PHOTOPRODUCT FORMATION IN DNA AT LOW TEMPERATURes.

Abstract— The temperature dependence of thy mine photoproduct formation in Escherichia culi DNA dissolved either in water or in a 50 per cent ethylene glycol solution was studied at temperatures between + 25 and — 196°C. At low temperatures, the formation of thymine dimer was strongly inhibited. A dose of 1 × 10⁴ ergs/mm² at 280 nm converted 2 per cent of the thymine to dimer at 25°C as compared with 0.2 per cent at — 196°C. In addition, a new thymine photo-product which was both nonphotoreversible and nonphotoreactivable was found at low temperatures. On the basis of its chromatographic mobility, this new photoproduct was assumed to be the same as that isolated from irradiated spores of Bacillus megaterium . Extensive irradiation at 254 nrn of DNA at — 120°C resulted in a yield of > 23 per cent for the 'spore-type' photoproduct as compared with 6 per cent for the thymine dimer. In poly d(AT), irradiated at low temperature, no spore-type photoproduct was found; this suggests that adjacent thymine residues are necessary for the formation of the spore-type photoproduct.     

ELECTRON SPIN RESONANCE STUDIES OF THE TRIPLET STATES OF MONOANIONIC THYMINE*

Abstract— Ultraviolet irradiation of the thymine anion tautomers 1-HT- and 3-HT^- at -196°C generates excited triplet states having δm=±2 resonances at 1067 and 1260 G respectively (with v= 9.0 GHz). Excitation at 270nm gave predominantly the low field signal while excitation at 300 nm produced only the high field resonance. These results when compared with the absorbance properties of the monoanions are consistent with the low field resonance being due to 1-HT^- and the high field resonance being due to 3-HT^-.     

Flash photolysis and inactivation of aqueous lysozyme

Abstract— –Flash photolysis of aqueous lysozyme has shown that the initial photochemical products are photo-oxidized tryptophan residues (Λ_max= 500 nm), hydrated electrons (Λ_max= 720 nm), and the cystine residue electron adduct (Λ_max= 420 nm). Comparisons with mixtures of the chromophoric amino acids show that 1 to 2 tryptophan residues provide electrons at a quantum yield of 0.018 (25 per cent). Part of the ejected electrons are captured by cystine residues via a short-range, intramolecular process with essentially unit efficiency. The remainder become hydrated and back react with oxidized tryptophan residues before 10^-4sec. The cystine residue electron adduct decays with 2 msec halftime (25°C) and 1.5 kcal/mole activation energy. The surviving oxidized tryptophan residues decay with a comparable time constant in a hydroxyl ion catalyzed process. In acid solutions the oxidized tryptophan residue and long-lived H atom adduct are observed (Λ_max= 380 nm). The quantum yield of lysozyme inactivation induced by xenon flash irradiation above 250 nm is 0.023 (20 per cent), which is not sensitive to oxygen or pH. Comparison to the primary photochemical reactions indicates that electron ejection from the essential tryptophan residues inactivates the enzyme, irrespective of the electron trap and subsequent reactions. On the basis of the structure and supporting information it is proposed that the tryptophan residues of the active site are involved. Direct disruption of cystine residues does not contribute more than 10 per cent to the inactivation quantum yield in this wavelength region. Lysozyme inactivation may differ from other enzymes because the chromophores include essential residues located in the active center.     

UV-induced free radicals in oriented DNA.

Abstract— Samples of oriented DNA containing 30% water were UV-irradiated at 77 K and investigated by electron paramagnetic resonance (EPR). The EPR spectra recorded in directions parallel and perpendicular to the DNA fibre direction showed that at low UV doses the induced free radicals are very similar to those induced by γ-irradiation at the same temperature. The γ-induced free radicals have previously been analysed and found to consist mainly of anionic free radicals on thymine and cationic free radicals on guanine. At higher UV doses or by suitable annealing of the samples given a low UV dose, significant amounts of hydrogen-addition radicals on thymine were observed. The quantum yield of free radical formation for irradiation at 300nm ± 10nm was estimated to 10^--⁴. We also made a quantitative determination of the UV-induced free radicals inside an optically effective volume of the sample. The following free radical induction frequencies at 77 K were estimated: γ-rays: 2 × 10^--¹² free radicals per rad per dalton and UV (300nm): 6 × 10^--¹² free radicals per J/m² per dalton.     

The Photochemistry of Thymine in Frozen Aqueous Solution: Trimeric and Minor Dimeric Products

Early work identified three compounds, namely the c,s cyclobutane dimer, the so‐called (6‐4) photoproduct (5‐hydroxy‐6‐4′‐(5‐methylpyrimidin‐2′‐one)‐5,6‐dihydrothymine) and a trimer hydrate, as products formed upon UV irradiation of thymine in frozen aqueous solution. More recent work has shown that an (α‐4) product, namely α‐4′‐(5′‐methylpyrimidine‐2′‐one)‐thymine, is a likely product formed under these reaction conditions. During a thorough reinvestigation of the photochemistry of Thy in ice at ?78.5°C, we found that a variety of other products could be detected. In addition to the c,s dimer, the other three known cyclobutane dimers, namely the c,a, t,s and t,a forms, are produced, although in considerably smaller amounts. The so‐called “spore product” of thymine (5,6‐dihydro‐5‐(α‐thyminyl)thymine) is likewise formed. Two other dimers have been identified as minor products; one of these has been determined to be 5‐(thymin‐3‐yl)‐5,6‐dihydrothymine and the other has been tentatively assigned to be a (5‐4) adduct (6‐hydroxy‐5‐4′‐(5‐methylpyrimidin‐2′‐one)‐5,6‐dihydrothymine). Compounds with the behavior expected of true trimeric compounds have been isolated via HPLC and characterized by mass spectrometry and photochemical behavior. One of these materials, putatively containing an oxetane ring, decomposes thermally to a secondary trimeric product that is then converted into the known trimer hydrate.     

X-ray- and U.V.-induced excitation of adenine, thymine and the related nucleosides and nucleotides in solution at 77 degrees K

Abstract— The light emitted by adenine and thymine and the related nucleosides and nucleotides in ethylene glycol/water glass during irradiation at 77°K with X-rays and 270 nm U.V. light has been studied. It was found that the same radiative levels were excited by both types of radiation. However, the phosphorescence to fluorescence ratios observed with X-rays were considerably enhanced relative to those obtained with U.V. light. This observation was taken to mean that a major fraction of the excitation results from ion recombination, although direct excitation of the triplet manifold as well as intersystem crossing between upper excited levels may have contributed to the enhancement as well. The yields of the X-ray-induced excitation of radiative levels. which were calculated by combining the yields of X-ray-induced emission and the quantum yields observed with u.v.-excitation, corresponded to: G≥ 4.0 excitations per 100 eV of dose absorbed in the solute for adenine and G≥ 1.8 for thymine. The excitation yields obtained for the nucleosides and nucleotides were somewhat smaller than those observed for the corresponding bases. It was inferred from the yields that a substantial fraction of the dose absorbed in the ribose of the nucleosides was transferred to the radiative levels of the constituent bases.     

VARIATION IN THE PHOTOCHEMICAL REACTIVITY OF THYMINE IN THE DNA OF B. SUBTILIS SPORES, VEGETATIVE CELLS AND SPORES GERMINATED IN CHLORAMPHENICOL*,†

Abstract— The chief photoproduct of thymine produced in u.v. irradiated (2537Å) vegetative cells of B. subtilis is the cyclobutane-type dimer while in spores very little of this dimer is produced (maximum yield 2·6 per cent of thymine) but a new photoproduct is produced in high yield (maximum of 28·4 per cent of thymine). This difference in photochemical response appears to be due, at least in part, to a difference in uydration of the DNA. The photochemistry of thymine in isolated DNA irradiated in solution is similar to that of DNA in irradiated vegetative cells, but differs markedly from that of isolated DNA irradiated dry. The yield of cyclobutane-type thymine dimer is much reduced in isolated DNA irradiated dry but a new photoproduct of thymine. is produced which is chromatographically similar to the spore photoproduct. The yield of this photoproduct, however, is never as great as that obtained in irradiated spores. The photochemistry of the DNA thymine of spores germinated in the presence of chloramphenicol is very similar to that of normal vegetative cells. Except for hydration, the physical state of the DNA is probably not otherwise altered by germination in the presence of chloramphenicol since DNA replication is prevented by the presence of chloramphenicol. These results are also consistent with the hypothesis that the unique photochemistry of spores is due, at least in part, to the hydration state of the DNA. The acid stability of the spore photoproduct is indicated by the fact that it is isolated from irradiated spores after hydrolysis in trifluoroacetic acid at 155°C for 60 min. It still contains the methyl group of thymine as judged by the fact that for a given dose of u.v. the same yield of photoproduct was obtained whether the spores were labeled with thymine-2–C-14 or -methyl-C-14. This photoproduct is stable to reirradiation (2537Å) in solution under condiditions where thymine dimers of the cyclobutane-type are completely converted back to monomeric thymine. On a column of molecular sieve material (Sephadex-G10), the spore photoproduct elutes in a region intermediate between the cyclobutanetype thymine dimers and monomeric thymine. Of the numerous compounds tested by paper chromatography, the spore photoproduct is most similar (but not identical) in several solvents to 5–hydroxyuracil and 5–hydroxymethyluracil. Our data do not allow us to decide if the product is a monomer or a dimer. Although the photochemistry of thymine in the DNA of spores differs markedly from that for vegetative cells, several lines of evidence make it seem doubtful that the enhanced resistance of spores to u.v. relative to that of vegetative cells can be explained solely on the basis of this difference in the photochemistry of DNA thymine.     

Photoinduzierte Cycloadditionen von aliphatischen 2H-Azirinen. 37. Mitteilung über Photoreaktionen

The purely aliphatic 2,3-dipropyl-2H-azirine ( 1 ) reacts on irradiation with a mercury high-pressure lamp through a Vycor filter with methyl trifluoroacetate or acetone to form 3-oxazolines 3a, b (65%) resp. 4 (14%) (Scheme 1). 9-Azabicyclo[6.1.0]non-1(9)-ene ( 5 ) on irradiation in the presence of the dipolarophiles methyl trifluoroacetate, methyl difluoroacetate, 1,1,1-trifluoro-propanone and acetone behaves in a similar way, whereby the corresponding bicyclic 3-oxazolines 7–10 result in yields of 60–20% (Scheme 2). By analogy with the photochemical behaviour of 3-aryl-2H-azirines it is assumed that nitrile-ylides 2 resp. 6 represent intermediates. In fact irradiation of 2,3-dipropyl-2H-azirine ( 1 , λ_max 239 nm, ? 240) at ?196° with light of wavelength 245 nm in a hydrocarbonglass gives rise to a pronounced maximum at 280 nm, for which an ? of ? 15000 can be estimated. The quantum yield for the formation of nitrile-methylide 2 is 0,8. Irradiation of the dipole 2 at ?196° or warming to ?150° causes the maximum at 280 nm to disappear.     

KINETICS OF THE ULTRAVIOLET-INDUCED DIMERIZATION OF THYMINE IN FROZEN SOLUTIONS*

Abstract— It is known that thymine forms dimers when aqueous solutions are irradiated with ultraviolet light while in the frozen state, but does not form dimers when solutions are irradiated in the liquid state. The eutectic point of aqueous thymine solutions was found to be. —0.02°C. Since the irradiation of frozen solutions is always carried out at lower temperatures, the dimerization must be occurring in the solid state. Activation energies and quantum yields for dimer formation were determined by irradiating 1–mm layers of thymine solution at —5°C to — 707deg;C for various lengths of time. As expected, the activation energy was zero. After measuring the amount of radiation scattered by samples of ice, the extreme values for the quantum yield were found to be 0.73 and 4.08. The lower limit assumed that all the scattered light was absorbed by thymine; the upper limit assumed that none was absorbed. Since the theoretical maximum quantum yield is 2, the best estimate of the quantum yield is considered to be between 1 and 2.     

Structural factors controlling the action of UV light in nucleic acids

Abstract— UV light induced conformational effects of different deoxyoligonucleotides and deoxypolynucleotides containing thymine and adenine residues are investigated by means of CD measurements and quantum yield calculations. UV-irradiation at the wavelengths 254 , 280 and 313 nm indicate that unsensitized irradiation at low doses leads to thymine photoproduct formation of non-cyclobutane type. In contrast to that irradiation at 313 nm in the presence of acetophenone causes different changes in the CD spectra due to the formation of thymine dimers of the cyclobutane type structure. Quantum yield calculations demonstrate a pronounced dependence of the photoproduct formation on the nucleotide sequence of the oligomers. Thus, clustering of thymine dimer formation can be neglected. Adenine photoproducts in the (A.T) containing oligomers are only formed at higher fluences. > 1.5 × 10⁴ J/m² and are biological less important events.     

Photoreactivation of killing in Streptomyces. II. Kinetics of formation and photolysis of pyrimidine dimers and adducts in S. coelicolor and S. griseus PHR-1

Abstract— A pyrimidine adduct, 6-4‘-[pyrimidine-2’-one] thymine (PO-T)?, observed in DNA hydrolysates of 254-nm ultraviolet (u.v.) irradiated conidia of Streptomyces coelicolor, increases linearly with u.v. dose up to 2 × 10⁵ ergs/mm². Yields of thymine dimer (T○) and uracil-thymine dimer (U○) level off at much lower doses. Initial relative rates of formation of these u.v. photoproducts are: 1:1.3:4.8 for PO-T, T○ and U○, respectively. Similar results were obtained with a Streptomyces griseus mutant, PHR-1. An equation is derived to estimate the ratio of the amount of PO-T to the total amount of thymine-derived photoproducts at low (biological) u.v. doses. The observed PO-T fractions compare well with the calculated values. Rapid photolysis of the precursor of PO-T was observed by post-u. v. treatment at 313 nm of conidia of S. coelicolor and of S. griseus PHR-1. The photolysis was much slower at 365 nm and did not occur at all at 405 nm. Pyrimidine dimers were not appreciably affected by post-u. v. treatment at the above wavelengths in these Streptomyces strains. Both of these strains are phenotypically photoreactivation-deficient, and the present results indicate that they do not possess active photoreactivating enzyme. In earlier papers[3,4,5], the pyrimidine adduct found in acid hydrolysates of DNA was loosely referred to as “uracil-thymine adduct (U-T adduct)”. Such terminology is not strictly correct. The pyrimidine adduct in acid hydrolysates is PO-T (sometimes called P₂B), which could theoretically result from removal of ammonia from a C-T adduct or removal of water from a U-T adduct (see [6]).     

PROPERTIES OF THE ULTRAVIOLET-LIGHT-MEDIATED BINDING OF BOVINE SERUM ALBUMIN TO DNA*

Abstract— The binding of DNA to protein mediated by U V (254 nm) radiation has been investigated using binding of the complex to Millipore membrane filters as an assay technique. The reaction proceeds through an activated protein intermediate which then reacts with the DNA. The activated protein has a half-life of about 75 min at 0°C and about 18 min at 37°C. Short wavelengths are more efficient in forming the complex than wavelengths in the 250–280 nm range. N-ethyl maleimide treatment of protein before irradiation markedly inhibits the reaction.     

Stabilization of phytochrome intermediates by low temperature

Abstract— The photocon versions between the red-absorbing form (P_r) and the far-red absorbing form (P_fr) of phytochrome were examined at low temperatures. Partially purified preparations of the chromoprotein were examined in phosphate buffer and in 25 per cent buffer plus 75 per cent glycerol. Actinic irradiation of P, below – 150°C produces an intermediate with maximum absorbance near 695 nm, R₆₉₅. Actinic irradiation of R₆₉₅ converts it back to P. Above – 150°C R₆₉₅ decays to a low extinction form of phytochrome, R, which in turn decays to P_fr upon further warming. Light absorption by P_fr below – 150°C results in the formation of an intermediate form of phytochrome with maximum absorbance near 660 nm, FR₆₆₀. FR₆₆₀ decays upon warming to a lower extinction form, FR'. which in turn decays to P_r on continued warming. No evidence was obtained to suggest that any of the observed intermediate states are involved in more than one direction of phytochrome photocon version.     

Excision of cytosine-thymine adduct from the DNA of ultraviolet-irradiated Micrococcus radiodurans

Abstract— 6-4‘-[pyrimidin-2’-one]-thymine (PO-T), a deamination product of a cytosine–thymine adduct accounted for about 17 per cent of the total detectable thymine-derived photoproducts in u.v.-irradiated Micrococcus radiodurans. On incubation of the irradiated cells, oligonucleotides containing the photoproducts were released into the medium. After various periods of incubation the different photoproducts were isolated both from the cells and from the medium. Analysis of these different photoproducts showed that during the excision process the ratio of PO–T to total thymine-derived products remained constant both in the cells and in the medium. This shows that the precursor to PO–T is excised at the same rate as the other thymine-derived photoproducts by the dark repair mechanisms exhibited by this radiation-resistant organism.     

IDENTIFICATION OF ULTRAVIOLET-INDUCED THYMINE DIMERS IN DNA BY ABSORBANCE MEASUREMENTS

Abstract— The irradiation of native DNA's by ultraviolet radiation of different wave lengths changes their absorption spectra. The changes are similar to those found for the formation of dimers between adjacent thymines in polynucleotide chains. The decreases in absorbance at 270 mµ produced by 280 mµ irradiation are reversed to a large extent by subsequent 239 mµ irradiation. The magnitude of the absorbance changes produced by large doses of 280 mµ correspond to the formation of dimers between approximately 50 per cent of all the TT sequences in the DNA. An incident dose of 100 erg/mm² of 280 mµ radiation forms about one dimer per molecule of calf thymus DNA of molecular weight 6 times 10⁶. The irradiation of heat-denatured DNA produces larger absorbance changes than are observed in native DNA. The absorbance changes in denatured DNA arise in part from a heat-reversible reaction, presumably involving cytidine, part from the formation of thymine dimers, and part from some unknown photoproducts. The reversal of thymine dimers by short wave length irradiation does not pioduce an equivalent change in the melting temperature of the DNA.     

REVERSIBLE PHOTOCYCLOADDITION OF A 4',5'-DIHYDROPSORALEN DERIVATIVE WITH THYMINE

The photocycloaddition reaction between a 4′,5′-dihydropsoralen derivative and thymine was studied in solution using a synthetic bichromophoric model 8 in which the two rings are associated by a tetramethylene chain. In water this model molecule exhibits intramolecular ring-ring stacking interactions as evidenced by UV and NMR spectroscopies. Irradiation at 365 nm at usual concentrations ( 5.10^?-⁴M) leads exclusively to a regio- and stereo-selective dimerization reaction involving the 3,4 double bonds of the psoralen moities. Extreme dilutions (ca 2.10^?-⁵M) were necessary to observe the intramolecular reaction which results in the exclusive formation of a 3,4 cb-anti adduct. This reaction is completely reversed by irradiation at 254 nm. These results are discussed with regard to the behavior of the homologous models in which the furan part of the psoralen ring is not hydrogenated, These latter compounds also lead exclusively to a 3,4 cis-anti adduct. It appears that saturation of the furan ring increases strongly the quantum yield of the photaddition at 365 nm (0.01 → 0.18) and that the triplet excited state of the 4′,5′-dihydropsoralen is involved in the photoaddition.     

Electron spin resonance study of poly-3,3-bis(chloromethyl) oxetane irradiated with electron beams and ultraviolet light

Poly-3,3-bis(chloromethyl)oxetane (poly-BCMO) was irradiated at ?196°C with electron beams and ultraviolet light, and observed ESR spectra were compared. A three-line spectrum (coupling constant of about 21 gauss) and a two-line spectrum (coupling constant of about 18 gauss) were observed after irradiation with electron beams in vacuo. They were attributed to free radicals and respectively. On the other hand, a three-line spectrum (coupling constant of about 20 gauss) and an asymmetric singlet spectrum were observed after ultraviolet irradiation in vacuum. They were assigned to free radicals and ? CH₂? O·, respectively. Mechanisms of radical formation were discussed in each case. When poly-BCMO was irradiated with electron beams at ?196°C in the presence of air, peroxy radicals were produced after subsequent treatment at ?78°C. The reaction between alkyl radicals and oxygen molecules was found to be diffusion-controlled.     

Correlation of photoreactivation of ultraviolet-induced killing with dimer splitting before and after DNA replication in an excisionless strain of Escherichia coli

Abstract— Splitting of thymine-containing dimers was compared quantitatively with photoreactivation (PR) of killing induced by ultraviolet radiation (254 nm) in a uvrA (excisionless) strain of E. coli. Immediately after irradiation, the splitting rate (number of dimers split/genome/unit PR dose) agreed well with the PR rate of the cells (rate of recovery from photoreactivable lethal damage converted into an ‘estimated’ number of dimers split/genome/unit PR dose). After 4 h of incubation of cells in nutrient medium, the maximal fraction of splittable dimers decreased, as did the maximal fraction of photoreactivable lethal damage. However, the initial splitting rate after incubation was equal to that before incubation. During the 4-h incubation, the heavily irradiated uvrA cells did not divide but became filamentous and their DNA increased about 70 per cent. It is concluded that roughly half of the dimers in DNA that has replicated after ultraviolet irradiation are split as efficiently as those in DNA that has not replicated.     

Tieftemperaturbestrahlungen von 3-Phenyl-2H-azirinen

2, 2, 3-Triphenyl-2H-azirine ( 4a ) in a matrix of 2, 2-dimethylbutane/pentane 8:3 (DMBP) at ?185° gave rise on irradiation with light of 250–350 nm to a new UV.-maximum at 350 nm (Fig. 1). We assign the dipole benzonitrildiphenylmethylide ( 1a ) to this new maximum. Irradiation with monochromatic light of 366 nm destroyed this maximum and the initial absorption curve reappeared (Fig. 2). When the azirine 4a was photolysed in DMBP at ?185° in the presence of methyl trifluoracetate (TFEM), the maximum at 350 nm was obtained again. This maximum vanished upon increasing the temperature to ?160°. Through gas chromatography we were able to show that 5-methoxy-5-trifluormethyl-2, 2, 4-triphenyl-3-oxazoline ( 6a ) was produced. 6a was also obtained upon irradiation of 4a at room temperature in the presence of TFEM (scheme 1 and table 1). Modification of the previously described experiment, in which the maximum at 350 nm was extinguished in the matrix due to irradiation at 366 nm gave, after warm up, almost no dipole adduct 6a (table 1). From these experiments, an extinction coefficient of 17, 000 for the 350 nm maximum of 1a , was calculated. These experiments have shown that irradiation of triphenylazirine 4a leads to the dipole 1a , which can be reversed photochemically – but not tharmally – into azirine 4a. 1a reacts at less than ?160° with TFEM to give adduct 6a . The results which were obtained with triphenylazirine 4a could be correspondingly obtained with 2, 3-diphenyl-2H-azirine 4b (Fig. 3, scheme 2 and table 2). The dipole 1b showed two UV.-maxima at 330 nm (ε = 17, 500) and 343 nm (ε = 21, 000). Later experiments established, that the two maxima belonged to a single dipole species. The dipole 1c obtained upon irradiation of 2, 2-dimethyl-3-phenyl-2H-azirine ( 4c ) in DMBP at ?190°, appears to absorb in the same region as the azirine 4c . The presence of the dipole 1c was univocally established by low temperature trapping experiments with TFEM. The dipole 1a showed no ESR.-spectrum characteristic for a triplet state. We assume therefore, that 1a is in a singlet state. Photolysis of oxazolinone 7 at ?190° in DMBP led to the dipole 1a with loss of CO₂. 1a recombines apparently in considerable amount with the CO₂ trapped in the matrix to give starting oxazolinone 7 because the 350 nm-maximum of 1a appeared with low extinction. Irradiation with light of 366 nm into this matrix produccd triphenylazirine 4a . Low temperature trapping experiments with TFEM led to small amounts of 5-methoxy-5-trifluormethyl-triphenyl-3-oxazolinc ( 6a ).     

ESR spectra of poly(methacrylic acid) and poly(methyl methacrylate): Reinterpretation of the 9-line spectrum

The temperature dependence of the ESR spectra of poly(methacrylic acid) and poly-(methyl methacrylate) γ-irradiated at room temperature was studied between ?196°C and +25°C. The conventional 9-line spectrum was observed throughout this range with no significant spectral change, in contrast to the propagating radical ··· CH₂? °C(CH₃)COOR found in methacrylic acid monomer or barium methacrylate dihydrate irradiated at ?196°C. In addition, the irradiation of methacrylic acid monomer with a low dose at 0°C gave the same 13-line spectrum as that of the propagating radical obtained by the irradiation at ?196°C, while prolonged irradiation at 0°C gave the same conventional 9-line spectrum as that of poly(methacrylic acid) or poly(methyl methacrylate). The conventional 9-line spectrum has a much weaker 4-line component than that of the propagating radical. The difference comes from the surrounding matrix, and the conventional 9-line spectrum is well interpreted by introducing the concept of the distribution of the conformational angle in the irregular polymer matrix. From simulation of the ESR spectrum, it was found that the intensity of the 4-line component is very sensitive to the distribution, and that the observed 9-line spectrum is well reproduced assuming a Gaussian distribution (half-height width of 5–6°) around the most probable conformation which is nearly the same as that of the propagating radical, where the conformational angles of the two C? H_β bonds to the half-filled p-orbital are 55° and 65°.