ESR of
The Journal of Physical Chemistty, Vol. 83, No. 22,
Peptide Radicals in Frozen Solutions
1979 2887
An Electron Spin Resonance Study of y-Irradiated Frozen Aqueous Solutions Containing Dipeptides. Mechanisms of Radical Reaction Michael D. Seviila, James B. D’Arcy, and Klm M. Morehouse Department of Chemistty, Oakland University, Rochester, Michigan 48063 (Received May 3, 1979) Publication costs assisted by the U.S. Army Natick Research and Development Center and the US.Department of Energy
The y irradiation at 77 K of frozen aqueous solutions of over 14 peptides was investigated by ESR (electron spin resonance) spectroscopy. The investigation was composed of three parts. The first part consists of a study of simple dipeptides of glycine and alanine, Le., Gly-Ala, Ala-Gly, and Ala-Ala. Detailed analyses were made of the radicals produced by the irradiation, their relative concentrations, and their stability as a function of temperature. The results show evidence for anion formation, primary deamination, and decarboxylation initially. This is followed by abstraction from the parent compound to form the a-carbon radical at the C-terminal residue. In the case of Ala-Ala there is evidence for “hole” stabilization at low temperature. In the case of Gly-Ala some secondary deamination is noted. In the second part the analysis of the final spectra for 11dipeptides are reported. The final radicals were all found to be a result of abstraction from the a carbon of the C-terminal residue to produce the radical NH3+CHRCONHCR’C0f.Radicals with R’ = H or CH2R”produced a hyperfine coupling due to one proton of 18-19 G. Since 15 of the 20 common amino acid residues have a structure with R’ = H or CH2R”,the dominant “final” radical in proteins should produce a doublet spectrum. In the final portion, three dipeptides with aromatic side groups were studied in frozen HzO solutions. It was found that electron attachment to the aromatic ring effectively competed with deaminationof the primary amine group. The electron attachment to the aromatic ring was followed by protonation at a carbon site on the ring. The results for Gly-His were typical. Here we found a spectrum indicative of 25% protonation, 30% deamination, and 45% decarboxylation. For Gly-Tyr, phenoxy1 radical formation was found to dominate decarboxylation as a decomposition pathway for the hole.
Introduction The radiation chemistry of peptides has been investigated by a variety of techniques including ESR spectro~copy,’-’~ pulse r a d i o l y ~ i s , ’ ~and - ~ ~product analysis.24 These studies have been carried out in irradiated aqueous solutions and single crystals as well as aqueous glasses. The major reaction pathway of the primary anionic intermediate has been characterized for irradiated simple peptides with nonreactive side groups and does not greatly depend on the m a t r i ~ . ’ ~ ~ Thus, ~ ~ ~ *all J ~techniques J~ agree on the following pathway (where R is an aliphatic side group):
have attempted to characterize and quantify the various radical intermediates and to study their decay mechanisms.
Experimental Section The peptides employed in this work were obtained from Sigma. The experimental techniques employed in this work are similar to those employed by Gregoli, Olast, and Bertinchamps in studies of frozen solutions of DNA cons t i t u e n t ~ .In~ ~our work, solutions of peptides in D20 or HzO,ranging in concentration from 20 to 200 mg/mL, were frozen quickly in liquid nitrogen. The samples were opaque and polycrystalline in nature. The samples are NH3+CHRC(O-)NHCHR’CO2assumed to be composed of microcrystals of ice as well as frozen out solute containing various amounts of water NH3 + CHRC(=O)NHCHR’C02- (1) (“frozen puddles”). These samples were than y irradiated CHRC(=O)NHCHR’COz- + (Co-60) with a dose of 0.5 Mrd at 77 K. Dose studies showed the radical production was linear with dose to over NH3+CHRC(=O)NHCHR’C022 Mrd. Concentration studies showed that radical proCHzRC(=O)NHCHR’CO2- + duction was also linear with solute concentration. From NH3+CHRC(=O)NHCR’COz- (2) these studies and those of Gregoli et al., we conclude that the major effect of radiation in these systems is the direct Secondary deamination of the anion has been found to effect. Apparently, radicals formed by the radiolysis of compete with primary deamination in aqueous solutions the ice microcrystals cannot migrate to the solute. All a t room t e m p e r a t ~ r e . ~ samples were annealed to 120 K to remove the background The major reaction pathway for the “hole” has been signal due to the irradiation of the matrix, D20 or H20. suggested to be decarboxylation. This reaction, (3), has The ESR spectrometer used was a Varian V-4500-10A NH3+CHRC(=O)NHCHR’COZ. unit, operating at X-band (-9.26 GHz) fitted with a dual cavity by using a 100-kHz sample channel detection and NH,+CHRC(=O)NHCHR’* + COZ (3) a 400-Hz reference channel. Fremy’s salt (potassium been studied in only a few amino acids, peptides, or peroxylaminedisulfonate) was used as reference for peptide analogues in single crystal^.^^^^^ measuring hyperfine splittings and g values (AN = 13.1 G, For peptides containing reactive side groups, initial g = 2.0056). A Tektronix 4051 minicomputer was used for reactions at the side group must be c ~ n s i d e r e d . ~ summing ~ ~ ~ ~ ~and ~~ ~ ~ ~of spectra on magnetic tapes. The storing In this work we have investigated the y irradiation of three peak positions of the Fremy’s salt were included in peptides in a new matrix frozen aqueous ~olution.’~ We all stored ESR spectra. +
-
-+
0022-3654/79/20a3-2aa7$01
.oo/o 0 1979 American Chemical Society
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The Journal of Physical Chemistry, Vol. 83, No. 22, 1979
Flgure 1. ESR spectra found in aqueous glasses for radicals of form RCH,. (A) CH2CO; (I) produced by electron attachment to chloroacetic acld in 12 M LiCI-D20 at 120 K. (6)-CH2CONDCH2C02(11) produced by electron attachment to N-chloroacetylglycine in 12 M LiCl at 120 K. (C) ND3+CHRCONDCH2. (111) produced by photolytic decarboxylation of tryptophylglycine in 8 M NaCI0,-D,O at 77 K (ref 14). The markers in the spectra represent the components of the Fremy salt spectrum. They are separated by 13.09 G with g = 2.0056.
The ESR spectra, for each sample, were normally recorded at temperatures between 100 and 220 K with a Varian V4540 variable low-temperature accessory.
Sevilla, D'Arcy, and Morehouse
Figure 2. ESR spectrum found in aqueous glasses for radials of form RCHCH,. (A) CH&HCO,- (IV) produced by electron attachment to 2-bromopropionic acld in 12 M LICI-D,O at 120 K. (6) CH,CHCONDCHRCO,- (V) produced by electron attachment to Lalanylglycine In 12 M LiCI-D20 at 120 K. (C) ND,+CHRCONDCHCH, radical (VI) produced by photolytic decarboxylation of L-phenyialanyl-L-alanine at 77 K in 8 M NaCIO4 (ref 14).
A
Results and Discussion A. Glycyl- and Alanyl-Containing Dipeptides. 1. Radicals Expected. From a consideration of previous studies of dipeptides and N-acetyl peptides in aqueous glasses'&l5 and single crystals,@we expect radicals I-VI11 .CH,CO,-
.CH,(=O)NDCHRCO,-
I I1 ND,+CHRC(=O)NDCH,. .CHCH,CO,I11 IV CHCH,C(=O)NDCHRCO,- ND,+CHRC(=O)NDCHCH, V
VI
ND ,+CHRC(=o )NDCR#co >- ND ,+cHRC( 0-INDCHR'co 2VI1 VI11
in y-irradiated dipeptides containing glycyl and alanyl residues. Each of these species have been observed previously in our work in aqueous glasses.1F15 We show the spectra of radicals of types I, 11, and I11 in parts A-C of Figure 1,respectively, and we show those for types IV, V, and VI in parts A-C of Figure 2, respectively. These spectra are of radicals in aqueous glasses and were produced for this investigation by our previously described technique^.^^'^ As can be seen in the figures, although the major hyperfine interactions are similar for radicals I, 11, and 111, the spectra are easily distinguishable by the anisotropic hyperfine couplings, the overall width, and the isotropic splittings. The same argument applies for radicals IV, V, and VI. For example, the overall width of the spectra in parts B and C of Figure 1are 43 (2 H at 21.5 G) and 39 G (2 H at 19.5 G), respectively, whereas in parts B and C of Figure 2 the values are 96 (4H at 24 G) and 85 G (4 H at 21.3 G ) . The differences in lineshape and anisotropic structure should also be noted. Spectra from these radical species are not expected too differ signifi-
Figure 3. ESR spectra found after annealing to various temperatures for y-irradiated frozen aqueous solutions of L-alanylglycine (200 mg/mL DZO): (A) 120 K, (B) 160 K, (C) 180 K, (D) 210 K.
cantly in the frozen aqueous solutions employed in this work. For example, a detailed comparison of Figures 1A and 2A with the same species produced in frozen ice matrices show only very slight changes. Thus, these spectra are used as a basis for the analysis of results found from glycylalanyl dipeptides in this work. 2. Alanylglycine, Glycyl-L-alanine, and L-Alanyl+ alanine. In Figures 3-5, we report the results found after y irradiation of 200 mg/mL D20 samples of Ala-Gly, Ala-Ala, and Gly-Ala. Each of the spectra found at various temperatures has been analyzed in terms of the radical species expected from our previous work. Table I reports the results from the computer analyses of these spectra. The fractions of radicals present at each temperature have been determined by subtraction of the spectra in Figures 1 and 2 from those in Figures 3-5. The estimates of percent composition are considered to be accurate to within 10%. Although this technique assumes the radical
The Journal of Physical Chemistty, Vol. 83, No. 22, 1979 2889
ESR of Peptide Radicals In Frozen Solutions
TABLE I: Estimates of Relative Radical Yields for y-Irradiated Frozen Aqueous Solutions of Glycylalanyl Dipeptides yield,b % dipeptide Ala-Gly
processa DC DA Ab An
radical
III ( R = CH,) V ( R = H) VI1 ( R = CH,, R’ = H) VI11 ( R = CH,, R = H)
120 K 55 25 15 5, est 120 K
Ala-Ala
Gly-Ala
DA DC Ab An Ho
V (R = CH,) VI ( R = CH,) VI1 ( R = R’ = CH,) V I I I ( R = R ’ = CH,) I X ( R = CH,)
DA DC Ab SDA
I1 ( R = CH,) VI (R = H) VI1 (R = H, R’ = CH,) IV
30 25
160K
180K
210K
50 30 20
30 30 40
100
165KC
205KC
215K
45 40 5
45 20 25
100
I45
35 40 10 10
10
40 35 10
100
a These terms have the following meanings: DC, decarboxylation; DA, deamination; Ab, abstraction, An, anion; Ho, Yields are relative at each temperature, however, the total radical concentration did hole; SDA, secondary deamination. The totals do not add t o 100% in these cases because of the presence not decrease appreciably until warmed to >ZOO K. of unidentified species.
n
Flgure 4. ESR spectra found after annealing to various temperatures for y-irradlated frozen aqueous solutlons of L-alanyl-L-alanine (200 mg/mL of D,O): (A) 120 K, (B) 165 K, (C) 205 K, (D) 215 K.
types before analysis, it should be noted that in each case there is excellent evidence for these radicals. For Ala-Gly (Figure 3A) a 97-G wide quintet is found as expected for radical V (R = H). In addition, the outer components show the hyperfine structure which is characteristic of this species (see Figure 2B). For Gly-Ala (Figure 5A) the observed 88-G wide quintet can only arise from radical VI (R = H). For Ala-Ala an overlap of two quintets is apparent with an 87-G wide quintet easily observed at 120 K (Figure 4A) and a 96-G wide quintet increasing in relative intensity at 205 K (Figure 4C). This is considered good evidence for the presence of both radical VI (R = CH,) and radical V (R = CH,). Below, we give an example of the computer subtraction technique followed by a more detailed discussion of each dipeptide. As an example of the computer subtraction technique we show the analysis of Figure 3B in Figure 6. Figure 6A is the same spectrum as in Figure 3B. Figure 6B shows the spectrum after subtraction of 30% of the spectrum of radical V (R = H) (Figure 2B). Figure 6C shows the spectrum after subtraction of 20% of the spectrum of radical VI1 (R = CH,, R’ = H)(Figure 3D).This spectrum
Flgure 5. ESR spectra found after the y irradiation of frozen aquaous solutlons of glycyl-L-alanine (225 mg/mL): (A) 130 K, (B) 190 K, (C) 215 K.
(6C) shows a triplet of 39-G total width which is identified as radical I11 (R = CH3) (see Figure 1C for comparison). The data in the table result from similar analyses. Ala-Gly. The results for Ala-Gly given in Table I and in Figure 3 clearly show the following: The radicals due to decarboxylation (111, R = CH3) and deamination (V, R = H)are produced initially. As the sample is warmed, the decarboxylated radical (111) converts to the abstracted radical (VII, R = CH,, R’ = H). For example, notice that the doublet from radical VI1 has increased in intensity at 180 K in Figure 3C while the fraction of radical I11 decreases from 55 to 30%. Radical V does not decrease as the temperature is raised to 180 K. In fact, we believe the slight increase shown in Table I is the conversion of residual anion to deaminated radical (V). At 210 K radical V also converts to the abstracted radical (VII). Ala-Ala. In the case of Ala-Ala we initially observe spectra of the deaminated radical (V, R = CH3), the decarboxylated radical (111,R = CH,), and a large fraction
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The Journal of Physical Chemistry, Vol. 83, No. 22, 1979
Flgure 6. ESR spectra showing the computer subtraction technique for Ala-Gly. (A) Irradiated Ala-Gly at 120 K. (B) Spectrum after subtraction of 30% of radical V (R = H) (Figure 2B) from (A). (C) Spectrum after subtraction of 20% of the spectrum of radical VI1 (R = CH,, R’ = H) (Figure 3D) from (B). This species is identified as radical I11 (R = CH,). Compare with Figure 1C.
of radicals, 45% which produce the central component in Figure 4A. Upon warming both the amount of deaminated and decarboxylated radicals increase. We, thus, believe that this central component is due to the anion (VIII) and the “hole” (IX, R = CH3). ND3+CHRCONDCHCH&202* IX If true, this is surprising, since radicals such as IX are Further warming to normally unstable above 77 K.7*9~28 205 K results in a loss of the decarboxylated radical with a corresponding increase in abstraction radical VI1 (R = R’ = CH,). The deamination radical remains stable. Warming to 215 K results in all radicals converting to the abstracted species (VII). Gly-Ala. The results given in Table I for Gly-Ala show two unusual features. First, there is evidence (Figure 5A,B) for secondary deamination (IV). This is a small fraction of the primary deamination radical (11)but IV is clearly observed. Second, we find that the deaminated radical (11) abstracts before the decarboxylated radical VI. The reverse was found for Ala-Ala and Ala-Gly. We believe that both of these findings are due to the increase in stability provided by secondary radicals such as IV and VI (R = H) relative to the primary radical I1 (R = CH3). The final radical formed in Gly-Ala was studied as a function of the pD of the original solution in Figures 5C and 7A,B. We show the spectra found at elevated temperatures (210 K) for pD 6, 9, and 12, respectively. What the spectra show is, as the pD increases, the 19-G quartet is replaced by a more poorly resolved 16-G doublet structure with an anisotropic nitrogen coupling parameters (All= 21.5 G and g, - gil = 2 X lo-,). This leads us to conclude that the abstraction site shifts at higher pH to form a radical such as X or XI. ND2CHCONDCHCH3C02X NDzCH2C(=O)NCHCH3C02XI Reaction Mechanisms. The results for Ala-Gly, Gly-Ala, and Ala-Ala show evidence for anion production (reaction
Sevilla, D’Arcy, and Morehouse
Flgure 7. ESR spectra found after the y irradiation of frozen aqueous solutions of glycyl-L-alanine under basic condltlons: (A) 200 K, pD 9; (B)200 K, pD 12.
4), primary deamination (reaction 51, and decarboxylation (reaction 6). In the case of Gly-Ala we find evidence for ND,+CHRC(eo)NDCHR’C02-
+e-
[ND3+CHRC(=O)NDCHR’COz-](4) VI11
-
[ND,+CHRC (EO) NDCHR’C02-1-
+
ND3+ + .CHRC(=O)NDCHR’CO,- (5a) 11, v ND,+CHRC(=O)ND- t CHR’COZIV (5b)
-e-
ND3+CHRC(=O)NDCHR’COZND,+CHRC(=O)NDCHR’ t COz (6) II1,VI a small fraction of radical IV which must be produced by secondary deamination of the anion (reaction 5B). This reaction (reaction 5B) is clearly less favorable than (5A) in all cases investigated. However, the reaction has important implications for the radiolysis of proteins, since at room temperature this reaction has been suggested to effectively compete with primary d e a m i n a t i ~ n . ~ Radicals I11 and VI are found to undergo the abstraction reaction (reaction 7) starting with temperatures near 180 ND,+CHRC(=O)NDCHR’ + 111, VI ND3CHRC(=O)NDCHR’C02ND3+CHRC(=O)NDCHZR’ + ND3+CHRC(=O)NDCR’C02- (7) VI1 K. Radical V undergoes the abstraction reaction (reaction
-
CHRC(=O)NDCHR’CO,- + 11, v ND,+CHRC(=O)NDCHR’CO2CH2RC(=O)NDCHR’C02- + ND,+CHRC(=O)NDCR’C02- (8) VI1 +
8) at temperatures above 200 K. The exception to the above is in the case of Gly-Ala where radical I1 (R = CH3) abstracts before radical VI (R = H).
ESR of Peptide Radicals in Frozen Solutions
The Journal of Physical Chemistry, Vol, 83, No. 22, 1979
TABLE 11: Final Radicals Observed in ylrradiated Dipeptide Solutions concn (D,O), dipeptide mdmL radical suggested Gl y-Gly 200 ND; CH, C( = O)NDCHCO,Ala-Gly 200 ND,~CHCH,C(=O)NDCHCO,Gly-Ala 180 ND,+CH,C(= O)NDCCH,CO; Ala-Ala 180 ND;CHCH,C(=O)NDCCH,CO,100 ND,+CH, CH,C( = O)NDCHCO,P-Ala-Gly 200 ND;CH,C(= O)N(CH,)CHCO,G1y-Sar 21 ND,+CH,C(= O)NDC(CH,CO,H)CO,Gly-Asp 31 ND,TH,C(=O)NDC(CH,CH,CO,H)CO,Gly-Glu 60 ND,+CH,C(= O)NDC(CH,CH,SCH,)CO,Gly-Met 220 ND,+CH,C(= O)ND~(CH,OH)CO,Gly-Ser 125 ND,+CH[ (CH,),ND,’ IC(=O)NDC[(CH,),ND,+]CO,~ Lys-Lys
2891
hyperfine splitting, G 18.1 (1H ) 18.4 (1H) 19.2 (3 H ) 19.1 (3 H) 18.4 (1 H) 19.2 (1 H) 19.4 (1H ) 18.3 (1 H ) 18.3 (1H ) 14.2 (1H) 1 9 . 2 (1H)
Figure 8. ESR spectra found after the y irradiation of frozen aqueous (D20) solutions of various dipeptides and warming to 220 K; (A) giycyk-aspartic acid (21 mg/rnL), (B)g1ycyl-L-glutamic acid (31 mg/mL), (C)glycyl-L-serine (220 mg/mL), (D) L-lysyl-L-lysine (125 mglmL).
The results suggest that the decarboxylated species I11 and VI are less stable toward abstraction than radical V. However, radical VI is more stable than radical 11. The final radicals observed (VII) are produced by abstraction from the C-terminal residue (reactions 7 and 8). Only upon increasing the pD to where the N-terminal amine is dedeuterated (in Gly-Ala) was there evidence for abstraction from another site possibly the N-terminal residue. B. Other Dipeptides. Other dipeptides investigated were glycylarcosine, glycylglycine, glycyl-L-aspartic acid, P-alanylglycine, glycyl-L-glutamicacid, glycylc-methionine, glycyl-L-serine,and L-lysyl-blysine. Detailed investigations of the initial radicals formed in these species were not made. Only the radicals produced upon warming the irradiated samples to -50 “C were studied. The results are reported in parts A-D of Figure 8 for glycylaspartic acid, glycylglutamic acid, glycylserine, and lysyl-L-lysine, respectively. The results for all the peptides are given in Table I1 (including the peptides studied in section A). As can be seen in Figure 8 and Table 11, nearly all the final spectra observed are doublets of approximately 18-19 G. The exceptions are those with alanyl residues at the C-terminal position which show a quartet and Gly-Ser which shows a small doublet. The radicals found from peptides with glycine residues at the C terminal have been shown to be produced in other media.zv8J5 The 19-G quartet observed for peptides containing alanyl residues at the C terminal is also that expected from previous work in other media.6J3 All other dipeptides listed in Table I1
Figure 9. ESR spectra found after y irradiatlon of frozen aqueous (H20) solutions of various dipeptides containing aromatic residues: (A) glycyl-L-histidine at 140 K, (B) glycyl-L-phenylalanine at 140 K, (C) glycyl-L-tyrosine at 140 K.
-
have the structure CHzR for the side group. To produce a doublet one proton of the CH2group must have a 19-G splitting while the other must have a splitting less than the line width. Such hyperfine splittings would arise if the /3 protons were at dihedral angles (e) near 40 and -80”. This orientation has been found for similar radicals in aqueous glassed3 and polycrystalline state.29 The significance of the results in Table I1 for protein radiolysis is readily apparent. It suggests that the dominant “fin+” radical observed in proteins should be of the form (NDCRCO) and this structure will in 15 of the 20 common amino acid residues produce a doublet splitting near 19 G. It should be noted that results for H 2 0 ices were the same as found for D20 ices, i.e., 18-19-G doublets were found. C. Dipeptides with Aromatic Side Groups. Three dipeptides with aromatic side groups were investigated in frozen H 2 0 solutions. These were glycyl-L-histidine (130 mg/mL), glycyl-L-phenylalanine (40 mg/mL), and glycyl-L-tyrosine (110 mg/mL). The results for these compounds after y irradiation are shown in parts A-C of Figure 9, respectively. Gly-His. The analysis of Figure 9A for glycyl-L-histidine shows two signals due to electron attachment. These are the protonated histidine ring anion (XII) which consists of a triplet with 47-G spacing and the glycyl deamination
2892
The Journal of Physical Chemistry, Vol. 83, No. 22, 7979
A
Sevilla, D’Arcy, and Morehouse
The analysis of the spectrum in Figure 10B gives two protons at 19.5 G and one proton at 39.5 G. These splittings are in very good agreement with that expected for the relaxed conformation of radical XV. Gly-Tyr. Analysis of the spectrum in Figure 9C gives strong evidence for radical XVI. NH3’CHzCONHCHCO,
H
H
XVI
XVII Flgure 10. (A) ESR spectrum produced by the subtraction of 25% protonation radical XI1 and 30% deamination radical I1 from Figure 7A. This species is suggested to arise from the decarboxylated radical XIII. (B) ESR spectrum produced by the subtractlon of 25% Protonation radical XIV and 25% deamination radical I1 from the spectrum shown In Figure 78. This species is suggested to arise from the decarboxylated radical XV.
radical (11) which is a triplet of 21.5-G spacing and was shown previously in Figure 1B. Radical XI1 has been NH
2C H 2 CON H C HC 02I CH2T
$ c \\ H/ H
I
H
XI1
independently produced in aqueous glasses12and its ESR spectrum has been employed in the computer analysis of Figure 9A. Computer analysis suggests the spectrum in Figure 9A consists of 25% protonation (XII), 30% deamination (II), and 45% of the spectrum shown in Figure 10A. This spectrum shows five components and can be interpreted in terms of the decarboxylated radical (XIII) with two NH:CH,CONH;HCH,
T;> I
H
XI11
protons at 23.5 G and one proton at 26.5 G. This broad spectrum suggests the presence of an underlying resonance. However, similar radicals produced in aqueous glasses show multiple conformations which can produce such broad line shapes. Gly-Phe. The computer analysis of the spectrum in Figure 9B shows a similar distribution of radicals to that found for Gly-His. In this case we find about 25% deamination (11, R = CH,Ph), 25% ring protonation (XIV), and 50% decarboxylation radical (XV) as shown
XIV
in Figure 10B.
The hyperfine splittings are 45 G due to two protons and 1 2 G due to two protons. The broad central structure shows some resolution which detailed analysis clearly indicates the presence of the tyrosylphenoxyl radical (XVII). This species shows coupling to three protons at 6 G and one at 11G and was produced in previous work in aqueous g1a~ses.l~ In this previous work, XVII was found to arise from the decay of the phenol n cation. In this work XVII is also an oxidation product whereas XVI is a product of reduction. The fact that there is little evidence for decarboxylation in Figure 9C suggests the possibility of hole transfer from other portions of the Gly-Tyr peptide to the phenol ring.
Acknowledgment. The authors thank the Food Engineering Laboratory of the US. Army Natick Development Center and the U.S. Department of Energy for support of this work. The authors also thank Dr. Irwin Taub of the US.Army Natick Research and Development Center for helpful discussions.
References and Notes (1) P. Neta and R. W. Fessenden, J. Phys. Chem., 74, 2263 (1970). (2) R. Livlngston, D. G. Doherty, and H. Zeldes, J. Am. Chem. SOC., 97, 3198 (1975); 98, 7717 (1976). (3) S. Rustgi and P. Riesz, I n f . J. Radiat. Biol., 34, 449 (1978). (4) T. B. Melo, Int. J. Radiat. Biol., 23, 247 (1973). (5) G. Saxebol, T. B. Melo, and T. Henriksen, Radiat. Res., 51, 31 (1972). (6) J. Sinclair and P. Codelia, J. Chem. Phys., 59, 1569 (1973). (7) H. C. Box, E. E. Budzinski, and K. T. Liiga, J. Chem. Phys., 57, 4295 (1972). (8) F. Ngo, E. E. Budzinski, and H. C. Box, J. Chem. Phys., 60, 3373 (1974). (9) S. Kominami, K. Akasaka, H. Umegaki, and H. Hatano, Chem. Phys. Lett., 9, 510 (1971). (10) M. D. Sevilla, J. Phys. Chem., 74, 669, 2096, 3366 (1970). (11) M. D. Sevilia and V. L. Brooks. J. Phvs. Chem.. 77. 2954 11973). (12j M. D. Sevilla, C. Van Paemei, H. Frumk, V. L. Brooks; and R.~Faiio;, J. Phys. Chem., 79, 839 (1974). (13) I. A. Taub, J. W. Halilday, and M. D. Sevilla, Adv. Chem. Ser., in press. (14) M. D. Sevlila and J. B. D’Arcy, J . Phys. Chem., 82, 338 (1978). (15) M. D. Seviliaand J. B. D’Arcy, Radiat. Phys. Chem., 13, 119 (1979). (16) M. G. Simic, Agri. Food Chem., 26, 6 (1978). (17) R. Braams, Radiat. Res., 27, 319 (1966). (18) P. Neta, M. Simic, and E. Hayon, J . Phys. Chem., 74, 1214 (1970). (19) Y. Tal and M. Faraggi, Radiat. Res., 62, 337 (1975). (20) M. Faraggi and Y. Tal, Radiat. Res., 62, 347 (1975). (21) P. S. Rao and E. Hayon, J . Phys. Chem., 78, 1193 (1974). (22) M. Farraggi and A. Bettelheim, Radiat. Res., 71, 311 (1977). (23) M. Farraggi and A. Bettelheim, Radiat. Res., 72, 81 (1977). (24) W. M. Garrison, Radiat. Res. Rev., 3, 305 (1972). (25) W. Flossman and E. Westhoff, Int. J. Radiat. Bid., 33, 139 (1978). (26) W. Flossmann and E. Westhoff, Radiat. Res., 73, 75 (1978). (27) S. Gregoli, M. Oiast, and A. Bertinchamps, Radiat. Res., 72, 201 (1977). (28) H. C. Box, “Radlation Effects: ESR and ENDOR Analysis”, Academic Press, New York, 1977. (29) R. C. Drew and W. Gordy, Radiat. Res., 18, 552 (1963).
