Reaction of Hydroxyl Radicals with Oligopeptides
Acknowledgment. The support for this study by the National Science Foundation is gratefully acknowledged. Appendix We wish to mention here two observations pertinent to our studies. (1) It has been reported that many labile radical anions are stable in HMPA.2 Therefore, we investigated the possibility of generating Py- in this solvent by electron transfer from sodium biphenylide (Na+,B.-). Surprisingly, there was no reaction on adding dry, purified Py to a M solution of Na+,B.- in HMPA, the concentration of P y ranging from to 10-1M.It could be argued that this may be due to an unfavorable equilibrium, the electron affinity of Py being too low in this solvent or, alternatively, that the dimerization of the free Py- anions is too slow. It is more likely, however, that a specific interaction of HMPA with Py prevents or reduces the electron transfer. This anomalous behavior is currently being investigated. (2) The behavior of Li+ salts is often different from those of other alkali metals. We investigated, therefore, the dimerization of Py- induced in DME or in THF by Tr.-,Li+. The reaction was too fast to be followed and a
100
pale yellow precipitate appeared. Apparently Li+(-PyPy-)Li+ is insoluble in these solvents since addition of a slight excess of lithium tetraphenylboride to yellow solution of the sodium salt in THF and DME produced similar precipitation.
References and Notes (1) C. L. Talcott and R. J. Meyers, Mol, Phys., 12, 549 (1967). (2) J. Chaudhuri, S. Kume, J. Jagur-Grodzinski, and M. Szwarc, J. Amer. Chem. SOC.,90, 6421 (1968). (3) A. R. Buick, T. J. Kemp, G. T. Neal, and T. J. Stone, J. Chem. SOC.A, 1609 (1969). (4) C. R. Smlth, J. Amer. Chem. Soc., 46,414 (1924). (5)R. L. Ward, J. Amer. Chem. SOC.,83, 3623 (1961). (6) C. D. Schmulbach, C. C. Hlnckley, and D. Wasmund, J. Amer. Ghem. SOC.,90,6600 (1968). (7) A. Carrington and J. Santos-Velga, Mol. Phys., 5, 21 (1962). (8) R. Setton, C.R. Acad. Sci., Ser. AB, 244, 1205 (1957). (9) J. W. Dodd, F. J. Horton, and N. S. Hush, Proc. Chem. Soc., 61 (1962). (10) N. M. Atherton, F. Gerson, and J. N. Murrell. Mol. Phys., 2, 509 (1962). (11) A. Rainis, R. Tung, and M. Szwarc. J. Amer. Chem. SOC., 95, 659 (1973). (12) Y. Karasawa, G. Levln, and M. Szwarc, Proc. Roy. SOC.,Ser. A, 326, 53 f1971). (13) Since Hush, et a/., attributed the absorption to the radical anion, Py-, and not to its dimer, the value quoted in their paper has to be multiplied by a factor of 2. (14) M. Szwarc, "Carbanions, Living Polymers and Electron-Transfer Processes," Wlley, New York, N.Y., 1968, pp 314-316.
Reaction of Hydroxyl Radicals with Oligopeptides in Aqueous Solutions. A Pulse Radiolysis Study P. S. Rao and E. Hayon* Pioneering Research Laboratoty, U.S.Army Natick Laboratories, Natick, Massachusetts 0 1760 (ReceivedAugust 22, 1974) Publication costs assisted by Natick Laboratories
The reaction rate constants of OH radicals and the spectral characteristics of the free-radical intermediates produced from various oligopeptides in water have been studied in detail using the technique of pulse radiolysis. These include the amides of glycine, glycylglycine, tetraglycine, N- acetylglycylglycine, and glycyl/3-alanine, as well as glycylsarcosine and the N- acetyl derivatives of triglycine, trialanine, and trisarcosine. The k OH values are dependent, in particular, on the state of protonation of the terminal amino group: the rate constants increase on deprotonation of the -NH3+ group. The site(s) of attack by OH radicals are also dependent upon the state of protonation of the terminal amino group: on deprotonation of the -NH3+ group, radicals in an a position to the -NH2 group are preferentially formed. The transient absorption spectra of the free-radical intermediates are strongly dependent on the pK, of the parent molecules, as well as on the pK, of the free radicals produced. Based on these results, it is concluded that ionization of the peptide hydrogen occurs for various peptide radicals -CONHC(R)-CON-c(R)H+.
+
Introduction The fast-reaction technique of pulse radiolysis and kinetic absorption spectrophotometry is an important tool in the study and understanding of the free-radical chemistry of amino acids and peptides in aqueous solutions. It can provide information on the nature of the free radicals produced on reaction with eaq- and OH radicals, the acid-base properties of the peptide radicals, the effect of oxygen and
pH on the formation and reactions of the peroxy peptide radicals, and the redox properties of peptide radicals. The above information is necessary to provide reaction mechanisms for the formation of the products observed on exposure.of peptides to ionizing radiations. Using this technique, the interaction of eaq- with amino acids,l simple peptides,2 ~ligopeptides,~ and the peptide linkage,4?5as well as amides,6 have been studied in this laboratory. Reductive deamination and electron addition to The Journal of Physical Chemistry, Voi. 79, No. 2, 1975
P. S.Rao and E.
110
the carbonyl peptide to form ketyl-type radicals are the major observed processes. The reduction of sulfur amino acids7p8lead to different chemical reactions. The reactions of OH radicals with amides,Qamino acids,lJO grid simple peptides2s4have also been examined and the sites of attack to form carbon-centered free radicals have been suggested. The redox reaction of these radicals have been observed11-14 in the presence of various electron acceptors. Esr studies15J6 have provided valuable information on the interaction of the odd electron with the substituents on the peptide molecule. The detailed studies by Garrison and coworkers on the identification of the products *formed from these reactions have recently been reviewed17 by him. A great deal of important information is presented therein. Reported below is a detailed examination of the reactions of OH radicals with vqrious oligopeptides in water, as a function of pH. The aim of this work was an attempt to characterize the sites of attack by OH radicals on these molecules, and to determine the ionization constants of the free radicals formed. The chemical reactions of the acidbase forms of free radicals can be significantly different,18J9 leading to different permanent products.
Experimental Section The pulse radiolysis set-up and experimental conditions used have been described.20*21Single pulses of 2.3-MeV electrons and -30-nsec duration were used. The experiments were carried out in the presence of 1 atm of NzO (-2.2 X loR2M ) in order to convert eaq- into OH radicals eaq- + NzO Nz + OH + OH(1) where k l = 8.7 X lo9 M-l sec-l (ref 22). The concentrations of the peptides and oligopeptides used were calculated on the basis of their reactivity toward eaq- such that >95% of the hydrated electrons reacted with N20. The OH radicals are knownz3to ionize OH ---L 0- + H' (2) in alkaline solutions with pK, = 11.9. Many experiments reported below were carried out at pH >11.0. It was assumed that the reactions of OH and 0- radicals with oligopeptides gave rise to similar free-radical intermediates, albeit with slightly different reaction rate constants. This assumption may not be valid in all cases. Extinction coefficients were derived based on G (eaq-) = G(0H) = 2.8 and using the KCNS dosimetry.20 The E values given were calculated on the basis that only one radical intermediate is formed from the reaction of OH with peptides. As is indicated below, this is not the case in a number of systems. Hence, in those cases, the E values given are too low. The chemicals used were the highest research grade commercially available. They were obtained from Cyclochemicals, Miles Laboratory, Fox Chemical Co., and Sigma Chemicals. The reagents used were obtained from Eastman Chemicals, Aldrich, Mdlinckrodt, and Baker and Adamson. Solutions were buffered with perchloric acid, potassium hydroxide, and -0.5-1.0 mM phosphates and tetraborate. Solutions were prepared just previous to use and were not stored overnight.
-
Results and Discussion Reactivity toward e aq-. The reaction rate constants of eaq- with all the peptides and oligopeptides studied here The Journal of Physical Chemistry, Vol. 79, No. 2, 1975
Hayon
were determined at different pH values, based on the pK, of the terminal amino group (-NH3+) of these molecules. These rates are given el~ewhere.5~3J9~~~ In all cases, the k values decrease on ionization of the -NH3+ groups. Reactivity toward OH Radicals. The reaction rate constants of hydroxyl radicals were f o ~ n d 1 , 2 J 9to~be ~ ~markedly dependent on the state of protonation of the terminal amino groups of the simple amino acids and peptides studied. On ionization of the -NH3+ groups, the reactivity of. these molecules toward OH radicals increased. These rate constants were derived by competition kinetics using CNS- ions, and were based on k (OH CNS-) = 1.1X 1O1O M-l sec-l (ref 24). The reactivity of OH radicals with the oligopeptides studied here was determined by measuring the formation kinetics of the free radical formed from this reaction, at the appropriate wavelength. From these pseudo-first-order rate constants, the second-order rateB were calculated and are given in Table 1:With a few systems, the OH rates were also determined by competition kinetics using CNS- ions. The values obtained are also shown in Table I. In almost all cases, the kOH rates derived by competition kinetics were higher. One of the main reasons for this difference lies in the method used: the competition kinetics method determines the reaction of the whole molecule toward OH radicals, whereas the rate constants derived by monitoring the formation kinetics of the free radicals give a value for the reaction of OH at this particular site in the molecule. Clearly, the latter method is expected to give an apparent lower k OH value.
+
Free-Radical Intermediates Glycinamide. The reaction of OH radicals with glycinamide (pK a = 7.9) forms free-radical intermediates whose absorption spectra are strongly dependent upon pH, see Figure 1. Three distinct species appear to be formed, and their spectra were determined at pH 3.2, 7.2, and 13.2. On monitoring the change in absorbance at 270 nm with pH, titration curves are observed (see insert in Figure 1) from which pKa (radical) values of 4.3 f 0.2 and 113.0 can be derived. The following reactions are suggested to occur in acid solutions: h3CHCONHz + HzO (3)
OH
+
--L
~H,CH,CONH~
h3CH,CONH
hH3CHCONH2
NH2CHCONHz
+
H'
+
HzO (4)
PKa4.3 ( 5 )
The +NH&HzCONH radical is expectedg to absorb weakly below -280 nm, and the extent of its formation cannot be established. The absorption spectrum of the +NH&HCONHz radical with a ,A,, -330 nm resembles the spectra of amide radicalsg RCHCONH2, indicating a stronger interaction of the odd electron with the -CONHz group. The spectrum of NH&HCONHz resembles more closely the spectrum of the NH2CHCOO- radical1 (see more below). In alkaline solutions, the following reactions are suggested: OH
+
NHzCHzCONHz
NHzCHCONHz
===
-
NH2CHCONH,
NHCHCONHz
+
H'
+
HzO (6)
pK, 2 13.0 (7)
Similar reactions have been proposed for amino acids1 and peptide2s4radicals.
Reaction of Hydroxyl Radicals with Oligopeptides
111
TABLE I: Rate Constants for the Reaction of OH Radicals with Oligopeptides in Aqueous Solution Peptide
Ionic form
pH
k(OH
+ peptide), M"sec-'
b
5 .O 8.3 x 107 'NHsCHzCONH2 10.o 2.8 x 109 NHzCHzCONHz Glycylglycinamide 'H2-Gly-Gly-NH2 3.3 2.7 X lo8 5.5 5.5 x 1 0 8 (1.6 x 109) Tetraglycinamide *Hz (GlY)4"z 10.8 1.8 x 109 (1.2 x 109) H (G1Y)JW Glycine -6-alaninamide 'NH3CHZCONHCH2CH2 CONH, 5 .O 1.1 x 109 NH2 CH2 CONHCHzCHzCONHa 10.0 2.5 x 109 9 .o 2.4 x 109 N-Acetyltriglycine Ac(Gly),O' 9 .o 3.0 x 109 N- Acetyltrialanine Ac (Ala),O9.o 3.8 x 109 N-Acetyltrisarcosine Ac(Sar),O' 9 .o 2.9 x 109 N- Acetylhexaalanine Ac (Ala)60' 6 .O 7.6 X lo8 (2.3 X lo9) N -Acetylserinamide Ac-Ser-NH2 6 .O 8.6 X lo8 N- Acetyldigly cinamide AC( G ~)2"Y 2 5.5 i .I x 109 (1.3 x 109) Glycylsarcosine (3.O, 8.6) +Hz-Gly -Sar -0H- Gly Sar -0' 10.8 (1.0 x 109) Hexa-L -alanine +H2(Ala),OH 3.6 1.7 x 109 Numbers in parentheses are pKa values. Rates determined by following the formation kinetics of the intermediates at the appropriate wavelength; the rate values in parentheses were determined by the thiocyanate method (see text). Glycinamide (7.9)
-
r.
-CONHCHCONH-. The following reactions are suggested: OH +
+NH~CH~CONH~"CONH~
+
+
NH~CH~CONHCH~CONHZ
H2O
(8)
NH CH CONHCH2 CONH 2
+
H2O (9) The observed pK, of -6.6 could be due to either ionization of the peptide hydrogen, reaction 10, or change in the site of reaction of the OH radical due to deprotonation of the terminal -NH3+ group, pKa ~ 7 . 8reactions , 11,12. +",CH~CONHCHCONH~
+",CH2CONCHCONH2 X,nm
OH
Spectra of intermediates produced from the reaction of OH radicals with glycine amide (7 mM, 1 atm of N20) at pH 3.2, 0, pH 7.2, 0,and pH 13.2, A. A0D270 vs. pH. Total dose k 2 . 4 krads/ pulse. Figure 1.
It is interesting to compare the pKa (radical) for glycinamide with those o b s e r ~ e d ~for J ~glycine: ,~~ + .
NH,CHCOOH e NH2CHCOOH + H'
NHzCHCOOH ====
NH~CHCOO-
NH2CHCOO-
== 'NHCHCOO-
+ +
H'
pK,
12.0
HI PIT,
The deprotonation of the -NH3+ group in glycine occurs with a pK of 9.6 and of the -COOH with a pKa of 2.3. In the glycine radical the inverse order occurs. Table I1 gives the extinction coefficients and decay kinetics of the radicals formed from glycinamide at different pH values. Glycylglycinamide. This peptide has a pK, 7.8. Three distinct transient absorptions are observed a t pH 3.2, 9.2, and 13.2, see Figure 2. At pH 3.2, a strongly absorbing species is formed with,,,A at 265 and 325 nm. This spectrum appears to resemble that of the peptide radical4
-
+
+
Ht pK,6.6 (10)
NH2CHCONHCH2CONH2
1
NH~CH~CONHCHZCONH~
+
H2O
(11)
NH~CH~CONHCHCONH~
+
HzO (12) The peptide radical in glycine anhydride* has a pKa = 9.6. It is conceivable that the PKa 6.6 observed for GlyGlyNH2, reaction 10, may be lower due to the inductive effect of the terminal -NH3+ group, which is absent in glycine anhydride. However, no firm conclusion can be reached on this point. The pK, 2 13.0 observed (see Figure 2 and Table 11) is suggested to be due primarily to the ionization of the radical formed in an a position to the terminal amino group:
-
NH~CHCONHCH~CONH, == NHCHCONHCH~CONH~ + H+ (13) N-Acetylglycylglycinamide. The terminal amino group in GlyGlyNH2 is replaced by an acetyl group in N-AcGlyGlyNHZ. This molecule has no ionizable group in the pH range 0-14. The reaction of OH radicals can attack this compound a t primarily two sites to form the AcGlyNHcHCONHz and CH3CONHCHCOGlyNH2 radicals in The Journal ot Physical Chemistry, Voi. 79, No. 2, 1975
P. S.Rao and E. Hayon
112
TABLE 11: AbsorptionlMaxima,Extinction Coefficients,Ionization Constants, and Decay Kinetics of Radicals Produced from the Reaction of Oligopeptides with OH Radicals in Aqueous Solution Emax.,
pH
Peptide" Glycinamide (7.9) Glycylgly cinamide N-Acetylglycylglycinamide
Glycylsarcosine
Tetraglycinamide
nm
A,,
3.2 7.2 13.2 3.3 9.2 13.2 6.5
mM-' cm-*
-270;330 2.1;4.0 265 5.6 265 9.6 265;325 11.8;3.9 265 5.5 -265 9.9 265;325 13.5;4.2
13.3
295
3.O 6.5 9.3 13.1 3.2
240;350 5.7;1.8 255;360 6.8;2.0 265;350 7.5;1.5 265;360 9.5;3.0 265;320 11.4;3.8
17.0
9.2
265
5.6
13.1
290
11.0
Glycyl-P-alaninamide
2k, M-' sec-' pKa (radical)
2.0 x 109 +",CHCONHz 4.2 x 109 4.3 * 0.2 NHzCHCONHz 7.4 x lo8 213.0 -NH~HCONH~ 3.8 X lo8 *NH3CH2CONHCHCONHzb 1.7.xlo9 6.6 f 0.2 NH~CHCONHCH~CONH~~ 1.2 x 109 213.0 -NH~HCONHCH~CONH~~ 6.9 x lo8 CH,CONH~H~O-GIY -NH2; AC-Gly yNHCHCONH2 -2.5 X lo8 11.8 i 0.2 CH~CO-NCHCO-GIY-NH~ ; Ac-Gly--NCHCONH2 8.5 X lo8 +"3CH2 CON (CH,)CHC02H 9.5 x 108 3.5 * 0.2 +"3.CH2CON(CH3)CHCOz3.6 x lo9 8.1 * 0.2 NHz CHCON (CH3 )CH2C0zmb 7,.1X lo8 213.0 -NHCHCON(CH3)CHzCOzmb 3.8 X lo8 WH3CH2CO-Gly -NHCHCO~ i-NHCH~ y CONH~~ 1.1 x 109 6.7 + 0.2 N H 6~ ~ co(GIY) 2 ~ - ~ ~ CONH~~ 2.1 x 109 213.0 -NHCHCO(G~~)~NHCH~CONHzb 5.5 x 108 C 1.3 x lo9 8.1;7.2 d 1.4 X lo9 213 .O d d 4.8 X lo8 7.2 x lo8 - CONHCH1.1 x 109 11.5 i: 0.2 - C O - N ~ H 6.2 X lo8 d
3.2 240;370 7.8;2.6 10.0 250 4.8 13.2 260 10.2 N - Acetyltrigly cine 3.2 270;320 12.O;1.4 265;330 12.0;1.8 6.5 13.O 295 17.0 N - Acetyltrialanine 3.1 265;325 12.0;2.9 260;330 11.5;2.8 6.6 12.7 290 5.1 N-Acetyltrisarcosine 3.0-12.1 -245;355 5.6i2.1 N -Acetylhexaalanine 3.1 270;335 8.0;2.8 13 .O 295;350 9.1;5.4 a Numbers in parentheses are the pKa values. * Mainly this radical species.
I
I
250
300
I
- ,
350
I
I
400
X,nm
Figure 2. Spectra of intermediates produced from the reaction of OH radicals with glycylglycinamide (4 and 8 mM, 1 atm of N20) at pH 3.2, 0, pH 9.2, 0,and pH 13.2, A. AOD285 vs. pH. Total dose -2.4 krads/pulse. The Journal of Physical Chemistry, Vol. 79, No. 2, 1975
Suggested radical
.
7.0 X
lo8
-
CONH.C(CH,)-
1.5 x 109 10.9 * 0.2 -CO-NC(CH$3.4 x 108 -CON(CH,)CH3.4 x 108 -CONH~(CH,)3.6 x lo8 12.1 * 0.2 - CO-Nk (CH3)Mixture of radicals, see text. See text.
neutral solution. The spectra observed at pH 6.5 and 13.3, Figure 3, resemble closely the spectra of the neutral and ionized peptide radicals formed in glycine anhydride? Accordingly, the pK, = 11.8 f 0.2 found for the radical from N - AcGlyGlyNHg is suggested to be due to the ionization of the peptide hydrogen to form the radical AcGly-NCHCONHz and CH3CO-NCHCOGlyNH2. This pK, is to be compared to the pK, = 9.6 for the ionization of the CH2CONHCHCOyH radical.4 Glycylsarcosine. The reaction of OH radicals with glycylsarcosine (pK, = 3.0 and 8.6) can'abstract an H atom from different sites in the molecule, dependent upon the pH. As was done above, only the main radicals formed are 'indicated. Figure 4 shows the spectra of the intermediates produced at pH 3.0, 6.5, 9.3, and 13.1. Also shown are the titration curves (insert in Figure 4) and the pK, (radical) values of 3.5 f 0.2,8.1 f 0.2, and 113.0 derived, see also Table 11. The pK, 3.5 is close to that of the -COOH group in the parent molecule, and is suggested to be due to the equilibrium 'NH,CHzCON(CH3)CHCOOH -L "NH3CHzCON(CH3)CHC0O.+ H' pK,3.5 (14)
-
z
Reaction of Hydroxyl Radicals with Oligopeptides
113
AC-Gly-Gly-NHe 0.250.4
-
0.20 0.3 -
ri 0
OIS0.2 -
d 0
0.100.1-
005 I
I
1
I
300
350 X,nm
400
450
Flgure 3. Spectra of intermediates produced from the reaction of OH radicals with Kacetylglycylglycinamide (4 mM, 1 atm of N 2 0 ) at and pH 13.3, 0.AOD295vs. pH. Total dose -2.4 krads/ pH 6.5, 0, pulse.
On ionization of the -NH3+ group in GlySar, a change in the site of attack by OH radicals occurs leading to the formation of a radical in an a position to the amino group, NH&HCON(CH3)CH2COO-. Hence the pKa = 8.1 follows the pK, = 8.6 of the parent molecule, i.e., it does not represent the ionization of a free radical. In very alkaline solutions, the transient spectrum observed and the pK, (radical) 2 13.0 is assigned to the -NHcHCON(CH3)CH2COO- radical, see Table 11. At all pH values, some H atom abstraction from the N CH3 group in GlySar by OH radicals is expected, based on the reactions observedg with amides. The -CON(CHz)radicals have spectra9 with maxima in the 350- and 250-nm wavelength regions. Tetraglycinamide. This molecule has four peptide groups. Hydroxyl radicals are expected to form various peptide radicals by abstraction of H atoms at different -CH2- groups within this oligopeptide. When the amino group is protonated (PKa 7.6 for this peptide) the -CONHCH- radicals formed are furthest away from it. The pK, 6.7 f 0.2 observed (Figure 5 and Table 11) is suggested to be due to (a) change in the site of attack by OH radicals due to the ionization of the terminal -NH3+ group and the formation of the NH2CHCO(Gly)3NH2 radical, and (b) the ionization of the peptide hydrogen in, for example, the +NH3CH2CONHCHCO(Gly)2NH2 radical. This latter suggestion is tentatively proposed on the basis of the "wide stretch" in the titration curve (insert in Figure 5) in the pH range -6-9.5. The titration curve may be indicating the ionization of the peptide hydrogen in the radical which then merges into the formation of another radical (due to the ionization of the -NH3+ group and the change in the site of OH radical attack). At higher pH values, the pK, (radical) 2 13.0 is again suggested to be due to the ionization of the NHzCHCO(Gly)3NH2 radical to form -NHCHCO(Gly)3 "2. Glycyl- P-alaninamide. In solutions a t pH lower than the pKe 8.2 (expected for this molecule), two radicals are
i I
I
I
I
-
-
-
X, nrn Figure 5. Spectra of intermediates produced from the reaction of OH radicals with tetraglycinamide (1 mM, 1 atm of N20) at pH 3.2, 0,pH 9.2, 0 ,and pH 13.1, A. AODnTovs. pH. Total dose -2.4 kraddpulse.
probably formed: +NH~CH~CONHCHCHZCONHZ and +NH&HzCONHCHZ~HCONHZ.The transient absorption spectrum observed, Figure 6, is not inconsistent with this assignment. The apparent pKa 8.1 observed (insert in Figure 6) is due to the ionization of the -NH3+ group and a change in the site of attack by OH radicals, with the formation of the NH~CHCONHCH~CH~CONHZ radical. The different pK, 7.2 observed on monitoring the radicals at 370 nm is more difficult to explain. It would be tempting to suggest that it represents the ionization of the peptide hydrogen in the +NH3CH2CONHCHCH2CONHz radical. But no evidence is available to support this suggestion.
-
-
The Journal of Physical Chemistry, Vol. 79, No. 2, 7975
P. S. Rao and E. Hayon
114
0 20
0 15
d d
\.i
0.10
I
a#\\
- 1
0.05
250
300.
350
400
O'OT
450
X , nm
*bo
0'
Spectra of intermediates produced from the reaction of OH radicals with glycine-&alaninamide (1 and 5 mM, 1 atm of N20) at pH 3.2, 0, pH 10.0, 0, and pH 13.2, A. A00265 and A00370 vs. pH. Total dose ~ 2 . krads/pulse. 4
I
I
300
350
450
400
A, n m
Figure 6.
Figure 8.
Spectra of intermediates produced from the reaction of
OH radicals with Kacetyltrialanine (2 mM, 1 atm of N20) at pH 3.1, 0,pH 6.6, 0 , and pH 12.7, A. AOD265 vs. pH. Total dose ~ 2 . 4
krads/pulse.
r-
I
0.3
0.I
250
300
-
0
250
300
350
X, n m
400 '
I
Spectra of intermediates produced from the reaction of OH radicals with Kacetyitrigiycine (2 mM, 1 atm of N20) at pH'3.2, 0, pH 6.5, A, and pH 13.0, 0.AOD265 vs. pH. Total dose -2.6 krads/pulse.
0.2
s
-
N0.2--
t
I I I POLYSARCOSINE
-
c
0
u
-
l i
6
d
N-Acetyl Tripeptides. The reactions of OH radicals with N - acetyltriglycine, N - acetyltrialanine, and N - acetyltrisarcosine have been studied at various pH values, see Figures 7-9 and Table 11. Three types of radicals are primarily formed from these peptides, e.g., CH&ONHCHCO(Gly)2COOH, CHsCO(Gly)NHCHCO(Gly)COOH, and CH&O (G1y)zNHCHCOOH for N-acetyltriglycine. The small difference observed in the transient spectra of these peptides between pH -3.2 and 6.5 is probably due to the ionization of the -COOH in the -NHCHCOOH radical. A second pK a is observed for these ridicals in alkaline solution. N - Acetyltriglycine and N - acetyltrialanine give pK, (radical) values of 11.5 f 0.2 and 10.9 f 0.2,respectively. No ionization of the radical from N - acetyltrisarcosine was observed up to pH 12.5. These results indicate that
I
5
b
Flgure 7.
The Journal ot Physical Chemistry, Vol. 99, No. 2, 1975
nm
J
450
400
350
A,
d
4
0.1
I
u
6
8
-
0
IO
12
PH
250
300
350
400
X,nm
Spectra of intermediates produced from the reaction of OH radicals with (a) 2 mM N-acetyitrisarcosine at pH 3.0, 0, pH 8.2, 0,and pH 12.1, A, and (b) 0.4 mM polysarcosine at pH 6.6, 0, and pH 11.2, 0.Solutions saturated with N20.OD250 vs. pH. Total dose Figure 0.
-4.0
and 2.3 krads/pulse.
the observed pK, (radical) in alkaline solutions are due to the ionization of the peptide hydrogen in the radicals
Reaction of Hydroxyl Radicals with Oligopeptides 0.3
I
I
0 l .3~
115
" " ' " _ I
'C
g 0.2 0.2
d d 0.1
0
I
I
I
I
250
300
350
400
X,nm Figure 10. Spectra of intermediates produced from the reaction of OH radicals with Kacetylhexaalanine (2.5 X M, 1 atm of N20) at pH 3.1, 0,and pH 13, 0.A0D296 vs. pH. Total dose ~ 2 . krads/ 4 pulse.
CH&O(Gly)NHCHCO(Gly)COO- and CH&O(Ala)NHC(CH3)CO(Ala)C00-. In support of this conclusion, the reaction of OH radicals with polysarcosine (mol w t 1500) was examined. No radicals at either end of this polypeptide chain in an a position to the terminals -NH2+ and -COOH are statistically likely to be formed. The transient spectrum produced, Figure 9b, is typical of the -N(CH3)CHCO- radical. Furthermore, no change in the spectrum was observed over the pH range 5-13, in support of this assignment. The transient spectrum formed from N- acetyltrialanine a t pH 12.7, Figure 8, is quite different from most of the spectra observed. Its nature is uncertain. Rupture of N-C or C-C bonds may occur on ionization of the peptide hydrogen in the N - acetyltrialanine radical. The transient spectra formed from the reaction of OH radicals with N acetylhexaalanine, Figure 10, do not show this peculiar effect. Some impurity present in N - acetyltrialanine cannot be excluded.
-
Conclusions A detailed study of the reactions of OH radicals with oligopeptides in water has been undertaken. Based on the spectral characteristics of the free-radical intermediates produced, and their dependence upon pH and the ionization constants of the -NH3+ and -COOH functional groups, the site(s) of attack by OH radicals on the oligopeptides have been suggested. Evidence has been presented for the ionization of the peptide hydrogen in peptide radicals -CONHCH2-. It is interesting to point out that with aromatic amino acids and peptides, the reactions of OH radicals occur predominantly with the aromatic ring, whereas the reactions of eaq- are more selective and can lead to d e a r n i n a t i ~ n . ~ ~ ~ ~ ~ References and Notes (1)P. Neta, M. Slmic, and E. Hayon, J. Phys. Chem., 74, 1214 (1970). (2)M. Slmic, P. Neta, and E. Hayon, J. Amer. Chem. Soc., 92, 4763 (1970). (3)M. Simic and E. Hayon, Radiat. Res.. 48,244 (1971). (4)E. Hayon and M. Slmic, J. Amer. Chem. Soc., 93,6781 (1971). (5) P. S.Raoand E. Hayon, J. Phys. Chem., 78, 1193 (1974). (6)M. Simlc and E. Hayon, J. fhys. Chem., 77,996 (1973). (7)M. 2. Hoffmanand E. Hayon, J. Amer. Chem. Soc., 94, 7950 (1972). (8) M. 2. Hoffmanand E. Hayon, J. phys. Chem., 77, 990 (1973). (9)E. Hayon, T. ibata, N. N. Lichtin, and M. Simic, J. Amer. Chem. SOC., 92,3898(1970); 93, 5388 (iwi). (10)P. Neta, M. Simic, and E. Hayon, J. fhys. Chem., 78,3507 (1972). (11) M. Simlc and E. Hayon. Inf. J. Radlat. 8/01., 22, 507 (1972). (12)P. S.Rao and E. Hayon, Biochem. Biophys. Acta, 292,516(1973). (13)E. Hayon and M. Sirnic, J. Amer. Chem. SOC., 95,6681 (1973). (14)P. S.pao and E. Hayon, J. phys. Chem., to be submitted for publication. (15)P. Neta and R. Fessenden, J. fhys. Chem., 74,2263(1970). (16)M. D. Sevllla, J. Phys. Chem., 74, 2096,3366 (1970). (17)W. M. Garrison, Radiaf. Res. Rev., 3,305 (1972). (18)E. Hayon and M. Sirnic, Accounts Chem. Res., 7, 114 (1974). (19)E. Hayon and M. Sirnic, htra-Sci. Chem. Rep., 5,357(1971). (20)M. Simic, P. Neta, and E. Hayon, J. fhys. Chem., 73,3794 (1969). (21)J. P. Keene, E. D. Clack, and E. Hayon, Rev. Sci. Insfrum., 40, 1199 (1969). (22)M. Anbar, M. Bambenek, and A. B. Ross, Nat. Stand. Ref. Data Ser., N8t. Bur. Stand., NO. 43 (1973). (23)J. Rabani and M. S. Matheson, J. Phys. Chem., 70, 761 (1968). (24)L. M. Dorfrnan and G. E. Adams. Nat. Stand. Ref. Data Sef., Nat Bur. Stand., No. 48 (1973). (25)H. Paul and H. Fischer, Helv. Chim. Acta, 54,485 (1971). (26)J. Feitelson and E. Hayon, J. fhys. Chem., 77, 10 (1973). (27)J. P. Mlttal and E. Hayon, J. fhys. Chem., 78, 1790 (1974).
The Journalof Physical Chemistry, Vol. J9, No. 2, 1975
