ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 186, No. 1, February, pp. 189-195, 1978

Generation of Superoxide Radical during Autoxidation of Hydroxylamine and an Assay for Superoxide Dismutase

YASUHISA KONO

Department of Bacteriology, Tottori University, School of Medicine, Yonugo, Tottori 683, Japan

Received July 6, 1977; revised November 1, 1977

Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of at&oxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by euperoxide dismutaee, indicating the partici- pation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide diemutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.

Hydroxylamine is an intermediate in tion of hydroxylamine, an assay procedure the reduction of nitrate to ammonia by for superoxide dismutase is also described. halotolerant bacteria (1, 2) and in the oxidation of ammonia to nitrite by Nitro- MATERIALS AND METHODS

somonus (3-5). Hydroxylamine is autoxi- Cu,Zn-superoxide dismutase from spinach leaves

dized rapidly in the presence of trace met- was a generous gift of Dr. Asada, Kyoto University.

als at high pH. Although extensive studies The enzyme concentration was determined from an

on the mechanism of the autoxidation extinction coefticient at 258 nm (c = 9920 M-I *cm-‘)

have been reported (64, there has not (20). Superoxide dismutase was assayed from an

been a report on the univalent reduction inhibition of cytochrome c reduction by O,- gener-

of oxygen in this process. On the other ated with a xanthine-xanthine oxidase system us-

hand, Elstner et al. (9) and Elstner and ing the method of Asada et al. (21). NBT’ was obtained from Sigma. Hydroxylamine hydrochloride

Heupel (10) have reported that at neutral and Triton X-100 were obtained from Nakarai pH hydroxylamine is oxidized by superox- Chemical Co. (Kyoto, Japan). An aqueous solution ide radical (0,-j to nitrite whose produc- of hydroxylamine was prepared daily and its pH tion is inhibited by superoxide dismutase. was adjusted to 6.0 with 2 M sodium hydroxide. The

However, the detailed mechanism of nitrite solution was kept in a tightly stoppered test tube

formation from hydroxylamine by O,- until use. Other chemicals were reagent grade.

is not revealed yet. Glass-distilled water was used throughout.

O,- has been shown to be generated Absorbance measurements were carried out with

during autoxidation of ferredoxin (ll), fla- a Shimazu multipurpose MPSdOL epectrophotome- ter at 25°C. The reaction was initiated by the addi-

vins and quinones (12), epinephrine (13), tion of hydroxylamine to the reaction mixture and tiron (14), dopamines (15), tetrahydropter- the reduction of NBT was followed by an absorbance idines (16), thiols (17), pyrogallol (18), and increase at 560 nm under aerobic conditions. Nitrite phenylhydrazine (19). Therefore, the au- was determined calorimetrically by the diaxo-cou-

toxidation of hydroxylamine would also produce 02-, and we confirmed in the

1 Abbreviations used: NBT, nitroblue tetrazo-

present paper that this is the case. Using lium; EDTA, ethylenediaminetetraacetic acid; SOD, superoxide dismutase; Me, transition metal; FMN,

the formation of 02- during the autoxida- flavin mononucleotide.

189

0003-9861/78/1861-0189802.00/O Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

190 YASUHISA

pling procedure as described by Elstner and Heupel (10).

RESULTS AND DISCUSSION

Nitrite Formation and Reduction of NBT during Auto&l&ion of Hydroxylamine When hydroxylamine was added to an

aerobic solution containing 50 mM sodium carbonate, pH 10.2, and 0.1 mM EDTA, hydroxylamine was at&oxidized producing nitrite linearly at least for 30 min. Under these conditions the production of nitrite is first order with respect to the concentra- tion of hydroxylamine, with a rate con- stant of 1.9 ‘X 10e5 s-l. Nitrate was not detected using the brucine method (22).

The addition of NBT to the above reac- tion mixture induced an increase in ab- sorbance at 560 nm due to the accumula- tion of blue formazan (Fig. lc). When Triton X-100 was added, an early slow phase in the reduction of NBT disappeared and the reduction was apparently stimu- lated (Fig. la). The addition of Triton X- 100 for the assay of formazan formation has been recommended by Nishikimi (16) and brings about the stabilization of colloi- dal product. In the following experiments, 0.03% (v/v) Triton X-100 was added. The

1

AAs6cmm =0.01

rz

FIG. 1. The time courses of NBT reduction dur- ing autoxidation of hydroxylamine and the effects of Triton X-100 and superoxide dismutase. The re- action mixture contained, in a total volume of 1.0 ml, 50 mM sodium carbonate, pH 10.2, 24 PM NBT, 0.1 rnr+r EDTA, and 1 mM hydroxylamine which was added at the arrow point [line (c)l. In lines (a) and (b), in addition to the above mixture, 0.03% (v/v) Triton X-100 or 0.03% (v/v) Triton X-100 plus 9.5 nM superoxide dismutase were added, respectively.

KONO

0 I NeOH :rnM)

3

FIG. 2. The initial reduction rate of NBT as a function of hydroxylamine concentration. Hydrox- ylamine at the indicated concentration was added to 50 mM sodium carbonate, pH 10.2,O.l mM EDTA, 24 PM NBT, and 0.03% (v/v) Triton X-100 in a final volume of 1.0 ml.

initial reduction rate of NBT (O-30 s) as a function of the concentration of hydroxyl- amine is shown in Fig. 2.

As shown in Fig. 1, curves a and b, the reduction of NBT was inhibited by super- oxide dismutase to the same degree whether the enzyme was added during the reaction or prior to the initiation of reac- tion. The inhibition was also observed in the absence of Triton X-100. The results indicate the participation of O,- in the autoxidation of hydroxylamine. However, nitrite formation was not affected by su- peroxide dismutase (Table III).

Under anaerobic conditions where the formation of nitrite was not detected, no NBT reduction was observed (Table III), indicating that the reduction was not in- duced by hydroxylamine, but by 02-.

Two substituted hydroxylamines, O- methylhydroxylamine and N-methylhy- droxylamine, were examined to confirm the reactive site of the hydroxylamine molecule. While 0-methylhydroxylamine did not reduce NBT and did not produce nitrite, N-methylhydroxylamine was au- toxidized at a four times faster rate than that of the same concentration of nonsub- stituted hydroxylamine, in both the ab- sence and the presence of EDTA, pH 10.2.

Effects of Transition Metals, EDTA-Metal Complexes, and EDTA on Autoxidation of Hydroxylamine Mn2+ and EDTA-Mn2+ inhibited NBT

reduction during autoxidation of hydrox-

HYDROXYLAMINE AUTOXIDATION AND 02-

ylamine, but did not affect nitrite forma- tion (Table I). As previously reported, Mn2+-pyrophosphate is oxidized by 02- at a second-order rate of 6 x log M-’ 6 s-l (23). Thus, the effects of Mn2+ and EDTA-Mn2+ are attributed to competition with NBT for O,- in a similar manner to superoxide dismutase. While Fe2+, Fe3+, and EDTA- Cu2+ did not significantly affect the pro- duction of nitrite and NBT reduction, EDTA-Fe2+ and Cu2+ stimulated both re- actions to a great extent, as summarized in Table I. Under the present conditions,

TABLE I EFFECTS OF TRANSITION METALS AND EDTA-

METAL COMPLEXES ON AUTO~IDATION OF HYDROXYLAMINE’

Additions Relative NBT Relative ni- reduction rata trite forma-

tion rate

Control” MnCl*, 100 PM CUSO,, 1 PM FeSO,

1 PM 10 /LM

Fe(N03h 50 p.M

100 &LLM EDTA-Mn*+, 100 PM EDTA-Cu2+, 10 PM EDTA-Fez+

0.01 /hM

0.05 @I 0.1 PM

CuSO,, 1 /LM, + SOD, 47 nM

FeSO,, 10 PM, + SOD, 47 nM

EDTA-Fe*+, 0.1 PM, + SOD, 47 nM

SOD, 47 mu

100 100 41 108

331 235

103 138 89

101 102 113 108 80 102

104 105

206 244 336 259 89

56

123

13 91

a The reaction mixture contained, in a total vol- ume of 1.0 ml, 50 mM sodium carbonate, pH 10.2, 24 PM NBT, 1 mM hydroxylamine, and 0.03% (v/v) Triton X-100. NBT reduction was followd by an absorbance increase at 560 nm. Nitrite was deter- mined calorimetrically, as described in Fig. 3, &er 20-min reaction using a mixture containing 1 mM hydroxylamine and 50 mM sodium carbonate, pH 10.2, in a total volume of 1.0 ml. The reaction mixture contained the additions indicated.

b Control rates were 0.0064 AA,,, Jmin and 16.5 nmol of nitrite formed/20 min.

FIG. 3. Effect of EDTA on the rate of autoxida- tion of hydroxylamine and NBT reduction accompa- nying autoxidation. The reaction mixture con- tained. in a total volume of 1.0 ml, 50 mM sodium carbonate, pH 10.2, 24 paa NBT, 1 mM hydroxyla- mine, 0.03% (v/v) Triton X-100, and the indicated concentration of EDTA. NBT reduction was followed by an absorbance increase at 560 nm. After reaction for 20 min, initiated by the addition of 1 mM hydrox- ylamine to the reaction mixture (containing 50 mM sodium carbonate, pH 10.2, and the indicated con- centration of EDTA, in a total volume of 1.0 ml), nitrite was determined as described by Elstner and Heupel (10). The rates in the absence of EDTA were 0.0064 AA %,, ,,,/min and 16.5 nmol of nitrite formed/20 min.

probably due to Triton X-100, no precipi- tate of metal hydroxides was formed.

EDTA up to 10 PM inhibited rates of both NBT reduction and nitrite formation by about 50%. However, in the presence of 100 GM EDTA both reaction rates were enhanced about 1.8 times compared with those in the absence of EDTA (Fig. 3). These results suggest that the autoxida- tion of hydroxylamine is a free radical chain reaction initiated not only by aquo metal ions but also by EDTA-metal com- plexes and that the EDTA complex is more effective than free metal. The dual role of EDTA in the oxidation of sulfite (24) and epinephrine (13) was discussed by Fridov- ich et al. They suggested that free metal ion probably reacts with oxygen by an inner-sphere mechanism, whereas EDTA- metal complex reacts by an outer-sphere mechanism, which generates O,- into the solution. It seems likely that the mecha- nism of autoxidation of hydroxylamine is similar to that of sulfite and epinephrine.

192 YASUHISA KONO

Effect of pH on Autoxidation of Hydrox- ylamine

As shown in Fig. 4, the rate of autoxi- dation of hydroxylamine, detected by its ability to reduce NBT, was sharply de- creased with lowering pH and was not observed below pH 8.0. The rate of nitrite production also increased above pH 8.0, as presented in Table II. Hydroxylamine is stable in acid solution, presumably be- cause its protonated form does not react with metal ion. The pK value for the dissociation (NH30H+ C$ NH,OH + H+) is 5.9 (25). Although the pK value for the dissociation (NH,OH e NH,O- + H+) has

PH

FIG. 4. Effect of pH on the initial rate of NBT reduction. The reaction mixture consisted of 24 PM NBT, 0.1 mre EDTA, 1 mM hydroxylamine, and 0.03% (v/v) Triton X-100 in the following buffer system: pH 7.4 and 7.8, 50 mM potassium phosphate (O-O); pH 8.4, 9.2, and 9.6, 50 mM Tris-HCl (0-O); pH 9.6 and 10.2, 50 mM sodium carbonate (A-A), in a total volume of 1.0 ml.

TABLE II

EFFECT OF pH ON AUTOXIDATION OF HYDROXYLAMINIP

Buffers Nitrite formation (50 mhi) (nmoU20 min)

Potassium phosphate, pH 7.4 0.4 Potassium phosphate, pH 7.8 0.4 Tris-HCl, pH 8.4 1.0 Tris-HCl, pH 9.2 1.5 Tris-HCl, pH 9.6 2.0 Sodium carbonate, pH 9.6 1.9 Sodium carbonate, pH 10.2 25.5

D The reaction mixture contained, in a total vol- ume of 1.0 ml, 1 mM hydroxylamine and 0.1 rnre EDTA in the indicated buffered solutions.

not been reported, using interpolation a value of 14 is calculated (26). Therefore, under the present conditions, almost all of the hydroxylamine occurs in a form of NH,OH and we cannot account for the increase in autoxidation above pH 8.0 by a form of hydroxylamine. Superoxide dis- mutase inhibited NBT reduction at any pH and maximum inhibition was slightly increased at low pH. At pH 9.6, a saturat- ing concentration of superoxide dismutase (47 nM) inhibited the reduction by 98%, in contrast to 85% at pH 10.2.

Participation of H,O, in the Autoxidu- tion of Hydroxylamine The addition of 0.1 mM H,O, augmented

1.6 and 1.8 times the rates of NBT reduc- tion and nitrite production, respectively. H202, per se, did not reduce NBT. This result suggests that H,Oz formed through spontaneous disproportionation of 02- oxi- dizes hydroxylamine. The addition of ben- zoate, mannitol, and formate, scavengers of -OH (27-291, did not afkt the autoxi- dation, as summarized in Table III, indi- cating that the hydroxyl radical formed by the Harber-Weiss reaction (30) does not participate in the reaction. In order to assure the involvement of H,O, in the autoxidation, the effect of Na,S,O,, which decomposes H202, was examined because, unfortunately, catalase is inhibited by hy-

NBT (mM)

FIG. 5. Effect of the concentration of NBT on its reduction induced with autoxidation of hydroxyla- mine. The reaction mixture contained, in a total volume of 1.0 ml, 50 mM sodium carbonate, pH 10.2, 0.1 mM EDTA, 0.1 mM hydroxylamine, 0.03% (v/v) Triton X-100, and the indicated concentration of NBT.

HYDROXYLAMINE AUTOXIDATION AND OS- 193

droxylamine. Na.&O, at 5 mM inhibited the rates of NBT reduction and nitrite formation by 50 and 60% respectively, in the case of 24 PM NBT. However, when the concentration of NBT was high enough to saturate (1.2 mM, cf. Fig. 51, where all of the 02- formed during autoxidation of hydroxylamine reduced NBT and did not produce HzOz, the inhibition by Na&O, was not detected. Na,S,O, up to 100 mM did not affect the NBT reduction by 02- generated by the FMN-photochemical sys- tem at pH 7.8 (23). As summarized in Table III, superoxide dismutase inhibited the H,O,-induced reduction of NBT, indi- cating the participation of H,Oz and gen- eration of O,- in the autoxidation of hy- droxylamine.

Mechanism

The reduction of NBT and the inhibition by superoxide dismutase indicate the gen- eration of O,- during autoxidation ofahy- droxylamine. The reaction is a free radical chain reaction initiated by a free metal

TABLE III EFFWTS OF BENZOATE, MANNITOL, FORMATE,

THIOBULFATE, CYANIDE, HYDR~CXN PEROXIDE, AND SUPEROXIDE DISMUTABE ON AU~XIDATION OF

HYDROXYLAMINE” Additions Relative NBT Relative ni-

reduction rat.45 trite forma- tion rate

Control* 100 100 Benzoate, 0.2 mM 100 108 Mannitol, 0.2 mM 100 84 Formate, 0.2 mM 96 94 Na&03

1rnM 86 - 5rnM 50 39

10 rnM 52 HzOz, 0.1 mM 158 178 H,O,, 0.1 mM, + SOD, 18

47 nM SOD, 47 nM 14 88 Anaerobic 0.08 0.04 KCN, 2 mM 95

0 Reaction conditions were the same as in Table I, except for the addition of the compounds indicated and of 0.1 mM EDTA. Anaerobic conditions were obtained by bubbling nitrogen gas through the re- action mixture.

* Control rates were 0.0086 AA,,, Jmin and 20.7 nmol of nitrite formed/20 min.

ion and/or metal complex such as Cu2+ and EDTA-Fe2+ because autoxidation is enhanced. The observations described above are inferred by the following se- quences of reactions.

NH,OH + Me” + NH,0 . + H+ + Me”-’ (a) NH,0 . + 0, + NO- + Ob- + 2H+ (b) Men-1 + O9 + Men + Ox- (c) 20,- + 2H+ + Hz02 + 02 Cd) NH,O. + H,O, + NO- + H+ + H,O (4 2NO- + Op + 2NO,- U-J

A metal (Me”) oxidizes hydroxylamine to the hydroxylamine radical [reaction (a)]. 02- is generated by the univalent reduction of 0, by a hydroxylamine radical [reaction (b)l and by a reduced metal ion [reaction (c)l. O,- dismutates sponta- neously or catalytically with superoxide dismutase [reaction (d)]. H202 participates in the autoxidation [reaction (e)] because the scavenging H202 by thiosulfate in- hibits the autoxidation. O,- formed in re- actions (b) and (c) reduces NBT and this reduction was inhibited by superoxide dis- mutase. However, a minor part of the NBT reduction might be induced by the hydroxylamine radical since about 14% of the reduction is insensitive to superoxide dismutase. At neutral pH, hydroxylamine is oxidized by 02- to nitrite according to reaction (g>, as confirmed by Elstner et al. (10).

NH,OH + 20,- + H+ + NO,- + H,O, + H,O (g)

Superoxide dismutase inhibited the nitrite production by only 12%, indicating that reaction (g) is not a major reaction of autoxidation at pH 10.2. Hughes and Nick- lin have proposed that an intermediate (NO-) is first formed by attack of an oxy- gen atom to hydroxylamine (31). K,[Ni(CN),I, which is known to react with NO- to form K,[Ni(CN),NO] (32), slightly inhibited the autoxidation of hydroxyla- mine, indicating that the species NO- would be formed during the reaction.

Hydroxylamine is a powerful mutagenic and phage inactivating agent that specifi- cally attacks cytosine. Its mechanism has been explained by the addition of NHOH- to the 6-position of the double bond (33). The data mentioned above suggest that the radicals formed during the autoxida-

194 YASUHISA KONO

tion involve these biochemical effects by hydroxylamine.

Assay of Super-oxide Dismutase Activity by Means of Inhibition of NBT Reduction with Hydroxylamine A&oxidation Most measurements of superoxide dis-

mutase activity are based on its ability to inhibit the O,--dependent reaction. When the concentration of a compound (A), which reacts with 02-, is the saturation level to prevent a spontaneous dismuta- tion of 02- in the absence of superoxide dismutase, the following relationship holds (21, 34, 35):

V/v = 1 + k’[SODl,

where v and V are the reaction rates of (A) in the presence and absence of super- oxide dismutase, respectively, and k’ is k,/K, [Al (k, and k, are the second-order rate constants of the reaction between O,- and A and between 02- and superoxide dismutase, respectively). In the present system, (A) is NBT. Figure 5 shows the initial rate of NBT reduction with 0.1 mM hydroxylamine as a function of the concen- tration of NBT. The rate was saturated at 1.2 mM and is 0.0070 min-‘. The inhibition of NBT reduction by superoxide dismutase in Fig. 6 was measured at the saturating level of NBT. The maximum inhibition was 85%. A replot of these data shows that V/v is proportional to the concentra- tion of superoxide dismutase. The enzy- matic unit of superoxide dismutase has generally been defined as the amount of the enzyme required to inhibit the reduc- tion by 50%. In the present system, one unit corresponds to 5.1 nM spinach Cu,Zn- superoxide dismutase, in a toal volume of 1.0 ml. This method is as sensitive as the method based on cytochrome c reduction by xanthine-xanthine oxidase for Cu,Zn- superoxide dismutase (21). Cu,Zn-super- oxide dismutase is efficiently inhibited by cyanide (361, whereas Mn (37)- and Fe (38)-enzymes are unaffected. Thus, cya- nide may be used as a means of distin- guishing between different types of super- oxide dismutase. Potassium cyanide did not afikct the rate of NBT reduction (Table

v-----l mo- o/O-O - / 2 0 s =‘O- / 0’ Q 4

;40- O s / ts ii /fy m: /

:*

rt* ‘I

OO

Oo SOD ‘“(“w l O

IO 20 40 20 S~oxlda tiZLY.d”“,

FIG. 6. Effect of superoxide dismutase on the initial reduction rate of NBT induced with autoxi- dation of hydroxylamine. The reaction mixture con- tained, in a total volume of 1.0 ml, 50 mM sodium carbonate, pH 10.2, 0.1 mM EDTA, 1.2 mM NBT, 0.1 mre hydroxylamine, 0.03% (v/v) Triton X-100, and superoxide dismutase as indicated. V and v are the at&oxidation rates in the absence and presence of superoxide dismutase. The rate in the absence of superoxide dismutase was 0.0070 AA%,, ,,,/min.

III). This method has the advantage of simplicity and has found favor in labora- tories for situations in which multiple as- says must be performed, as in monitoring column eluates for super-oxide dismutase activity.

ACKNOWLEDGMENTS

I would like to thank Professor A. Takagi for hia encouragement during the course of this work. I would also like to express my sincere thanks to Dr. K. Asada of Kyoto University for his valuable discussions and suggestions during the preparation of this manuscript.

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