6 Pulse Radiolysis Studies on Reactive Intermediates in Organic Chemical Processes By G.E. ADAMS and R. L. WILLSON Cancer Research Campaign Research Unit in Radiobiology Mount Vernon Hospital Northwood Middlesex 1 Introduction Application of the pulse radiolysis technique to the study of short-lived organic intermediates continues. Interest has been maintained in organic free-radical equilibria reactions of hydrated electrons hydrogen atoms and hydroxyl radicals in aqueous organic media general properties of organic radicals and in effects of molecular structure.’*’ Although there appears to be a temporary lull in activity in the field concerned with the detection and reactions of excited states there has been a notable increase in interest in the study of short-lived intermediates in biochemical and biological processes.These include reduction and oxidation processes in amino-acids simple peptides enzymes and the nucleic acids mechanisms of protein binding free-radical mechanisms in cellular radiosensitisation and various examples of biochemically-related electron-transport systems. With reference to advances in techniques some early results have been pub- lished on the application of the picosecond pulse radiolysis te~hnique~,~ showing for example that free electrons in both water and alcohols become solvated in times less than lo-“ s. However in the presence of high concentrations of acceptors such as acetone electron capture can occur earlier i.e. before solvation. Future applications of this fast technique to the general study of organic inter- mediates will be valuable.Another interesting advance involves the detection and recording of transient spectra by self-absorption of Cerenkov light produced during nanosecond pulse radiolysis.’ Some spectra of solvated electrons and also a benzene excimer have been confirmed. Relevant review literature published during the year includes a book ‘The Hydrated Electron’ which gives a comprehensive account of the physical chemistry of this important species‘ and a textbook ‘Principles of Radiation ‘ G. E. Adams Ann. Reports (B) 1968 65 223. G. E. Adams Ann. Reports (B) 1969,66,210. R. K. Wolff M. J. Bronskill and J. W. Hunt J. Chem. Phys. 1970,53,4201. M. J. Bronskill R. K. Wolff and J. W. Hunt J.Chem. Phys. 1970,53,4211. D. C. Walker and S. C. Wallace Chem. Phys. Letters 1970 6 11 1. E. J. Hart and M. Anbar ‘The Hydrated Electron’ Wiley New York 1970. G. E. Adams and R. L. Willson Chemi~try'.~Other general articles discuss the nature and reactions of initial species in radioly~is,~,~ electron transfer and protonation,".' ' reactions of excited states in liquids,' and the use of pulse radiolysis in general biochemical and enzyme studies.l3 2 Aqueous Organic Systems Hydroxyl radicals formed in the radiolysis of water add to the double bonds of vinylmethylketone acrolein and crotonaldehyde at diffusion-controlled rates. l4 The respective radicals can disproportionate to form enols of acetoacetaldehyde and malondialdehyde.The hydrated enol of crotonaldehyde a reaction product dehydrates spontaneously in a base-catalysed reaction MeC(OH)=CH-€H(OH) + MeC(OH)=CH<HO + H,O (1) The respective rate constants for the decay of the neutral molecule and its conju- gate base are 54 s-' and lo4s-'. Transient spectra have been obtained on pulse radiolysis of acrylamide in aqueous s~lution.'~ These have been resolved and assigned to the H atom-and OH radical-adducts at the double bond and to the protonated form of the electron adduct. It is proposed that the latter species is of the form CH -CH-C(NH,)OH. The radicals decay rapidly with bimolecular rate con- stants of the order of lo91mol-' s-l although in alkaline solution the unpro- tonated electron-adduct decays unimolecularly to produce the radical CH =CH-CH( 0)NH -.Simultaneous measurements of transient changes in the optical absorption and the electrical conductivity of pulse-irradiated solutions of formic acid and carbon monoxide have provided information on the chemistry of the carboxyl radical.16 As shown previ~usly'~ this radical can be formed either by OH addition to CO,.or by hydrogen abstraction from formic acid or its ion OH + HCOOH -+ H,O + COOH (2) Both COOH and CO (pK = 3.9 k0-3)reduce nitrobenzene by simple electron ' J. H. O'Donnell and D. F. Sangster 'Principles of Radiation Chemistry' Arnold London 1970. B. Cercek E. J. Land and A. J. Swallow 'Large Radiation Sources for Industrial Processes' International Atomic Energy Agency Vienna 1969 p. 51. G.E. Adams in 'Quaderni de La Ricerca Scientifica'. No. 68 Consiglio Nazionale delle Ricerche Rome 1970 p. 27. lo A. J. Swallow Proc. 9th Japan Conference on Radioisotopes Atomic Energy Society of Japan 1970 p. 571. L. M. Dorfman Accounts Chem. Res. 1970,3 224. j2 J. K. Thomas Ann. Rev. Phys. Chem. 1970,21 17. l3 G. E. Adams R. B. Cundall and R. L. Willson 'Chemical Reactivity and Biological Role of Functional Groups in Enzymes' ed. R. M.S. Smellie Academic Press 1970 p. 171. l4 J. Lillie and A. Henglein Ber. Bunsengesellschaft Phys. Chem. 1970 74 388. Is K. W. Chambers E. Collinson and F. S. Dainton Trans. Faraday SOC.,1970,66 142. l6 A. Fojtik G. Czapski and A. Henglein J. Phys. Chem. 1970 74 3204. J. P. Keene J. Raef and A. J. Swallow in 'Pulse Radiolysis' ed.M. Ebert J. P. Keene A. J. Swallow and J. H. Baxendale Academic Press London and New York 1965 p. 99. Pulse Radiolysis Studies on Reactiue Intermediates 209 transfer and CO reacts rapidly with oxygen as shown previously." Other examples of the use of the carboxyl radical as an electron donor are discussed later. Transient absorption spectra have been reported for some hydroxyperoxyl radicals formed from irradiated aqueous solutions of aliphatic alcohols cyclo- hexane and diethylether.' The acid dissociation constants for the methanol ethanol and isopropanol species are respectively 7.1 8.0 and 8.5. or-Hydroxy-peroxyl radicals dimerise to form dibasic acids which decay spontaneously with rates which are pH-dependent. It is suggested that these acid-intermediates are a-hydroxy-hydrotetroxides.The reactivities of the basic form of the hydroxyl radical 0-,with methanol and ethanol have been redetermined by absolute measurement of the rates of formation of the product radical-ions CH20-and C2H,0-.20 Measured rate constants of 5.8 0-8 x lo8 1mol-' s-' and 11.3 f1.7 x lo8 1 mol-'s-' agree reasonably well with earlier indirect deter- minations.2',22 Two reports from Hengelin and c~-workers~~*~~ are concerned with the pulse radiolysis of solutions of poly(ethy1ene oxide) (PEO) poly(vinylpyrrolidone) and dextran. The hydroxyl radical reacts rapidly with these polymers although the rate constants change with chain-length and polymer concentration. Rate constants for bimolecular reaction of PEO radicals decrease with increasing chain-length as expected.The data are compared with theoretical models for polymer kinetics. The large number of papers summarised in the reports for 1968 and 1969 demonstrates the wide interest in reactions involving addition of hydroxyl radicals to aromatic molecules and also in the chemistry of the hydroxycyclo- hexadienyl radicals which result. Michael and Hart have obtained rate con- stants for reaction of OH H and e.& with benzene the 1,3- and 1,4-cyclohexa- dienes and cy~lohexene.~~ The dienes react with OH partly by addition and partly by hydrogen abstraction to form the C6H7.radical. The proportions abstracting were 30 % and 45 % for the 1,3- and 1,4-dienes respectively. Although the rate constants for reaction of OH and H are all in the range 3-10 x lo91 mol-'s-',electron reactivities vary considerably in cyclohexene and the 1,4- diene the rate constants are less than lo61 mol-'s-l whereas for the 1,3- derivative in which conjugation is present the rate constant is much larger (lo91mol-' s-').The trend is reversed in benzene where the rate constant falls to 1.2 x 10' 1 mol-' s-I an effect which is attributed to the large resonance energy of this compound. A similar study has been made of radical reactions in G. E. Adams and R. L. Willson Trans. Furaday Soc. 1969,65,2981. l9 K. Stockhausen A. Fojtik and A. Henglein Ber. Bunsengesellschaft Phys. Chem. 1970 74 34. 2o R. Wander B. L. Gall and L. M. Dorfman J. Phys. Chem. 1970,74 1819.'' G. E. Adams J. W. Boag and B. D. Michael Proc. Roy. Soc. 1966 A289 321. 22 B. L. Gall and L. M. Dorfman J. Amer. Chem. Soc. 1969,91 2199. " U. Borgwardt W. Schnabel and A. Henglein Makromol. Chem. 1969 127 176. 24 A. Behzadi U. Borgwardt A. Henglein E. Schamberg and W. Schnabel Ber. Bun-sengesellschuft Phys. Chem. 1970,74 649. '' B. D. Michael and E. J. Hart J. Phys. Chem. 1970,74,2878. 210 G. E. Adams and R. L. Willson pulse-irradiated aqueous solutions of ben~onitrile.'~ Both H and OH react rapidly by addition (kH = 3.1 x 10' 1 mol-'s-' and k, = 8.5 x lo91 mol-' s-'). The high rate-constant for reaction with e (1.7 x 10'O I mol-' s-l) illustrates the influence on the reaction cross-section of the electronegative CN group.The reaction which is non-dissociative produces the benzonitrile radical-anion which absorbs strongly in alkaline solution with maxima at 315 and 410 nm. Protonation occurs at pH 7.2 as is shown by a shift in the absorption spectrum. Below pH 3 H atoms which are formed by preferential electron scavenging by H,O+ react with the solute to yield a mixture of isomeric adduct-radicals. Nitrobenzene reacts with ea& to form the intensely absorbing anion C6H,N0, an intermediate in the reduction to nitro~obenzene.~' It is now reported" that short-lived hydrates of these nitrose derivatives are formed in the radiation- induced reduction of aromatic nitro-compounds. The mechanism involves the disproportionation of two protonated nitro-radicals e.g. XC6H,N0 H to form the hydrate OH / 2XC6H4NO2H -+ XC6H4N02 + XC6H4N (3) \ OH followed by the unimolecular elimination of water OH / XC,H,N \ -+ XC6H4N0 + HZO (4) OH The reaction proceeds through the protonated form and it was found that the acid equilibrium constant is related to the Hammett constant for the substituent X.Kinetic data from the pulse radiolysis of pyrollidine suggest that OH abstracts hydrogen to form the C,H,NH radical.29 Electron attack which is less efficient occurs at the nitrogen atom. In the presence of the electron scavenger CO, carboxylation occurs presumably by interaction between C4H,NH and the radical COY H The absorption spectrum of the radical intermediate of the ascorbic acid redox couple has been observed.The maximum is at 360nm and the extinction co- efficient 3.7 x lo31 mol-' cm-1.30 26 B. Chutny and A. J. Swallow Trans. Faraday SOC. 1970 66 284. " W. Grunbein and A. Henglein Ber. Bunsengesellschaft Phys. Chem. 1969 73 376. 28 W. Grunbein A. Fojtik and A. Henglein Monatsh. 1970 101 1243. 29 N. Getoff and F. Schworer Radiation Res. 1970 41 1. 30 B. H. J. Bielski and A. 0.Allen J. Amer. Chem. SOC.,1970 92 3793. Pulse Radiolysis Studies on Reactice Intermediates 21 1 3 Non-aqueous Systems Investigations by Sauer and co-workers into reactions of H atoms with organic molecules in the gas phase continues with a paper concerning substituent effects in monosubstituted benzene^.^ ’ Transient spectra and H atom reactivities have been obtained for some benzene and xylene derivatives naphthalene and pyridine.The rate constants for the reactions can be correlated with the Hammett CT values of the substituents and as in aqueous solutions of substituted benzenes,32 the form of the relationship suggests that H atoms are electrophilic in these reactions. This is in contrast with their reaction with olefins processes which have been classified as ele~troneutral.~~ An interesting application of microwave conductivity methods is the study of electron reactivity in pulse-irradiated fluorocarbon gases.34 This paper adequately illustrates the advantage of the technique for studying compounds of low reactivity. An important application of the pulse radiolysis techniques is the determination of free-ion yields in irradiated organic liquids.While this field is central to pro- gress in radiation chemistry much information of general relevance to inter- mediates in organic reactions derives from this work. Large differences can occur in free-ion yields from irradiated organic liquids. This is again illustrated by the values obtained by a pulse conductivity technique35 for neopentane (0-9) and n-hexane (0.13). The difference is attributed to the lower efficiency of electron localisation in the latter liquid a parameter which is a function of the geometry of the hydrocarbon molecule. Galvinoxyl or Coppinger’s radical has been used to measure ion-yields in a range of organic liquids including hydrocarbons alcohols and nitro-com- pounds.36 This stable free radical previously shown to be an efficient electron acceptor,37 absorbs strongly with a maximum near 600 nm.Where available free-ion yields obtained by electrical conductivity methods agree well with the yields of galvinoxyl anion. From the data relative values of the mean charge- separation distance were calculated and found to decrease with increasing polarity of the medium. Lithium aluminium hydride has been used as a positive-ion scavenger in the pulse radiolysis of tetrahydr~furan.~’ In this system the tran- sient spectrum of the solvated electron was observed. In the presence of pyrene or anthracene the anions are formed with yields of 0.66 and 0.70 when measured on microsecond time scales. However on nanosecond time scales yields are much higher.This rapid fall in yield is due to ion-recombination in regions of high local concentration (spurs). LiAlH, by functioning as a cationic scavenger gives increased yields and larger lifetimes of the anions. A primary ion yield of 2.6 ” M. C. Sauer jun. and I. Mani J. Phys. Chem. 1970,74 59. 32 M. Anbar D. Meyerstein and P. Neta Nature 1966 209 1348. 33 R. J. Cvetanovic Adu. Photochem. 1963 1 115. 34 R. W. Fessenden and K. M. Bansal J. Chem. Phys. 1970,53,3468. 35 P. H. Tewari and G. R. Freeman J. Chem. Phys. 1969,51 1276. 36 C. Capellos and A. 0.Allen J. Phys. Chem. 1970,74 840. 37 M. S. Karasch and B. S. Joshi J. Org. Chem. 1957,22 1435. 38 J. H. Baxendale D. Beaumond and M. A. J. Rodgers Trans. Furuduy Soc. 1970 66 1996. 212 G.E. Adams and R. L. Willson was measured and in anthracene solutions. emission due to singlet anthracene was recorded. The low singlet yields (G = 0.1) and also that of the triplet (G = 0.04)are both concentration-invariant in contrast with the behaviour in benzene di~xan,~~ and dimeth~xyethane~' where the excited state yields are much higher. A possible explanation is discussed. Electron-trapping in low-temperature organic glasses have been studied by Richards and Thomas41 using 3-methyl pentane 3-methyl hexane 2-methyl tetrahydrofuran cumene and ethanol. Changes in short-lived transient spectra have been attributed to reorganisation of trapping sites of variable depth. Annealing of pulse-irradiated 3-methyl hexane containing naphthalene revealed the formation of excited states arising from ion-recombination reactions.Fast infra-red detection was used to record the absorption spectra of solvated electrons in ethylene diamine-water and amrn~nia-water.~~ In pure diamine or ammonia the spectra resemble those of the solvated electron in alkali-metal solutions. The dependence of the spectral characteristics on the composition of the two-component systems suggests that the electron is delocalised with proper- ties determined by the aggregate properties of the mixed solvent and not by solvation with a small number of solvent molecules. In pulse-irradiated ethanol43 the free-electron yield derived from the data (G = 1.7) is considerably higher (60%) than that found previously in both stationary-state and pulse experiments.Reasons for this discrepancy are discussed. An unusual series of experiments involved the nanosecond pulse radiolysis of molten napthalene (100 "C)and benzophenone (35 "C)containing 1,2-benz- anthra~ene.~~ Singlet- and triplet-excited solute spectra were formed by excita- tion transfer from the solvent. Ions do not appear to be involved in the transfer. An excited state of all-trans-j3-carotene assumed to be the lowest triplet can be produced by photosen~itisation.~~ The absorption spectrum of this species has now been observed without a sensitiser present in pulse-irradiated hexane solutions.46 The lifetime is rather long (9 ,us) and it is suggested therefore that failure to observe this species in previous flash-spectroscopic studies is due to the very small efficiency for singlet-triplet conversion.Excitation by high- energy radiation in hydrocarbons permits the formation of some triplets inde- pendent of singlet formation. The systematic studies of Dorfman and co-workers on electron- and proton- transfer reactions involving aromatic radical-ions have been extended to include some cationic electron-transfer processes.47 Some aromatic compounds were 39 J. H. Baxendale and M. A. J. Rodgers J. Phys. Chem. 1968,72,3849. 40 T. J. Kemp and P. J. Roberts Trans. Faraday SOC., 1968,64,'2106. 41 J. T. Richards and J. K. Thomas J. Chem. Phys. 1970,53 218. 42 J. L. Dye M. G. DeBacker and L. M. Dorfman J. Chem. Phys. 1970,52,6251. 43 J. W. Fletcher P. J. Richards and W.A. Seddon Canad. J. Chem. 1970,48 1647. 44 R. A. Holroyd L. M. Theard and F. C. Peterson J. Phys. Chem. 1970,74 1895. 45 M. Chessin R. Livingston and T. G. Truscott Trans.Faraday SOC.,1966 62 1519. 4b E. J. Land A. Sykes and T. G. Truscmt Chem. Comm. 1970 p. 332. 47 N. E. Shank and L. M. Dorfman J. Chem. Phys. 1970,52,4441. Pulse Radiolysis Studies on Reactive Intermediates 213 + pulse-irradiated in dichloroethane (DCE) solution. Charged species (DCE ) react with the aromatic solute by electron transfer DCE+ + Ar -+DCE + Ar+ (6) The system was used to investigate cationic electron transfer between pairs of aromatic molecules e.g. to p-terphenyl+ from anthracene and to biphenyl' from p-terphenyl and pyrene. Transfer constants are or are near diffusion- limited values in the range 5.1-9-9 x lo91mol-'s-' at 25 "C.The rate of electron transfer from the aromatic molecules to the solvent cation is so high as to suggest that migration of the solvent cation may involve an electron-jump process in the solvent. Biihler in a recent review,48 has summarised his work on pulse-radiolysis studies of transient charge-transfer complexes with halogen atoms. A current paper reports4' the observation of an absorption band at 560nm formed on pulse radiolysis of deaerated bromobenzene. It is assigned to the charge-transfer complex of a bromine atom with bromobenzene. Alter- native assignment to ions or excited states are ruled out. 4 Systemsof Biological and Biochemical Interest Further work is reported on the reaction of primary-water free-radicals with derivatives of the nucleic acids although owing to the complexity of the systems the assignment of transient spectra has depended principally on the results of product analysis from stationary-state system^.^^^^ Acautionary note has been struck by the observation of transient spectral changes which arise from reaction of pyrimidines with hydroxide ions formed by the radiation pulse.52 The spectral changes associated with this process are not necessarily related to the nature and yield of the final products of the radiolysis.The phenomenon has been observed in solutions of thymine and uracil but not thymidine thymidylic acid or 1,3- dimethyl uracil where the spectra of the tautomeric forms of the solutes are similar.It is well established that in neutral solution the hydroxyl radical adds to the 5,6-double bond in pyrimidine^.'^ It has now been shown that the ionised radical 0-, behaves similarly in alkaline solutions of uracil and cytosine where the pyrimidine structures are i~nised.~~'~ However for thymine and 5-methyl 48 R. E. Buhler in 'Quaderni de La Ricerca Scientifica' No. 68 Consiglio Nazionale delle Ricerche Rome 1970 p. 79. 49 J. M. Bossy R. E. Buhler and M. Ebert J. Amer. Chem. SOC.,1970 92 1099. 50 J. D. Zimbrich J. F. Ward and L. S. Myers jun. Internat. J. Radiation Biol. 1969,16 505. 51 J. D. Zimbrich J. F. Ward and L. S. Myers jun. Internat. J. Radiation Biol. 1969 16 525. 52 E. M. Fielden G. C. Stevens J.M. Phillips G. Scholes and R. L. Willson Nature 1970 225 632. 53 G. Scholes J. F. Ward and J. Weiss J. Mol. Biol. 1960,2 379. 54 L. S. Myers jun. A. Warnick M. L. Hollis J. D. Zimbrich L. M. Theard and F. C. Peterson J. Amer. Chem. SOC.,1970 92 2871. 55 L. S. Myers jun. M. L. Hollis L. M. Theard F. C. Peterson and A. Warnick J.Amer. Chem. SOC.,1970,92,2875. 2 14 G. E. Adam and R.L. Willson cytosine transient spectra were assigned to radicals formed by H-atom abstrac- tion from the methyl group. The H-adduct of thymine is not identical with the protonated electron-adduct. The transient spectrum produced by H-atom abstraction following OH attack on dihydrothymine is almost identical with that formed by H-atom addition to thymine.55 The spectra differ considerably 0 0 0 U Me H H from that observed for thymine after direct electron attachment and protonation thus supporting earlier suggestions that electron addition is probably localised on the carbonyl groups.Although electron adducts of most pyrimidines have low extinction coefficients that for orotic acid 6-carboxypyrimidine (OA) is an exception. At 330 nm the extinction coefficient is 8.5 x lo31mol- 'cm".57 This spectrum is also quite different from that of the H-adduct. The effect of the carboxy-group on the elec- tron affinity of the molecule is illustrated by its ability to oxidise the ethanol radical by simple electron transfer MeCHOH + OA -+ MeCHO + OA-+ H,O+ (8) The suggestion that OA- forms a peroxyl radical with oxygen contrasts with the conclusion that the thymine electron adduct reacts with O2 in a fast electron- transfer reaction,58 (k = 8 x lo91 mol-' s-').This agrees with earlier findings that the reaction of e with thymine in oxygenated solution does not lead to thymine destruction. In general rate constants for reaction of oxygen with OH-adducts of various DNA derivatives tryptophan and histidine appear to be diffusion controlled.60 Data for thymine agree reasonably well with earlier values.6 1,62 Reactivities of nucleotide and nucleoside radicals are about half those of the radicals derived from the corresponding free bases. Radical reactivity studies63 with the cellular radiosensitiser 2,2,6,6-tetramethylpiperidin-4-oneN-oxide (triacetoneamine N-oxyl) have been extended to include various DNA derivatives.In particular the reaction of the OH-adduct of calf thymus DNA has been observed. In general 56 L. S. Myers jun. and L. M. Theard J. Amer. Chem. Soc. 1970,92,2868. '' C. L. Greenstock Trans. Faraday Soc. 1970,66 2541. 58 H. Loman and M. Ebert. Internat. J. Radiation Biol.. 1970 18. 369. 59 G. Scholes and R. L. Willson Trans. Faraday SOC.,1967 63 2983. 6c R. L. Willson Internat. J. Radiation Biol.,1970 17 349. " P. T. Emmerson and R. L. Willson J. Phys. Chem. 1968 72 3669. '' L. M. Theard and F. C. Peterson in 'Radiation Chemistry' ed. R. F. Gould (Advances in Chemistry Series No. 81) 1968 vol. 1 p. 603. 63 R. L. Willson and P. T. Emerson 'Radiation Protection and Sensitisation' ed.H. Moroson and M. Quintiliani Taylor and Francis London 1970 p. 73. Pulse Radiolysis Studies on Reactive Intermediates 215 the reaction rate constants are an order of magnitude lower than the correspond- ing values for oxygen. Several publications have appeared dealing with the reactivities of e, H and OH with substituted amides peptides and enzymes. Hydrogen abstraction by OH from the N-methyl group in N-methyl amides is at least an order of magnitude faster than H-atom abstraction from the a-methyl The marked pH effect on the OH reactivity of the simple peptides glycylglycine and glycylglycylglycine had been previously attributed to the deactivating effect of NH protonation on the rate of electrophilic OH attack.65 This has now been by the demonstration that the pH effect is removed by prior acetyla- tion of both glycine and glycylglycine.The use of narrow band-widths and hence high spectral resolution in the analysis of transient spectra from pulse-irradiated tryptophan solutions has revealed the existence of radical- isomer^.^ The fine structure of the transient absorption band in the 300-350 nm region which is assigned to the OH-adduct shows three subsidiary maxiqa. It was found by comparing OH-adduct spectra from several methyl-substituted tryptophans that the three maxima represent attack at C-2 (A,, = 345 nm) at C-3 (350 nm) and some site on the aromatic ring (310nm). This use of fine resolution indicates obvious future applications for the analysis and identification of isomeric transient species where multiple possibilities exist for the location of the radical centre.Pulse radiolysis studies6* on solutions of glutathione show that H atoms add to the sulphur bond to form a species which is identical to that formed by protona- tion of the electron adduct of disulphide corn pound^.^^ The spectrum of the uncomplexed RS. radical is not observed indicating that dissociation of the RSSR.H radical does not occur. An interesting reaction has been observed in irradiated solutions of ~elenourea.~' A transient absorption similar to that observed previously for thiourea has been assigned to the radical-ion RSeSeR-. It is concluded that the reaction probably involves abstraction of a hydrogen atom from the -SeH group of the enol structure followed by association with another solute molecule.H,N-C=NH H2N-C=NH -I I Se' 1 ] SeH -H+ (9) I H2N-C=NH -[H,N-C=NH " E. Hayon T. Ibata N. N. Lichtin and M. Simic J. Amer. Chem. Soc. 1970,92 3898. h5 G. Scholes P. Shaw R. L. Willson and M. Eberi in 'Pulse Radiolysis' ed. M. Ebert J. P. Keene A. J. Swallow and J. H. Baxendale Academic Press London and New York 1%5 p. 151. 66 M. Simic P. Neta and E. Hayon J. Amer. Chem. Soc. 1970 92 4763. 67 R. C. Armstrong and A. J. Swallow Radiation Res. 1969 40,.563. h8 M. Simic and M. Z. Hoffman J. Amer. Chem. Soc. 1970,92,6096. '* G. E. Adams. G. S. McNaughton and B. D. Michael in 'The Chemistry of Ionization and Excitation' ed. G. R. A. Johnson and G. Scholes Taylor and Francis London 1967 p.28 1. R. Badiello and E. M. Fielden. internat. J. Radiation Biol. 1970 17 I. 216 G. E. Adams and R. L. Willson Spectra similar to the RSSR-ion-complex have now been observed on pulse radiolysis of the enzymes ribonuclease chymotrypsin and papain and the coenzyme lip~ate.'~?~ These have been assigned to electron adducts where the odd-electron is localised on the sulphur bridges. The absorptions are long- lived relative to those from simple disulphides such as cystamine which suggests that the RSSR-radical is stabilised by the overall structure of the macromolecules. Pulse radiolysis continues to provide a kinetic method for the study of protein binding irrespective of whether metachromasia is present or not.7 2-7 Overall reactivities of e,; in solutions of penicillin G in the presence of lysozyme or bovine serum albumen deviate from values calculated from the reactivities of the indi- vidual components.On addition of salt equivalence is restored thus indicating the presence of binding in the salt-free medium. No effect was observed however with pencillin G methyl ester or with lysozyme containing lysine residues which had been chemically modified with 2,4,6-trinitrobenzene sulphonic acid. This suggests that the carboxy-group of penicillin G and the lysine residues in lysozyme participate in the binding process.73 Similar studies are reported on the binding of eosin to protein7' and on the effect of temperature on the electron reactivity of methylene blue and various polyanion~.~~ Attempts were made to estimate thermodynamic parameters for the polyanion-dye reaction.Although to date work on aqueous systems has been concerned principally with the study of reactions of the primary water radicals e,; H and OH the pulse radiolysis technique provides a convenient method for the systematic study of a wide range of free radicals in solution. By the appropriate use of radical scavengers experimental systems can be designed in which a given free radical can be studied in isolation. Some recent investigations of reactions of halide radical-ions e.g. X; illustrate potential applications. In N,O-saturated solu-tions where e,; is effectively replaced by OH ea; + N,O -+ N + OH+OH-(10) the halide radical-ion is formed by subsequent reaction between OH and the halide.It has been shown that the radicals Br; (CNS) react with some enzymes including lysozyme ribonuclease and chymotrypsin.' In addition there is some indication that simple iodine atoms can react with alcohol dei~ydrogenase.~~ There is increasing evidence that some of these reactions are specific for certain amino-acids and when inactivation data are also available are useful for investi- gating enzyme structure. 71 R. L. Willson Chem. Comm. 1970 1425. 72 J. S. Moore G.0.Phillips J. V. Davies and K. S. Dodgson Carbohydrate Res. 1970 12 253. 73 G. 0.Phillips D. M. Power C. Robinson and J. V. Davies Biochim. Biophys. Acta 1970 215 491. J. S. Moore G.0.Phillips D. M. Power and J. V. Davies J. Chem. Sor. (A),1970,1155. '' A.Husain J. Ovadia and L. I. Grossweiner Trans. Faraday Soc. 1970,66 1472. 76 J. V. Davies M. Ebert and M. Quintiliani 'Radiation Protection and Sensitisation' ed. H. L. Moroson and M. Quintiliani Taylor and Francis London 1970 p. 87. Pulse Radiolysis Studies on Reactive Intermediates 217 Chemiluminescence occurring after pulse radiolysis of aqueous solutions of acriflavin can be enhanced three-fold in the presence of halide ions.77 Reaction of e; with the product of reaction between acriflavin and the halide radical is believed to be the cause of the increase in luminescence. The radical-ion Br; also reacts with the coenzyme nicotinamide adenine dinucleotide (NADH) to form the NAD. radical.78 The reaction of NAD. with O2is thought to proceed by simple electron transfer since complementary stationary-state studies show the formation of the oxidised product NAD+.79 The formate radical-ion COY has been used as a one-electron donor in several systems including the reduction of riboflavin and lipoate (RSSR).71 COY + RSSR -+ COz + RSSR-(11) A similar reaction occurs on pulse radiolysis of lysozyme solutions containing formate ion.13 The rate of formation of the RSSR- absorption indicating elec- tron transfer to the disulphide bridges is much slower than the corresponding reaction with e;.Pulse radiolysis studies on quinones continue to be of interest. Reaction of ubiquinone with the methanol radical CH20H yields the semiquinone ion which rapidly protonates in acid solution.80 From pH studies the pK of this equilibrium was found to be 6-45 & 0.15 a value significantly larger than that for the simple benzsemiquinone radical.*’ Following irradiation of an aqueous solution of acetone isopropanol NAD oxygen and benzoquinone sequential electron + transfer along the chain was observed.82 Some fast one-electron oxidation reactions have been observed in pulse-irradiated solutions of simple nucleotides and some cellular radiosensitisers including N-ethylmaleimide (NEM) and 2-methyl naphthaq~inone.~~ Electron transfer from the electron-adducts of the nucleotides was indicated by the formation of the transient spectra of the NEM electron-adduct and the naphthasemiquinone anion.Further information relevant to excitation or electron transfer and to radio-sensitisation mechanisms in organic and biological matrices is provided by measurements of luminescence following pulse radiolysis of solid mixtures.Incorporation of 5-bromodeoxyuridine (5-BUdR) into DNA notably affects the luminescence characteristics of DNA consistent with extensive energy or electron migration to the halogenated base.84 This result is interesting with respect to 77 W. A. Prutz and E. J. Land J. Phys. Chem. 1970,74,2107. 78 E. J. Land and A. J. Swallow Biochem. J. 1969,116 16P. 79 E. J. Land and A. J. Swallow Abstracts of the Fourth International Congress on Radiation Research Evian 1970 p. 127. E. J. Land and A. J. Swallow J. Biol. Chem. 1970,245 1890. G. E. Adams and B. D. Michael Trans. Furuduy SOC.,1967,63 1171.82 R. L. Willson Chem. Comm. 1970 1005. 83 C. L. Greenstock G. E. Adams and R. L. Willson ‘Radiation and Protection and Sensitization’ ed. H. L. Moroson and M. Quintiliani Taylor and Francis London p. 65. 84 E. M. Fielden and S. C. Lilhcrap in ‘Radiation Protection and Sensitisation’ ed. H. L. Moroson and M. Quintiiiani Taylor and Francis London 1970 p. 81. G. E. Adams and R. L. Willson suggested mechanisms for radiosensitisation of organisms in which 5-BUdR is in~orporated.~’.~ General application of pulse radiolysis studies on lumin- escence in the solid state has been reviewed.86 G. E. Adams in ‘Current Topics in Radiation Research’ ed. M. Ebert and A. Howard North-Holland Pub. Co. 1967 p. 35. 86 E. M. Fielden in ‘Quaderni de La Ricerca Scientifical Consiglio Nazionale delle Ricerche Rome 1970 p.63.