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 participation 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 the reduction of nitrate to ammonia by halotolerant bacteria (1, 2) and in the oxidation of ammonia to nitrite by Nitrosomonus (3-5). Hydroxylamine is autoxidized rapidly in the presence of trace metals at high pH. Although extensive studies on the mechanism of the autoxidation have been reported (64, there has not been a report on the univalent reduction of oxygen in this process. On the other hand, Elstner et al. (9) and Elstner and Heupel (10) have reported that at neutral pH hydroxylamine is oxidized by superoxide radical (0,-j to nitrite whose production is inhibited by superoxide dismutase. However, the detailed mechanism of nitrite formation from hydroxylamine by O,is not revealed yet. O,- has been shown to be generated during autoxidation of ferredoxin (ll), flavins and quinones (12), epinephrine (13), tiron (14), dopamines (15), tetrahydropteridines (16), thiols (17), pyrogallol (18), and phenylhydrazine (19). Therefore, the autoxidation of hydroxylamine would also produce 02-, and we confirmed in the present paper that this is the case. Using the formation of 02- during the autoxida-

tion of hydroxylamine, an assay procedure for superoxide dismutase is also described.

MATERIALS AND METHODS Cu,Zn-superoxide dismutase from spinach leaves was a generous gift of Dr. Asada, Kyoto University. The enzyme concentration was determined from an extinction coefticient at 258 nm (c = 9920 M-I *cm-) (20). Superoxide dismutase was assayed from an inhibition of cytochrome c reduction by O,- generated with a xanthine-xanthine oxidase system using the method of Asada et al. (21). NBT was obtained from Sigma. Hydroxylamine hydrochloride and Triton X-100 were obtained from Nakarai Chemical Co. (Kyoto, Japan). An aqueous solution of hydroxylamine was prepared daily and its pH was adjusted to 6.0 with 2 M sodium hydroxide. The solution was kept in a tightly stoppered test tube until use. Other chemicals were reagent grade. Glass-distilled water was used throughout. Absorbance measurements were carried out with a Shimazu multipurpose MPSdOL epectrophotometer at 25C. The reaction was initiated by the addition of hydroxylamine to the reaction mixture and the reduction of NBT was followed by an absorbance increase at 560 nm under aerobic conditions. Nitrite was determined calorimetrically by the diaxo-cou1 Abbreviations used: NBT, nitroblue tetrazolium; EDTA, ethylenediaminetetraacetic acid; SOD, superoxide dismutase; Me, transition metal; FMN, flavin mononucleotide. 189 0003-9861/78/1861-0189802.00/O Copyright Q 1978 by Academic Press, All rights of reproduction in any form
Inc. reserved.

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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 concentration of hydroxylamine, with a rate constant of 1.9 X 10e5 s-l. Nitrate was not detected using the brucine method (22). The addition of NBT to the above reaction mixture induced an increase in absorbance at 560 nm due to the accumulation 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 stimulated (Fig. la). The addition of Triton X100 for the assay of formazan formation has been recommended by Nishikimi (16) and brings about the stabilization of colloidal product. In the following experiments, 0.03% (v/v) Triton X-100 was added. The

NeOH :rnM)

FIG. 2. The initial reduction rate of NBT as a function of hydroxylamine concentration. Hydroxylamine 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.

AAs6cmm =0.01 1 rz

FIG. 1. The time courses of NBT reduction during autoxidation of hydroxylamine and the effects of Triton X-100 and superoxide dismutase. The reaction mixture contained, in a total volume of 1.0 ml, 50 mM sodium carbonate, pH 10.2, 24 PM NBT, 0.1 rnr+rEDTA, 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.

initial reduction rate of NBT (O-30 s) as a function of the concentration of hydroxylamine is shown in Fig. 2. As shown in Fig. 1, curves a and b, the reduction of NBT was inhibited by superoxide dismutase to the same degree whether the enzyme was added during the reaction or prior to the initiation of reaction. 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 superoxide 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 induced by hydroxylamine, but by 02-. Two substituted hydroxylamines, Omethylhydroxylamine and N-methylhydroxylamine, 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 autoxidized at a four times faster rate than that of the same concentration of nonsubstituted hydroxylamine, in both the absence 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 formation (Table I). As previously reported, Mn2+-pyrophosphate is oxidized by 02- at a second-order rate of 6 x log M- 6s-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 EDTACu2+ did not significantly affect the production of nitrite and NBT reduction, EDTA-Fe2+ and Cu2+ stimulated both reactions to a great extent, as summarized in Table I. Under the present conditions,
TABLE I
EFFECTS OF TRANSITION METALS AND METAL COMPLEXES ON AUTO~IDATION HYDROXYLAMINE

EDTAOF

Additions Control MnCl*, 100 PM

CUSO,, 1 PM

Relative NBT Relative nireduction rata trite formation rate 100 41 331 103 138 101 113 80 104 206 244 336 89 56 123 13 91 100 108 235 89 102 108 102 105

FeSO, 1 PM 10 /LM
Fe(N03h

FIG. 3. Effect of EDTA on the rate of autoxidation of hydroxylamine and NBT reduction accompanying autoxidation. The reaction mixture contained. in a total volume of 1.0 ml, 50 mM sodium carbonate, pH 10.2, 24 paa NBT, 1 mM hydroxylamine, 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 hydroxylamine to the reaction mixture (containing 50 mM sodium carbonate, pH 10.2, and the indicated concentration 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.

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

259

a The reaction mixture contained, in a total volume 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 determined 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.

probably due to Triton X-100, no precipitate 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 autoxidation of hydroxylamine is a free radical chain reaction initiated not only by aquo metal ions but also by EDTA-metal complexes 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 Fridovich et al. They suggested that free metal ion probably reacts with oxygen by an inner-sphere mechanism, whereas EDTAmetal complex reacts by an outer-sphere mechanism, which generates O,- into the solution. It seems likely that the mechanism of autoxidation of hydroxylamine is similar to that of sulfite and epinephrine.

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Effect of pH on Autoxidation of Hydroxylamine As shown in Fig. 4, the rate of autoxidation of hydroxylamine, detected by its ability to reduce NBT, was sharply decreased 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 because 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

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 dismutase inhibited NBT reduction at any pH and maximum inhibition was slightly increased at low pH. At pH 9.6, a saturating 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 Autoxidution of Hydroxylamine The addition of 0.1 mM H,O, augmented 1.6 and 1.8 times the rates of NBT reduction and nitrite production, respectively. H202, per se, did not reduce NBT. This result suggests that H,Oz formed through spontaneous disproportionation of 02- oxidizes hydroxylamine. The addition of benzoate, mannitol, and formate, scavengers of -OH (27-291, did not afkt the autoxidation, as summarized in Table III, indicating 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-

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 pH ON AUTOXIDATION OF HYDROXYLAMINIP Nitrite formation (nmoU20 min) pH 7.4 pH 7.8 0.4 0.4 1.0 1.5 2.0 1.9 25.5

EFFECT

OF

Buffers (50 mhi) Potassium phosphate, Potassium phosphate, Tris-HCl, pH 8.4 Tris-HCl, pH 9.2 Tris-HCl, pH 9.6 Sodium carbonate, pH Sodium carbonate, pH

NBT

(mM)

9.6 10.2

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

FIG. 5. Effect of the concentration of NBT on its reduction induced with autoxidation of hydroxylamine. 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 02generated by the FMN-photochemical system at pH 7.8 (23). As summarized in Table III, superoxide dismutase inhibited the H,O,-induced reduction of NBT, indicating the participation of H,Oz and generation of O,- in the autoxidation of hydroxylamine.
Mechanism

ion and/or metal complex such as Cu2+ and EDTA-Fe2+ because autoxidation is enhanced. The observations described above are inferred by the following sequences of reactions. NH,OH + Me + NH,0 . + H+ + Me- (a)
NH,0 . + 0, Men-1 + O9 20,- + 2H+ NH,O. + H,O, 2NO- + Op NO- + Ob- + 2H+ Men + OxHz02 + 02 NO- + H+ + H,O + 2NO,+ + + + (b) (c) Cd) (4
U-J

The reduction of NBT and the inhibition by superoxide dismutase indicate the generation of O,- during autoxidation ofahydroxylamine. 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
OF

SUPEROXIDE DISMUTABEON AU~XIDATION HYDROXYLAMINE Additions Relative NBT Relative

reduction rat.45

nitrite formation rate

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 spontaneously or catalytically with superoxide dismutase [reaction (d)]. H202 participates in the autoxidation [reaction (e)] because the scavenging H202 by thiosulfate inhibits the autoxidation. O,- formed in reactions (b) and (c) reduces NBT and this reduction was inhibited by superoxide dismutase. 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)

Control* Benzoate, 0.2 mM Mannitol, 0.2 mM Formate, 0.2 mM Na&03 1rnM 5rnM 10 rnM HzOz, 0.1 mM H,O,, 0.1 mM, + SOD, 47 nM SOD, 47 nM Anaerobic KCN, 2 mM

100 100 100 96 86 50 52 158 18 14 0.08 95

100 108 84 94 39 178 88 0.04

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

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 Nicklin have proposed that an intermediate (NO-) is first formed by attack of an oxy(31). gen atom to hydroxylamine K,[Ni(CN),I, which is known to react with NO- to form K,[Ni(CN),NO] (32), slightly inhibited the autoxidation of hydroxylamine, indicating that the species NOwould be formed during the reaction. Hydroxylamine is a powerful mutagenic and phage inactivating agent that specifically attacks cytosine. Its mechanism has been explained by the addition of NHOHto the 6-position of the double bond (33). The data mentioned above suggest that the radicals formed during the autoxida-

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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 dismutase 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 dismutation 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 superoxide 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 concentration 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 concentration of superoxide dismutase. The enzymatic unit of superoxide dismutase has generally been defined as the amount of the enzyme required to inhibit the reduction by 50%. In the present system, one unit corresponds to 5.1 nM spinach Cu,Znsuperoxide 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,Znsuperoxide dismutase (21). Cu,Zn-superoxide dismutase is efficiently inhibited by cyanide (361, whereas Mn (37)- and Fe (38)-enzymes are unaffected. Thus, cyanide may be used as a means of distinguishing between different types of superoxide dismutase. Potassium cyanide did not afikct the rate of NBT reduction (Table

;40ts ii rt*

mo- o/O-O / 2 /0s 0 Q =O/fym:

v-----l
O :* I 4 s / / OO SOD (w Oo IO 20 S~oxlda lO 40 20 tiZLY.d,

FIG. 6. Effect of superoxide dismutase on the initial reduction rate of NBT induced with autoxidation of hydroxylamine. The reaction mixture contained, 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 laboratories for situations in which multiple assays 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. REFERENCES 1. KONO, M., AND TANIGUCHI, S. (1960) Bbchim.
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AUTOXIDATION

AND

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