[23] Spin trapping of superoxide and hydroxyl radicals

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F O R M A T I O N OR REMOVAL OF OXYGEN RADICALS

[23]

inhalator equipped with a Fluotec attachment. The dose is checked using a Hewlett-Packard gas-liquid chromatograph with integrator. Acknowledgments The work at the University of Guelph was supported by the Natural Sciences and Engineering Research Council Canada. Grateful acknowledgment is hereby made. The author is thankful for an invitation to spend November 1982 at the Oklahoma Medical Research Foundation Research Laboratory where this chapter was written. Help from Gregory A. Coulter, who checked the manuscript, and Uwe M. Oehler, who made the drawings, is appreciated.

[23] S p i n T r a p p i n g o f S u p e r o x i d e a n d H y d r o x y l Radicals

By GERALD M. ROSEN and ELMER J. RAUCKMAN A free radical is by definition a species containing an unpaired electron, and is therefore paramagnetic. Paramagnetism forms the basis for the detection of free radicals by electron paramagnetic resonance (EPR) spectrometry, whereby the magnetic moment exerted by the unpaired electron is directly detected. This high degree of selectivity for only parRmagnetic species renders EPR useful in complex biological systems. The theoretical lower limit of free radical detection by EPR in aqueous solutions using existing instruments is approximately 10 nM. ~ In most circumstances, however, if hyperfine splittings are to be resolved, the practical limit of detection is about 1 /xM. t Thus, it is only possible to detect radicals which accumulate to these measurable concentrations. There are many examples of such radicals being directly detected in biological systems. Such studies have recently been reviewed by Mason. 2 Many free radicals of biological interest are highly reactive and never reach a concentration high enough to be detected by EPR. An example of this is the hydroxyl radical, which reacts with itself or with most organic molecules at diffusion controlled rates. 3 Its rate of reaction is limited mainly by the frequency with which it collides with other species. Thus, the direct detection of hydroxyl radicals by EPR in a biologic system is impossible. For short-lived radicals of lesser reactivity compared to the hydroxyl radical, there are various means of detection using EPR. A simple method D. C. Borg, Free Radicals Biol. 1, 69 (1976). z R. P. Mason, Reo. Biochem. Toxicol. 1, 151 (1979). 3 L. M. Doffman and G. E. Adams, Nat. But'. Standards NSRDS No. 46 (1973).

METHODS IN ENZYMOLOGY. VOL. 105

Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182005-X

[23]

SPiN TRAPPINGOF RADICALS

199

is to slow the rate of disappearance of the radical by rapidly freezing the sample. This has the disadvantage that the radical is no longer in a fluid environment, and the resultant anisotropic effects can obscure the identification of the radical. This technique is further limited by the concentration of the radical present before freeezing and by the length of time required to freeze the sample, which is about 5 to I0 msec. 4 One can improve the sensitivity of free radical detection in biological samples by lyophilization; this decreases microwave absorption by water and increases signal intensity. Artifactual radicals, however, such as that due to ascorbate, are often seen in lyophilized samples exposed to air. ~Continuous flow EPR, in conjunction with signal averaging techniques, improves the detectability of short lived radicals, and enabled Yamazaki and Piette to detect the ascorbate semiquinone free radical in the EPR studies of ascorbate oxidase) However, such studies are time consuming and require large quantities of enzyme. In theory, spin trapping can overcome many of these difficulties. This technique consists of using a spin trap, i.e., a compound that forms a stable free radical by reacting covalently with an unstable free radical. Thus, the radical species is "trapped" in a long-lived form which can be observed at room temperature using conventional EPR equipment. The hyperfine splitting of the adduct provides information which can aid in the identification of the original radical. Since the stable free radical accumulates, spin trapping is an integrative method of measuring free radicals and is inherently more sensitive than procedures which measure only instantaneous or steady-state levels of free radicals. Nitrones and nitroso compounds are the spin traps most commonly used, however, only nitrones detect oxygen centered radicals like superoxide and hydroxyl radicals at room temperature. 6 Both nitrones and nitroso compounds react covalently with numerous free radicals to produce a nitroxide (N-:-"O) spin adduct. The spectrum of a nitroxide gives a characteristic triplet which can exhibit further hyperfine splittings if nuclei having a magnetic moment are bonded nearby. The magnitude and nature of this interaction is dependent upon the nuclear quantum spin number as well as resonance, inductive, and steric effects. 7 Unless a conjugated system is present, magnetic nuclei farther than three bond lengths away from the nitroxide, where the unpaired electron is localized, 4 j. R. Bolton, D. C. Borg, and H. M. Swartz, in "Biological Applications of Electron Spin Resonance" (H. M. Swartz, J. R. Bolton, and D. C. Borg, eds.), p. 63. Wiley (Interscience), New York, 1972. 5 I. Yamazaki and L. H. Piette, Biochim. Biophys. Act 50, 62 0961). 6 j. A. Wargon and F. Williams, J. Am. Chem. Soc. 94, 7917 (1973). 7 E. G. Janzen, Ace. Chem. Res. 4, 31 (1971).

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FORMATION OR REMOVAL OF OXYGEN RADICALS

[23]

will not cause further resolvable splittings. With nitrone spin traps, the trapped radical is bonded to the a-carbon, and magnetic nuclei present in the trapped radical are far away from the nitroxide nitrogen. Thus, hyperfine splitting due to the original radical is less readily resolved. Spin traps possessing a a-hydrogen, such as 5,5-dimethyl-l-pyrroline N-oxide (DMPO), will yield adducts with hyperfine splitting due to both the ahydrogen and the nitroxide nitrogen. In these spin-trapped adducts, the magnitudes of AN and AH are very sensitive to the nature of the trapped radical, and this can serve as a means to help identify the trapped species. 7-9 In this chapter we discuss the spin trapping of the biologically important free radicals: superoxide and hydroxyl. Chemistry of Nitrone Spin Traps Nitrones are highly reactive compounds, which can participate in a wide variety of reactions other than radical trapping. Thus, it is not surprising to note that nitroxides can be generated from nitrones by methods other than radical trapping. For this reason, a knowledge of nitrone chemistry is essential in understanding how artifactual radicals are generated from these compounds. Nitrones can be reduced or oxidized into a variety of products.l° Interconversions between nitrones, hydroxylamines, oximes, imines, hydroxamic acids, nitroxides, and nitroso compounds are possible, depending upon the conditions and reagents used. Metal ions commonly encountered in biological systems, such as iron and copper, can often carry out or catalyze such reactions. For example, aqueous ferric chloride is known to oxidize DMPO and related nitrones into the corresponding hydroxamic acid. ~ Oxidation of the hydroxamic acid would produce the corresponding nitroxide 5,5-dimethylpyrrolidone-(2)-oxyl-(l) (DMPOX). This compound has also been observed in a biochemical system containing hematin and cumene hydroperoxide. 12 However, in this case DMPOX arises by spin trapping cumene hydroperoxyl radicals followed by base-catalyzed rearrangement.~3 We have also found that DMPOX can be produced from DMPO by the action of oxidizing agents such as lead dioxide, m3Thus, the production of DMPOX from DMPO is undoubtedly a common artifact in many oxidizing systems. 8 E. G. Janzen and J. I. P. Liu, J. Magn. Reson. 9, 510 0973). 9 E. G. Janzen, C. A. Evans, and J. I. P. Liu, J. Magn. Reson. 9, 513 0973). J0 j. Hamer and A. Macaluso, Chem. Rev. 64, 473 0964). u j. F. EIsworth and M. Lamchen, J. S. Aft. Chem. Inst. 24, 196 0971). n R. A. Floyd and L. A. Soong, Biochem. Biophys. Res. Commun. 74, 79 (1977). 13 G. M. Rosen and E. J. Rauckman, Mol. Pharmacol. 17, 233 0980).

[23]

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201

Chelated iron can also produce radicals from nitrones. In phosphate buffer, iron-EDTA oxidizes DMPO into a nitroxide, AN = 15.3, An = 22.0. J4 The spectrum is due to an oxidative product of DMPO itself, as the same signal is observed in Tris buffer containing iron, without EDTA. Only trace amounts of iron are required to generate enough of this species to be observed by EPR. The iron present in phosphate buffer as an impurity is usually sufficient to produce a detectable signal. We have suggested that the spectrum is due to formation of a DMPO dimer. J5 Copper ions can also give rise to artifacts in spin trapping experiments. For example, the air oxidation of hydroxylamines is greatly accelerated by cupric salts.J° Therefore, we recommend that buffers used in spin trapping be passed through a Chelex 100 (Bio-Rad, Richmond, CA) column to remove polyvalent metal ion impurities. The use of diethylenetriaminepentaacetic acid (DETAPAC), a chelating agent which renders iron and copper incapable of oxidizing DMPO, is highly recommended. J4,J6 Nitrones are prone to hydrolysis in aqueous solution, forming an aldehyde and a hydroxylamine. J0 The hydrolysis is pH dependent, being more rapid at low pH, ~° and is structure specific. Acyclic nitrones are very susceptible to hydrolysis, while aryl nitrones are less so, and cyclic nitrones are reportedly very resistant to hydrolysis. I° For example, one report claimed that there was little decomposition of an aqueous DMPO solution stored in the dark for 5 months, jj as measured by its UV absorbante. In contrast, the half-life of the aryl nitrone, a-(4-pyridyl l-oxide)-Nt-butylnitrone (4-POBN) is 13.8 min at pH 2, although it is stable for 32 hr at neutral pH.17 Hydrolysis of nitrones can also give rise to nitroxides. Janzen e t al. J7 have shown the addition of water across the double bond of 4-POBN, followed by oxidation, produces 4-POBN-OH which is the same species generated by the spin trapping of the hydroxyl radical by 4-POBN. Thus, under certain conditions, hydrolysis and air oxidation can lead to the erroneous assumption that the hydroxyl radical has been spin trapped. Synthesis of Spin Traps Nitrones used in spin trapping experiments should he of the highest purity, and should be free from nitroxide or hydroxylamine impurities. ~4E. Finkeistein, G. M. Rosen, E. J. Rauckman, and J. Paxton, Mol. PharmacoL 16, 676 (1979). t5 R. F. C. Brown, V. M. Clark, M. Lamchen, and A. Todd, J. Chem. Soc. 2116 (1959). 16G. R. Buettner, L. W. Oberley,and S. W. H. G. Leuthauser, Photochem. Photobiol. 28, 693 (1978). 17E. G. Janzen, Y. Y. Wang, and R. V. Shetty, J. Am. Chem. Soc. 100, 2923 (1978).

202

FORMATION OR REMOVAL OF OXYGEN RADICALS

NO2 I CH2=CHCHO CH3--C H--CH3 + NoOEt

[23]

CHO NO2

,L •F•H

<

Zn

H"°7

NO2 ~0]

I

o-

DMPO FIG. 1. The synthesis of 5,5-dimethyi-l-pyrroline N-oxide (DMPO).

Commercially available aryl nitrones such as 4-POBN are usually of sufficient purity and appear to be stable for a long period of time; however, DMPO and related spin traps have shorter shelf lives as they are more susceptible to decomposition by light and oxygen. Storage of cyclic nitrones like DMPO should always be at - 2 0 °, under nitrogen and away from light. Commercially available DMPO usually requires further purification. The method of choice, especially for large quantities, is fractional vacuum distillation. DMPO purified by this method is a colorless solid, mp 25°. An alternative method of purification is column chromatography using charcoal-Celite, or filtration of an aqueous DMPO solution through charcoal, as described by Buettner and Oberley. ~8Charcoal-Celite behaves as a true reverse phase chromatographic medium, in that polar solvents elute only the nitrone, while nonpolar solvents will elute both nitrone and impurities. ~9Elution of DMPO can be conveniently monitored by its UV absorbance (DMPO, ,emax(234) 7700 M -] c m - I ) ° The synthesis of DMPO is a three-step procedure which depends upon the reduction of 4-methyl-4-nitro-l-pentanal with zinc (Fig. 1). =

Preparation of 4-Methyl-4-nitro-l-pentanal A solution of sodium methoxide was prepared by dissolving 3.7 g (0.16 tool) sodium metal in 250 ml anhydrous methanol. To this mixture was ,8 G. R. Buettner and L. W. Obcrley, Biochem. Biophys. Res. Commun. 83, 69 (1978). 19G. M. Rosen, E. Finkeistein, and E. J. Rauckman, Arch. Biochem. Biophys. 215, 367

(1982).

[23]

sPl~ TRAPPINGOF RADICALS

203

added 106.8 g (1.2 mol) 2-nitropropane (Aldrich Chemical Company, Milwaukee, Wl). The addition was at such a rate that the temperature of the reaction mixture did not exeed 10.. After the additon of 2-nitropropane was completed, the temperature of the reaction was reduced to - 2 0 °, and a solution of 9 g (0.16 mol) of freshly distilled acrolein (Aldrich Chemical Company, Milwaukee, WI) and 26.7 g (0.3 mol) 2-nitropropane was added dropwise over a 3-hr period such that the temperature was kept at or below -20*. After the addition was completed, the mixture was stirred for 30 min, acidified with gaseous HCI (to pH 1.0), maintaining a temperature in the reaction flask no greater than 10°, and then dried with anhydrous sodium sulfate. Evaporation of the solution to dryness gave a liquid which was vacuum distilled to yield 9.5 g (41%) 4-methyl-4-nitro-l-pentanal, bp 85-88 ° at 3 mm Hg, lit. ref. 88.3-89.5 ° at 33 m m H g . 2°

Preparation of 2-(3-Methyl-3-nitrobutyi)-l,3-dioxolane To 9.5 g (0.066 mol) 4-methyl-4-nitro-l-pentanal in 50 ml dry benzene was added 4.26 g (0.069 mol) dry ethylene glycol and a catalytic amount (0.2 g) p-toluenesulfonic acid. This reaction was refluxed under a DeanStark trap until 1.1 ml water was collected. The benzene solution was cooled, treated with aqueous sodium hydrogen carbonate, dried over anhydrous sodium sulfate, and evaporated to dryness to give, after distillation, 9.2 g (75%) 2-(3-methyl-3-nitrobutyl)-l,3-dioxolane, bp 102-105 ° at 0.5 mm Hg, lit. ref. bp 105° at 0.5 mm Hgfl

Preparation of S,5-Dimethyl-l-pyrroline-N-oxide To a rapidly stirred solution of 9.2 g (0.057 mol) 2-(3-methyl-3-nitrobutyl)-l,3-dioxolane and 2.76 g (0.052 mol) ammonium chloride in 56 mi water at 10. was added 12.9 g (0.197 mol) zinc dust over a 20-min period of time. During the addition, the temperature was maintained below 15° . The reaction was stirred for 15 min, filtered, and the filter cake washed with hot water (at approximately 70*). The filtrate and washings were acidified with concentration hydrochloric acid, let stand overnight, and then heated to 75° for 1 hr before evaporation to one-third the original volume. The solution was then made alkaline, evaporated to near dryness, and extracted with chloroform. The chloroform solution was dried over anhydrous sodium sulfate and evaporated to dryness. Vacuum distillation gave 20 H. Schechter, D. E. Ley, and L. Zeldin, J. Am. Chem. Soc. 74, 3664 (1951). 2t R. Bonnett, V. M. Clark, A. Giddy, and A. Todd, J. Chem, Soc. 2087 (1959).

204

FORMATION OR REMOVAL OF OXYGEN RADICALS

[23]

5,5-dimethyl-l-pyrroline N-oxide, bp 64-66 ° at 0.6 mm Hg, lit. ref. bp 6667° at 0.6 mm Hg. 22 Experimental Considerations Spin trapping is easily conducted directly in an EPR cell so that the progress of the reaction may be continuously monitored by observing the EPR spectrum. A flat quartz cell is most frequently used because of its large surface area, and small volumes (less than 0.4 ml is sufficient). Generally, the reactants are mixed in a test tube, poured into the cell and immediately placed in the EPR cavity. Since spin trapping is an integrative technique, it invariably takes several minutes to reach sufficiently high levels that the nitroxide can be observed spectrometrically. Optimal conditions for spin trapping superoxide requires high concentrations of DMPO since the rate constants for the reaction of DMPO with superoxide are small. 23 Concentrations of DMPO between I0 and 100 mM are recommended. Under these conditions, we have not found that DMPO inhibits the activity of such enzymes as cytochrome/-450. Nevertheless, because of the high concentrations required, it is important to determine whether or not DMPO is a promoter or an inhibitor of the enzymic process under study. As expected, the spin trapping of hydroxyl radicals by DMPO is quite rapid with a rate constant of 1.8 x 109 M -~ sec -j, and thus, lower concentrations of this spin trap may be employed. Because of the disparity between the rates of spin trapping superoxide and hydroxyl radical, care must be taken to prevent the formation of hydroxyl radical by artifactual means. The pH of the buffer will greatly affect the ability of DMPO to spin trap superoxide. This is due to two separate phenomena. First, the rate of spontaneous dismutation of superoxide is pH dependent, with superoxide having a longer half-life at higher pH. Second, the second order rate constant for the reaction of HOP- with DMPO is 6.6 x 103 M -j sec -j, while that for OO;- is only 10 M -~ sec-~. 23 Thus, reaction of superoxide with DMPO favors acidic conditions. Unfortunately, most biological systems in which the detection of superoxide is desirable have pH optima in the range of 7.0-8.0. Detection of DMPO-OH does not necessarily mean that the hydroxyl radical has been spin trapped. One method of verification is to utilize the 2z R. Bonnett, R. F. C. Brown, V. M. Clark, I. O. Sutherland, and A. Todd, J. Chem. Soc. 2094 (1959). 2~ E. Finkelstein, G. M. Rosen, and E. J. Rauckman, J. Am. Chem. Soc. 102, 4994 0980).

[23]

SPIN TRAPPING OF RADICALS

DMPO

•OH Me

C HE OH

Me-(~--OH I H

>

DMPO >

205

~N"~ I° 0

0H

~/~C I"

H-OH I~e

0

FIo. 2. Reaction of hydroxyl radical with either ethanol or DMPO to give a-hydroxyethyl radical or DMPO-OH. DMPO then spin traps ct-hydroxyethyi radical, which has an EPR spectrum distinctively different from that of DMPO-OH.

ability of spin trapping techniques to distinguish between different radical species. For example, hydroxyl radicals react with ethanol to produce cthydroxyethyl radicals.24 These secondary radicals then react with the spin trap to produce an adduct with an EPR spectrum distinguishable from that of the hydroxyl radical. In some biological systems, ethanol cannot be used, so dimethyl sulfoxide or sodium formate are excellent agents to employ as sources for secondary radical trapping experiments. Thus, if the production of DMPO-OH is due to the spin trapping of hydroxyl radicals, the addition of ethanol (dimethyl sulfoxide or sodium formate) should both inhibit the production of DMPO-OH and result in the appearance of a new signal due to the spin trapping of o~-hydroxyethyl radical. This is demonstrated in Fig. 2. The quartet spectrum of DMPO-OH is not unique to DMPO-OH, since any nitroxide with hyperfine splitting constants with AN = An = 14.4 G has the same or a similar spectrum. 25,26Therefore, verification of hydroxyl radical trapping by independent means is a necessity. Spin trapping of oxygen-centered radicals other than superoxide can likewise result in spectra similar to the spectrum of DMPO-OOH. For example, the spectrum of benzyloxyl radical adduct of DMPO is similar to that of DMPO-OOH (Fig. 3). Verification of superoxide trapping can be obtained by proper design of experiments. For example, in homogenous preparations, the addition of superoxide dismutase will inhibit the formation of DMPO-OOH. Also certain sulfhydryl agents, such as diethyldithiocarbamate rapidly reduce DMPO-OOH to DMPO-OH. la Finally, u E. G. Adams and P. Wardman, Free Radicals Biol. 3, 53 (1977). 25 F. P. Sargent and E. M. Gardy, Can. J. Chem. 54, 275 (1976). 26 B. Kalyanaraman, E. Perez-Reyes, and R. P. Mason, Biochim. Biophys. Acta 630, 119 (1980).

206

F O R M A T I O N OR REMOVAL OF O X Y G E N RADICALS

[23]

I0 O

~o(~

FIG. 3. Similarity between superoxide and benzyioxy radical adduct of DMPO, in aqueous solution. The upper scan is DMPO-OOH produced by the reaction of DMPO with superoxide generated by a xanthine/xanthine oxidase-generating system. The lower spectrum is that of benzyloxy radical adduct of DMPO, generated by thermal decomposition of benzoyl peroxide in a 1 : 1 mixture of ethanol and water, which was dissolved in water.

[23]

SPIN TRAPPING OF RADICALS

f

[ i!

I

"" "'", ,,"" """

A/¢ =14.3

1

II

if

!!i:

" :'

"" "'"

207

A~ =11.7

I '

F=G. 4. Computed (stick) spectrum for DMPO-OOH which shows the formation of the 12-line spectrum.

as discussed earlier, the stability of DMPO-OOH is pH dependent and is greater at acidic pH. Thus, the chemical properties of DMPO-OOH can serve to distinguish it from other species. During our studies into the mechanism of oxygen radical production by human neutrophils, we observed that, no matter how diligent we were in removing metal ion impurities from our reaction mixtures, we invariably spin trapped a small but quantifiable amount of hydroxyl radical. Upon extensive investigation, we discovered that during the spin trapping of superoxide by DMPO, de n o v o production of hydroxyl radical takes place. 27 It appears that once DMPO-OOH is formed, one of its decomposition products is hydroxyl radical which is then spin trapped by DMPO to give DMPO-OH. Thus, there will be a background level of approximately 3% D M P O - O H relative to DMPO-OOH. Therefore, the detection of hydroxyl radical by means of the spin trap DMPO must be interpreted with caution if the level of hydroxyl radical is less than 3% of the rate of superoxide generation. Analysis of Spectrum The EPR spectrum of DMPO-OOH displays three hyperfine splittings, one from the nitroxide nitrogen (with a multiplicity of 3) and two from nonequivalent protons (with a multiplicity of 2 each). The result of these hyperfine splittings is to give a 12-line spectrum as depicted in Fig. 4. Because an EPR spectrum is displayed as the first derivative (having both positive and negative components), and because the lines have finite widths and arc extensively overlapped, the resulting spectrum appears to have lines of variable intensity. This observation is misleading in that all the lines are of equal size. The EPR spectrum of D M P O - O H displays only two hyperfine splittings as shown in Fig. 5. The splittings for the nitrogen and/3-hydrogen 27 E. Finkelstein, G. M. Rosen, and E. J. Rauckman, Mol. Pharmacol. 21, 262 (1982).

208

FORMATION

OR R E M O V A L O F

OXYGEN RADICALS

[23]

.

f'" ,--., .

f

i

Ii ,--

"'1 A .,,87 AH =14.81

, -.

1

F I o . 5. Computer (stick) spectrum for DMPO-OH which illustrates the formation of the 6-line (with the overlap of two lines spectrum.

are equal causing overlap of the central lines. This results in a four line spectrum with a 1 : 2 : 2 : 1 intensity ratio. Because DMPO-OOH rapidly decomposes into DMPO-OH and because the time required to obtain an EPR spectrum is measured in minutes, the observed spectrum is invariably a combination of DMPO-OOH and DMPO-OH. Determining relative ratios of each species can only be conducted with the assistance of a computer. A potential problem with spin trapping in biological systems is the reduction, either enzymically or chemically, of the nitroxide into its hydroxylamine, which cannot be detected by EPR. Nitroxides can be reduced by various biological systems such as ascorbic acid, 2s sulfhydryl agents, 29 the mitochondrial electron transport chain, 3° cytochrome P450, 3~,32 and bacterial electron transport systems. 33 Although superoxide and other radical species have been detected by spin trapping techniques in hepatic microsomes, 3.'37 none of these studies has considered whether or not reduction actually has taken place. In general, superoxide and hydroxyl radical adducts of aryl nitrones are less stable than their cyclic nitrone counterparts. For example, the half-lives of DMPO-OH and 4-POBN-OH are 2.6 hr and 23 see, respecI. C. P. Smith, in "Biological Applications of Electron Spin Resonance" (H. M. Swartz, J. R. Bolton, and D. C. Borg, eds.), p. 483. Wiley (lnterscience), New York, 1972. 29 j. M. Jallow, A. Difranco, F. Leterrier, and L. Piette, Biochem. Biophys. Res. Commun. 74, 1186 (1977). 3o V. K. Koltover, L. M. Reichman, A. A. Jasaitis, and L. A. Blumenfeld, Biochim. Biophys. Acta 234, 306 (1971). 3J A. Stier and E. Sackman, Biochim. Biophys. Acta 311, 400 (1973). 3z G. M. Rosen and E. J. Rauekman, Biochem. Pharmacol. 26, 675 (1977). 33 j. S. Goldberg, E. J. Rauckman, and G. M. Rosen, Biochem. Biophys. Res. Commun. 79, 198 (1977). R. C. Scaly, H. M. Swartz, and P. L. Olive, Biochem. Biophys. Res. Commun. 82, 680 (1978). 35 G. M. Rosen and E. J. Rauckman, Proc. Natl. Acad. Sci. U.S.A. 78, 7346 (1981). 36 C. S. Lai and L. H. Piette, Biochem. Biophys. Res. Commun. 78, 51 (1977). 37 B. Kalyanaraman, R. P. Mason, E. Perez-Reyes, and C. F. Chignell, Biochem. Biophys. Res. Commun. 89, 1065 (1979).

[24]

REACTIONOF "OH

209

tively.14 The half-lives of DMPO-OH and 4-POBN-OOH are approximately 8 min and 30 see, respectively.'4 An alternative integrative EPR technique for the detection of superoxide in biological systems is the "spin-exchange" of superoxide with hydroxylamines. \ /

N--OH + HO0' ~

\ /

N "--:-0 + H202

The second-order rate constant for reaction of certain hydroxylamines with superoxide is much greater than for the covalent reaction of superoxide with DMPO) 9 Thus, this technique offers an alternative to the spin trapping method for the detection of superoxide.

[24] R e a c t i o n of .OH

By GIDON CZAPSKI Histmy The first evidence for a free OH radical was observed in the absorption spectra of H2-O2 flames by W. W. Watson in 1924.~ In later studies other spectral lines of OH were observed in the gas phase, in pyrolyzed vapor and in electric discharges of water vapor or H2/O2 mixtures. As a result of these observations, Bonhoeffer and Haber2 proposed that OH. and H atoms are the chain carriers in the chain reaction of H2-O2 combustion. It was first suggested by Haber and Willstiitte# that in aqueous media the OH" radical plays a role in the mechanism of decomposition of H202 by catalase. Later Haber and Weiss 4 proposed that OH. is an intermediate in the Fenton reaction. Since then, OH. is known to participate in many reactions in aqueous solutions and is known to be formed in chemical, photochemical, electrochemical, and radiolytic reactions. More recently, in the last decade it was shown that OH. is formed also in biological systems and is involved as an active intermediate in physiological processes,

i W. W. Watson, Astrophys. J. 60, 145 (1924). 2 K. F. Bonhoeffer and F. Haber, Z. Phys. Chem. (Leipzig) A137, 263 (1928). 3 F. Haber and R. Willstiitter, Bet. Dseh. Chem. Ges. 64, 2844 (1931). 4 F. Haber and J. Weiss, Naturwissenschaften 20, 948 (1932).

METHODS IN ENZYMOLOGY, VOL. 105

Copyright © 1984by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182005-X