[48]
IRON REDOX REACTIONS AND LIPID PEROXIDATION
457
are pooled and concentrated to 5-10 ml using an Amicon ultrafiltration apparatus with an YM10 membrane. This fraction is applied on a Sephadex G-50 column (140 x 5.5 cm) equilibrated with 25 m M Tris-HCl (pH 7.4), 300 m M KSCN, 5 m M mercaptoethanol, 10% (v/v) glycerol. The active portion is eluted 160 ml after the void volume of the column. The active fractions are pooled and concentrated with the same ultrafiltration apparatus as before. This fraction is dialyzed against 10 mM potassium phosphate (pH 6.5), 0.1 M KC1, 5 m M mercaptoethanol. Some proteins precipitate during dialysis and are eliminated by centrifugation. At this purification stage, PHGPX accounts for 40-70% of the proteins. A preparation at this level of purification is useful for routine purposes such as measuring phospholipid hydroperoxides. If PHGPX is stored at this purification step, 10% (v/v) glycerol should be added to the last dialysis buffer. This preparation is very stable (several months at
-20°). The final purification step is carried out by HPLC using either gelpermeation or ion-exchange columns, e.g., TSK SW 2000 (gel permeation), TSK DEAE (weak anion exchanger), Mono Q (strong anion exchanger), and TSK CM (weak cation exchanger). We describe here the chromatographic conditions for a TSK CM column. Buffer A: l0 mM potassium phosphate, 100 mM KCI, 5 mM mercaptoethanol (pH 6.5); buffer B: l0 m M potassium phosphate, 300 m M KCI, 5 mM mercaptoethanol (pH 6.5). Flow rate: 1 ml/min. The gradient from 0 to 100% buffer B is developed in 25 rain after 3 min in isocratic conditions. Detection is at 280 nm; injection volume is less than 0.2 ml. PHGPX is eluted as a single peak when KC1 is approximately 200 mM. Peaks isolated from several runs are pooled, and 10% (v/v) glycerol is added. The preparation is then concentrated by ultrafiltration to a final protein concentration no higher than 0.3 mg/ml to avoid aggregation.
[48] I r o n R e d o x R e a c t i o n s a n d L i p i d P e r o x i d a t i o n By STEVEN D. AUST, DENNIS M. MILLER, and VICTOR M. SAMOKYSZYN
Introduction Iron-catalyzed lipid peroxidation has been studied in many in vitro model systems. While the mechanism of iron-catalyzed lipid peroxidation is not completely understood, it is well established that the redox chemisMETHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
458
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[48]
try of iron influences both the occurrence and the rate of lipid peroxidation. For example, iron-catalyzed lipid peroxidation in systems comprised initially of Fe(II) and phospholipid liposomes requires some Fe(II) oxidation.l Conversely, iron-catalyzed lipid peroxidation in systems containing Fe(III) and phospholipid liposomes requires some Fe(III) reduction. 2 However, complete Fe(II) oxidation or complete Fe(III) reduction results in conditions that do not promote lipid peroxidation. Thus, measurements of the rate and extent of Fe(II) oxidation or Fe(III) reduction aid the interpretation of experimental lipid peroxidation data. While several methods are available, only methods pertinent to lipid peroxidation are detailed here. Two important factors which influence both the rates and extents of Fe(II) oxidation or Fe(III) reduction are chelation and pH. Chelators which preferentially bind Fe(II) tend to prevent or slow the rate of Fe(II) autoxidation as well as the rates of Fe(II) oxidation by peroxides. Chelators which bind Fe(III) with greater affinity than Fe(II), however, have the opposite effect, that is, they increase the rate of Fe(II) autoxidation as well as Fe(II) oxidation by peroxides. In addition, the rate of Fe(II) autoxidation, particularly unchelated Fe(II), increases with increasing pH. Fe(III) Reduction
Enzymatic Fe(III) Reduction Numerous electron-transport enzymes are able to catalyze the reduction of Fe(III) to Fe(II). Perhaps the easiest method for determining the rate of enzymatic Fe(III) reduction is to measure the rate of Fe(II) formation spectrophotometrically using one of the Fe(II) chelators listed in Table I.
Continuous Fe(III) Reduction Assay Reaction mixtures should contain the enzyme of interest, appropriate reducing cosubstrate, Fe(III) chelated to the compound(s) of interest [owing to the strong absorbances of some of the Fe(II) chelators in Table I, the total iron concentration should be less than 100 t~M], and one of the Fe(II) chelators in Table I at a concentration at least 3-fold greater than the total iron concentration [because these chelators form a tris complex with Fe(II)]. ff the assay is conducted in the presence of microsomes, liposomes, or fatty acid micelles, an antioxidant should be included [e.g., G. Minotti and S. D. Aust, J. Biol. Chem. ?,62, 1098 (1986). 2 M. Tien, J. R. Bueher, and S. D. Aust, Biochem. Biophys. Res. Commun. 107, 279 (1982).
[48]
IRON REDOX REACTIONS AND LIPID PEROXIDAT1ON
459
TABLE I FE(II) CHELATORS Chelator 4,7-Diphen yl- 1,10-phenanthroline a (bathophenanthroline) 4,7-Diphenyl-l,10-phenanthroline disulfonate 1,10-Phenanthroline (o-phenanthroline) 3-(2-Pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-tr~azine (ferrozine) a
Xm~ (nm)
e ( n -1
cm -~)
534
22,200
534
22,100
510 564
11,100 27,900
Bathophenanthroline is insoluble in water but soluble in most organic solvents.
butylated hydroxytoluene, 0.03% (w/v) final concentration] to prevent lipid peroxidation. Comments. There are several complications and artifacts that may hamper accurate determination of Fe(III) reduction. For example, the rate of Fe(III) reduction is often directly proportional to total iron concentration. In addition, the presence of other redox-active transition metals may interfere with chromophore formation or unpredictably effect iron reduction. Since most reagents are contaminated with iron and other redox-active transition metals, Chelex 100 treatment of these reagents, to remove trace metal contaminants, is essential. Chelex 100 treatment can be performed as a batch method or, preferably, by column chromatography. We routinely use a glass column (2.5 × 40 cm, flow rate - 1 0 ml/min) of Chelex 100 (pH approximately 6-8) to remove trace metal contaminants from saline solutions. A simple test for redox-active metal ion contamination, based on the rate of metal-catalyzed ascorbate oxidation, should be used to determine the level of metal contamination. 3 Briefly, the reagent of interest is incubated with 100/zM ascorbate, and the decrease in absorbance at 265 nm is monitored for 15 min. Based on the extinction coefficient (e 14,500 M -~ cm-1), a loss of ascorbate concentration of greater than 0.5% within 15 min indicates significant metal contamination. Note that there is a fairly common misconception that ascorbate autoxidizes; however, this is incorrect as its oxidation actually results from metal reduction followed by autoxidation of the reduced metals. Typically it is sufficient to treat the assay buffer with Chelex 100. However, some buffers, such as phosphate, are inherently resistant to metal removal and may, in fact, absorb addi3 G. R. Buettner, J. Biochem. Biophys. Methods 16, 27 (1988).
460
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[48]
tional metals from the Chelex 100 resin. Thus, this test should be performed both before and after Chelex 100 treatment of reagents. Additionally, many frequently used buffers, such as phosphate, or any of the Good buffers (e.g., HEPES) catalyze rapid autoxidation of Fe(II). 4 These buffers, apparently by chelating or ligating to iron, affect iron redox chemistry and hence the sensitivity of the Fe(III) reduction assay. Careful selection of buffer systems can reduce complications in the assay, or, if possible, saline solutions of desired ionic strength should be used to avoid buffer artifacts altogether. In systems which rely on superoxide (O2-) as the Fe(III) reductant (e.g., xanthine oxidase), the addition of catalase to remove H202 produced by 02- dismutation or by other sources increases the sensitivity of the technique. Many electron transport enzymes use reducing substrates which have characteristic absorbances, such as the pyridine nucleotides. The rate of reducing substrate oxidation should always be used as an additional source of information to estimate the rate of Fe(III) reduction, providing that the stoichiometry of reducing substrate and Fe(III) is known. If this is unknown, this method coupled with the method described above may be used to determine the stoichiometry.
Discontinuous Fe(llI) Reduction Assays Iron chelators which preferentially bind Fe(III) (e.g., EDTA) often make accurate determination of the rate of Fe(III) reduction difficult owing to rapid Fe(II) autoxidation catalyzed by these chelators. One possible solution for this problem is to conduct the Fe(III) reduction assay under anaerobic conditions as follows. All reactants (except the enzyme) are purged of dioxygen by exhaustive argon bubbling of the solution (argon is preferred over nitrogen because it has less dioxygen contamination and is denser than dioxygen). The reaction is started with the addition of the enzyme, and anaerobiosis is maintained during the time course of the assay by continuously purging the head space of the reaction vessel (e.g., a 13 x 100 mm test tube) of dioxygen with argon. At regular time intervals, 0.5-ml aliquots of the reaction mixture are removed and mixed with 1 ml of 15 mM 1,10-phenanthroline and 0.5 ml of 30% trichloroacetic acid, and the phenanthroline-Fe(II) complex is extracted with 2 ml of n-amyl alcohol. Since this procedure requires the use of solvent extraction, it may be necessary to compare the amount of Fe(II) formed to a standard curve of FeCI3 and excess thioglycolic acid subjected to identical assay conditions. 4 D. O. Lambeth, G. R. Ericson, M. A. Yorek, and P. D. Ray, Biochim. Biophys. Acta 719, 501 (1982).
[48]
IRON REDOX REACTIONS AND LIPID PEROXIDATION
461
Comment. Obviously, some enzymes, such as xanthine oxidase, require dioxygen for Fe(III) reduction, and thus cannot be assayed anaerobically. However, this method may be used aerobically to assay for Fe(III) reduction, providing rapid Fe(II) autoxidation is not a problem. Chemical Fe(III) Reduction Determination of the rate of Fe(III) reduction by chemical reductants can be performed using the continuous reduction assay, essentially as described in the previous section. Fe(III), complexed to the chelator(s) of interest, is incubated with the reductant in the presence of any of the Fe(II) chelators in Table I. In addition, it is often possible to measure the rate of Fe(III) reduction by monitoring the rate of oxidation of the reductant. For instance, as mentioned above, ascorbate exhibits a characteristic absorbance at 265 nm; thus, by following the decrease in absorbance at 265 nm, an indication of the rate of Fe(III) reduction can be obtained. Other reductants, such as cysteine or glutathione, while lacking characteristic UV-visible absorbances, react with 5,5'-dithiobis-2-nitrobenzoic acid to yield the corresponding 5-thio-2-nitrobenzoate derivatives which absorb at 412 nm. 4 Thus, by following the decrease in 5,5'-dithiobis-2nitrobenzoic acid-detectable thiols over time, an indication of the rate of thiol oxidation, and hence Fe(III) reduction, is obtained.
Fe(II) Oxidation Depending on the system under study, a variety of oxidants are present in many in vitro lipid peroxidation systems that can oxidize Fe(II) to Fe(III). These include dioxygen, O2-, H202, and other peroxides. The oxidation of Fe(II) by dioxygen [Eq. (1)], termed autoxidation, results in the generation of Oz-. Superoxide is itself an oxidant of many Fe(II) Fe(II) + O2--~ Fe(IlI) + O2-
complexes [e.g., Fe(II)--citrate, Fe(II)-EDTA] [Eq.
(1) (2)]. 5,6
Fe(II) + O2 v + 2 H + --~ Fe(Ill) + H202
In addition, (2)
various peroxides including HzO2 [Eq. (3)] and lipid (LOOH) or other alkyl (ROOH) hydroperoxides [Eq. (4)] are able to oxidize Fe(II) to Fe(III). Fe(II) + HzOz ~ Fe(IIl) + .OH + OH Fe(II) + L(R)OOH --~ Fe(lIl) + L(R)O. + O H -
5 G. Minotti and S. D. Aust, J. Free Radicals Biol. Med. 3, 379 (1987). 6 j. Butler and B. Halliwell, Arch. Biochem. Biophys. 218, 174 (1982).
(3) (4)
462
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[48]
The Fenton Reaction [Eq. (3)] generates the hydroxyl radical (.OH), which also is an oxidant of Fe(II) [Eq. (5)]. Fe(II) + ' O H - - 9 Fe(III) + O H -
(5)
As with the redox chemistry of Fe(III), chelation can greatly affect the rates of Fe(II) oxidation. For example, unchelated Fe(II) does not readily autoxidize in saline solution at neutral pH and ambient 02 tension; however, incubation of Fe(II) with metal chelators results in variable rates of Fe(II) autoxidation, depending on the nature of the chelator. Similarly, as stated in the previous section, many common buffers can influence the rates of unchelated Fe(II) autoxidation. Chelation of iron also can influence its reactivity with the other oxidants. For example, Fe(II)-EDTA reacts much more readily with hydroperoxides compared with ADP-chelated Fe(II). 7 To measure the rates of Fe(II) autoxidation a continuous method involving measurement of rates of dioxygen consumption is used. In addition, we have developed a discontinuous assay which is applicable for determining rates of Fe(II) oxidation in complex reaction mixtures. Continuous Assay for Fe(II) Autoxidation
The continuous assay method involves polarographic measurement of dioxygen consumption using a Clark-type electrode. Typically, chelated or unchelated Fe(II), prepared anaerobically, is injected into a waterjacketed reaction chamber maintained at a constant temperature. The initial rates of dioxygen consumption are used to determine the rate of Fe(II) autoxidation. It should be noted that the concentration of dissolved dioxygen decreases with increased temperature or increased ionic strength. Thermographs are available to calculate the dissolved dioxygen concentration under the conditions employed. 8 Comments. The kinetics of Fe(II) autoxidation are very complex, often dependent on the total Fe(II) and dioxygen concentrations. In addition, many competing side reactions often make accurate determination of the rate of Fe(II) autoxidation difficult. For example, 027, generated during Fe(II) autoxidation [Eq. (1)], can undergo several fates including oxidation of the Fe(II) chelate [Eq. (2)], reduction of the Fe(III) chelate generated by autoxidation, and dismutation yielding dioxygen at a rate which is second order with respect to 02 -~ concentration [Eq. (6)]. Fur2 0 2 - + 2 H + -.o HzOz + 02
(6)
thermore, the H202 generated by the dismutation reaction may also func7 B. A. Svingen, J. A. Buege, F. O. O'Neal, and S. D. Aust, ,I. Biol. Chem. 254, 5892(1979). 8 M. J. Green and H. A. O. Hill, this series, Vol. 105, p. 3.
[48]
IRON REDOX REACTIONS AND LIPID PEROXIDATION
463
tion as an Fe(II) oxidant. Thus, depending on the rate constants of the competing reactions, the initial rates of dioxygen consumption may not represent the true rate of Fe(II) autoxidation.
Discontinuous Fe(II) Oxidation Assay Alternatively, the rate of Fe(II) oxidation can be measured by sampling an aliquot of the reaction mixture at regular time intervals, mixing with one of the Fe(II) chelators in Table I, and measuring the absorbance of the corresponding Fe(II) chromophore. This assay has the advantages of being sensitive and applicable to complex reaction systems where the continuous assay described above is unsuitable because of rapid dioxygen depletion owing to other processes (e.g., lipid peroxidation, dioxygenutilizing enzymes). At regular time intervals, a 0.5-ml aliquots of the reaction mixture are mixed with 0.5 ml of the desired Fe(II) chelator at a stock concentration of 15 mM. To obtain accurate absorbance values, dilution may be necessary depending on the total Fe(II) concentration in the sample [typically this should be less than 100/zM Fe(II) concentration]. The absorbance of the samples should be determined as soon as possible or the samples stored in the dark until the absorbances can be determined because fluorescent lights cause artifactually higher absorbances. The turbidity inherent in reaction mixtures containing phospholipid can be eliminated by filtering the samples through 0.22-/~m filters. Turbidity associated with samples containing fatty acids can be eliminated by dispersion of the fatty acids with Chelex 100-treated detergent (e.g., Tween 20, 1 mM final concentration).
IRON REDOX REACTIONS AND LIPID PEROXIDATION
457
are pooled and concentrated to 5-10 ml using an Amicon ultrafiltration apparatus with an YM10 membrane. This fraction is applied on a Sephadex G-50 column (140 x 5.5 cm) equilibrated with 25 m M Tris-HCl (pH 7.4), 300 m M KSCN, 5 m M mercaptoethanol, 10% (v/v) glycerol. The active portion is eluted 160 ml after the void volume of the column. The active fractions are pooled and concentrated with the same ultrafiltration apparatus as before. This fraction is dialyzed against 10 mM potassium phosphate (pH 6.5), 0.1 M KC1, 5 m M mercaptoethanol. Some proteins precipitate during dialysis and are eliminated by centrifugation. At this purification stage, PHGPX accounts for 40-70% of the proteins. A preparation at this level of purification is useful for routine purposes such as measuring phospholipid hydroperoxides. If PHGPX is stored at this purification step, 10% (v/v) glycerol should be added to the last dialysis buffer. This preparation is very stable (several months at
-20°). The final purification step is carried out by HPLC using either gelpermeation or ion-exchange columns, e.g., TSK SW 2000 (gel permeation), TSK DEAE (weak anion exchanger), Mono Q (strong anion exchanger), and TSK CM (weak cation exchanger). We describe here the chromatographic conditions for a TSK CM column. Buffer A: l0 mM potassium phosphate, 100 mM KCI, 5 mM mercaptoethanol (pH 6.5); buffer B: l0 m M potassium phosphate, 300 m M KCI, 5 mM mercaptoethanol (pH 6.5). Flow rate: 1 ml/min. The gradient from 0 to 100% buffer B is developed in 25 rain after 3 min in isocratic conditions. Detection is at 280 nm; injection volume is less than 0.2 ml. PHGPX is eluted as a single peak when KC1 is approximately 200 mM. Peaks isolated from several runs are pooled, and 10% (v/v) glycerol is added. The preparation is then concentrated by ultrafiltration to a final protein concentration no higher than 0.3 mg/ml to avoid aggregation.
[48] I r o n R e d o x R e a c t i o n s a n d L i p i d P e r o x i d a t i o n By STEVEN D. AUST, DENNIS M. MILLER, and VICTOR M. SAMOKYSZYN
Introduction Iron-catalyzed lipid peroxidation has been studied in many in vitro model systems. While the mechanism of iron-catalyzed lipid peroxidation is not completely understood, it is well established that the redox chemisMETHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
458
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[48]
try of iron influences both the occurrence and the rate of lipid peroxidation. For example, iron-catalyzed lipid peroxidation in systems comprised initially of Fe(II) and phospholipid liposomes requires some Fe(II) oxidation.l Conversely, iron-catalyzed lipid peroxidation in systems containing Fe(III) and phospholipid liposomes requires some Fe(III) reduction. 2 However, complete Fe(II) oxidation or complete Fe(III) reduction results in conditions that do not promote lipid peroxidation. Thus, measurements of the rate and extent of Fe(II) oxidation or Fe(III) reduction aid the interpretation of experimental lipid peroxidation data. While several methods are available, only methods pertinent to lipid peroxidation are detailed here. Two important factors which influence both the rates and extents of Fe(II) oxidation or Fe(III) reduction are chelation and pH. Chelators which preferentially bind Fe(II) tend to prevent or slow the rate of Fe(II) autoxidation as well as the rates of Fe(II) oxidation by peroxides. Chelators which bind Fe(III) with greater affinity than Fe(II), however, have the opposite effect, that is, they increase the rate of Fe(II) autoxidation as well as Fe(II) oxidation by peroxides. In addition, the rate of Fe(II) autoxidation, particularly unchelated Fe(II), increases with increasing pH. Fe(III) Reduction
Enzymatic Fe(III) Reduction Numerous electron-transport enzymes are able to catalyze the reduction of Fe(III) to Fe(II). Perhaps the easiest method for determining the rate of enzymatic Fe(III) reduction is to measure the rate of Fe(II) formation spectrophotometrically using one of the Fe(II) chelators listed in Table I.
Continuous Fe(III) Reduction Assay Reaction mixtures should contain the enzyme of interest, appropriate reducing cosubstrate, Fe(III) chelated to the compound(s) of interest [owing to the strong absorbances of some of the Fe(II) chelators in Table I, the total iron concentration should be less than 100 t~M], and one of the Fe(II) chelators in Table I at a concentration at least 3-fold greater than the total iron concentration [because these chelators form a tris complex with Fe(II)]. ff the assay is conducted in the presence of microsomes, liposomes, or fatty acid micelles, an antioxidant should be included [e.g., G. Minotti and S. D. Aust, J. Biol. Chem. ?,62, 1098 (1986). 2 M. Tien, J. R. Bueher, and S. D. Aust, Biochem. Biophys. Res. Commun. 107, 279 (1982).
[48]
IRON REDOX REACTIONS AND LIPID PEROXIDAT1ON
459
TABLE I FE(II) CHELATORS Chelator 4,7-Diphen yl- 1,10-phenanthroline a (bathophenanthroline) 4,7-Diphenyl-l,10-phenanthroline disulfonate 1,10-Phenanthroline (o-phenanthroline) 3-(2-Pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-tr~azine (ferrozine) a
Xm~ (nm)
e ( n -1
cm -~)
534
22,200
534
22,100
510 564
11,100 27,900
Bathophenanthroline is insoluble in water but soluble in most organic solvents.
butylated hydroxytoluene, 0.03% (w/v) final concentration] to prevent lipid peroxidation. Comments. There are several complications and artifacts that may hamper accurate determination of Fe(III) reduction. For example, the rate of Fe(III) reduction is often directly proportional to total iron concentration. In addition, the presence of other redox-active transition metals may interfere with chromophore formation or unpredictably effect iron reduction. Since most reagents are contaminated with iron and other redox-active transition metals, Chelex 100 treatment of these reagents, to remove trace metal contaminants, is essential. Chelex 100 treatment can be performed as a batch method or, preferably, by column chromatography. We routinely use a glass column (2.5 × 40 cm, flow rate - 1 0 ml/min) of Chelex 100 (pH approximately 6-8) to remove trace metal contaminants from saline solutions. A simple test for redox-active metal ion contamination, based on the rate of metal-catalyzed ascorbate oxidation, should be used to determine the level of metal contamination. 3 Briefly, the reagent of interest is incubated with 100/zM ascorbate, and the decrease in absorbance at 265 nm is monitored for 15 min. Based on the extinction coefficient (e 14,500 M -~ cm-1), a loss of ascorbate concentration of greater than 0.5% within 15 min indicates significant metal contamination. Note that there is a fairly common misconception that ascorbate autoxidizes; however, this is incorrect as its oxidation actually results from metal reduction followed by autoxidation of the reduced metals. Typically it is sufficient to treat the assay buffer with Chelex 100. However, some buffers, such as phosphate, are inherently resistant to metal removal and may, in fact, absorb addi3 G. R. Buettner, J. Biochem. Biophys. Methods 16, 27 (1988).
460
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[48]
tional metals from the Chelex 100 resin. Thus, this test should be performed both before and after Chelex 100 treatment of reagents. Additionally, many frequently used buffers, such as phosphate, or any of the Good buffers (e.g., HEPES) catalyze rapid autoxidation of Fe(II). 4 These buffers, apparently by chelating or ligating to iron, affect iron redox chemistry and hence the sensitivity of the Fe(III) reduction assay. Careful selection of buffer systems can reduce complications in the assay, or, if possible, saline solutions of desired ionic strength should be used to avoid buffer artifacts altogether. In systems which rely on superoxide (O2-) as the Fe(III) reductant (e.g., xanthine oxidase), the addition of catalase to remove H202 produced by 02- dismutation or by other sources increases the sensitivity of the technique. Many electron transport enzymes use reducing substrates which have characteristic absorbances, such as the pyridine nucleotides. The rate of reducing substrate oxidation should always be used as an additional source of information to estimate the rate of Fe(III) reduction, providing that the stoichiometry of reducing substrate and Fe(III) is known. If this is unknown, this method coupled with the method described above may be used to determine the stoichiometry.
Discontinuous Fe(llI) Reduction Assays Iron chelators which preferentially bind Fe(III) (e.g., EDTA) often make accurate determination of the rate of Fe(III) reduction difficult owing to rapid Fe(II) autoxidation catalyzed by these chelators. One possible solution for this problem is to conduct the Fe(III) reduction assay under anaerobic conditions as follows. All reactants (except the enzyme) are purged of dioxygen by exhaustive argon bubbling of the solution (argon is preferred over nitrogen because it has less dioxygen contamination and is denser than dioxygen). The reaction is started with the addition of the enzyme, and anaerobiosis is maintained during the time course of the assay by continuously purging the head space of the reaction vessel (e.g., a 13 x 100 mm test tube) of dioxygen with argon. At regular time intervals, 0.5-ml aliquots of the reaction mixture are removed and mixed with 1 ml of 15 mM 1,10-phenanthroline and 0.5 ml of 30% trichloroacetic acid, and the phenanthroline-Fe(II) complex is extracted with 2 ml of n-amyl alcohol. Since this procedure requires the use of solvent extraction, it may be necessary to compare the amount of Fe(II) formed to a standard curve of FeCI3 and excess thioglycolic acid subjected to identical assay conditions. 4 D. O. Lambeth, G. R. Ericson, M. A. Yorek, and P. D. Ray, Biochim. Biophys. Acta 719, 501 (1982).
[48]
IRON REDOX REACTIONS AND LIPID PEROXIDATION
461
Comment. Obviously, some enzymes, such as xanthine oxidase, require dioxygen for Fe(III) reduction, and thus cannot be assayed anaerobically. However, this method may be used aerobically to assay for Fe(III) reduction, providing rapid Fe(II) autoxidation is not a problem. Chemical Fe(III) Reduction Determination of the rate of Fe(III) reduction by chemical reductants can be performed using the continuous reduction assay, essentially as described in the previous section. Fe(III), complexed to the chelator(s) of interest, is incubated with the reductant in the presence of any of the Fe(II) chelators in Table I. In addition, it is often possible to measure the rate of Fe(III) reduction by monitoring the rate of oxidation of the reductant. For instance, as mentioned above, ascorbate exhibits a characteristic absorbance at 265 nm; thus, by following the decrease in absorbance at 265 nm, an indication of the rate of Fe(III) reduction can be obtained. Other reductants, such as cysteine or glutathione, while lacking characteristic UV-visible absorbances, react with 5,5'-dithiobis-2-nitrobenzoic acid to yield the corresponding 5-thio-2-nitrobenzoate derivatives which absorb at 412 nm. 4 Thus, by following the decrease in 5,5'-dithiobis-2nitrobenzoic acid-detectable thiols over time, an indication of the rate of thiol oxidation, and hence Fe(III) reduction, is obtained.
Fe(II) Oxidation Depending on the system under study, a variety of oxidants are present in many in vitro lipid peroxidation systems that can oxidize Fe(II) to Fe(III). These include dioxygen, O2-, H202, and other peroxides. The oxidation of Fe(II) by dioxygen [Eq. (1)], termed autoxidation, results in the generation of Oz-. Superoxide is itself an oxidant of many Fe(II) Fe(II) + O2--~ Fe(IlI) + O2-
complexes [e.g., Fe(II)--citrate, Fe(II)-EDTA] [Eq.
(1) (2)]. 5,6
Fe(II) + O2 v + 2 H + --~ Fe(Ill) + H202
In addition, (2)
various peroxides including HzO2 [Eq. (3)] and lipid (LOOH) or other alkyl (ROOH) hydroperoxides [Eq. (4)] are able to oxidize Fe(II) to Fe(III). Fe(II) + HzOz ~ Fe(IIl) + .OH + OH Fe(II) + L(R)OOH --~ Fe(lIl) + L(R)O. + O H -
5 G. Minotti and S. D. Aust, J. Free Radicals Biol. Med. 3, 379 (1987). 6 j. Butler and B. Halliwell, Arch. Biochem. Biophys. 218, 174 (1982).
(3) (4)
462
ASSAY AND REPAIR OF BIOLOGICAL DAMAGE
[48]
The Fenton Reaction [Eq. (3)] generates the hydroxyl radical (.OH), which also is an oxidant of Fe(II) [Eq. (5)]. Fe(II) + ' O H - - 9 Fe(III) + O H -
(5)
As with the redox chemistry of Fe(III), chelation can greatly affect the rates of Fe(II) oxidation. For example, unchelated Fe(II) does not readily autoxidize in saline solution at neutral pH and ambient 02 tension; however, incubation of Fe(II) with metal chelators results in variable rates of Fe(II) autoxidation, depending on the nature of the chelator. Similarly, as stated in the previous section, many common buffers can influence the rates of unchelated Fe(II) autoxidation. Chelation of iron also can influence its reactivity with the other oxidants. For example, Fe(II)-EDTA reacts much more readily with hydroperoxides compared with ADP-chelated Fe(II). 7 To measure the rates of Fe(II) autoxidation a continuous method involving measurement of rates of dioxygen consumption is used. In addition, we have developed a discontinuous assay which is applicable for determining rates of Fe(II) oxidation in complex reaction mixtures. Continuous Assay for Fe(II) Autoxidation
The continuous assay method involves polarographic measurement of dioxygen consumption using a Clark-type electrode. Typically, chelated or unchelated Fe(II), prepared anaerobically, is injected into a waterjacketed reaction chamber maintained at a constant temperature. The initial rates of dioxygen consumption are used to determine the rate of Fe(II) autoxidation. It should be noted that the concentration of dissolved dioxygen decreases with increased temperature or increased ionic strength. Thermographs are available to calculate the dissolved dioxygen concentration under the conditions employed. 8 Comments. The kinetics of Fe(II) autoxidation are very complex, often dependent on the total Fe(II) and dioxygen concentrations. In addition, many competing side reactions often make accurate determination of the rate of Fe(II) autoxidation difficult. For example, 027, generated during Fe(II) autoxidation [Eq. (1)], can undergo several fates including oxidation of the Fe(II) chelate [Eq. (2)], reduction of the Fe(III) chelate generated by autoxidation, and dismutation yielding dioxygen at a rate which is second order with respect to 02 -~ concentration [Eq. (6)]. Fur2 0 2 - + 2 H + -.o HzOz + 02
(6)
thermore, the H202 generated by the dismutation reaction may also func7 B. A. Svingen, J. A. Buege, F. O. O'Neal, and S. D. Aust, ,I. Biol. Chem. 254, 5892(1979). 8 M. J. Green and H. A. O. Hill, this series, Vol. 105, p. 3.
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tion as an Fe(II) oxidant. Thus, depending on the rate constants of the competing reactions, the initial rates of dioxygen consumption may not represent the true rate of Fe(II) autoxidation.
Discontinuous Fe(II) Oxidation Assay Alternatively, the rate of Fe(II) oxidation can be measured by sampling an aliquot of the reaction mixture at regular time intervals, mixing with one of the Fe(II) chelators in Table I, and measuring the absorbance of the corresponding Fe(II) chromophore. This assay has the advantages of being sensitive and applicable to complex reaction systems where the continuous assay described above is unsuitable because of rapid dioxygen depletion owing to other processes (e.g., lipid peroxidation, dioxygenutilizing enzymes). At regular time intervals, a 0.5-ml aliquots of the reaction mixture are mixed with 0.5 ml of the desired Fe(II) chelator at a stock concentration of 15 mM. To obtain accurate absorbance values, dilution may be necessary depending on the total Fe(II) concentration in the sample [typically this should be less than 100/zM Fe(II) concentration]. The absorbance of the samples should be determined as soon as possible or the samples stored in the dark until the absorbances can be determined because fluorescent lights cause artifactually higher absorbances. The turbidity inherent in reaction mixtures containing phospholipid can be eliminated by filtering the samples through 0.22-/~m filters. Turbidity associated with samples containing fatty acids can be eliminated by dispersion of the fatty acids with Chelex 100-treated detergent (e.g., Tween 20, 1 mM final concentration).
