The mechanism of liver microsomal lipid peroxidation

232

Biochimica et Biophysica Acta, 385 ( 1 9 7 5 ) 2 3 2 - - 2 4 1 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27611

THE MECHANISM O F L I V E R MICROSOMAL LIPID P E R O X I D A T I O N

T H O M A S C. P E D E R S O N * a n d S T E V E N D. A U S T

Department of Biochemistry, Michigan State University, East Lansing, Mich. 48824 (U.S.A.) (Received August 16th, 1974)

Summary In the presence of Fe 3÷ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : c y t o c h r o m e c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase b u t produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1,3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation b u t strongly inhibited the peroxidation p r o m o t e d b y xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, b u t it significantly increased the rate of malondialdehyde formation in the reaction p r o m o t e d b y xanthine and xanthine oxidase. These results demonstrate that NADPH-dependent lipid peroxidation is p r o m o t e d by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe 3÷ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe 2÷ b y oxygen.

Introduction NADPH-dependent peroxidation of microsomal lipid can be inhibited by a variety of free-radical trapping agents [1--3], indicating a general similarity with the autooxidation of unsaturated fats and oils, which is primarily an * P r e s e n t address: S c h o o l o f C h e m i c a l S c i e n c e s , University of Illinois, Urbana, Ill. 61801, U.S.A. A b b r e v i a t i o n s : ADP • Fe, Fe 3÷ c h e l a t e d b y a 17-fold m o l a r e x c e s s o f ADP~ EDTA • Fe, Fe 3÷ c h e l a t e d by equimolar EDTA.

233 autocatalytic radical chain oxidation promoted by the radical-generating reaction between lipid hydroperoxides and trace levels of transition metal ions [4,5]. However, May and McCay [2] have shown that microsomal NADPHdependent peroxidation does not proceed as an autocatalytic process but depends on the continuous oxidation of NADPH. Apparently, the extent of the chain reaction is limited by other microsomal components such as the phosphoryl bases of phospholipids, some of which have been shown to have antioxidant properties [6]. The dependence on continuous NADPH oxidation suggests an enzyme-mediated m~chanism, which initiates the formation of hydroperoxides, may be an essential part of the overall peroxidation process resulting in the production of the characteristic by-product, malondialdehyde. Fong and coworkers [7] have suggested that the NADPH-dependent peroxidation of liver subcellular membranes is initiated by hydroxyl free radical. They implied that superoxide anion, generated by flavin oxidation, would dismutate to form hydrogen peroxide which in turn would react with additional superoxide to form the hydroxyl radical. The hydroxyl radical would then abstract a hydrogen from a methylene carbon atom of the fatty acids to initiate hydroperoxide formation. We have recently shown [8] that the generation of the superoxide anion, by xanthine oxidase, will promote peroxidation of microsomal lipid in a reaction mechanism involving the singlet state of molecular oxygen. Singlet oxygen, presumably generated by the non-enzymatic dismutation of superoxide [9,10], will readily react with unsaturated fatty acids to form hydroperoxides [11,12] allowing the subsequent oxidative reactions which result in malondialdehyde production. We reported preliminary results which showed that NADPH-dependent peroxidation of microsomal lipid could be partially inhibited by erythrocuprein suggesting that superoxide may be involved in microsomal lipid peroxidation [13]. Evidence has also been presented by ourselves [14] and other investigators [15--17 ] which suggests that both intact microsomes and purified NADPH : cytochrome c reductase catalyze NADPH-dependent superoxide production. However, further investigation described in this paper has established that NADPH-dependent peroxidation in both intact microsomes and a model system does not depend on the production of superoxide or any other of the expected reactive intermediates generated by reduction of oxygen. It has been demonstrated that addition of Fe 2÷ alone will promote peroxidation of the lipids of microsomes and other subcellular organelles [18--20]. It is therefore conceivable that the initiation of microsomal lipid peroxidation involves the reduction of iron. Poyer and McCay [21] have shown an absolute requirement for iron in microsomal lipid peroxidation and when ADP • Fe is added to intact microsomes, the rate of NADPH oxidation is increased [22,23] suggesting that it serves as an electron acceptor. However, purified NADPH : cytochrome c reductase, the flavoprotein involved in microsomal lipid peroxidation [24], will not readily reduce ADP • Fe, but will rapidly reduce EDTA • Fe [24,25]. We have described a model NADPH-dependent peroxidation system in which a purified preparation of the reductase promotes peroxidation of extracted microsomal lipid providing EDTA • Fe is also included in the reaction mixture [13]. In addition, it was shown that the microsomal flavoprotein,

234 NADH : c y t o c h r o m e bs reductase, which also reduces EDTA ' Fe will promote NADH-dependent peroxidation of microsomal lipid when both EDTA • Fe and ADP" Fe are present [24]. In this communication we will show that the NADPH-dependent lipid peroxidation in intact microsomes and in the model system apparently involve the same mechanism, which is different from the mechanism involved in the x a n t h i n e / x a n t h i n e oxidase system. The data support the conclusion that lipid peroxidation is initiated through the reduction of iron to form some type of reactive iron-oxygen complex. Methods and Materials Microso mal prepara tio ns Microsomes from the livers of male rats (250 g), fed water containing 0.1% phenobarbital for 10 days prior to being killed, were prepared as previously described [26]. NADPH : c y t o c h r o m e c reductase was purified from these microsomes by methods previously described [24] using bromelain digestion to solubilize the enzyme, followed by gel filtration and DEAE-cellulose affinity chromatography. The total lipid fraction was extracted from microsomes under anaerobic conditions by the m e t h o d of Folch et al. [27], and stored under nitrogen at --20 ° C. Liposomes were prepared from the extracted lipid immediately prior to use by sonication under anaerobic conditions as previously described [24]. Isolation o f erythrocuprein Erythrocuprein was purified from bovine erythrocytes by the m e t h o d of McCord and Fridovich [28]. The purified protein was dialyzed against water, lyophilized, and stored at --20 ° C. The copper c o n t e n t of the purified protein was determined by atomic absorption spectroscopy with a Perkin Elmer Model 303 atomic absorption spectrophotometer. The preparation was found to contain 22 000 mg of protein per mmol of copper. The a m o u n t of erythrocuprein was calculated assuming 2 g atoms o f copper per mol of enzyme [29]. Lipid peroxidation assays The extent of lipid peroxidation in reaction mixtures containing either extracted microsomal lipid or intact microsomes was assayed as previously described [ 24], by using the thiobarbituric acid assay to measure the formation of malondialdehyde. The assays for peroxidation of extracted microsomal lipid were performed in reaction mixtures containing 0.25 M Tris • HC1 (pH 6.8 at 37 °C), 0.25 M NaC1, liposomes at a concentration of 0.5 pmol of lipid phosphorous per ml, and usually, both A D P . Fe (2.0 mM ADP and 0.12 mM Fe(NO3)3 ) and EDTA • Fe (0.10 mM EDTA and 0.10 mM Fe(NO3 )3). The reaction mixtures indicated to include NADPH and NADPH : cytochrome c reductase contained 0.2 mM NADPH. The reaction mixtures indicated to include xanthine and xanthine oxidase contained 0.33 mM xanthine. The xanthine oxidase was dissolved in 0.05 M Tris • HC1 (pH 7.5) and passed through a Sephadex G-50 column immediately prior to use. The reaction mixtures for the assay of lipid peroxidation in intact microsomes contained either 0.4 mM NADPH or 0.33 mM xanthine where indicated. The reaction mixtures were

235 incubated at 37°C under an atmosphere of air in a Dubnoff shaker and the procedures by which the reaction was initiated and the rate of malondialdehyde formation determined have been previously described [24]. Activities, expressed as nmol of malondialdehyde formed per min per ml of incubation mixture, were linear with respect to enzyme concentration and varied less than 1%. The addition of 1,3-diphenylisobenzofuran to the reaction mixtures containing extracted microsomal lipid was accomplished by adding the compound to the lipid extract in chloroform/methanol (2 : 1, v/v), removing the solvents under a stream of nitrogen, and then preparing liposomes. In the reaction mixtures containing intact microsomes, 1,3-diphenylisobenzofuran was added to the microsomes prior to their addition to the reaction mixture by the following procedure. A finely divided suspension of the compound was prepared by rapidly diluting an appropriate aliquot of 0.1 M 1,3-diphenylisobenzofuran in acetone into Tris • HC1 buffer. Microsomes were then added to the mixture at a final concentration of 5 mg of protein per ml. Care was taken in these operations to minimize the exposure to light. Other methods Protein was determined by the method of Lowry et al. [30]. The rate of hydrogen peroxide formation, generated by glucose and glucose oxidase, was assayed by measuring the decomposition of o-dianisidine in the presence of horseradish peroxidase according to the method of Maehly and Chance [31]. Materials Xanthine oxidase (Sigma, Grade I), catalase (Sigma, two times recrystallized), glucose oxidase (Sigma, Type II), horseradish peroxidase (Sigma, Type I), ADP, o-dianisidine, NADPH, and xanthine were all obtained from Sigma Chemical Co., St. Louis, Missouri. 1,3-Diphenylisobenzofuran was obtained from the Aldrich Chemical Co., Milwaukee, Wisconsin. All other reagents used were reagent grade. Results

Role o f complexing anions We have previously described the requirements for complexing anion in the various peroxidation systems presented in this paper [8,13,24]. In all peroxidation systems, the addition of EDTA at a concentration in excess of the Fe 3+ concentration effectively inhibits lipid peroxidation. However, the rate of malondialdehyde formation is greatest in all systems when the reaction mixture includes EDTA . Fe in addition to either FeC13 or ADP • Fe. The only system which requires EDTA • Fe is the NADPH-dependent peroxidation of extracted microsomal lipid promoted by the purified reductase. The involvement of superoxide We observed that a commercial preparation of erythrocuprein would partially inhibit the NADPH
236 TABLE I THE EFFECT OF ERYTHROCUPREIN All c o n d i t i o n s

Reaction

ON LIPID PEROXIDATION

axe t h e s a m e as t h o s e d e s c r i b e d

under Methods.

mixture

M i c r o s o m e s , 0 . 4 m g p r o t e i n p e r m l , N A D P H a n d A D P ~ Fe in 0 . 0 5 M Tris - H C I ( p H 6 , 8 ) P l u s 0 . 2 pM e r y t h r o c u p r e i n Plus 1.0 p M e r y t h r o c u p r e i n M i c r o s o m e s , 0 . 4 m g p r o t e i n p e r m l , N A D P H a n d A D P • Fe in 0 . 2 5 M NaCl and 0.25 Tris • HCI (pH 6.8) Plus 0 . 2 pM e r y t h r o c u p r e i n P l u s 1.0 /2M e r y t h r o c u p r e i n NADPH : cytochrome c reductase, 0.17 #g/ml, N A D P H and extracted lipid in t h e p r e s e n c e o f A D P • Fe a n d E D T A • F e P l u s 0 . 2 pM e r y t h r o c u p r e i n Plus 0.4/2M e r y t h r o c u p r e i n Plus 1.0 p M e r y t h r o c u p r e i n X a n t h i n e o x i d a s e , 2 5 p g / m L x a n t h i n e a n d e x t r a c t e d l i p i d in t h e p r e s e n c e o f A D P • Fe Plus 0 . 2 ~zM e r y t h r o c u p r e i n Plus 1.0 pM e r y t h r o c u p r e i n X a n t h i n e o x i d a s e , 25 p g / m l , x a n t h i n e a n d e x t r a c t e d l i p i d in t h e p r e s e n c e o f A D P - Fe a n d E D T A • Fe Plus 0 . 2 pM e r y t h r o c u p r e i n Plus 1.0 pM e r y t h r o c u p r e i n

Malondialdehyde

formed

n m o l / m in per ml

% Inhibition

2.73 2.59 2.44

5 11

3.32 3.19

5 10

3.06 2.58 2.42 1.95

16 21 37

1.17 0.49 0.29

57 75

2.42 1.17 0.58

52 76

peroxidation under the same conditions [8]. The inhibition of NADPHdependent lipid peroxidation by a purified preparation of erythrocuprein was examined and compared to the inhibition of the peroxidation activity promoted by xanthine and xanthine oxidase. But as shown in Table I, NADPHdependent lipid peroxidation in both the model system and in intact microsomes is considerably less sensitive to inhibition by erythrocuprein than the peroxidation activity promoted by xanthine and xanthine oxidase. Strobel and Coon [32] observed that erythrocuprein would inhibit drug hydroxylation in a reconstituted microsomal P-450 system, but the inhibition was complete only in the presence of a high salt concentration. We found that the inhibition by erythrocuprein of microsomal NADPH-dependent peroxidation was not increased by the presence of high ionic strength, as shown in Table I. It is not possible to discern whether the observed inhibition of NADPH-dependent peroxidation by erythrocuprein is due to the superoxide dismutase activity or some other inhibitory property because the Cu 2÷ and Zn 2÷ released during the heat inactivation of erythrocuprein produce an even greater inhibition of lipid peroxidation [8].

The involvement of singlet oxygen We have demonstrated the involvement of singlet oxygen in lipid peroxidation promoted by superoxide [8]. It has also been suggested that peroxidation of adrenal mitochondrial lipids promoted by the mitochondrial NADPH-

237 TABLE

II

THE EFFECT

OF 1,3-DIPHENYLISOBENZOFURAN

ON LIPID PEROXIDATION

All conditions

are the same as those described under Methods.

Reaction mixture

Malondialdehyde formed nmol/min per ml

Microsomes, 0.2 mg protein per ml and NADPH in the presence of ADP • Fe Plus 0.2 pmol 1,3-diphenylisobenzofuxan per mg microsomal protein Plus 1.0 pmol 1,3-diphenylisobenzofuran per mg microsomal protein M i c r o s o m e s , 0 . 2 m g p r o t e i n p e r m l a n d N A D P H in t h e p r e s e n c e o f A D P • F e and EDTA • Fe Plus 0.2 pmol 1,3-diphenyllsobenzofuran per mg microsomal protein Plus 1.0 pmol 1,3-diphenylisobenzofuran per mg microsomal protein M i c r o s o m e s , 0 . 2 5 m g p r o t e i n p e r m l , p l u s x a n t h i n e o x i d a s e l 5 0 pg/ml, a n d xanthine in the presence of ADP • Fe and EDTA • Fc Plus 1.0 pmol 1,3-diphenylisobenzofuran per mg microsomal protein NADPH : cytochrome c reductase, 0.14 pg/ml, NADPH, and extracted lipid in t h e p r e s e n c e o f A D P • F e a n d E D T A - F e Plus 0.2 pmol 1,3-diphenylisobenzofuran per mg mierosomal protein Plus 1.0 pmol 1,3-diphenylisobenzofuran per mg microsomal protein Xanthine oxida6e, 25 pg/ml, xanthine, and extracted lipid in the presence of ADP • Fe and EDTA • Fe Plus 0.2 pmol 1,3-diphenylisobenzofuran per mg microsomal protein Plus 1.0 gmol 1,3-diphenylisobenzofuran per mg microsomat protein

% Inhibition

1.46 1.51 1.56

0 0

2.49 2.63 2.73

0 0

2.49 1.22

51

2.44 2.44 2.52

0 0

1.46 0.59 0.29

60 80

dependent electron transport system involves singlet oxygen [33]. We found that 1,3-diphenylisobenzofuran, which reacts very rapidly with singlet oxygen, is a suitable trapping agent because of its lack of effect on the generation of superoxide by xanthine oxidase or on the chain oxidation and production of malondialdehyde p r o m o t e d b y ascorbic acid [8]. The results in Table II show the effect of 1,3-diphenylisobenzofuran on NADPH-dependent lipid peroxidation in b o t h the model system and in intact microsomes. In contrast to its ability to inhibit lipid peroxidation p r o m o t e d by xanthine and xanthine oxidase, 1,3-diphenylisobenzofuran had no effect on NADPH-dependent lipid peroxidation. NADPH-dependent peroxidation in intact microsomes in the presence of either A D P . Fe and E D T A . Fe, or only A D P . Fe, was not inhibited by the singlet-oxygen trapping agent. In order to establish that superoxide, via the generation of singlet oxygen, would also p r o m o t e lipid peroxidation in intact microsomes, and to demonstrate that 1,3-diphenylisobenzofuran was a suitable singlet-oxygen trapping agent in intact microsomes as well as in extracted microsomal lipid, we examined the ability of xanthine and xanthine oxidase to p r o m o t e the peroxidation of lipid in intact microsomes. As shown in Table II, xanthine and xanthine oxidase will readily p r o m o t e peroxidation in intact microsomes and the activity is inhibited b y the presence of 1,3-diphenylisobenzofuran, whereas NADPH-dependent lipid peroxidation in the same microsomes is not inhibited. The involvement o f HOOH or hydroxyl radicals Liver microsomes form H O O H in the presence of NADPH [34,35] and it

238 has been suggested that the H O O H may be involved in initiating the peroxidation of lipid [35]. It has been previously found that HOOH and Fe "~÷ fail to p r o m o t e peroxidation of lipid in mitochondrial membranes [36]. We similarly found that no malondialdehyde was produced when the NADPH and NADPH : c y t o c h r o m e c reductase in the reaction mixture containing microsomal lipid were replaced by glucose and glucose oxidase at concentrations generating between 7 and 175 nequivalents of HOOH per min per ml. We had previously observed that the NADPH-dependent peroxidation of extracted microsomal lipid catalyzed by NADPH : c y t o c h r o m e c reductase was inhibited to some degree by the addition of catalase to the reaction mixture [13]. HOOH may be formed during the reoxidation of Fe 2+ by oxygen and subsequently reduced by additional Fe 2+ to generate hydroxyl radicals [ 3 7 - - 4 0 ] . Therefore, the possible involvement of hydroxyl radicals was examined. The presence of either 2-methyl-2-propanol (0.1 M), sodium formate (0.5 M) or mannitol (0.25 M), which are effective scavengers of hydroxyl radicals [ 4 1 - - 4 3 ] , had no effect on the rate of NADPH-dependent lipid peroxidation in either intact microsomes or in the model system containing the purified reductase. Since NADPH-dependent lipid peroxidation does n o t depend on the generation of hydroxyl radicals, the inhibitory effect of catalase on NADPHdependent lipid peroxidation was reexamined. The catalase was passed through a short Sephadex G-50 column prior to use to remove the antioxidant t h y m o l included in commercial preparations of catalase as a stabilizing agent. In contrast to previous results, the thymol-free catalase had no effect on the NADPHdependent peroxidation activity, as can be seen in Table III. It has been suggested by other investigators that HOOH and superoxide react to form a reactive intermediate, such as the hydroxyl radical or singlet oxygen, which may p r o m o t e the peroxidative damage of biological organelles [7,44,45]. Therefore, we investigated the effect of catalase on the peroxidation of microsomal lipid p r o m o t e d by xanthine and xanthine oxidase, which has been demonstrated to involve b o t h superoxide and singlet oxygen [8]. As can be seen in Table III, catalase produced no inhibition of the xanthine oxidase-promoted peroxida-

TABLE EFFECT

III OF CATALASE

ON THE PEROXIDATION

OF EXTRACTED

MICROSOMAL

LIPID

The catalase was passed through a Sephadex G-50 column immediately prior to use, The reaction mixture containing glucose oxidase also contained ADP - Fe and EDTA • Fe and generated 140 nequivalents of HOOH per rain per ml. All other conditions are the same as those described in Methods.

Reaction

mixture

NADPH : cytochrome c reductase, 0.09 pg per ml, and NADPH Plus 10 #g/ml catalase Plus 100 ttg/ml catalase Xanthine oxidase, 25 ttg/ml and xanthine Plus 10 pg/ml catalase P l u s 1OO t t g / m l e a t a l a s e Glucose oxidase, 100 ttg/ml and glucose Plus 100 pg/ml catalase

Maiondialdehyde formed (nmol/min per ml) 1.43 1.45 1.44 1.17 2.15 2.15 0.00 0.00

239 tion, but rather increased the rate of malondialdehyde formation. The possibility that this increase in peroxidation activity was promoted by an interaction between HOOH and catalase was proven not likely since the combination of catalase and hydrogen peroxide generated by glucose and glucose oxidase promoted no peroxidation activity. Discussion

In this paper, we have demonstrated that the NADPH-dependent peroxidation of rat liver microsomal lipid is promoted by a reaction mechanism different from that by which superoxide, generated by xanthine and xanthine oxidase, promotes lipid peroxidation. Erythrocuprein inhibits NADPH-dependent lipid peroxidation much less effectively than the peroxidation promoted by xanthine and xanthine oxidase. The inhibition of NADPH
240

peroxidation p r o m o t e d by Fe ~* also required such a mechanism. The existence of such a mechanism would not be unlikely since an aqueous solution of Fe 2÷ and oxygen is able to promote a variety of other oxidative reactions such as aromatic h y d r o x y l a t i o n [47--49], modification of proteins [50], and the generation of bio- and chemiluminescence [51,52]. These reactions also either require or are strongly activated by strong complexing anions such as EDTA, pyrophosphate, or phosphate. There are a variety of reactive intermediates which may be produced during the oxidation of Fe 2+ by molecular oxygen. Oxygen is known to combine with Fe 2÷ to form the perferryl ion FeO22+ as an intermediate in the oxidation reaction [38,39]. Strong complexing anions increase the rate of oxidation and change the reaction from second to first order with respect to Fe 2.. Although superoxide may be generated during the oxidation of Fe 2* [53,54], the failure to show evidence of its involvement in NADPH-dependent peroxidation may be due to the rapid rate at which Fe 3. is reduced, since Michelson [52] observed that the chemiluminescence from superoxide generated by the oxidation of Fe 2÷ was decreased at higher concentrations of Fe 2÷, apparently because the excess Fe 2÷ rapidly reduces superoxide.

Acknowledgements These studies were supported in part by N.I.H. Training Grant GM 1091 from the National Institute of General Medical Sciences and by National Science Foundation Grant No. GB-40711. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

H o c h s t e i n , P. a n d E r n s t e r , L. ( 1 9 6 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 12, 3 8 3 - - 3 9 4 M a y , H . E . a n d M c C a y , P.B. ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 2 2 9 6 - - 2 3 0 5 P h e i f e r , P.M. a n d M c C a y , P.B. ( 1 9 7 1 ) J . Biol. C h e m . 2 4 6 , 6 4 0 1 - - 6 4 0 8 F a r m e r , E.H. ( 1 9 4 6 ) T r a n s . F a r a d a y Soc. 42, 2 2 8 - - 2 3 6 Uri, N. ( 1 9 6 1 ) in A u t o x i d a t i o n a n d A n t i o x i d a n t s ( L u n d b e r g . W . O . , e d . ) , Vol. I, p. 55, I n t e r s c i e n c e Publishers, New York Tsai, L.-S. a n d S m i t h , L.M. ( 1 9 7 1 ) L i p i d s 6, 1 9 6 - - 2 0 2 F o n g , K . - L . , M c C a y , P.B., P o y e r , J . L . , K e e l e , B.B. a n d Misra, H. ( 1 9 7 3 ) J. Biol, C h e m . 2 4 8 , 7 7 9 2 - 7797 P e d e r s o n , T.C. a n d A u s t , S.D. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 52, 1 0 7 1 - - 1 0 7 8 Khan, A.U. (1970) Science 168, 476--477 S t a u f f , J., S a n d e , U. a n d J a e s c h k e , W. ( 1 9 7 3 ) in C h e m i l u m i n e s c e n c e a n d B i o l u m i n e s c e n c e ( C o r m i e r , M.J., e d . ) , p. 1 3 1 , P l e n u m Press, N e w Y o r k R a w l s , H , R . a n d v a n S a n t e n , P.J. ( 1 9 7 0 ) A n a l . N.Y. A c a d . Sci. 1 7 1 , 1 3 5 - - 1 3 8 D o w t y , B., L a s e t e r , J . L . , G r i f f i n , G.W., P o l i t z e r , I.R. a n d W a l k i n s h a w , C.H. ( 1 9 7 3 ) S c i e n c e 1 8 1 , 669--670 P e d e r s o n , T.C. a n d A u s t , S.D. ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 4 7 , 1 1 3 3 - - 1 1 3 7 A u s t , S.D., R o e r i g , D . L . a n d P e d e r s o n , T.C. ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n , 4 7 , 1 1 3 3 - - 1 1 3 7 C o o n , M.J., S t r o b e l , H.W. a n d B o y e r , R . G . ( 1 9 7 3 ) D r u g Met. Disp. 1, 9 2 - - 9 7 D e b e y , P. a n d B a l n y , C. ( 1 9 7 3 ) B i o c h e m i e 55, 3 2 9 - - 3 3 2 N a k a m u r a , S. a n d Y a m a z a k i , I. ( 1 9 6 9 ) B i o c h i m . B i o p h y s . A c t a 1 8 9 , 2 9 - - 3 7 B e l o f f - C h a i n , A., S e r l u p i - C r e s c e n z i , G., C a t a n z a r o , R., V e n e t t a c c i , D. a n d Belliano, M, ( 1 9 6 5 ) Biochim. Biophys. Acta 97, 416--421 O t t o h e n g h i , A. ( 1 9 5 9 ) A r c h . B i o c h e m . B i o p h y s . 7 9 , 3 5 5 - - 3 6 3 M c K n i g h t , R . C . , H u n t e r , J r , F.E. a n d O e h l e r t , W.H. ( 1 9 6 5 ) J. Biol. C h e m . 2 4 0 , 3 4 3 9 - - 3 4 4 6 P o y e r , J . L . a n d M c C a y , P.B. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 2 6 3 - - 2 6 9 B e l o f f - C h a i n , A., C a t a n z a r o , R. a n d S e r l u p i - C r e s c e n z i , G. ( 1 9 6 3 ) N a t u r e 1 9 8 , 3 5 1 - - 3 5 4

241

23 E r n s t e r , L. a n d N o r d e n b r a n d , K . ( 1 9 6 7 ) M e t h o d s E n z y m o l . 1 0 , 5 7 4 - - 5 8 0 2 4 P e d e r s o n , T . C . , B u e g e , J . A . a n d A u s t , S.D. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 7 1 3 4 - - 7 1 4 1 2 5 K a m i n , H. a n d M a s t e r s , B.S.S. ( 1 9 6 8 ) in E n z y m a t i c O x i d a t i o n o f T o x i c a n t s ( H o d g s o n , E., e d . ) , p. 5, North Carolina State University, Raleigh 2 6 P e d e r s o n , T.C. a n d A u s t , S.D. ( 1 9 7 0 ) B i o c h e m . P h a x m a c o l . 1 9 , 2 2 2 1 - - 2 2 3 0 27 F o l c h , J., Less, M. a n d S l o a n e - S t a n l e y , G . H . ( 1 9 5 6 ) J. Biol. C h e m . 2 2 6 , 4 9 7 - - 5 0 9 2 8 M c C o r d , J . M . a n d F r i d o v i c h , I. ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 6 0 4 9 - - 6 0 5 5 29 Keele, J r , B.B., M c C o r d , J . M . a n d F r i d o v i c h , I. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 2 8 7 5 - - 2 8 8 0 3 0 L o w r y , O . H . , R o s e b r o u g h , N . J . , Farr~ A . L . a n d R a n d a l l , R . J , ( 1 9 5 1 ) J. Biol. C h e m . 1 9 3 , 2 6 5 - - 2 7 5 31 M a e h l y , A . C . a n d C h a n c e , B. ( 1 9 5 4 ) in M e t h o d s o f B i o c h e m i c a l A n a l y s i s ( G l o c k , D., e d ) , Vol. I, p. 3 5 7 , I n t e r s c i e n c e P u b l i s h e r s , N e w Y o r k 32 S t r o b e l , H.W. a n d C o o n , M . J . ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 7 8 2 6 - - 7 8 2 9 3 3 G o d a , K., C h u , J., K i m u r a , T. a n d S c h a a p , P.A. ( 1 9 7 3 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 5 2 , 1 3 0 0 - 1306 3 4 T h u r m a n , R . G . , L e y , H . G . a n d S c h o l z , R. ( 1 9 7 2 ) E u r . J. B i o c h e m . 2 5 , 4 2 0 - - 4 3 0 3 5 Boveris, A., O s h i n o , N. a n d C h a n c e , B. ( 1 9 7 2 ) B i o c h e m . J. 1 2 8 , 6 1 7 - - 6 3 0 3 6 H u n t e r , J r , F . E . , S c o t t , A., H o f f s t e n , P.E., G u e r r a , F., W e i n s t e i n , J., S c h n e i d e r , A., S c h u t z , B., F i n k , J . , F o r d , L. a n d S m i t h , E. ( 1 9 6 4 ) J. Biol. C h e m . 2 3 9 , 6 0 4 - - 6 1 3 37 K h a r a s c h , M.S., A r i m o t o , F.S. a n d N u d e n b e r g , W. ( 1 9 5 1 ) J. O r g . C h e m . 1 6 , 1 5 5 6 - - 1 5 6 5 3 8 Weiss, J. ( 1 9 5 3 ) E x p e r i e n t i a 9, 6 1 - - 6 2 39 B a s o l o , F. a n d P e a r s o n , R . C . ( 1 9 5 8 ) in M e c h a n i s m s o f I n o r g a n i c R e a c t i o n s , p. 3 3 7 , J. Wiley a n d S o n s , Inc., N e w Y o r k 4 0 I n g r a i n , L . L . ( 1 9 6 6 ) in C o m p r e h e n s i v e B i o c h e m i s t r y ( F l o r k i n , M. a n d S t o t z , E . H . , eds), p. 4 2 4 , Elsevier P u b l i s h i n g Co., A m s t e r d a m 41 A n b a r , M. a n d P e c h t , I. ( 1 9 6 4 ) J. P h y s . C h e m . 6 8 , 3 5 2 - - 3 5 5 4 2 P e c h t , I. a n d F a r a g i i , M. ( 1 9 7 2 ) P r o c . N a t l . A c a d . Sci. U.S. 6 9 , 9 0 2 - - 9 0 6 4 3 F r i d o v i c h , I. ( 1 9 7 2 ) A c t . C h e m . Res. 5, 3 2 1 - - 3 2 6 4 4 Fee, J . A . a n d T e i t e l b a u m , D . H . ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 4 9 , 1 5 0 - - 1 5 7 4 5 Z i m m e r m a n , R., F l o b e , L., Weser, U. a n d H a r t m a n n , H . J . ( 1 9 7 3 ) F E B S L e f t . 2 9 , 1 1 2 - - 1 2 0 4 6 B e a u c h a m p , C. a n d F r i d o v i c h , I. ( 1 9 7 0 ) 3. Biol. C h e m . 2 4 5 , 4 6 4 1 - - 4 6 4 6 47 U d e n f r i e n d , S., C l a r k , C., A x e l r o d , J. a n d B r o d i e , B.B. ( 1 9 5 4 ) J. Biol. C h e m . 2 0 8 , 7 2 1 - - 7 3 9 4 8 N o f r e , C., Cier, A. a n d L e f i e r , A. ( 1 9 6 1 ) Bull. S o c . C h i m . F t . 4 3 0 - - 4 3 5 4 9 G o s c i n , S.A. a n d F r i d o v i c h , I. ( 1 9 7 2 ) A r c h . B i o c h e r n . B i o p h y s . 5 3 , 7 7 8 - - 7 8 3 50 T a b o r s k y , G. ( 1 9 7 3 ) B i o c h e m i s t r y 1 2 , 1 3 4 1 - - 1 3 4 8 51 B e h a r , D., C z u p s k i , G., R a b a n i , J., D o r f m a n , L.M. a n d S c h w a r z , H . A . ( 1 9 7 0 ) J. P h y s . C h e m . 7 4 , 3 2 0 9 52 M i c h e l s o n , A.M. ( 1 9 7 3 ) B i o c h e m i e 55, 3 6 5 - - 3 7 9 53 G e o r g e , P. ( 1 9 5 4 ) J. C h e m . S o c . 4 3 4 9 - - 4 3 5 9 5 4 H u f f m a n , R . E . a n d D a v l d s o n , N. ( 1 9 5 6 ) J. A m . C h e m . S o c . 7 8 , 4 8 3 6 - - 4 8 4 2