhances electron-donating capacity. In Cu(2+)-induced lipid peroxidation of human LDL, the lag period preceding the commencement of lipid peroxidation in the presence of XH bearing OH at meta and para positions on the 3-phenyl ring is extended to twice that observed with a comparable concentration of quercetin, a reference antioxidant. These antioxidants are also superior to quercetin in protecting human skin keratinocytes against tert-butylhydroperoxide-induced oxidative stress. While XH antioxidant activity in model biological systems is consistent with the structure-activity relationship, their response is also modulated by the localization of XH and by structural factors.

Conjugates of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridinium-4-yl)porphyrin (P-H) are promising photoactive agents for medical applications. As their ultimate efficacy will depend on the behavior of initial excited states, photophysical parameters have been determined with conventional steady-state absorption and fluorescence as well as time-resolved femto- and nanosecond spectroscopies. The fluorescence quantum yield of P-H and P-H conjugated to uncharged groups increases from approximately 0.03 in pH 7 buffer to approximately 0.05 in Triton X100 micelles (TX100) and in ethanol and to 0.12 in sodium dodecyl sulfate (SDS) micelles. Corresponding (1)S(1) lifetimes are approximately 5-10 ns. In buffer, an equilibrium between P-H monomers and small-size aggregates is observed. Conjugation with poly-S-lysine (P-(Lys)(n)) results in fluorescence quenching in all solvents. Structural reorganization of conjugates bearing a Di-O-isopropylidene-alpha-d-galactopyranosyl or a alpha/beta-d-galactopyranosyl group occurs in ethanol (k approximately 0.15 ps(-1)) after (1)S(1) state solvation (approximately 700 fs). Relaxation of bulky P-(Lys)(n) polypeptide chains takes place on a longer time scale in all solvents (k

It is shown that the relationship between the alkyl chain length of 3-alkyl-3',4',5,7 tetrahydroxyflavones (FnH) bearing alkyl chains of n=1, 4, 6, 10 carbons and their capacity to counter oxidative damage varies markedly with the nature of the biological system. In Cu(2+)-induced lipid peroxidation of low-density lipoprotein (LDL), the less hydrophobic short-chain F1H and F4H are probably located in the outer layer of LDL and parallel the reference flavonoid antioxidant, quercetin (Q) as effective inhibitors of lipid peroxidation. A marked inhibition of haemolysis induced in red blood cells (RBC) suspensions by the membrane-permeant oxidant, tert-butylhydroperoxide (t-BuOOH), is observed with F4H and F6H present at concentration in the micromolar range. However, F10H the most hydrophobic FnH is even more effective than Q against both haemolysis and lipid peroxidation as measured by malondialdehyde (MDA) equivalents. In oxidation of RBC by H(2)O(2,) at least 50 times more F6H and F10H than by t-BuOOH are required to only partly inhibit haemolysis and MDA production. The F1H, F4H and Q are found rather inactive under these conditions. At concentrations in the micromolar range, a marked protection against the cytotoxic effects of the t-BuOOH-induced oxidative stress in human skin NCTC 2544 keratinocytes is also exhibited by the four FnH antioxidants and is comparable to that of Q. Thus, the four FnH species under study may be considered as potent antioxidants which manifest complementary anti-oxidative actions in biological systems of markedly different complexity.

Objective: Our aim was to determine the safety of BAL in young children <6 years with CF., Methods: As part of a multi-center study of BAL-directed therapy, children with CF < 6 years had one or more BALs between September 1999 and December 2005. Adverse events were recorded intraoperatively and for 24 hr thereafter. Clinical characteristics before BAL, findings at bronchoscopy and BAL results were assessed as risk factors for adverse events., Results: 333 BALs were conducted in 107 (56 males) children, median age 23.5 (range 1.6-67.5) months, including 170 (51%) for pulmonary exacerbation. 29 BALs (8.7%) were followed by fever >or=38.5 degrees C and 10 (3%) had clinically significant episodes (five intraoperative hemoglobin desaturations to <90% requiring intervention, one tachyarrhythmia, two needing post-operative supplemental oxygen, one hospitalization for stridor). Two contaminated bronchoscopes were detected. 180 minor adverse events were recorded in 174 (52%) BAL procedures (137 altered cough, 41 fever <38.5 degrees C). Low percentage BAL return (P = 0.002) and focal bronchitis (P = 0.02) were associated with clinically significant deterioration. Multivariable analysis identified Streptococcus pneumoniae (OR 22.3; 95% confidence interval (CI); 6.9,72), Pseudomonas aeruginosa (OR 2.4; 95% CI 1.0, 5.8), respiratory signs (OR 5.0; 95% CI 1.7, 14.6) and focal bronchitis (OR 5.9; 95% CI 1.2, 29.8) as independent risk factors for post-bronchoscopy fever >or=38.5 degrees C., Conclusions: Adverse events are common with BAL in young CF children, but are usually transient and well tolerated. Parents should be counseled that signs of a pre-existing lower respiratory infection are associated with increased risk of post-BAL fever., ((c) 2008 Wiley-Liss, Inc.)

In micellar solutions, one-electron reduction of *O2(-) radical-anions by 3-alkylpolyhydroxyflavones (FnH) with alkyl chains of n = 1, 4, 6, 10 carbons produces phenoxyl radicals ( (*Fn) identical to those obtained by one-electron oxidation by *Br2(-) radical-anions or by repair of tryptophan radicals. In cetyltrimethylammonium bromide (CTAB), F1H localizes in the Stern layer, and alkyl chains of other FnH solubilize in the hydrophobic interior, interacting with cetyl tails. This interaction produces more compact micelles with lower intramicellar fluidity, as suggested by the increase in the pseudo-first-order rate constant of *Fn formation ( k 1) from approximately 390 s (-1) for n = 1 to 610 s (-1) for n = 10, leading to an intramicellar bimolecular rate constant of 1 x 10 (5) M (-1) s (-1). Additionally, *F1 and *F4 decay by intermicellar bimolecular reaction (2 k = 20 and 2 x 10 (5) M (-1) s (-1), respectively) whereas other *Fn radicals are stable over seconds due to increased localization with regards to the Stern layer. In contrast, the thick uncharged hydrophilic palisade layer and the compact hydrophobic core of Triton X100 micelles are responsible for a much higher microviscosity resulting in a decrease in k 1 from approximately 15.6 s (-1) for n = 1 to 9.6 s (-1) for n = 10.

In the minor fraction of HDL3 containing alpha-tocopherol (alphaTocOH), selective one-electron oxidation of Trp and Tyr residues of apolipoproteins A-I and A-II by *Br2- radical-anions produces the corresponding semioxidized species, TyrO* and *Trp. Repair of TyrO* by endogenous alphaTocOH generates the alpha-tocopheroxyl radical (alphaTocO*). Fast spectroscopic studies show that two populations representing 80% of alphaTocO* initially formed are repaired over several seconds with rate constants of 3.0 x 10(6) and 1.5 x 10(5) M-1 s-1 by quercetin bound to human serum albumin (HSA) at physiologically relevant concentration. Formation of HSA-bound quercetin radicals (*Qb) is observed. In the major fraction of HDL3 particles lacking alphaTocOH, TyrO* and *Trp are repaired by free and HSA-bound quercetin. In LDL particles which all contain alphaTocOH, alphaTocO* radicals are formed in the millisecond time scale by repair of TyrO* radicals produced in apolipoprotein B. Then, 75% of initial alphaTocO* are repaired over seconds by HSA-bound quercetin (rate constant: 2.0 x 10(6) M-1 s-1). HSA-bound quercetin can also repair *Trp radicals. In O2-saturated solutions, the fraction of alphaTocO* radicals (more than 50%) not repaired by superoxide radical-anions can be repaired by HSA-bound quercetin with formation of *Qb but to a much lesser extent in LDL than in HDL.

It has been recently shown that the inhibition of apolipoprotein A-I (apoAI) reverse cholesterol transport activity during oxidation of HDL by myeloperoxidase may involve myeloperoxidase electron transfer pathways other than those leading to tyrosine chlorination. To better understand how such mechanisms might be initiated, the role of semioxidized Tyr and Trp residues in loss of apoAI and apolipoprotein A-II (apoAII) integrity has been assessed using selective Trp and Tyr one-electron oxidation by *Br2(-) radical-anions in HDL3 as well as in unbound apoAI and apoAII. Behavior of these radicals in apolipoprotein B of LDL has also been assessed. Formation of semioxidized Tyr in HDL3 is followed by partial repair during several milliseconds via reaction with endogenous alpha-tocopherol to form the alpha-tocopheroxyl radical. Subsequently, 2% of alpha-tocopheroxyl radical is repaired by HDL3 carotenoids. With LDL, a faster repair of semioxidized Tyr by alpha-tocopherol is observed, but carotenoid repair of alpha-tocopheroxyl radical is not. Only a small fraction of HDL3 particles contains alpha-tocopherol and carotenoids, which explains limited repair of semioxidized Tyr by alpha-tocopherol. All LDL particles normally contain multiple alpha-tocopherol and carotenoid molecules, and the lack of repair of alpha-tocopheroxyl radical by carotenoids probably results from hindered mobility of carotenoids in the lipid core. Western blots of gamma-irradiated HDL3 comparable to those reported for apoAI myeloperoxidase oxidation show that the incomplete repair of semioxidized Tyr and Trp induces apoAI and apoAII permanent damage including formation of a heterodimer of one apoAI with a monomeric apoAII at about 36 kDa.

Four polyhydroxylated 2-styrylchromones, structurally related to flavones and cinnamic acid, have been studied. An SC derivative with OH groups only at positions 3' and 4' on the styryl moiety and another SC bearing an additional OH group at position 5 on the benzopyrone ring were more potent inhibitors of the Cu2+-induced peroxidation of LDL than the flavonoid quercetin. Fluorescence and absorption spectroscopies suggested that one LDL particle may bind 40 SC molecules. A pulse radiolysis study in pH 7 buffered micellar solutions of neutral TX100 and positively charged CTAB demonstrated that one-electron oxidation by *Br2-, *O2- and tryptophan radicals (8Trp) depends strongly on the micellar microenvironment. All SCs were readily oxidized by *O2- in CTAB micelles (rate constants: 6-18 x 10(6) M(-1) s(-1)). In TX100 micelles only the SC derivative with OH groups in position 3' and 4' reacted with *O2- (rate constant: 1.1 x 10(6) M(-1)s(-1)). In CTAB, electron transfer to *Trp radicals was observed for all SCs with rate constants > or =3.2 x 10(7) M(-1) s(-1). In TX100 micelles, this reaction occurred solely with the derivative bearing OH groups only at positions 3' and 4'.

The 1:1 complex of copper (II) and human serum albumin (HSA) slowly reacts with radiolytically generated *O2- radical-anion at a rate constant of 6.1 x 10(6) M(-1) s(-1). Absorbance and fluorescence spectroscopies demonstrate that addition of an equimolar portion of quercetin (QH2) to the solution of the copper (II)-HSA complex induces a relocalization of the copper resulting in a ternary copper (II)-QH2-HSA complex. This form of quercetin slowly oxidizes in air-saturated solutions. A 10-fold excess urate, a plasma antioxidant, cannot displace copper (II) bound to HSA. In N2O-saturated solutions the ternary complex form of QH2 can repair the urate radical with a rate constant of 2.7 x 10(6) M(-1) s(-1) by an electron transfer reaction similar to that observed in the absence of copper (II). In O2-saturated solutions and in the absence of copper, HSA-bound QH2 fails to repair the urate radical because of the fast competitive reaction of *O2- with urate radicals. However, addition of equimolar copper (II) restores the electron transfer from QH2 to the urate radical. These contrasting results are tentatively explained either by an enhanced reactivity of copper (II) with *O2- in the ternary complex or by direct production of quercetin radicals via a copper-catalyzed reduction of the *O2- radicals by QH2.

Selective oxidative damage to apolipoprotein B in LDL can be effected radiolytically by (*)Br(2)(-) radicals. Twenty-seven Trp residues constitute major primary sites of oxidation, but two-thirds of oxidized Trps ((*)Trp) that are formed are repaired by intramolecular electron transfer from Tyr residues with formation of phenoxyl radicals (TyrO(*)). Analysis of (*)Trp and TyrO(*) transient absorbance changes suggests that other apolipoprotein B residues, probably Cys, are oxidized. LDL-bound quercetin can efficiently repair this damage. Absorption studies show that a LDL particle has the capacity to bind approximately 10 quercetin molecules through interaction with apolipoprotein B. The repair occurs by intramolecular electron transfer characterized by a rate constant of 2 x 10(3) s(-)(1). In contrast, rutin, a related flavonoid which does not bind to LDL, cannot repair oxidized apolipoprotein B. Urate is a hydrophilic plasma antioxidant which displays synergistic antioxidant properties with flavonoids. Urate radicals produced by (*)Br(2)(-) can also be repaired by LDL-bound quercetin. This repair occurs with a reaction rate constant of 6.8 x 10(7) M(-)(1) s(-)(1). Comparison with previous studies conducted with human serum albumin-bound quercetin suggests that quercetin analogues tailored to be carried preferentially by lipoproteins might be more powerful plasma antioxidants than natural quercetin carried by serum albumin.

Neutral tryptophan (*Trp) and tyrosine (TyrO(*)) radicals are repaired by certain flavonoids in buffer, in micelles and in human serum albumin (HSA) with corresponding formation of semioxidized flavonoid radicals. In deaerated buffer, *Trp but not TyrO(*) radicals react with catechin. In micelles, quercetin and rutin repair both *Trp and TyrO(*) radicals. In addition to amino acid reactivity, microenvironmental factors and nature of the flavonoids govern this repair. Electron transfer efficiencies from quercetin to negatively charged *Trp radicals are 100% in the micellar pseudophases of positively charged cetyltrimethylammonium bromide, (CTAB), and neutral Triton X100 (TX100), but 55% in negatively charged sodium dodecyl sulfate (SDS). In oxygen-saturated CTAB micelles, quercetin also reacts with the superoxide radical anion. When bound to domain IIA of HSA, quercetin repairs, by intra- or intermolecular encounter, less than 20% of oxidative damage to HSA. Quercetin can also repair freely circulating oxidized molecules with repair efficiencies falling to 7% for oxidized Trp, Tyr and alpha-MSH and to less than 2% for urate radical. This limited effectiveness is attributed both to the inaccessibility of bound quercetin and rutin toward radicals of circulating molecules and to the diffusion-controlled recombination of these radicals.

The kinetics of several processes involving the potential antioxidant role of urate in physiological systems have been investigated by pulse radiolysis. While the monoanionic urate radical, .UH-, can be produced directly by oxidation with .Br2- or .OH, it can also be generated by oxidation with the neutral tryptophan radical, .Trp, with a rate constant of 2 x 10(7) M-1s-1. This radical, .UH-, reacts with .O2- with a rate constant of 8 x 10(8) M-1s-1. Also, .UH- is reduced by flavonoids, quercetin and rutin in CTAB micelles at rate constants of 6 x 10(6) M-1s-1 and 1 x 10(6) M-1s-1, respectively. These results can be of value by providing reference data useful in further investigation of the antioxidant character of urate in more complex biological systems.

The 355 nm laser flash photolysis of argon-saturated pH 8 phosphate buffer solutions of the fluoroquinolone antibiotic flumequine produces a transient triplet state with a maximum absorbance at 575 nm where the molar absorptivity is 14,000 M(-1) cm(-1). The quantum yield of triplet formation is 0.9. The transient triplet state is quenched by various Type-1 photodynamic substrates such as tryptophan (TrpH), tyrosine, N-acetylcysteine and 2-deoxyguanosine leading to the formation of the semireduced flumequine species. This semireduced form has been readily identified by pulse radiolysis of argon-saturated pH 8 buffered aqueous solutions by reaction of the hydrated electrons and the CO2*- radicals with flumequine. The absorption maximum of the transient semireduced species is found at 570 nm with a molar absorptivity of 2,500 M(-1) cm(-1). In argon-saturated buffered solutions, the semireduced flumequine species formed by the reaction of the flumequine triplet with TrpH stoichiometrically reduces ferricytochrome C (Cyt Fe3+) under steady state irradiation with ultraviolet-A light. In the presence of oxygen, O2*- is formed but the photoreduction of Cyt Fe3+ by O2*- competes with an oxidizing pathway which involves photo-oxidation products of TrpH.

The kinetics of O2*- 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 Br2*- oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 x 10(8) M(-1) s(-1); but drops to 8 x 10(7) M(-1) s(-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 x 10(6) M(-1) s(-1)) at/or below pH 6.2 then drops to 1.3-1.6 x 10(6) M(-1) s(-1) at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semi-oxidized Trp* residues upon Br2*- reaction. The kinetics of Trp*(Lyz) with ascorbate, a reducing species rather larger than O2*- 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) + O2*- 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 O2*-. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O2*- reactivity. To our knowledge, this is the first report on the kinetics of a protein-O2*- reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.

The reaction of the superoxide radical anion (O2-*), with the semi-oxidized tryptophan neutral radical (Trp*) generated from tryptophan (Trp) by pulse radiolysis has been observed in a variety of functionalized Trp derivatives including peptides. It is found that the reaction proceeds 4-5 times faster in positively charged peptides, such as Lys-Trp-Lys, Lys-Gly-Trp-Lys and Lys-Gly-Trp-Lys-O-tert-butyl, than in solutions of the negatively charged N-acetyl tryptophan (NAT). However, the reactivity of O2-* with the Trp* radical is totally inhibited upon binding of these peptides to micelles of negatively charged SDS and is reduced upon binding to native DNA. By contrast, no change in reactivity is observed in a medium containing CTAB, where the peptides cannot bind to the positively charged micelles. On the other hand, the reactivity of the Trp* radical formed from NAT with O2-* is reduced to half that of the free Trp* in buffer but is markedly increased in CTAB micelles. The models studied here incorporate elements of the complex environment in which Trp* and O2-* may be concomitantly formed in biological system and demonstrate the magnitude of the influence such elements may have on the kinetics of reactions involving these two species.

Using the fluorescence of diphenylhexatriene (DPH), lipid peroxidation in monomolecular films of phospholipids has been monitored dynamically to elucidate the efficacy of such a probe and to elucidate the effects of molecular organization on such peroxidation processes. Behavior in well-controlled model systems may be used to obtain insight into oxidative processes in complex biological systems. Mixed monolayers of hexadecanoyl-DPH-phosphatidylcholine (HDPH-PC) and diarachidonoyl-PC (DAA-PC) 1/10 (mol/mol) were prepared on a Langmuir trough. With pure water as subphase, and under an atmosphere of N2/O2 (3/1, v/v), DPH fluorescence remained stable over several hours, even under continuous illumination. However, over copper sulfate solution, rapid decay in the fluorescence intensity was observed and correlated with both the copper ion concentration and film pressure. No change in fluorescence was observed in absence of subphase copper ions or under an N2 atmosphere. Substitution of dioleoyl phosphatidylcholine for DAA-PC in systems above gave no decrease in fluorescence intensity. This lipid alone is not susceptible to free radical chain reaction. Absorption spectra from HDPH-PC/DAA-PC monolayers confirm that DPH is actually destroyed during film oxidation. We thus propose this system as a new experimental model for studies on lipid peroxidation in organized systems.

From pulse radiolysis measurements in oxygenated aqueous solution, the semioxidized tryptophan radical (Trp.- formed by the one-electron oxidation of Trp by Br2- radical--has been shown to oxidize the superoxide radical anion with a rate constant of k = 2 x 10(9) M-1 s-1. Proof of this reaction is found in addition of superoxide dismutase (SOD) to the system, which totally eliminates the contribution of the Trp. + O2- mechanism to Trp. decay. Little, if any, reaction of molecular oxygen with Trp. may be observed on the time scale of the pulse radiolysis experiment.

Kinetics of photoinduced electron transfer from a lipid functionalized pyrene, 1-(10-(6(8)-octadecylpyrenyl) decanoyl)-2-hexanoyl-sn-glycero-3-phosphorycholine (OPyPC), to a two component viologen acceptor system have been measured by laser flash photolysis. N,N'-tetramethylene-2,2' -bipyridinium ion (DQ2+) and N,N'-dipropyl-4,4'-bipyridinium sulfonate (PVS), have been utilized as the primary and secondary acceptors. It has been shown that utilization of a lipid with a net negatively charged phosphatidylglycerol headgroup provides a driving force for localizing high concentrations of primary acceptor (DQ2+) in the region of donor. Subsequently, the charged interface can act to maintain long-term separation between the oxidized pyrene donor (OPyPC+) and the reduced secondary acceptor, PVS-. When a dioleoyl lipid is used, reaction of (OPypc+) with the double bond competes significantly with back reaction. However, substitution of diphytylphosphatidylglycerol for the dioleoyl analog results in a cation lifetime of about 0.5 ms and a continued very long-lived reduced species (approximately 4 h). Quantum yields of approximately 0.15 may be obtained in this system.

A new class of 2,5 dimethylpyrrole functionalized amino acid and peptide antioxidants has been synthesized. Pulse radiolysis studies have been carried out on these compounds to determine their reactivity in one-electron oxidation reactions with Br2- and (SCN)2-, as well as their reactivity toward the water radicals .OH and eaq-. When compared to similar reactions with parent amino acids and peptides, both rate constants and transient spectra indicate considerable interaction between the electronic states of the pyrrole moiety and amino acid or peptide. Where comparison data is available, radical reactions are significantly more rapid with these derivatives than with the parent compounds. Exceptions occur in some cases of .OH attack where steric hindrance may be evoked to explain a lower rate constant in the derivative compounds. Electron attachment rates are also found to be enhanced by the presence of the pyrrole ring.

Benzophenone-photosensitized peroxidation in linoleic acid micelles (Type I reaction) has been investigated and quantum yields of both sensitizer photolysis and lipid peroxidation have been obtained. Benzophenone as well as its derivative, benzophenone-4'-heptyl-4-pentanoic acid (BHPA) have been utilized as sensitizers in this study. A quantum yield of 1.2 for hydroperoxide formation, compared with a benzophenone disappearance quantum yield of 0.41 (in the same linoleate micellar solution) was obtained; yields of 1.3 and 0.6 were found for comparable systems in which BHPA was the sensitizer. Although such data are consistent with the peroxidation mechanism of chain reaction, it is necessary to know the yields of initiating species to arrive at chain reaction lengths in these systems. Such yields have been determined by flash photolysis. The dependencies of sensitizer photolysis and hydroperoxide yields on linoleate concentration have also been determined in order to elucidate the effects of micelle formation or molecular organization on the peroxidative process. Additionally, for comparison in a saturated surfactant (lipid) system hydrogen abstraction was investigated by photolysis techniques in sodium dodecyl sulfate alone using these two sensitizers.

Electron spin resonance and flash photolysis studies have been combined to determine, amid conflicting reports from radiolysis studies, the spectrum for the phenoxyl radical of alpha-tocopherol. Triplet state ketones were used to abstract the alpha-tocopherol phenolic hydrogen in order to obtain both the transient ESR spectrum and optical spectrum. Analysis of the ESR data yields g = 2.00469, a(H, 5-CCH3) = 6.04G, a(H,7-CCH3) = 4.51G, a(H, CH2) = 1.38G, a(H', CH2) = 1.49G, and a(H,8-CCH3) = 0.89G. The g factor shows large spin population on oxygen in this radical and demonstrates conclusively involvement of the phenoxyl radical. Both spectra were in agreement with those produced by radiolysis of alpha-tocopherol in N2 saturated EtOH. While the spectral characteristics of the phenoxyl radical now appear to be clarified, uncertainties remain concerning optical spectra for radiolysis of alpha-tocopherol in air- and oxygen-containing systems.

Laser photolysis techniques have been used to characterize the reactivity of triplet state lipoidal benzophenone derivatives toward fatty acids and glycerides in benzene solution. The reactivities of benzophenone-4-heptyl-4'-pentanoic acid (BHPA) toward fatty acid compounds having different configurations of olefinic bonds have been determined. The rates of hydrogen abstraction are found to be lower when compared with similar measurements using benzophenone alone. However, the contribution of physical quenching of the triplet derivative by double bonds also appears to be slightly lower than that found with benzophenone itself. The hydrogen abstraction efficiencies of three other benzophenone derivatives toward linoleic acid in benzene have also been measured. For benzophenone incorporated into a fatty acid molecule, there is a limited relationship between structure and photoreactivity. Finally, these sensitizers have been incorporated into mixed SDS/linoleate micelles to determine the effects of molecular organization on photochemical behavior of the sensitizer and lipid.

Both eaq- and .OH have been found to react with 8-methoxypsoralen (8-MOP), giving rate-constants of 1.1 X 10(10) M-1 s-1. Transient spectra of products from the reactions of eaq-, .OH with 8-MOP have been characterized. Rate-constants for the oxidation by 8-MOP of reduced and oxidized DNA bases have also been measured and found to lie in the range 3-6 X 10(9) M-1 s-1. Oxidation of reduced bases occurs by electron transfer with 100 per cent efficiency in all cases. However, for oxidized bases, only approximately 25 per cent of the intermediate yield produced by OH attack undergoes electron transfer; the balance of the oxidized base appears to form adducts with 8-MOP.

Ozone concentrations of 0.03 ppm are shown to initiate physical disruption of both oleic and linoleic acid spread monolayers in a few seconds. Approximately equal yields of water soluble products in nitrogen- and oxygen-containing environments as well as in the two acid systems suggest that the dominant reaction pathway involves direct attack on fatty acid double bonds and not oxidative chain reactions. A possible explanation for the observed loss of monolayer material is provided by the Criegee mechanism for ozonide rearrangement following ozone attachment to double bonds. In this scheme polar intermediates are generated which could pass into the aqueous subphase before recombination can occur. alpha-Tocopherol exhibits a protective effect only when present at a high fraction in the fatty acid monolayer (6 mole%). These systems provide an efficient elementary model for studying the physical effects of ozone on lipid membrane structure.

To gain an understanding of the mechanism by which the hydroxyl free radical can arise in superoxide generating systems and learn how different chelaters of iron can inhibit this reaction, a pulse radiolysis kinetic study of the reaction of O;- with Fe(III)EDTA, Fe(III)HEDTA and Fe(III)DETAPAC (or DTPA)was undertaken. Superoxide reacts readily with Fe(III)EDTA and Fe(III)HEDTA with a pH-dependent second-order rate constant having values of 1.9 x lo6 M-l. s-’ and 7.6 x 16 M-‘. s-’ at pH 7,respectively. However, the rate constant for the reaction of O;- with Fe(III)DETAPAC was found to be much slower, the upper limit for the rate constant being 104 M-l. s-l. These results in conjunction with spin-trapping experiments with Fe(II)EDTA, Fe(II)HEDTA, Fe(II)DETAPAC and Hz02 suggests that DETAPAC inhibits the formation of 'OH by slowing the reduction of Fe(II1) to Fe(I1) and not by inhibiting the Fenton reaction.

Pulse radiolytic studies of alpha-tocopherol (alpha TH) oxidation-reduction processes were carried out with low doses (5 Gy) of high-energy electrons in O2-, N2-, and air-saturated ethanolic solutions. Depending on the concentration of oxygen in solution, two different radicals, A . and B ., were observed. The first, A ., was obtained under N2 and results from alpha TH reaction with solvated electron (k alpha TH + e-solv = 3.4 X 10(8) mol-1 liter s-1) and with H3C-CH-OH, (R.) (k alpha TH + R. = 5 X 10(5) mol-1 liter s-1). B., observed under O2, is produced by alpha TH reaction with RO2 . peroxyl radicals (k alpha TH + RO2 . = 9.5 X 10(4) mol-1 liter s-1).

The toxicity of halogenated alkanes originates from their metabolism by cytochrome P-450 which leads to the formation of reactive intermediates. In particular, peroxyl radicals derived from the halogenated compounds are believed to induce peroxidative chain degradation of lipids. To examine this hypothesis, radical reactions in a system involving FeIII-deuteroporphyrin as a model of cytochrome P-450, fatty acids or cholesterol, and carbon tetrachloride or the anesthetic agent halothane are studied by means of pulse radiolysis. It is shown that haloperoxyl radicals react with the fatty acids in competition with their reaction with the ferriporphyrin. Moreover, the secondary fatty acid peroxyl radicals also react efficiently with the porphyrin. A model for halogenated alkane toxicity is discussed in terms of these new findings. The importance of local oxygen concentration and structural arrangement of fatty acids around cytochrome P-450 are emphasized.

Laser photolysis techniques have been used to measure the reactivity of benzophenone triplet (3BZP) toward various fatty acids and two glycerides in benzene solution. Eight compounds varying both in number and in the configuration of olefinic bonds have been examined. It has been found that the rate constant for hydrogen abstraction from these compounds by 3BZP may be related to the number of secondary, allylic and doubly allylic hydrogens in each molecule by the equation: kH = [0.023[H sec] + 0.112[H allylic] + 1.78[H doubly allylic]] x 10(7) M-1s-1