[2]
PULSE RADIOLYSIS IN OXYGEN RADICALS
89
[2] P u l s e R a d i o l y s i s in S t u d y o f O x y g e n R a d i c a l s
By MICHAEL G. S1M1C Oxygen Radicals and Pulse Radiolysis Because of the unpaired electron, oxygen radicals, like most other free radicals, are highly reactive not only with numerous and varied substrates but also with each other.l Consequently, their lifetimes either in model aqueous systems or especially in biosystems are very short. This particular characteristic renders studies of their physicochemical properties complex. Studies of their structural features can be simplified by generating them in a solid or a frozen matrix where their lifetimes may extend indefinitely. Their kinetics and energetics properties, however, can be determined only in liquid systems where the lifetimes are short. Hence, physicochemical properties of oxygen radicals can be studied only with detection techniques such as kinetic spectrophotometry, kinetic conductivity, time-resolved Raman and electron spin resonance (ESR) spectroscopy, and many other techniques with short time resolution. 2,3 In addition to adequate monitoring systems, studies of free radicals require convenient free radical-generating techniques and methods. These techniques and methods must satisfy some basic prerequisites. First, the radicals should be generated in a short period of time, before they start reacting with each other and the substrates. Second, the concentrations of radicals generated should be high enough to allow accurate monitoring. Third, only a specific radical should be generated without the presence of other interfering radicals. Finally, the capability of generating diverse free radicals as required, and sometimes a combination of radicals, should exist. Such features can be found in a technique known as pulse radiolysis. 3.4 Pulse radiolysis is accomplished by a combination of a pulsed-radiationgenerating machine (electron accelerator) and a variety of time-resolved detecting systems. In many respects pulse radiolysis, in which free radiW. A. Pryor (ed.), " F r e e Radicals in Biology," Vols. I - V I . A c a d e m i c Press, N e w York, 1976-1985. 2 L. M. Dorfman, J. Chem. Educ. 58, 82 (1981). 3 Farhataziz and M. A. J. Rodgers (eds.), "Radiation C h e m i s t r y . " Verlag C h e m i e , N e w York, 1987. 4 C. v o n Sonntag, " T h e Chemical Basis o f Radiation Biology." Taylor & Francis, L o n d o n , 1987.
METHODS IN ENZYMOLOGY.VOL. 186
Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
90
PRODUCTION, DETECTION, AND CHARACTERIZATION
[2]
cals are generated by ionizing radiations, is similar to laser and flash photolysis, 5 in which free radicals are generated by photonic processes. In this chapter pulse radiolysis studies of hydroxyl (. OH), peroxyl (HROO-), superoxide (. 02-), alkoxyl (HRO.), and phenoxyl radicals (ArO -), as well as their properties, are briefly discussed. Underlying principles, attributes, and limitations are emphasized; detailed accounts of pulse radiolysis have been published recently. 3.4 Generation of Free Radicals Ionizing radiations (high-energy electrons, X- and y-rays, high-energy nuclei, etc.) ionize the matter through which they pass. In aqueous media, the first step is ionization of water, H_,O---~ H_~O+" + e-
(1)
Both H20 +" and e - a r e , in principle, free radicals because they do not have paired electrons. Within a few picoseconds (10 -12 sec) they undergo the following reactions: H:O +-+ H20 ~ . OH + H30+ e- + n H,O--~ eaq
(2) (3)
Hence, if the radiation pulse were 1 nsec long, by the end of the pulse the reaction cell through which the radiation passed would contain relatively homogeneously distributed hydroxyl radicals and hydrated electrons, e~q. In addition to these two extremely reactive radicals, a small amount of. H is generated. Therefore, radiation splits water into primary water radicals (yields are indicated in parentheses): HzO --> • OH (2.8) + e~q (2.8) + • H (0.6)
(4)
In pure water the primary water radicals recombine at diffusion-controlled rates, for example, • OH + e,q --9 O H -
(5)
k - - 3 × 10 I°M -l sec l Water radicals react with solutes when present to give a second generation of radicals, the solute radicals. The reactivities of diverse solutes with all three primary water radicals have been tabulated. 6 The simultaneous occurrence of • OH and eaq reactions may interfere with the measurements of free radical properties. To resolve this experi5 R. V. Bensasson, E. J. Land, and T. G. T r u s c o n , " F l a s h Photolysis and Pulse Radiolysis." Pergamon, New York, 1983. 6 G. V. Buxton, C. L. Greenstock, W. P. Helman, and A. B. Ross, J. Phys. Chem. R~f~ Data 17, 513 (1988).
[2]
PULSE RADIOLYSIS IN OXYGEN RADICALS
91
mental difficulty, two methods m a y be used. H y d r a t e d electrons m a y be c o n v e r t e d to hydroxyl radicals in the presence of gaseous N_,O (usually ! arm): e~q + N_,O ~ ' O H + OH + N_, (6) k = 9 x l 0 9 M -I sec -j At 1 atm and r o o m temperature, [N20] = 25 mM. Hence, reaction (6) is completed in about 1 nsec. This rapid generation of the radiolytic • O H has critical advantages o v e r the much slower generation of • O H via the H a b e r - W e i s s reaction, H_,O2 + Fe(II)EDTA--* . O H + OH
k = 104 M -t
+ Fe(III)EDTA
(7)
sec -f
Unfortunately, there is no convenient method to convert hydroxyl radical to hydrated electron. The experimental conditions can be simplified, h o w e v e r , by removing • O H with solutes that give redox-unreactive radicals. E x a m p l e s of such solutes are tert-butanoi and some allylic compounds: •OH
+ (CH3)3COH --) k = 6 × •oil
+
H20 + • CH2(CH3)2COH
\ C=C / --, \ c _ ~ OH / \ / I
k = 2
x
(8)
108 M -~ s e c - j (9)
109 M -I sec -I
Neither radical is a good hydrogen a t o m or electron donor nor exhibits a c i d - b a s e properties. Both radicals have weak absorption spectra below 250 nm, an important feature in kinetic spectroscopy. When reducing radicals are required, the oxidizing • O H can be converted to a reducing radical, for example, -OH + HCO_,---~ H_,O + .CO_, k = 3.2 × l 0 9 M -j sec i
(10)
With an E0 value of - 1.9 V, the carboxyl radical is a powerful reductant. It is used to reduce disulfide bonds, c y t o c h r o m e s , quinones, nitro compounds, etc. Since superoxide has an E7 value of - 0 . 3 3 V, • CO2- readily reduces oxygen: • co_,
+ o_, ~
co_, + - o_,-
( l I)
k = 4 × 109M -I sec -I In practice, solute concentrations of the order of 1 to 100 m M are generally used. The actual concentrations must be adjusted t o satisfy specific experimental requirements, particularly when multiple solutes
92
PRODUCTION, DETECTION, AND CHARACTERIZATION
[2]
are used. At concentrations greater than 1 M, direct ionization of the solute (approximately equal to the weight percent of the solute) must be taken into account. The dose per pulse may be varied, and concentrations of free radicals from 1 nM to 0. l mM per pulse can be generated easily. The time scale of free radical monitoring in most experiments is 0.1/zsec to 1 sec. If required, however, special equipment allows measurements on a time scale of picoseconds. Physicochemical Properties of Radicals
Absorption Spectra Free radical absorption spectra are usually red shifted compared with parent compounds, an important feature in monitoring. The spectra can be obtained point by point by measuring absorption at different wavelengths selected by a monochromator at a constant dose/pulse) The absorbances are calculated from actual concentrations of radicals corresponding to the dose/pulse. Instead of multiple pulsing, an optical multichannel analyzer (OMA) records absorption spectra at a preselected time after a single pulse. 7 The technique is based on spectrographic resolution of analyzing light and a diode array instead of a monochromator and photomultiplier as in kinetic spectroscopy. Hydroxyl and alkoxyl radicals have poorly defined spectra; peroxyl radicals absorb at 240 to 250 nm, • O2H at 225 nm, • 02- at 245 nm, and p-substituted phenoxyl radicals at about 400 nm.
Acid-Base Properties Acid-base properties 8 are determined from spectral changes with pH. For example, a-hydroxy radicals are stronger acids than their parent alcohols:
/
C--OHm-
pKa ~ 10-12
/
C--O- + H +
(12)
(for aliphatic)
The absorption spectra of the deprotonated forms are usually red shifted, as already indicated for the • O2H and • 02- forms.
7 E. P. L. Hunter, M. G. Simic, and B. Michael, Rev. Sci. lnstrum. $6, 2199 (1985). 8 E. Hayon and M. G. Simic, Acc. Chem. Res. 7, 114 (1974).
[2]
PULSE RADIOLYSIS IN OXYGEN RADICALS
93
Redox Properties Redox properties are determined from interactions of free radicals with oxidants and reductants. Redox potentials can be determined if a satisfactory equilibrium can be achieved between the radical under investigation, • X, and a radical, • A, with a known redox potential, kf
-X+A ~ X kr
+.A
(13)
using the Nernst equation: AE = E(. A/A-) - E(. X/X-) = 0.059 log K
(14)
The equilibrium constant K can be determined kinetically, K = kf/kr, or spectroscopically from relative absorptions of two radicals at the equilibrium, provided their spectra do not overlap. The redox potential of superoxide radical, E7 = -0.33 V, was determined this way, using quinones (Q) with a known redox potential, 9 "02- + Q ~ 02 + "Q-
(15)
It is important to realize that the redox potentials of free radicals are oneelectron redox potentials, and they should not be confused with twoelectron redox potentials. Miscellaneous Properties Some other properties of free radicals are crucial for their proper identification and monitoring. Radicals may be neutral or charged, which is easily discernible by kinetic conductivity. They may be long lived, for example, the viologen radicals. They all have an unpaired electron and are consequently amenable to ESR investigations (flow or kinetic).l° In addition to structural features, ESR provides information about the distribution of the unpaired electron within radicals. Kinetics and Mechanisms of Oxygen Radicals Hydroxyl Radical, • OH Reactions o f - O H radicals are invariably studied in the presence of NzO in order to eliminate hydrated electrons [reactions (6)]. In addition to 9 D. Meisel and G. Czapski, J. Phys. Chem. 79, 1503 (1975).
x0A. D. Trifunac, in "Study of Fast Processes and Transient Species by Electron Pulse Radiolysis"(J. H. Baxendaleand F. Busi, eds.), p. 163.Reidel,Hingham,Massachusetts, 1982.
94
PRODUCTION, DETECTION,AND CHARACTERIZATION
[2]
hydrogen-atom abstraction [reaction (8)] and addition to double bonds [reaction (9)], hydroxyl radical adds readily to aromatic rings. For example, phenylalanine, Phe, reacts with • O H to give three transients (o-, m-, and p - P h e - - O H ) , which absorb in the 300 to 350 nm region. ~ Reaction of these transients can be followed and measured by monitoring their absorption. F r o m both pulse radiolytic and product measurements the following scheme has been developed:
R ~OH
R
©
R -.~ I ~ O H
R
+ *OH
=
[~°)~
o-Tyr
R
OH
---
II
1
~OH
m-Tyr
(16)
Phe R
R
OH
OH
In the absence of oxygen, P h e - - O H transients disappear by disproportionation and combination,tl 2/'he--OH [
, Tyr + PheH--OH , HO--Phe--Phe--OH
(17a) (17b)
The hydrated benzene ring readily loses water both in the dimers and monomers, for example, PheH--OH ~ Phe + H:O
(18)
In the presence of oxygen, OH adducts react with oxygen, /'he--OH + 02---,' OOPhe--OH
(19)
The reaction mechanisms of this peroxyl radical are not fully determined. Some of the products are known, however, namely, the three isomers of tyrosine. Consequently, • OOPhe--OH ---~----~o-, m-. and p-Tyr + products
(20)
it M. G. Simic, E. Gajewski, and M. Dizdaroglu, Radiat. Phys. Chem. 24, 465 (1985).
[2]
PULSE RADIOLYSIS IN OXYGEN RADICALS
95
Hence, both in the absence and in the presence of oxygen, • OH radicals lead to formation of the tyrosine isomers. Since o-Tyr has not been found in normal proteins, its presence is indicative o f . OH radical processes.*2 Formation of o-Tyr in proteins can be used as a proof of radiation processing of foods. Hydroxyl radical is a potent oxidant (E0 = 2.6 V) capable of oxidizing numerous metal ions and their complexes• The redox component in a biosystem may be diminished owing to competition of abstraction and addition reactions. Superoxide Radical, • 02-
In addition to being formed by reactions (10) and (11), superoxide radical can be generated by the reaction of oxygen with hydrated electrons: e~q + 02 ~
- O2
(21)
or hydrogen atoms: • H + O2-->'O2H
(22)
since • O2H ~
-O2
+ H+
(23)
pKa = 4.8
as determined from spectral changes as a function of pH.13 Superoxide radical is extremely long lived on its own because the electron transfer reaction is very s l o w , j3 + • 02 --, 0 2 -~ k -< 1 M - l sec -I • 02
(24)
The reaction between the acid and the base form is much faster, • O2- + ' O 2 H ~
H O 2- + 02
(25)
k = 8 x 10 7 M -l sec -l Because the pKa value for • 02- is only 2 units below biological pH, superoxide would decay quite rapidly in biosystems, even in the absence of any other reactions, such as those with cytochrome c, • 02
+ cyt c(lll)--~ 02 + cyt c(ll)
k-
105M -j sec -l
~-"L. R. Karam and M. G. Simic, Anal• Chem. 60, 1842 (1988). i~ B. H. J. Bielski, Photochem. Photobiol. 28, 645 (1978)•
(26)
96
PRODUCTION, DETECTION~ AND CHARACTERIZATION
[2]
or cobalt macrocyclic complexes, 14 for example (M is 1,3,8,10-tetraene N4), 15 • 02-
+ MCo(II) ~
k = 1.6
MCo(II)O2-
× 10 9 M -1 s e c -1
(27)
Peroxyl Radicals, RO0. Peroxyl radicals (alkylperoxyl radicals) and their chemistry are amenable to pulse radiolytic studies. A variety of alkyl radicals can be generated in aqueous and nonaqueous solutions. In aqueous media, where most studies have been conducted, hydroxyl radical is used as an initiator. In the presence of oxygen (NzO : 02 = 4 : 1) and solute (H2R), the following consecutive reactions occur: • OH + H2R ~ H20 + HR. k ~ 1 0 9 M -1 s e c - I
(28)
HR. + 02 ~ HROO. k ~ 109 M -1 s e c - I
(29)
Of particular interest are peroxyl radicals of amino acids, sugars, DNA bases, and fatty acids. 4 Peroxyl radicals interact rapidly with each other, and the rate constant may vary considerably. One of the mechanisms of interaction is formation of tetroxides, which may decay according to the Russell mechanism, ~5 2 HROO. --~ HROOOORH ~ RO + HROH + 02 k --- 107 M -1 s e c -1 k ~ 0 . 1 - 1 s e c -I
(30)
The a-halogenated peroxyl radicals, which are much more reactive than their nonhalogenated counterparts (e.g., E7 -- 0.6 V for CH3OO .,~6 whereas E7 -> 1.1 V for CC13OO • 17), have been of considerable interest. The increase in the redox potential is paralleled by an increase in reactivity with electron donors (phenolic antioxidants, ascorbate). Poor electron donors, such as linoleic acid (H2L), react fairly slowly and are not amenable to pulse radiolytic studies, as for example, HROO. + H2L--~ HROOH + H L .
(31)
k ~ 60 M -1 sec -1 14 M. G. Simic and M. Z. Hofman, J. Am. Chem. Soc. 99, 2370 (1977). t~ G. A. Russell, J. Am. Chem. Soc. 79, 3871 (1957). 16 R. E. Huie and P. Neta, Int. J. Chem. Kinet. 18, 1185 (1986)• t7 S. V. Jovanovic and M. G. Simic, in "Oxygen Radicals in Biology and Medicine" (M. G. Simic, K. A. Taylor, J. F. Ward, and C. von Sonntag, eds.), p. 115. Plenum, New York, 1988. 18j. A. Howard and K. V. Ingold, Can. J. Chem. 45, 785 (1967).
[2]
PULSE RADIOLYSIS IN OXYGEN RADICALS
97
An interesting reaction of some peroxyl radicals is elimination of • 02-. For example, elimination o f . 02- from a-hydroxyperoxyl radicals
at pH values approaching the pKa value for the hydroxyl group can be followed by kinetic conductivity because a neutral species is converted to two charged species, OO'
on~
/
/
C=O+.O2 + n +
(32)
Alkoxyl Radicals, HRO. Alkoxyl radicals (alkyloxy radicals) have not been investigated by pulse radiolysis because of their instability. The only convenient way of generating them is via 19 HROOH + eaq--'-> HRO. + OH-
(33)
k -'= 10l° M -1 sec -l Alkoxyl radicals decay rapidly 19 via the so-called fl-scission mechanism, O.
- - ~ - - ~ - - ~ - - ~ ' + O=C / I
I
k ~ 107 s e c -I
I
(34)
\
(in water)
Alkoxyl radicals are studied more conveniently by laser photolysis, which allows faster generation. 2° Alkoxyl and hydroxyl radicals are generic species, and their properties should be similar. For example, the following reaction, HRO. + H2R ~ HROH + HR. k ~ 107 M - l s e c - l
(35)
is similar to reaction (28).
Phenoxyl and Aroxyl Radicals, ArO. Phenoxyl radicals are derivatives of the primary phenoxyl radical originating from phenol. Aromatic oxyl radicals (aroxyl radicals, ArO .) are a more general form of these important species, and no special effort will be made to distinguish them. Phenoxyl radicals originate from phenolic antioxidants [butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ot-tocopherol]. Aroxyl radicals originate from, for example, heterocyclic physiological antioxidants (5-OH-Trp and ( C 6 H 5 0 ")
19 p. Neta, M. Dizdaroglu, and M. G. Simic, lsr. J. Chem. 24, 25 (1984). 20 j. C. Scaiano, in "Oxygen Radicals in Biology and Medicine" (M. G. Simic, K. A. Taylor, J. F. Ward, and C. yon Sonntag, eds.), p. 59. Plenum, New York, 1988.
98
PRODUCTION, DETECTION, AND CHARACTERIZATION
[2]
serotonin, which are 5-hydroxyindole derivatives) and numerous other aromatic hydroxy derivatives (naphthol, 8-hydroxyguanine, uric acid). Phenoxyl radicals can be generated from phenol using either • OH or oxidizing radicals (oxidants). The reactions are somewhat different2J: (36)
C6HsOH + . OH --~ • C6Hs(OH)2
k ~ 6
x
10 9
M -t sec -j
• C6Hs(OH)2--~ C6HsO" + OH
(37)
+ H+
k ~ 103 secHaving a high oxidation potential [ET(C6HsO "/C6HsOH) = 0.95 V],17 reactions of phenol with oxidants are slow: C6HsOH + • B r , --~ C6H50" + 2 Br k = 106M -I sec -I
(38)
+ H+
The rate is increased considerably by deprotonation of the hydroxyl group or by electron-donating substituents, such as the methoxyl group (CH30), which reduces the redox potential to 0.73 V. 9 Hence, 17 p-CH3OC6H4OH + • Br_, --~ p-CH3OC6H40. + 2 Br
+ H+
(39)
k = 8.8 x 107M -I sec J The effect of the p-methoxyl substituent is enhanced considerably by the formation of a five- or six-membered ring between oxygen of the methoxyl group and the adjacent position on the benzene ring. 22 Vitamin E (EOH) is a well-known example of a six-membered ring. The redox potential of vitamin E (E7) is reduced to 0.48 V, ~7 and its reactivity with oxidants is greatly enhanced. In cyclohexanefl 3 E O H + H R O O . --~ E O . + HROO
(40)
k = 7.9 x 106 M -1 sec -J Ea = 2.8 kcal mol -l Rate constants for selected phenolic antioxidants and oxyl radicals are shown in Table 1. Aroxyl radicals absorb strongly (A = 4 - 6 x 103 M -~ cm ~) in the 400 nm region or, for some substituted phenols, even at longer wavelengths. 24 Hence, pulse radiolysis is an excellent experimental technique to study the properties of aroxyl radicals. One property is the decay kinetics. Most aroxyl radicals (BHA, B H T , phenol) decay rapidly, '_t E. J. Land and M. Ebert, Trans. Faraday, Soc. 63, 1181 (1967). -,2 G. W. Burton, T. Doba, E. J. Gabe, L. Hughes, F. L. Lee, L. Prasad, and K. U. Ingold, J. Am. Chem. Soc. 107, 7053 (1985). 23 M. G. Simic and E. P. L. Hunter, in "'Radioprotectors and Anticarcinogensis" (O. F. Nygaard and M. G. Simic, eds.), p. 449. Academic Press, New York, 1983. _,4 S. Steenken and P. Neta, J. Phys. Chem. 86, 3661 (1982).
[2]
99
PULSE RADIOLYSIS IN OXYGEN RADICALS TABLE l RATE CONSTANTS FOR REACTION OF SOME RADICALS WITH ANTIOXIDANTS" Substance
-R
a-Tocopherol
PULSE RADIOLYSIS IN OXYGEN RADICALS
89
[2] P u l s e R a d i o l y s i s in S t u d y o f O x y g e n R a d i c a l s
By MICHAEL G. S1M1C Oxygen Radicals and Pulse Radiolysis Because of the unpaired electron, oxygen radicals, like most other free radicals, are highly reactive not only with numerous and varied substrates but also with each other.l Consequently, their lifetimes either in model aqueous systems or especially in biosystems are very short. This particular characteristic renders studies of their physicochemical properties complex. Studies of their structural features can be simplified by generating them in a solid or a frozen matrix where their lifetimes may extend indefinitely. Their kinetics and energetics properties, however, can be determined only in liquid systems where the lifetimes are short. Hence, physicochemical properties of oxygen radicals can be studied only with detection techniques such as kinetic spectrophotometry, kinetic conductivity, time-resolved Raman and electron spin resonance (ESR) spectroscopy, and many other techniques with short time resolution. 2,3 In addition to adequate monitoring systems, studies of free radicals require convenient free radical-generating techniques and methods. These techniques and methods must satisfy some basic prerequisites. First, the radicals should be generated in a short period of time, before they start reacting with each other and the substrates. Second, the concentrations of radicals generated should be high enough to allow accurate monitoring. Third, only a specific radical should be generated without the presence of other interfering radicals. Finally, the capability of generating diverse free radicals as required, and sometimes a combination of radicals, should exist. Such features can be found in a technique known as pulse radiolysis. 3.4 Pulse radiolysis is accomplished by a combination of a pulsed-radiationgenerating machine (electron accelerator) and a variety of time-resolved detecting systems. In many respects pulse radiolysis, in which free radiW. A. Pryor (ed.), " F r e e Radicals in Biology," Vols. I - V I . A c a d e m i c Press, N e w York, 1976-1985. 2 L. M. Dorfman, J. Chem. Educ. 58, 82 (1981). 3 Farhataziz and M. A. J. Rodgers (eds.), "Radiation C h e m i s t r y . " Verlag C h e m i e , N e w York, 1987. 4 C. v o n Sonntag, " T h e Chemical Basis o f Radiation Biology." Taylor & Francis, L o n d o n , 1987.
METHODS IN ENZYMOLOGY.VOL. 186
Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
90
PRODUCTION, DETECTION, AND CHARACTERIZATION
[2]
cals are generated by ionizing radiations, is similar to laser and flash photolysis, 5 in which free radicals are generated by photonic processes. In this chapter pulse radiolysis studies of hydroxyl (. OH), peroxyl (HROO-), superoxide (. 02-), alkoxyl (HRO.), and phenoxyl radicals (ArO -), as well as their properties, are briefly discussed. Underlying principles, attributes, and limitations are emphasized; detailed accounts of pulse radiolysis have been published recently. 3.4 Generation of Free Radicals Ionizing radiations (high-energy electrons, X- and y-rays, high-energy nuclei, etc.) ionize the matter through which they pass. In aqueous media, the first step is ionization of water, H_,O---~ H_~O+" + e-
(1)
Both H20 +" and e - a r e , in principle, free radicals because they do not have paired electrons. Within a few picoseconds (10 -12 sec) they undergo the following reactions: H:O +-+ H20 ~ . OH + H30+ e- + n H,O--~ eaq
(2) (3)
Hence, if the radiation pulse were 1 nsec long, by the end of the pulse the reaction cell through which the radiation passed would contain relatively homogeneously distributed hydroxyl radicals and hydrated electrons, e~q. In addition to these two extremely reactive radicals, a small amount of. H is generated. Therefore, radiation splits water into primary water radicals (yields are indicated in parentheses): HzO --> • OH (2.8) + e~q (2.8) + • H (0.6)
(4)
In pure water the primary water radicals recombine at diffusion-controlled rates, for example, • OH + e,q --9 O H -
(5)
k - - 3 × 10 I°M -l sec l Water radicals react with solutes when present to give a second generation of radicals, the solute radicals. The reactivities of diverse solutes with all three primary water radicals have been tabulated. 6 The simultaneous occurrence of • OH and eaq reactions may interfere with the measurements of free radical properties. To resolve this experi5 R. V. Bensasson, E. J. Land, and T. G. T r u s c o n , " F l a s h Photolysis and Pulse Radiolysis." Pergamon, New York, 1983. 6 G. V. Buxton, C. L. Greenstock, W. P. Helman, and A. B. Ross, J. Phys. Chem. R~f~ Data 17, 513 (1988).
[2]
PULSE RADIOLYSIS IN OXYGEN RADICALS
91
mental difficulty, two methods m a y be used. H y d r a t e d electrons m a y be c o n v e r t e d to hydroxyl radicals in the presence of gaseous N_,O (usually ! arm): e~q + N_,O ~ ' O H + OH + N_, (6) k = 9 x l 0 9 M -I sec -j At 1 atm and r o o m temperature, [N20] = 25 mM. Hence, reaction (6) is completed in about 1 nsec. This rapid generation of the radiolytic • O H has critical advantages o v e r the much slower generation of • O H via the H a b e r - W e i s s reaction, H_,O2 + Fe(II)EDTA--* . O H + OH
k = 104 M -t
+ Fe(III)EDTA
(7)
sec -f
Unfortunately, there is no convenient method to convert hydroxyl radical to hydrated electron. The experimental conditions can be simplified, h o w e v e r , by removing • O H with solutes that give redox-unreactive radicals. E x a m p l e s of such solutes are tert-butanoi and some allylic compounds: •OH
+ (CH3)3COH --) k = 6 × •oil
+
H20 + • CH2(CH3)2COH
\ C=C / --, \ c _ ~ OH / \ / I
k = 2
x
(8)
108 M -~ s e c - j (9)
109 M -I sec -I
Neither radical is a good hydrogen a t o m or electron donor nor exhibits a c i d - b a s e properties. Both radicals have weak absorption spectra below 250 nm, an important feature in kinetic spectroscopy. When reducing radicals are required, the oxidizing • O H can be converted to a reducing radical, for example, -OH + HCO_,---~ H_,O + .CO_, k = 3.2 × l 0 9 M -j sec i
(10)
With an E0 value of - 1.9 V, the carboxyl radical is a powerful reductant. It is used to reduce disulfide bonds, c y t o c h r o m e s , quinones, nitro compounds, etc. Since superoxide has an E7 value of - 0 . 3 3 V, • CO2- readily reduces oxygen: • co_,
+ o_, ~
co_, + - o_,-
( l I)
k = 4 × 109M -I sec -I In practice, solute concentrations of the order of 1 to 100 m M are generally used. The actual concentrations must be adjusted t o satisfy specific experimental requirements, particularly when multiple solutes
92
PRODUCTION, DETECTION, AND CHARACTERIZATION
[2]
are used. At concentrations greater than 1 M, direct ionization of the solute (approximately equal to the weight percent of the solute) must be taken into account. The dose per pulse may be varied, and concentrations of free radicals from 1 nM to 0. l mM per pulse can be generated easily. The time scale of free radical monitoring in most experiments is 0.1/zsec to 1 sec. If required, however, special equipment allows measurements on a time scale of picoseconds. Physicochemical Properties of Radicals
Absorption Spectra Free radical absorption spectra are usually red shifted compared with parent compounds, an important feature in monitoring. The spectra can be obtained point by point by measuring absorption at different wavelengths selected by a monochromator at a constant dose/pulse) The absorbances are calculated from actual concentrations of radicals corresponding to the dose/pulse. Instead of multiple pulsing, an optical multichannel analyzer (OMA) records absorption spectra at a preselected time after a single pulse. 7 The technique is based on spectrographic resolution of analyzing light and a diode array instead of a monochromator and photomultiplier as in kinetic spectroscopy. Hydroxyl and alkoxyl radicals have poorly defined spectra; peroxyl radicals absorb at 240 to 250 nm, • O2H at 225 nm, • 02- at 245 nm, and p-substituted phenoxyl radicals at about 400 nm.
Acid-Base Properties Acid-base properties 8 are determined from spectral changes with pH. For example, a-hydroxy radicals are stronger acids than their parent alcohols:
/
C--OHm-
pKa ~ 10-12
/
C--O- + H +
(12)
(for aliphatic)
The absorption spectra of the deprotonated forms are usually red shifted, as already indicated for the • O2H and • 02- forms.
7 E. P. L. Hunter, M. G. Simic, and B. Michael, Rev. Sci. lnstrum. $6, 2199 (1985). 8 E. Hayon and M. G. Simic, Acc. Chem. Res. 7, 114 (1974).
[2]
PULSE RADIOLYSIS IN OXYGEN RADICALS
93
Redox Properties Redox properties are determined from interactions of free radicals with oxidants and reductants. Redox potentials can be determined if a satisfactory equilibrium can be achieved between the radical under investigation, • X, and a radical, • A, with a known redox potential, kf
-X+A ~ X kr
+.A
(13)
using the Nernst equation: AE = E(. A/A-) - E(. X/X-) = 0.059 log K
(14)
The equilibrium constant K can be determined kinetically, K = kf/kr, or spectroscopically from relative absorptions of two radicals at the equilibrium, provided their spectra do not overlap. The redox potential of superoxide radical, E7 = -0.33 V, was determined this way, using quinones (Q) with a known redox potential, 9 "02- + Q ~ 02 + "Q-
(15)
It is important to realize that the redox potentials of free radicals are oneelectron redox potentials, and they should not be confused with twoelectron redox potentials. Miscellaneous Properties Some other properties of free radicals are crucial for their proper identification and monitoring. Radicals may be neutral or charged, which is easily discernible by kinetic conductivity. They may be long lived, for example, the viologen radicals. They all have an unpaired electron and are consequently amenable to ESR investigations (flow or kinetic).l° In addition to structural features, ESR provides information about the distribution of the unpaired electron within radicals. Kinetics and Mechanisms of Oxygen Radicals Hydroxyl Radical, • OH Reactions o f - O H radicals are invariably studied in the presence of NzO in order to eliminate hydrated electrons [reactions (6)]. In addition to 9 D. Meisel and G. Czapski, J. Phys. Chem. 79, 1503 (1975).
x0A. D. Trifunac, in "Study of Fast Processes and Transient Species by Electron Pulse Radiolysis"(J. H. Baxendaleand F. Busi, eds.), p. 163.Reidel,Hingham,Massachusetts, 1982.
94
PRODUCTION, DETECTION,AND CHARACTERIZATION
[2]
hydrogen-atom abstraction [reaction (8)] and addition to double bonds [reaction (9)], hydroxyl radical adds readily to aromatic rings. For example, phenylalanine, Phe, reacts with • O H to give three transients (o-, m-, and p - P h e - - O H ) , which absorb in the 300 to 350 nm region. ~ Reaction of these transients can be followed and measured by monitoring their absorption. F r o m both pulse radiolytic and product measurements the following scheme has been developed:
R ~OH
R
©
R -.~ I ~ O H
R
+ *OH
=
[~°)~
o-Tyr
R
OH
---
II
1
~OH
m-Tyr
(16)
Phe R
R
OH
OH
In the absence of oxygen, P h e - - O H transients disappear by disproportionation and combination,tl 2/'he--OH [
, Tyr + PheH--OH , HO--Phe--Phe--OH
(17a) (17b)
The hydrated benzene ring readily loses water both in the dimers and monomers, for example, PheH--OH ~ Phe + H:O
(18)
In the presence of oxygen, OH adducts react with oxygen, /'he--OH + 02---,' OOPhe--OH
(19)
The reaction mechanisms of this peroxyl radical are not fully determined. Some of the products are known, however, namely, the three isomers of tyrosine. Consequently, • OOPhe--OH ---~----~o-, m-. and p-Tyr + products
(20)
it M. G. Simic, E. Gajewski, and M. Dizdaroglu, Radiat. Phys. Chem. 24, 465 (1985).
[2]
PULSE RADIOLYSIS IN OXYGEN RADICALS
95
Hence, both in the absence and in the presence of oxygen, • OH radicals lead to formation of the tyrosine isomers. Since o-Tyr has not been found in normal proteins, its presence is indicative o f . OH radical processes.*2 Formation of o-Tyr in proteins can be used as a proof of radiation processing of foods. Hydroxyl radical is a potent oxidant (E0 = 2.6 V) capable of oxidizing numerous metal ions and their complexes• The redox component in a biosystem may be diminished owing to competition of abstraction and addition reactions. Superoxide Radical, • 02-
In addition to being formed by reactions (10) and (11), superoxide radical can be generated by the reaction of oxygen with hydrated electrons: e~q + 02 ~
- O2
(21)
or hydrogen atoms: • H + O2-->'O2H
(22)
since • O2H ~
-O2
+ H+
(23)
pKa = 4.8
as determined from spectral changes as a function of pH.13 Superoxide radical is extremely long lived on its own because the electron transfer reaction is very s l o w , j3 + • 02 --, 0 2 -~ k -< 1 M - l sec -I • 02
(24)
The reaction between the acid and the base form is much faster, • O2- + ' O 2 H ~
H O 2- + 02
(25)
k = 8 x 10 7 M -l sec -l Because the pKa value for • 02- is only 2 units below biological pH, superoxide would decay quite rapidly in biosystems, even in the absence of any other reactions, such as those with cytochrome c, • 02
+ cyt c(lll)--~ 02 + cyt c(ll)
k-
105M -j sec -l
~-"L. R. Karam and M. G. Simic, Anal• Chem. 60, 1842 (1988). i~ B. H. J. Bielski, Photochem. Photobiol. 28, 645 (1978)•
(26)
96
PRODUCTION, DETECTION~ AND CHARACTERIZATION
[2]
or cobalt macrocyclic complexes, 14 for example (M is 1,3,8,10-tetraene N4), 15 • 02-
+ MCo(II) ~
k = 1.6
MCo(II)O2-
× 10 9 M -1 s e c -1
(27)
Peroxyl Radicals, RO0. Peroxyl radicals (alkylperoxyl radicals) and their chemistry are amenable to pulse radiolytic studies. A variety of alkyl radicals can be generated in aqueous and nonaqueous solutions. In aqueous media, where most studies have been conducted, hydroxyl radical is used as an initiator. In the presence of oxygen (NzO : 02 = 4 : 1) and solute (H2R), the following consecutive reactions occur: • OH + H2R ~ H20 + HR. k ~ 1 0 9 M -1 s e c - I
(28)
HR. + 02 ~ HROO. k ~ 109 M -1 s e c - I
(29)
Of particular interest are peroxyl radicals of amino acids, sugars, DNA bases, and fatty acids. 4 Peroxyl radicals interact rapidly with each other, and the rate constant may vary considerably. One of the mechanisms of interaction is formation of tetroxides, which may decay according to the Russell mechanism, ~5 2 HROO. --~ HROOOORH ~ RO + HROH + 02 k --- 107 M -1 s e c -1 k ~ 0 . 1 - 1 s e c -I
(30)
The a-halogenated peroxyl radicals, which are much more reactive than their nonhalogenated counterparts (e.g., E7 -- 0.6 V for CH3OO .,~6 whereas E7 -> 1.1 V for CC13OO • 17), have been of considerable interest. The increase in the redox potential is paralleled by an increase in reactivity with electron donors (phenolic antioxidants, ascorbate). Poor electron donors, such as linoleic acid (H2L), react fairly slowly and are not amenable to pulse radiolytic studies, as for example, HROO. + H2L--~ HROOH + H L .
(31)
k ~ 60 M -1 sec -1 14 M. G. Simic and M. Z. Hofman, J. Am. Chem. Soc. 99, 2370 (1977). t~ G. A. Russell, J. Am. Chem. Soc. 79, 3871 (1957). 16 R. E. Huie and P. Neta, Int. J. Chem. Kinet. 18, 1185 (1986)• t7 S. V. Jovanovic and M. G. Simic, in "Oxygen Radicals in Biology and Medicine" (M. G. Simic, K. A. Taylor, J. F. Ward, and C. von Sonntag, eds.), p. 115. Plenum, New York, 1988. 18j. A. Howard and K. V. Ingold, Can. J. Chem. 45, 785 (1967).
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PULSE RADIOLYSIS IN OXYGEN RADICALS
97
An interesting reaction of some peroxyl radicals is elimination of • 02-. For example, elimination o f . 02- from a-hydroxyperoxyl radicals
at pH values approaching the pKa value for the hydroxyl group can be followed by kinetic conductivity because a neutral species is converted to two charged species, OO'
on~
/
/
C=O+.O2 + n +
(32)
Alkoxyl Radicals, HRO. Alkoxyl radicals (alkyloxy radicals) have not been investigated by pulse radiolysis because of their instability. The only convenient way of generating them is via 19 HROOH + eaq--'-> HRO. + OH-
(33)
k -'= 10l° M -1 sec -l Alkoxyl radicals decay rapidly 19 via the so-called fl-scission mechanism, O.
- - ~ - - ~ - - ~ - - ~ ' + O=C / I
I
k ~ 107 s e c -I
I
(34)
\
(in water)
Alkoxyl radicals are studied more conveniently by laser photolysis, which allows faster generation. 2° Alkoxyl and hydroxyl radicals are generic species, and their properties should be similar. For example, the following reaction, HRO. + H2R ~ HROH + HR. k ~ 107 M - l s e c - l
(35)
is similar to reaction (28).
Phenoxyl and Aroxyl Radicals, ArO. Phenoxyl radicals are derivatives of the primary phenoxyl radical originating from phenol. Aromatic oxyl radicals (aroxyl radicals, ArO .) are a more general form of these important species, and no special effort will be made to distinguish them. Phenoxyl radicals originate from phenolic antioxidants [butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ot-tocopherol]. Aroxyl radicals originate from, for example, heterocyclic physiological antioxidants (5-OH-Trp and ( C 6 H 5 0 ")
19 p. Neta, M. Dizdaroglu, and M. G. Simic, lsr. J. Chem. 24, 25 (1984). 20 j. C. Scaiano, in "Oxygen Radicals in Biology and Medicine" (M. G. Simic, K. A. Taylor, J. F. Ward, and C. yon Sonntag, eds.), p. 59. Plenum, New York, 1988.
98
PRODUCTION, DETECTION, AND CHARACTERIZATION
[2]
serotonin, which are 5-hydroxyindole derivatives) and numerous other aromatic hydroxy derivatives (naphthol, 8-hydroxyguanine, uric acid). Phenoxyl radicals can be generated from phenol using either • OH or oxidizing radicals (oxidants). The reactions are somewhat different2J: (36)
C6HsOH + . OH --~ • C6Hs(OH)2
k ~ 6
x
10 9
M -t sec -j
• C6Hs(OH)2--~ C6HsO" + OH
(37)
+ H+
k ~ 103 secHaving a high oxidation potential [ET(C6HsO "/C6HsOH) = 0.95 V],17 reactions of phenol with oxidants are slow: C6HsOH + • B r , --~ C6H50" + 2 Br k = 106M -I sec -I
(38)
+ H+
The rate is increased considerably by deprotonation of the hydroxyl group or by electron-donating substituents, such as the methoxyl group (CH30), which reduces the redox potential to 0.73 V. 9 Hence, 17 p-CH3OC6H4OH + • Br_, --~ p-CH3OC6H40. + 2 Br
+ H+
(39)
k = 8.8 x 107M -I sec J The effect of the p-methoxyl substituent is enhanced considerably by the formation of a five- or six-membered ring between oxygen of the methoxyl group and the adjacent position on the benzene ring. 22 Vitamin E (EOH) is a well-known example of a six-membered ring. The redox potential of vitamin E (E7) is reduced to 0.48 V, ~7 and its reactivity with oxidants is greatly enhanced. In cyclohexanefl 3 E O H + H R O O . --~ E O . + HROO
(40)
k = 7.9 x 106 M -1 sec -J Ea = 2.8 kcal mol -l Rate constants for selected phenolic antioxidants and oxyl radicals are shown in Table 1. Aroxyl radicals absorb strongly (A = 4 - 6 x 103 M -~ cm ~) in the 400 nm region or, for some substituted phenols, even at longer wavelengths. 24 Hence, pulse radiolysis is an excellent experimental technique to study the properties of aroxyl radicals. One property is the decay kinetics. Most aroxyl radicals (BHA, B H T , phenol) decay rapidly, '_t E. J. Land and M. Ebert, Trans. Faraday, Soc. 63, 1181 (1967). -,2 G. W. Burton, T. Doba, E. J. Gabe, L. Hughes, F. L. Lee, L. Prasad, and K. U. Ingold, J. Am. Chem. Soc. 107, 7053 (1985). 23 M. G. Simic and E. P. L. Hunter, in "'Radioprotectors and Anticarcinogensis" (O. F. Nygaard and M. G. Simic, eds.), p. 449. Academic Press, New York, 1983. _,4 S. Steenken and P. Neta, J. Phys. Chem. 86, 3661 (1982).
[2]
99
PULSE RADIOLYSIS IN OXYGEN RADICALS TABLE l RATE CONSTANTS FOR REACTION OF SOME RADICALS WITH ANTIOXIDANTS" Substance
-R
a-Tocopherol
