[3] Characterization of singlet oxygen

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CHEMISTRY AND BIOCHEMISTRY OF OXYGEN

[3]

[3] C h a r a c t e r i z a t i o n o f S i n g l e t O x y g e n

By CHRISTOPHER S. FOOTE, F. C. SHOOK, AND R. B. ABAKERLI

Considerable recent interest has focused on the question of the intermediacy of singlet molecular oxygen (~O2) in biological processes, formed either as a result of natural processes or under the effect of exogenous agents such as photosensitizers. ~,2 In well-characterized chemical systems, singlet oxygen can be produced by both photochemical and nonphotochemical processes, and its intermediacy can be quantitatively determined. 3 In biological systems (which are often inhomogeneous) demonstration of its intermediacy in the oxidation of target species is not easy. The main problem is that singlet oxygen is likely to be accompanied by other reactive oxygen species such as superoxide ion (O2-), hydroxyl radical (OH.), and alkoxy and peroxy radicals. The reactions of these species may compete with or be confused with those of singlet oxygen. 4 The four main methods for detection of singlet oxygen in a reacting system are to compare observed products with those known to be produced by singlet oxygen, to determine the change in the amount of reaction or effect produced on adding substances that modify the lifetime of singlet oxygen, to measure the luminescence of singlet oxygen, or to produce singlet oxygen independently in the system and compare its reactions with those observed under the test conditions. All of these methods have difficulties; the greatest degree of certainty is obtained by using as many independent techniques as possible in combination. At the present state of development, no one method used alone is likely to provide definitive evidence for the intermediacy of singlet oxygen in a complex system, nor is it likely that a single technique will even be usable in all systems. Thus this chapter provides a brief overview of a few of the available methods, and specific directions for only one. It is hoped that the technique described will be used with these warnings in mind.

i N. 1. Krinsky, ht "Singlet Oxygen" (H. H. Wasserman and R. W. Murray, eds.), p. 597. Academic Press, New York, 1979. C. S. Foote, Free Radicals Biol. 2, 85-133 (1976). 3 R. W. Murray and H. H. Wasserman, eds,, "Singlet Oxygen." Academic Press, New York, 1979. 4 C. S. Foote, in "Biochemical and Clinical Aspects of Oxygen" (W. S. Caughey, ed.), p. 603. Academic Press, New York, 1979.

METHODS IN ENZYMOLOGY,VOL. 105

Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISBN 0-12-182005-X

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SINGLET OXYGEN

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Sources of Singlet Oxygen

Nonphotochemical Routes There are a number of nonphotochemical reactions that have been shown to produce singlet oxygen in good yield. This subject has been reviewed. 5 Most of these reactions use strong oxidizing species (e.g., sodium hypochlorite, triphenyl phosphite ozonide) that could react directly with oxidizable species present in a biological system. Probably the best choice for a nonchemical source of singlet oxygen is to use an aromatic endoperoxide that liberates singlet oxygen on warming6'7; in principle, these derivatives can be individually designed to reflect desired solubility characteristics and the temperature at which singlet oxygen is liberated, although not much has yet been done along these lines.

Photosensitized Reactions There are two fundamental types of sensitized photooxygenation, s They differ in that the triplet sensitizer reacts directly with the substrate in the first (the Type I reaction), while in the second (Type II), it reacts first with oxygen to produce singlet oxygen. Thus even when photooxygenation (the most studied source of singlet oxygen) is used, the reactive intermediate is not certain to be singlet oxygen, but must be demonstrated in each case. Sens ~ ISens ~ ~Sens Type I ~ 3Sens l Subs,. Radicals

O:

~ Type I1 [ ~O2

An important characteristic of these reactions is that the process observed is often very concentration dependent. The reason is that there is always a competition between substrate and oxygen for the sensitizer triplet, s If the substrate concentration is too low, or the oxygen concenR. W. Murray, h~ "Singlet Oxygen" (H. H. Wasserman and R. W. Murray, eds.), p. 59. Academic Press, New York, 1979. 6 N. J. Turro, M. F. Chow, and J. Rigaudy, J. Am. Chem. Soc. 103, 7218 (1981). H. H. Wasserman and J. R. Scheffer, J. Am. Chem. Soc. 89, 3073 (1967). 8 C. S. Foote, #a "Pathology of Oxygen" (A. P. Autor, ed.), p. 21. Academic Press, New York, 1982.

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tration is too high, the Type I reaction may become very inefficient, and the Type II reaction is favored. Of course, if there is no good substrate for singlet oxygen available at reasonable concentration, the Type II process will result in quenching of the excited sensitizer with no reaction observed. In systems where the sensitizer is bound to an easily oxidized biomolecule, the local concentration of substrate becomes very high and Type I reactions become highly favored. In the Type I reaction, the sensitizer interacts directly with the substrate (for example, a hydrogen or electron donor) with a resulting hydrogen atom or electron transfer to produce radicals. These radicals can subsequently react with oxygen to produce oxidized products or other reactive species. The products are often peroxides, which can in turn break down to induce free radical chain autoxidation, leading to further oxidation in a nonphotochemical step. R O O R ' ~ RO' + R ' O '

Sensitizers can produce superoxide ion by undergoing electron transfer processes with the substrate or oxygen, as shown below. 3Sens + Subs ~ S e n s - + Subsox S e n s - + Oz--~ Sens + 02-

or 3Sens + 02 ~ Sens + + Oz-

These reactions produce O2-, which can subsequently give the very reactive hydroxyl radical (OH.) by several pathways. These radicals can react with organic molecules in a variety of ways, or can initiate radical chain autoxidation. 9 The sorts of compounds that react in the Type I reaction are those that are electron rich or have easily abstraetable hydrogens. Particularly reactive, for example, are aromatic amines, phenols, and sulfhydryl compounds. In the Type II reaction, the sensitizer can transfer its excitation energy to a ground state oxygen molecule, s Singlet molecular oxygen is produced. Singlet oxygen is a metastable species with a lifetime varying from about 4 #.sec in water to 25 to 100/~sec in nonpolar organic media that are reasonable models for lipid regions of the cell. 10 It is quite reactive, and reacts with many organic molecules to give peroxides or other oxidized products; however, it is also quite selective and fails to react with molecules that are not electron rich enough and simply returns to the ground state. Probably the best photochemical source of singlet oxygen for use in 9 A. Singh, Can. J. Physiol. Pharmacol. 60, 1330 (1982). Io F. Wilkinson and J. G, B r u m m e r , J. Phys. Chem. Ref. Dat. 10, 809 (1981).

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SINGLET OXYGEN

39

complex systems is one of the polymer-bound rose bengal derivativest~.~2; these must be carefully extracted to remove unbound rose bengal, but they are less likely than free dyes to give Type I reactions in biological systems. 302 ~

102 Acceplor~ AO2

A number of compounds have been found to quench (i.e.,deactivate without reaction) singlet oxygen efficiently.~3 For example, //-carotene inhibitsphotooxidation of 2-methyl-2-pentene efficientlyat 10-4 M without itselfbeing appreciably oxidized. Certain amines are quenchers, e.g., D A B C O (I,4-diazabicyclooctane).Other amines both quench singletoxygen and react with it,depending on conditions. Azide ion is a somewhat better quencher; phenols also quench singlet oxygen; some also react chemically with it. Io2 + Quencher--->302 + Quencher The major biological targets for singlet oxygen are now well k n o w n ) 4,15 Membranes are peroxidized, leading to fragility and easy lysis. The initial hydroperoxides appear to break down in a subsequent slower step, probably involving formation of free radicals and subsequent radical chain autoxidation to cause increased degradation of unsaturated molecules in the membrane.16 Nucleic acids are also an important target for singlet oxygen; guanine is the major target of this reaction. Many proteins and enzymes are damaged by singlet oxygen; the major targets are histidine, methionine, and tryptophan (although tryptophan can probably react by a Type I mechanism also). Tyrosine and cysteine are also photooxidation targets, although these appear more likely to be Type I reactions. Determination of Mechanism In homogeneous solution, the problem of mechanism determination is at its easiest and a variety of kinetic and trapping techniques can be used. u A. P. Schaap, A. L. Thayer, E. C. Blossey, and D. C. Neckers, J. Am. Chem. Soc. 97, 3741 (1975). 12A. P. Schaap, A. L. Thayer, K. A. Zaklika, and P. C. Valenti, J. Am. Chem. Sac. 101, 4016 (1979). z3C. S. Foote, in "Singlet Oxygen" (H. H. Wasserman and R. W. Murray, eds.), p. 139. Academic Press, New York, 1979. 14 C. S. Foote, in "Oxygen and Oxy-Radicalsin Chemistryand Biology" (M. A. J. Rodgers and E. L. Powers, eds.), p. 425. Academic Press, New York, 1981. 15 j. D. Spikes, in "Oxygen and Oxy-Radicalsin Chemistryand Biology" (M. A. J. Rodgers and E. L. Powers, eds.), p. 421. Academic Press, New York, 1981. i~ A. A. Lamola, T. Yamane, and A. M. Trozzolo, Science 179, 1131 (1973).

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CHEMISTRY AND BIOCHEMISTRY OF OXYGEN

It is important to recognize that the simple detection of a reactive species such as singlet oxygen in a system does not necessarily mean that it is the reactive species in the oxidation of the target molecules; its presence is a necessary but not sufficient condition for its intermediacy. In other words, we need to know not only how much of the intermediate is produced, but what fraction of the target molecules are actually oxidized via that intermediate. In this case, a negatioe result is easier to make definitive: if singlet oxygen is absent, it cannot be the reactive intermediate. It is useful in this case to set upper limits for the fraction of the reaction proceeding via singlet oxygen if possible. It is of the utmost importance that the technique used for the detection of singlet oxygen be specific for that intermediate, since many chemical reagents can react with different oxidizing species to give similar products. 4 There are a number of standard tests for the intermediacy of reactive oxygen species other than singlet oxygen; these have been reviewed, 9 and will not be discussed here, but their use should be considered in conjunction with tests for singlet oxygen. Specificity of the tests for other species is also a matter of concern. Radical chain processes also need to be ruled out; these may occur following the production of an initial peroxide molecule, and can lead to the oxidation of many more molecules than formed in the initial step. Radical chain oxidations are usually detected by their inhibition by a chain terminator such as a phenol, but this is often difficult to do with specificity in the presence of other oxidizing agents. RO" + PhOH --~ ROH + PhO.

(Chain Termination)

Singlet Oxygen Tests

Traps A large number of traps for singlet oxygen have been developed that give isolable products. The obvious hope in using these traps is that they will give these products only with singlet oxygen, but this is often not the case. The first class of traps to be used in biological systems was the furans; unfortunately, these are the least specific since they are oxidized by almost all strong oxidants to diketones. 4

[°l

I

[3]

SINGLET OXYGEN

41

A more specific class of traps is provided by substituted anthracene derivatives; these can be made water soluble with suitable substituents (S) and an isolable endoperoxide is formed. 17,~8 Unfortunately, these compounds sensitize their own photooxidation, so that the production of small amounts of product is difficult to avoid except in the complete absence of light. Also, their usefulness in complex systems may be limited because of adsorption and compartmentalization problems. R

R

R

R

One of the more specific traps that has been developed is cholesterol; this gives a single product (the 5a-hydroperoxide) on reaction with singlet oxygen. 19,2°Radical oxidation gives a very complex mixture that contains none of the 5a product. Cholesterol is useful as a marker for the presence of singlet oxygen in biological systems; however, a major drawback is its low reactivity, which results in poor sensitivity. It is also nearly completely insoluble in aqueous systems. The system using radiolabeled cholesterol bound to dispersible polymer beadlets, 21,22 described below, is quantitatible and overcomes the solubility and sensitivity limitations. Kinetic Methods The second method of detecting singlet oxygen is to inhibit its reactions by adding something that changes its lifetime by reacting with it, quenching it, or modifying its decay rate. This method can be quite effective in homogeneous solution, since the rate constants for a large number of compounds are well known.l° To be most effective, inhibition studies should be carried out quantitatively, so that all rate constants in the system are determined. It is much less effective simply to carry out one 17 A. P. Schaap, A. L. Thayer, G. R. Faler, K. Goda, and T. Kimura, J. Am. Clwm. Soc. 96, 4025 (1974). J8 j. M. Aubry, J. Rigaudy, C. Ferradini, and J. Pucheault, J. Am. Chem. Soc. 103, 4965 (1981). 19 L. L. Smith, J. I. Teng, M. J. Kulig, and F. L. Hill, J. Org. Chem. 38, 1763 0973). 20 L. L. Smith, W. S. Matthews, J. C. Price, R. C. Bachmann, and B. Reynolds, J. Cromatogr. 27, 182 (1967). 21 C. S. Foote, R. B. Abakerli, R. L. CIough, and R. I. Lehrer, in "Bioluminescence and Chemiluminescence" (M. A. DeLuca and W. D. McEIroy, eds.), p. 81. Academic Press, New York, 1980. 22 C. S. Foote. F. C. Shook, and R. B. Abakerli, J. Am. Chem. Soc. 102, 2503 (1980).

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point inhibition experiments, because all singlet oxygen quenchers and reagents are compounds of low oxidation potential and will also react with other strong oxidants. The kinetic parameters provide a fingerprint for the reaction species being trapped by the inhibitor. Of course, it is much more difficult to use quantitative techniques in inhomogeneous systems, since targets and quenchers are localized, and their local concentrations are usually not known. The inhibition method, if carefully quantitated, allows the determination of the fraction of target molecules reacting with singlet oxygen. The methodology of the kinetic techniques has been reviewed.~3 Another way of testing for the intermediacy of singlet oxygen is to determine the effect on the rate of the observed reaction on substituting D20 for water. 23 This technique is based on the fact that the lifetime of singlet oxygen in D20 is longer than in H20, so that reactions of singlet oxygen with substrates may be more efficient, since more singlet oxygen survives to react. This method requires that the reaction be carried out in such a way that only a small fraction of the singlet oxygen reacts with the substrate or with other quenchers and solvent deactivation determines the lifetime. This technique can and should be quantitatively compared with the effect calculated, based on known rate constants. Another problem is that other reactive species may show solvent deuterium isotope effects on their lifetimes. For example, 02- is known to live substantially longer in D20 than in water. 24 Luminescence There are two types of singlet oxygen luminescence, the direct emission from a single molecule at 1.27/zm, and "dimor' luminescence at 634 and 704 nm. 25 Both are extremely inefficient in solution, 102 "-' hv 2(|02) ~ hv

(I .27/.tin) (634,704 nm)

Dimol luminescence has often been used for the identification of singlet oxygen in biological systems. Unfortunately, this weak luminescence is usually accompanied by light of other wavelengths in complex systems. Although these emissions have been assigned to higher vibrational states of singlet oxygen, 25 it seems more likely that the extraneous emission comes from excited carbonyls or other species in the system. Precise wavelength determination is essential if this technique is to be used for the 23 R. Nilsson and D. R. Kearns, Photochem. Photobiol. 17, 65 (1973). 24 B. H. J. Bielski and E. Saito, J. Phys. Chem. "/5, 2263 (1971). 25 M. Kasha and A. U. Khan, Ann. N.Y. Acad. Sci. 171, 5 (1970).

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SINGLETOXYGEN

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detection of singlet oxygen; it appears to be of qualitative usefulness at best, since the luminescence depends on a second order process. More hope exists for quantitation of the 1.27 p.m emission. This luminescence, though weak, can be detected, both in steady-state 26.27 and time-resolved systems, 28-31and the wavelength is specific. We have used this system to detect singlet oxygen produced by the fungal photosensitizer cercosporin. 32This method may be useful for the detection of singlet oxygen in biological systems, although sensitivity requirements will be extreme. Changes in the lifetime of singlet oxygen measured using this technique could be interpreted in terms of the degree to which it is reacting with host molecules. Sample Procedure The procedure given below is a sample of the technique using the cholesterol beadlet preparation. The formation of the 5a-cholesterol oxidation product, determined by counting after thin-layer chromatography (TLC) separation, is a measure of the amount of singlet oxygen production; the presence of the isomeric 7a and 7/3 products are indications of free radical processes.19.2° Included is a method for determining the efficiency with which singlet oxygen is trapped, which depends on the method of preparation of the beadlets and the concentration of the dispersion. Knowing the efficiency of singlet oxygen trapping and the amount of 5or product formed allows calculation of the absolute amount of singlet oxygen formed. However, this determination must be supplemented by an inhibition study to allow determination of the quantitative involvement of singlet oxygen in the oxidation of target species. Methods

Materials Purification of Radiolabeled Cholesterol. Labeled [4-14C]cholesterol (New England Nuclear, 50/xCi in 2.5 ml benzene) was applied to a 10 × 20 cm × 0.25 mm preparative thin layer (TLC) plate (silica gel HF-254, 26 A. U. Khan and M. Kasha, Proc. Natl. Acad. Sci. U.S.A. 76, 6047 (1979). z7 A. A. Krasnovskii, Jr., Photochem. Photobiol. 36, 733 (1982). 2s K. I. Salokhiddinov, I. M. Bytcva, and B. M. Dzhagarov, Opt. Spektrosk. 47,487 (1979). 29 .p.R. Ogilby and C. S. Foote, J. Am. Chem. Soc. 104, 2069 (1982). 30 j. R. Hurst, J. D. MacDonald, and G. B. Schuster, J. Am. Chem. Soc. 104, 2065 (1982). 31 j. G. Parker and W. D. Stanbro, J. Am. Chem. Soc. 104, 2067 (1982). 32 D. C. Dobrowolsky and C. S. Foote, Angew. Chem. in press (1983).

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Merck). Unlabeled cholesterol was applied to the same plate, in a different spot, as a standard. The plate was irrigated twice with benzene/ethyl acetate (17 : 8). The portion of the plate containing the unlabeled cholesterol was sprayed with 50% H2SO 4 and heated gently with a heat gun to develop the bright red color characteristic of cholesterol. The corresponding unsprayed region was excised from the plate; the labeled cholesterol was then extracted with dry diethyl ether. The ether was removed and the cholesterol redissolved in 2.5 ml benzene. This solution was stored under argon in a tightly capped vial at -28 °. Aliquots were analyzed frequently; when the presence of significant activity outside the cholesterol band was seen, the sample was rechromatographed. Cholesterol Beadlets. A benzene solution (0.740 ml) of TLC-purified [14C]cholesterol (17.6 p.Ci/ml) was dried under a nitrogen stream and redissolved in 1 ml methanol. To this solution, 0.5 ml of a 10% polystyrene latex beadlet suspension (Dow Diagnostics) was added and dried under nitrogen. The residue was resuspended in I ml of doubly distilled water and diluted to 2 ml with phosphate-buffered saline (PBS). The suspension was filtered through a capillary, homogenized, and 10/.d was checked for radioactivity. Standards. The 3fl,5a-diol was synthesized by the procedure of Kulig and Smith33; the 7-ketone by that of Nickon and Mendelson~4; the 3fl-, 7or-, fl-diols through lithium aluminum hydride (LAH) reduction of the 7ketone, and the 5a,6a-epoxide by reacting cholesterol and m-chloroperoxybenzoic acid at room temperature. All standards were purified by column chromatography. A mixture containing the 5a-, the 7or-, and the 7fl,3fl-diols was conveniently prepared as follows. Cholesterol (0.10 g) (MCB, recrystallized twice from methanol) was dissolved in 20 mi dry benzene; 5.0 mg of zinc tetraphenylporphine was added as sensitizer. The solution was photooxidized for 4 hr. To facilitate conversion of the 5cr-hydroperoxide to the 7-hydroperoxides, the reaction mixture was transferred to a stoppered flask and allowed to stand at ambient temperature for 48 hr. The hydroperoxides were reduced by addition of excess NaBH4 and stirring for 1 hr. The solution was filtered, the benzene removed in vacuo, and the residue taken up in ether. An aliquot was removed and analyzed by TLC. Development showed the presence of cholesterol and the 5t~-, 7or-, and 7fl-diols. The mixture was stored under N2 at -28 °.

33 M. J. Kulig and L. L. Smith, J. Org. Chem. 38, 3639 (1973). 34 A. Nickon and W. L. Mendelson, J. Am. Chem. Soc. 87, 3921 (1965).

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Chromatography Cholesterol and oxidation products were analyzed by thin-layer chromatography on 20 × 20 cm × 0.25 mm preparative TLC plates (silica gel HF-254, Merck). The sample was applied in a thin streak using a commercial applicator. The plate was irrigated twice with benzene/ethyl acetate (17 : 8). Development of a narrow strip with a 50% H2SO4 spray and gentle heating typically gave four bands: (Rf, color with H2SO4, composition) Band I" 0.70, red-violet, cholesterol; Band 2: 0.24, blue, cholesterol 5adiol; Band 3: 0.19, blue, cholesterol 7/3-diol; Band 4: 0.13, blue, cholesterol 7o~-diol. The assignments were made by comparison with Rr values of known samples. For scintillation counting, the appropriate bands were excised from the TLC plate. The silica gel was transferred to a glass scintillation vial. BioFluor (10 ml), a high efficiency cocktail (New England Nuclear), was added. Total ~4C activity was recorded; typical counting times were 5 or 10 rain. Quenching was determined by the internal standards method. 35

Photooxidation All photooxidations were performed using a Sylvania DWY tungsten halogen lamp (60-80 V). A I% K~Cr207 solution was used as a filter. Oxygen was passed through the solution, but 02 uptake was not measured. Unless otherwise stated, photooxidations were performed at ambient temperature.

Assay This procedure will need to be modified somewhat, depending on the system to be studied. This version was developed for use with polymorphonuclear leukocytes. The samples were reduced with 1 ml of a methanol solution of triphenylphosphine (1.0 x 10-3 M) for at least 1 hr, then extracted with methanol three times, followed by centrifugation. The combined extracts were dried under nitrogen and the residue redissolved in 0.2 ml of toluene and checked for recovered radioactivity. The samples were applied to a silica gel plate. The unlabeled diols and cholesterol were applied as standards, and the plate was developed and assayed as above. After the identification of the standards, autoradiography was done by allowing the TLC plate to stay in contact with the film (Kodak SB-5 with Dupont Cronex Quanta II intensifying screens) for approximately I week at - 7 0 °. 3s A. Dyer, "An Introduction to Liquid Scintillation Counting," p. 57. Heyden, New York, 1974.

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Quantitation of to2 Trapping Efficiency Fifty microliters of a solution of [4-~4C]cholesterol in benzene (purified by TLC) was transferred by syringe to a clean, dry Pyrex test tube, and 0.2 mi of a 10% aqueous suspension of polystyrene latex beads was added via pipet. Approximately 0.5 ml ether and 5.0 ml of 5 × 10-4 M histidine in pH 8 phosphate buffer (0.1 M) was added. The solution was stirred for 0.5 hr while N2 was passed over the surface to remove the ether. Then I0/zl of 1.0 × 10-3 M methylene blue was added to give a final concentration of methylene blue of 2 × 10-6 M. The test tube was irradiated for 10 rain, then transferred to a separatory funnel and extracted with four 10-ml portions of CH2CI~. The organic layers were combined and excess triphenylphosphine added. The solution was stirred for I hr. The organic layer was dried over anhydrous Na2SO4 and the solvent removed in vacuo. The residue was taken up in ether and chromatographed. The appropriate bands were excised and counted to determine the amount of cholesterol oxidation product. The aqueous layer was transferred to a centrifuge tube and centrifuged for 10 rain. At the end of that time, there had collected at the bottom of the tube a small amount of suspended CH2C!2 and beads, leaving the aqueous layer only very faintly turbid. This solution was treated with the Pauly reagent 3° as follows: to 5 ml of solution was added 1 ml 1% sulfanilic acid in 1.0 N HCI and 1 ml of 5% NaNO2. The solutions were mixed and allowed to stand for 5 rain, then 3 ml of 20% Na2CO3 was added. The absorption spectra of the photooxidized sample, as well as that of a similarly treated unreacted sample of histidine, was obtained and compared to a reagent blank. This measurement allowed the amount of histidine photooxidized to be determined. A small correction was required since the amount of light absorbed by the sensitizer was decreased by light scattering in the turbid beadlet solution. The amount of correction is determined by photooxidizing a beadlet-free sample of histidine in parallel. The trapping efficiency assay should be done on each beadlet-cholesterol preparation, and under the same concentration conditions used in the test system, since it has been found that the trapping efficiency depends on the concentration. Calculation of Trapping Efficiency. The trapping efficiency (TE) is obtained from the formula

TE = PH/AH(fl + H) where P is the amount of 5a product, H the histidine concentration, AH 36 H. T. M a c P h e r s o n , Biochem. J. 40, 470 (1946).

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its change,/3 the concentration of histidine in H20 at which half the singlet oxygen is trapped (3.0 × 10-3 M37.3s); CF is the correction factor for light scattering. A typical value for CF is 1.34 and for TE is 2.5 x 10-5. The amount of ~O2 formed in the reaction is then Amt. IO2 = (P/TE)CF Units of all the concentrations are molar. Acknowledgment This work was supported by NIH Grant GM-20080. 37L. A. A. Sluyterman,Rec. Trao. Chim. 80, 989 (1961). L. Weft,Arch. Biochem. Biophys. 110, 57 (1965).

[4] R o l e o f I r o n in O x y g e n R a d i c a l R e a c t i o n s By BARRY HALLIWELL and JOHN M. C. GUTTERIDGE

The discovery of the enzymatic production of the superoxide (Oi-) radical and of the presence of superoxide dismutase (SOD) enzymes in aerobic cells led directly to the proposal that 0 i- is a major factor in oxygen toxicity and that SOD constitutes an important defense against it.' Systems generating the O~- radical have been shown to have a number of damaging effects, some of which are summarized in Table I. The superoxide radical itself in organic solvents is a powerful base and nucleophile,2 which may have relevance to reactions taking place within the interior of cell membranes. In aqueous solution it is far less reactive, acting mainly as a reducing agent and undergoing the dismutation reaction, O~- + O~- + 2H + ~ H202 + 02

(1)

Yet all of the damage observed in Table I refers to reactions carried out in aqueous solution. In many cases damage is decreased by addition not only of SOD but also of catalase, and it was proposed 3 that Oi- and H202 can combine together directly to generate the highly reactive hydroxyl radical, OH. H202 + O i- ~ OH. + O H - + 02

(2)

Indeed, damage is often decreased by scavengers of this radical such as mannitol, sodium formate, and thiourea. It must be emphasized that in i I. Fridovieh, Annu. Rev. Biochem. 44, 147 (1975). 2 D. T. S a w y e r and M. T. Gibian, Tetrahedron 35, 1471 (1979). 3 C. Beauehamp and I. Fridovich, J. Biol. Chem. 2459 4641 (1970).

METHODS IN ENZYMOLOGY,VOL. 105

CopyriBht © 1984by Academic Press, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-182005-X