Tetramethyl-p-phenylenediamine Research Articles

In situ evidence of an alternative oxidase and an uncoupling protein in the respiratory chain of Aspergillus fumigatus

Aspergillus fumigatus is an unusual pathogen in immunocompetent individuals; its incidence has increased in the last decades in patients immunocompromised, like those with chronic granulomatosis disease and AIDS. The aim of this study was to identify differences between the respiratory chain of host and the fungus planning to use the later as a pharmacological target. We evaluated respiration, membrane potential and oxidative phosphorylation of mitochondria of the spheroplasts of A. fumigatus in situ, after permeabilization with digitonin. Firstly, a functional respiratory chain (complex I–V) was demonstrated: adenosine 5′-diphosphate (ADP) induced an oligomycin-sensitive transition from resting to phophorylating respiration in the presence of the oxidizable substrates malate, glutamate, α-ketoglutarate, pyruvate, dihydroorotate, succinate, N,N,N′ ,N′-tetramethyl- p-phenylenediamine (TMPD) and exogenous NADH. In addition, the ability of the fungus to oxidize exogenous NADH, as well as the insensitivity of its respiration to rotenone, in association with the sensitivity to flavone, indicate the presence of an alternative NADH–ubiquinone oxidoreductase; the partial sensitivity of respiration to antimycin A and cyanide, in association with the sensitivity to benzohydroxamic acid, indicates the presence of an alternative oxidase. The fatty acid-uncoupled respiration was partly reversed by bovine serum albumin (BSA) and guanosine 5′-triphosphate (GTP) and was insensitive to either carboxyatractyloside or ADP. These results, together with evidences obtained using antibodies raised against uncoupling protein (UCP) from potato, indicate in addition, the presence of an uncoupling protein in the respiratory chain of A. fumigatus.

Pulse radiolysis study of radical cations of polysilanes

Molar extinction coefficients of radical cations are quantitatively discussed for a variety of substituted polysilanes by nano-second pulse radiolysis. Polysilane solution in benzene under saturated oxygen exhibits a strong absorption band ascribed to polysilane radical cation in the transient spectra. The transient species react with N, N, N ′, N ′-tetramethyl- p-phenylene-diamine (TMPD) to produce TMPD radical cations. Using the molar extinction coefficient of TMPD radical cation, the molar extinction coefficients for the radical cations of polysilanes are found to increase in the range 3.8 × 10 4–15.0 × 10 4 M −1 cm −1 with an increase in the segment length of the polymers. This is the first report on the extinction coefficient and oscillator strength of polysilane radical cations.

Ionization and protonation of aromatic diamines by sorption in zeolites

In situ diffuse reflectance UV visible, Raman scattering and EPR spectroscopies were used to monitor spontaneous ionization of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) by direct exposure to dehydrated M n ZSM-5 zeolite (M=Na +, H +; n=0,3,6). The TMPD •+ radical cation is found to be generated in low yield in purely siliceous silicalite-1 whereas TMPD •+ is generated in high yield in aluminated Na n ZSM-5 and H n ZSM-5. The ejected electron is also characterized though electronic absorption spectra. The charge separation was found to be persistent over several months. The tight fit between the shape of TMPD and the pore size of straight channels of zeolites is considered to be the main factor responsible for the stabilization of the TMPD radical ion by preventing rapid electron back transfer. Within Na n ZSM-5, the amounts of TMPD •+ and trapped electron were found to decrease over long time and to recombine to molecular TMPD. In contrast, in acidic H n ZSM-5 zeolite the charge recombination generates diprotonated TMPDH 2 2+ occluded species. Furthermore, within H n ZSM-5, protonation appears competitive to the ionization efficiency of zeolite.

Characterisation and application of a novel cell for mechanistic electrochemistry at elevated temperatures

A novel high temperature cell capable of measuring electrochemical signals up to temperatures of 115 °C is reported. Characterisation of the cell has been undertaken by examining three differing systems. First the voltammetric response of a range of simple redox systems in both aqueous and non-aqueous media was recorded in order to deduce the activation energies for diffusion of these molecules. Next the steady state linear sweep voltammetric response of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) in dimethyl sulfoxide (DMSO) is assessed as a means of studying an inferred structural change of the solvent at elevated temperatures. A change in the activation energy for diffusion from 13 kJ mol−1 to 27 kJ mol−1 was observed as the temperature was raised. Finally, the electrochemically catalysed reaction of the radical cation TMPD+˙ with ascorbic acid is studied from 25 °C to 107 °C; the corresponding kinetic parameters are deduced.

The aerobic electron transport system of Eikenella corrodens.

The respiratory system of the fastidious beta-proteobacterium Eikenella corrodens grown with limited oxygen was studied. Membranes showed the highest oxidase activity with ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) or succinate and the lowest activity with NADH and formate. The presence of a bc1-type complex was suggested by the inhibition exerted by 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), myxothiazol, and antimycin A on respiration with succinate and by the effect of the latter two inhibitors on the succinate-reduced difference spectra. Respiration with succinate or ascorbate-TMPD was abolished by low KCN concentrations, suggesting the presence of a KCN-sensitive terminal oxidase. Cytochromes b and c were spectroscopically detected after reduction with physiological or artificial electron donors, whereas type a and d cytochromes were not detected. The CO difference spectrum of membranes reduced by dithionite and its photodissociation spectrum (77 K) suggested the presence of a single CO compound that had the spectral features of a cytochrome o-like pigment. High-pressure liquid chromatography analysis of membrane haems confirmed the presence of haem B; in contrast, haems A and O were not detected. Peroxidase staining of membrane type c cytochromes using SDS-PAGE revealed the presence of five bands with apparent molecular masses of 44, 33, 30, 26, and 14 kDa. Based on our results, a tentative scheme of the respiratory chain in E. corrodens, comprising (i) dehydrogenases for succinate, NADH, and formate, (ii) a ubiquinone, (iii) a cytochrome bc1, and (iv) a type-cbb' cytochrome c oxidase, is proposed.

Stimulation of menaquinone-dependent electron transfer in the respiratory chain of Bacillus subtilis by membrane energization.

At a pH of <or=7, respiration of Bacillus subtilis cells on endogenous substrates shut down almost completely upon addition of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone [CCCP]) and a K+-ionophore (valinomycin). The same effect was observed with cell spheroplasts lacking the cell wall. The concentration of CCCP required for 50% inhibition of the endogenous respiration in the presence of K+-valinomycin was below 100 nM. Either CCCP or valinomycin alone was much less efficient than the combination of the two. The inhibitory effect was easily reversible and depended specifically on the H+ and K+ concentrations in the medium. Similar inhibition was observed with respect to the reduction of the artificial electron acceptors 2,6-dichlorophenolindophenol (DCPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine cation (TMPD+), which intercept reducing equivalents at the level of menaquinol. Oxidation of the reduced DCPIP or TMPD in the bacterial cells was not sensitive to uncoupling. The same loss of the electron transfer activities as induced by the uncoupling was observed upon disruption of the cells during isolation of the membranes; the residual activities were not further inhibited by the uncoupler and ionophores. We conclude that the menaquinone-dependent electron transfer in the B. subtilis respiratory chain is facilitated, thermodynamically or kinetically, by membrane energization. A requirement for an energized state of the membrane is not a specific feature of succinate oxidation, as proposed in the literature, since it was also observed in a mutant of B. subtilis lacking succinate:quinone reductase as well as for substrates other than succinate. Possible mechanisms of the energy-dependent regulation of menaquinone-dependent respiration in B. subtilis are discussed.

Mechanistic studies of direct and sensitized photolysis of methyl (4-nitrophenyl)diazoacetate in the presence of an electron-donating amine: photochemical generation of the diazoalkane radical anion

The direct and perylene-sensitized photolysis of methyl (4-nitrophenyl)diazoacetate (1) in the presence of an electron-donating amine was investigated by the characterization of reaction products and by the direct observation of reactive species using a laser flash photolysis technique. In the direct photolysis of 1 in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), electron transfer from TMPD occurs and the photoproducts are obtained through an intermediate other than the carbene 3, which we suppose to be the carbene radical anion 3−˙. In the perylene-sensitized photolysis of 1 in the presence of an amine having a large electron-donating ability such as N,N-dimethylaniline and TMPD, the radical anion of 1 can be generated by perylene-mediated electron transfer from the amine to 1. The resulting 1−˙ appears to undergo extrusion of dinitrogen to give 3−˙ smoothly, the behavior of which is dependent on the amine employed as an additive. The product studies revealed that 3−˙ reacts readily with O2 to give the ketoester 5 or abstracts hydrogen atoms to give the ester 7, which is not inconsistent with the theoretical prediction that 3−˙ has a σ radical character in its electronic structure.

Reaction of oxide radical ion (O.–) with substituted pyrimidines

Pulse radiolysis technique has been used to investigate the reaction of oxide radical ion (O.–) with 4,6-dihydroxy-2-methyl pyrimidine (DHMP), 2,4-dimethyl-6-hydroxy pyrimidine (DMHP), 5,6-dimethyl uracil (DMU) and 6-methyl uracil (MU) in strongly alkaline medium. The second-order rate constants for the reaction of O.– with these compounds are in the range 2-5 × 108 dm3 mol–1 s–1. The transient absorption spectra obtained with DHMP have two maxima at 290 and 370 nm and with DMHP have maxima at 310 and 470 nm. The transient spectrum from DMU is characterized by its absorption maxima at 310 and 520 nm and that of MU by its single maximum at 425 nm. The intermediate species were found to react with N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) with high G(TMPD.+) values ranged between 3.9 × 10–7 molJ–1 and 4.8 × 10–7 molJ–1. These radicals undergo decay by second-order kinetics (2k/∈ = 1.0-1.7 × 106 s–1). The reaction of O.– with the selected pyrimidines is proposed to proceed through a hydrogen abstraction from the methyl group forming allyl type radicals. These are mainly oxidizing radicals and hence readily undergo electron transfer reactions with TMPD.

Probing the spontaneous ionization of aromatic amines by adsorption in activated acidic zeolite HZSM‐5

AbstractN, N, N′, N′‐tetramethyl‐p‐phenylenediamine (TMPD) and N, N, N′, N′‐tetramethylbenzidine (TMB) were used as probe molecules to examine their interactions with catalytically active sites generated after thermal treatment at 600 °C under O2 of acidic H6ZSM‐5 zeolite with chemical composition H6(AlO2)6(SiO2)90. The aromatic amines were merely exposed as powders at room temperature under argon to activated acidic H6ZSM‐5 molecular sieve. The course of adsorption and subsequent reactions were monitored by the application of diffuse reflectance, UV–visible absorption, electron paramagnetic resonance (EPR) and Raman scattering spectroscopy. The experimental investigations provided specific evidence of TMPD·+ or TMB·+ radical cations as major durable species and TMB2+ as minor species after complete adsorption. No direct evidence of trapped electrons within the zeolite framework resulting from the ionization was provided by EPR and electronic absorption spectroscopy. The aromatic amines through resonance Raman spectroscopy appear to be a sensitive and specific tool to probe the concentration, strength and accessibility of electron acceptor Lewis acid sites created during thermal treatment under O2. In contrast, no evidence of Brønsted acid sites of H6ZSM‐5 was found during the sorption of TMPD or TMB through the generation of protonated species. Copyright © 2002 John Wiley &amp; Sons, Ltd.

Electron transfer oxidation of tryptophan and tyrosine by triplet states and oxidized radicals of flavin sensitizers: a laser flash photolysis study

The riboflavin (RF, Vitamin B 2) and flavin adenine dinucleotide (FAD)-sensitized photooxidation of tryptophan (TrpH) and tyrosine (TyrOH) were studied by laser flash photolysis. TrpH and TyrOH quench triplet flavin sensitizers to produce reduced flavin radicals (FlH ) and oxidized radicals of TrpH or TyrOH (Trp and TyrO ). Although Trp and TyrO cannot be observed directly by the laser flash photolysis, N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD), as a probe, was added to the system to result in the formation of radical cations of TMPD (TMPD + ) via quenching of Trp and TyrO , which provides more definitive proof of electron transfer in the photosensitization process than only direct observation of reduced flavin radicals. Electron transfer from TrpH and TyrOH to oxidized radicals of riboflavin and FAD with similar rate constants to the triplet flavins was observed for the first time, which may be a new way of TrpH and TyrOH damage. These results may shed new light on future application of flavins in photodynamic therapy, and imply that flavins might be applied potentially to photosensitization of oxygen deficiency or under high-intensity pulsed laser irradiation.

Properties of the OH Adducts of Hydroxy-, Methyl-, Methoxy-, and Amino-Substituted Pyrimidines: Their Dehydration Reactions and End-Product Analysis

Reactions of hydroxyl radicals (•OH) with 2-amino-4-methyl pyrimidine (AMP), 2-amino-4,6-dimethyl pyrimidine (ADMP), 2-amino-4-methoxy-6-methyl pyrimidine (AMMP), 2-amino-4-hydroxy-6-methyl pyrimidine (AHMP), 4,6-dihydroxy-2-methyl pyrimidine (DHMP), 2,4-dimethyl-6-hydroxy pyrimidine (DMHP), 6-methyl uracil (MU), and 5,6-dimethyl uracil (DMU) have been studied by pulse radiolysis and steady-state radiolysis techniques at different pH values. The second-order rate constants of the reaction of •OH with these systems are of the order of (2−9) × 10^9 dm^3 mol^(-1) s^(-1) at near neutral pH. The difference in the spectral features of the intermediates at near neutral pH and at higher pH (10.4) obtained with these pyrimidines are attributed to the deprotonation of the OH adducts. The G(TMPD•+) obtained at pH ∼ 6, from the electron-transfer reactions of the oxidizing intermediates with the reductant, N,N,N‘,N‘-tetramethyl-p-phenylenediamine (TMPD), are in the range (0.2−0.9) × 10^(-7) mol J^(-1) which constituted about 3−16% oxidizing radicals. These yields were highly enhanced at pH 10.5 in the case of AHMP, DHMP, DMU, and MU (G(TMPD^(•+)) = 3.8−5.5 ≅ 66−95% oxidizing radical). On the basis of these results, it is proposed that a nonoxidizing C(6)-ylC(5)OH radical adduct is initially formed at pH 6 which is responsible for the observed transient spectra. The high yield of TMPD•+ at higher pH is explained in terms of a base-catalyzed conversion (via a dehydration reaction) of the initially formed C(6)-ylC(5)OH adduct (nonoxidizing) to C(5)-ylC(6)OH adduct which is oxidizing in nature. Among the selected pyrimidines, such a dehydration reaction was observed only with those having a keto (or hydroxy) group at the C(4) position of the pyrimidine ring. Qualitative analyses of the products resulting from the OH adducts of DHMP (at pH 4.5) and DMHP (at pH 6) were carried out using HPLC-ES-MS and a variety of products have been identified. Glycolic and dimeric products were observed as the major end-products. The product profiles of both DHMP and DMHP have shown that the precursors of the products are mainly the C(6)-ylC(5)OH and the H adduct radicals. The identified products are formed mainly by disproportionation and dimerization reactions of these radicals. The mechanistic aspects are discussed.

Charge transfer complexes of 2,4,6-tricyano-s-triazine with tetrathiafulvalene (TTF) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD)

Reaction of 2,4,6-tricyano-s-triazine (TCT) with tetrathiafulvalene (TTF) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) leads to 1∶1 charge transfer complexes. The crystal structures of the TTF[TCT], as well as the decomposition product due to hydrolysis of the [TMPD][TCT], i.e. [HTMPD]+[2,4-dicyano-6-oxy-s-triazine]−, are reported and spectroscopically characterized.

Photosensitized degradation and crosslinking of linear aliphatic polyesters studied by GPC and ESR

Photosensitized reaction on aliphatic polyesters, 3g4 and M 23g4, prepared from succinic acid and propanediol derivatives were investigated by spectrophotometry, GPC, and electron spin resonance (ESR) methods. From change of GPC curves for 3g4 polyester doped with N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) as a photosensitizer, it was confirmed that not only degradation but also crosslinking reaction occurred for 3g4, M 23g4, and poly(lactic acid), by UV irradiation. By detailed analysis of ESR spectra, it was confirmed that several radical species are produced through photosensitized reaction; TMPD radical cation, ester radial anion of polyester, main chain alkyl radical at the center of diol component, and acyl-type radical. Although there was no direct evidence for producing the main chain scission radical for 3g4 and M 23g4, it was strongly suggested that the photosensitized degradation and the crosslinking reactions were originated from the ester radical anion that was produced by electron capturing.

Application of Marcus cross-relation to mixed inorganic–organic redox couples. A stopped-flow study of the oxidation of N,N,N′,N′-tetramethyl-p-phenylenediamine with various oxidantsIn memory of Lennart Eberson.

The rate constants of the oxidation of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPPD) are measured in water by means of stopped-flow techniques using different inorganic ions, like Fe(CN)63−, MnO4−, Co(NH3)63+, Ru(OH)3+6. The rates do not depend on the different oxidation potentials, but can be correlated well with calculated rate constants from the Marcus cross-relation. The electron self-exchange rates of the inorganic oxidants range over nine orders of magnitude. Measurements were performed at T = 293 K within a pH-range of 7–9.

Differential sensitivities of plant and animal mitochondria to the herbicide paraquat.

Paraquat herbicide is toxic to animals, including humans, via putative toxicity mechanisms associated to microsomal and mitochondrial redox systems. It is also believed to act in plants by generating highly reactive oxygen free radicals from electrons of photosystem I on exposure to light. Paraquat also acts on non-chlorophyllous plant tissues, where mitochondria are candidate targets, as in animal tissues. Therefore, we compared the interaction of paraquat with the mitochondrial bioenergetics of potato tuber, using rat liver mitochondria as a reference. Paraquat depressed succinate-dependent mitochondrial Delta(psi), with simultaneous stimulation of state 4 O2 consumption. It also induced a slow time-dependent effect for respiration of succinate, exogenous NADH, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)/ascorbate, which was more pronounced in rat than in potato mitochondria. However, with potato tuber mitochondria, the Delta(psi) promoted by complex-I-dependent respiration is insensitive to this effect, indicating a protection against paraquat radical afforded by complex I redox activity, which was just the reverse of to the findings for rat liver mitochondria. The experimental set up with the tetraphenyl phosphonium (TPP+)-electrode also indicated production of the paraquat radical in mitochondria, also suggesting its accessibility to the outside space. The different activities of protective antioxidant agents can contribute to explain the different sensitivities of both kinds of mitochondria. Values of SOD activity and alpha-tocopherol detected in potato mitochondria were significantly higher than in rat mitochondria, which, in turn, revealed higher values of lipid peroxidation induced by paraquat.

Periplasmic oxygen reduction by Desulfovibrio species.

Washed cells of Desulfovibrio vulgaris strain Marburg (DSM 2119) reduced oxygen to water with H(2) as electron donor at a mean rate of 253 nmol O(2) min(-1) (mg protein)(-1). After separating the periplasm from the cells, more than 60% of the cytochrome c activity and 90% of the oxygen-reducing activity were found in the periplasmic fraction. Oxygen reduction and the reduction of cytochrome c with H(2) were inhibited by CuCl(2). After further separation of the periplasm by ultrafiltration (exclusion sizes 30, 50, and 100 kDa), oxygen reduction with H(2) occurred with the retentates only. Ascorbate plus tetramethyl-p-phenylenediamine (TMPD), however, were also oxidized by the filtrates. The stoichiometry of 1 mol O(2) reduced per 2 mol ascorbate oxidized indicated the formation of water. Our experiments present evidence that in D. vulgaris periplasmic hydrogenase and cytochrome c play a major role in oxygen reduction. Preliminary studies with other Desulfovibrio species indicated a similar function of periplasmic c-type cytochromes in D. desulfuricans CSN and D. termitidis KH1.

Probing the reactivity of the radiation sensitizer motexafin gadolinium (Xcytrin®) and a series of lanthanide(III) analogues in the presence of both hydroxyl radicals and aqueous electrons

The competition of the radiation sensitizer motexafin gadolinium (Xcytrin®, gadolinium(III) texaphyrin) and several other water-soluble metallotexaphyrin complexes with N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) for solvated electrons and hydroxyl radicals was studied using pulse radiolysis and by steady-state γ-radiolysis. It was found that the one-electron reduced forms ( M - Tex ·+) of the Gd(III) , Eu(III) , Dy(III) , Yb(III) , and Cd(II) texaphyrin complexes, after an initial reaction with hydrated electrons, do not compete with TMPD for hydroxyl radicals formed under pulse radiolytic conditions. By contrast, the reduced Y(III) , In(III) , Tm(III) , and Lu(III) texaphyrin complexes do. These differences in competitive reactivity toward . OH are rationalized in terms of the relative rates of protonation of the various singly reduced texaphyrins. In the case of Gd - Tex 2+ in particular, the one-electron reduced product, Gd - Tex ·+, protonates rapidly, producing a redox-inactive species that does not react appreciably with . OH . By contrast, the one-electron reduced product from, e.g., Lu - Tex 2+ (motexafin lutetium), does. These results may explain, at least in part, why the Gd(III) texaphyrin functions as a radiation sensitizer in vivo, while the analogous Lu(III) complex does not.

14N/15N Isotope Effect on the Electron Transfer Process betweenN,N,N‘,N‘-Tetramethyl-p-phenylenediamine and Wurster's Blue

Appreciable 14N/15N equilibrium isotope effect has been observed for the electron-transfer process between N,N,N‘,N‘-tetramethyl-p-phenylenediamine (TMPD) and its radical cation perchlorate (TMPD+•ClCO4-, Wurster's blue). The equilibrium constant for the reaction, TMPD + [15N2]TMPD+• ⇌ TMPD+• + [15N2]TMPD, was determined to be 0.85 ± 0.05 at 25 °C in acetonitrile by electron spin resonance (ESR) analysis of the line-broadening effect of Wurster's blue in the presence of its parent neutral molecule. Redox potentials of TMPD and [15N2]TMPD were determined by cyclic voltammetry to be E° = 0.107 and 0.103 ± 0.002 V vs SCE respectively, corresponding to K = 0.86 ± 0.06 for the reaction. Comparison of the infrared and Raman spectra of TMPD, [15N2]TMPD and their radical cations revealed significant vibrational frequency shifts caused by the heavy-atom substitution and radical cation formation, from which the free energy change for the electron-transfer process was estimated to be 300 J mol-1, corresponding to K = 0.89. These results demonstrate that 15N-substitution of TMPD decreases appreciably the ionization potential of the molecule, making it easier to lose an electron forming the corresponding radical cation in solution.

Dysfunctional mitochondrial respiration in the wobbler mouse brain

The involvement of mitochondrial dysfunction promoting neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), has been suggested. Histopathological and biochemical mitochondrial abnormalities have been reported in both sporadic and familial patients and suggest the contention that mitochondria may play a key role promoting ALS. Animal models of ALS provide a unique opportunity to study this incurable and fatal human disease. In the present study we tested the hypothesis that alterations in mitochondrial physiology occur in the brain of wobbler mice. No significant difference was found in the respiratory control index or adenosine diphosphate/oxygen ratio values between isolated mitochondria of wobbler and control mice. When pyruvate and malate were used as substrates, oxygen consumption was decreased significantly by approximately 33% in mitochondria isolated from wobbler mouse brain compared to controls. Oxygen consumption in the presence of ascorbate and N,N,N′,N′-tetramethyl- p-phenylenediamine (TMPD) was decreased significantly by approximately 21% in wobbler brain mitochondria compared to controls, which suggests impairment in the function of complex IV. These findings are the first demonstration of mitochondrial respiratory chain dysfunction in the brain of the wobbler mouse.

ESR studies of photosensitized degradation of poly(L-lactic acid) via photoionization of dopant

The photosensitized degradation of poly(L-lactic acid) (PLA) via an anionic reaction process was studied using spectrophotometry, electron spin resonance (ESR), and gel permeation chromatography (GPC) measurements. PLA film doped with N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was irradiated at 77 K using UV light (λc = 356 nm) by which the PLA matrix itself cannot be directly excited. After photoirradiation, a new broad absorption band appeared over the original spectrum due to TMPD+ ·, which was produced by two-photon ionization. The ESR spectrum of the irradiated sample indicated the presence of the TMPD+ · radical and main-chain scission radical of PLA. During the thermal annealing at 0 °C, the latter radical changed to another radical species by dehydrogenation of the alpha hydrogen of the PLA main chain. TMPD+ · was extremely stable at room temperature for 7 d. However, by thermal annealing at 40 °C, all the radicals decayed due to the enhanced molecular motions near Tg of PLA (58.7 °C). Spectral simulation for the obtained ESR spectra revealed the relative amounts of four radicals: TMPD+ ·, a main-chain scission radical, a main-chain tertiary radical, and an unknown radical. The last one was tentatively assigned to the PLA radical anion because of its short decay time. GPC measurements clearly indicated a decrease in the molecular weight of PLA after irradiation. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 706–714, 2001