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
MATERIALS AND METHODS
oxidation of ammonia to nitrite by Nitro-
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
extinction coefticient at 258 nm (c = 9920 M-I *cm-‘)
on the mechanism of the autoxidation (20). Superoxide dismutase was assayed from an
have been reported (64, there has not inhibition of cytochrome c reduction by O,- gener-
been a report on the univalent reduction ated with a xanthine-xanthine oxidase system us-
of oxygen in this process. On the other ing the method of Asada et al. (21). NBT’ was
hand, Elstner et al. (9) and Elstner and 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
until use. Other chemicals were reagent grade.
formation from hydroxylamine by O,-
Glass-distilled water was used throughout.
is not revealed yet. Absorbance measurements were carried out with
O,- has been shown to be generated a Shimazu multipurpose MPSdOL epectrophotome-
during autoxidation of ferredoxin (ll), fla- 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 1 Abbreviations used: NBT, nitroblue tetrazo-
produce 02-, and we confirmed in the lium; EDTA, ethylenediaminetetraacetic acid; SOD,
present paper that this is the case. Using 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 KONO
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, 0 I 3
NeOH :rnM)
hydroxylamine was at&oxidized producing
nitrite linearly at least for 30 min. Under FIG. 2. The initial reduction rate of NBT as a
these conditions the production of nitrite function of hydroxylamine concentration. Hydrox-
is first order with respect to the concentra- ylamine at the indicated concentration was added
tion of hydroxylamine, with a rate con- to 50 mM sodium carbonate, pH 10.2,O.l mM EDTA,
stant of 1.9 ‘X 10e5 s-l. Nitrate was not 24 PM NBT, and 0.03% (v/v) Triton X-100 in a final
volume of 1.0 ml.
detected using the brucine method (22).
The addition of NBT to the above reac- initial reduction rate of NBT (O-30 s) as a
tion mixture induced an increase in ab- function of the concentration of hydroxyl-
sorbance at 560 nm due to the accumula- amine is shown in Fig. 2.
tion of blue formazan (Fig. lc). When As shown in Fig. 1, curves a and b, the
Triton X-100 was added, an early slow reduction of NBT was inhibited by super-
phase in the reduction of NBT disappeared oxide dismutase to the same degree
and the reduction was apparently stimu- whether the enzyme was added during the
lated (Fig. la). The addition of Triton X- reaction or prior to the initiation of reac-
100 for the assay of formazan formation tion. The inhibition was also observed in
has been recommended by Nishikimi (16) the absence of Triton X-100. The results
and brings about the stabilization of colloi- indicate the participation of O,- in the
dal product. In the following experiments,
autoxidation of hydroxylamine. However,
0.03% (v/v) Triton X-100 was added. The
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
AAs6cmm the reactive site of the hydroxylamine
=0.01 molecule. While 0-methylhydroxylamine
1
rz did not reduce NBT and did not produce
nitrite, N-methylhydroxylamine was au-
toxidized at a four times faster rate than
FIG. 1. The time courses of NBT reduction dur- that of the same concentration of nonsub-
ing autoxidation of hydroxylamine and the effects stituted hydroxylamine, in both the ab-
of Triton X-100 and superoxide dismutase. The re- sence and the presence of EDTA, pH 10.2.
action mixture contained, in a total volume of 1.0
ml, 50 mM sodium carbonate, pH 10.2, 24 PM NBT, Effects of Transition Metals, EDTA-Metal
0.1 rnr+rEDTA, and 1 mM hydroxylamine which was Complexes, and EDTA on Autoxidation
added at the arrow point [line (c)l. In lines (a) and of Hydroxylamine
(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 Mn2+ and EDTA-Mn2+ inhibited NBT
nM superoxide dismutase were added, respectively. 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-’ 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 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, FIG. 3. Effect of EDTA on the rate of autoxida-
tion of hydroxylamine and NBT reduction accompa-
nying autoxidation. The reaction mixture con-
TABLE I tained. in a total volume of 1.0 ml, 50 mM sodium
carbonate, pH 10.2, 24 paa NBT, 1 mM hydroxyla-
EFFECTS OF TRANSITION METALS AND EDTA-
METAL COMPLEXES ON AUTO~IDATION OF
mine, 0.03% (v/v) Triton X-100, and the indicated
HYDROXYLAMINE’
concentration of EDTA. NBT reduction was followed
by an absorbance increase at 560 nm. After reaction
Additions Relative NBT Relative ni-
reduction rata trite forma- for 20 min, initiated by the addition of 1 mM hydrox-
tion rate ylamine to the reaction mixture (containing 50 mM
Control” 100 100 sodium carbonate, pH 10.2, and the indicated con-
MnCl*, 100 PM 41 108 centration of EDTA, in a total volume of 1.0 ml),
CUSO,, 1 PM 331 235 nitrite was determined as described by Elstner and
FeSO, Heupel (10). The rates in the absence of EDTA
103 were 0.0064 AA %,,,,,/min and 16.5 nmol of nitrite
1 PM
10 /LM 138 89 formed/20 min.
Fe(N03h
50 p.M 101 102
100 &LLM 113 108 probably due to Triton X-100, no precipi-
EDTA-Mn*+, 100 PM 80 102 tate of metal hydroxides was formed.
EDTA-Cu2+, 10 PM 104 105 EDTA up to 10 PM inhibited rates of
EDTA-Fez+ both NBT reduction and nitrite formation
0.01 /hM 206
0.05 @I 244 by about 50%. However, in the presence of
0.1 PM 336 259 100 GM EDTA both reaction rates were
CuSO,, 1 /LM, + SOD, 89 enhanced about 1.8 times compared with
47 nM those in the absence of EDTA (Fig. 3).
FeSO,, 10 PM, + SOD, 56 These results suggest that the autoxida-
47 nM tion of hydroxylamine is a free radical
EDTA-Fe*+, 0.1 PM, + 123 chain reaction initiated not only by aquo
SOD, 47 nM metal ions but also by EDTA-metal com-
SOD, 47 mu 13 91 plexes and that the EDTA complex is more
a The reaction mixture contained, in a total vol- effective than free metal. The dual role of
ume of 1.0 ml, 50 mM sodium carbonate, pH 10.2, 24 EDTA in the oxidation of sulfite (24) and
PM NBT, 1 mM hydroxylamine, and 0.03% (v/v) epinephrine (13) was discussed by Fridov-
Triton X-100. NBT reduction was followd by an ich et al. They suggested that free metal
absorbance increase at 560 nm. Nitrite was deter- ion probably reacts with oxygen by an
mined calorimetrically, as described in Fig. 3, &er
20-min reaction using a mixture containing 1 mM
inner-sphere mechanism, whereas EDTA-
hydroxylamine and 50 mM sodium carbonate, pH metal complex reacts by an outer-sphere
10.2, in a total volume of 1.0 ml. The reaction mechanism, which generates O,- into the
mixture contained the additions indicated. solution. It seems likely that the mecha-
b Control rates were 0.0064 AA,,, Jmin and 16.5 nism of autoxidation of hydroxylamine is
nmol of nitrite formed/20 min. similar to that of sulfite and epinephrine.
192 YASUHISA KONO

Effect of pH on Autoxidation of Hydrox- not been reported, using interpolation a

ylamine value of 14 is calculated (26). Therefore,
As shown in Fig. 4, the rate of autoxi- under the present conditions, almost all of
dation of hydroxylamine, detected by its the hydroxylamine occurs in a form of
ability to reduce NBT, was sharply de- NH,OH and we cannot account for the
creased with lowering pH and was not increase in autoxidation above pH 8.0 by
observed below pH 8.0. The rate of nitrite a form of hydroxylamine. Superoxide dis-
production also increased above pH 8.0, as mutase inhibited NBT reduction at any
presented in Table II. Hydroxylamine is pH and maximum inhibition was slightly
stable in acid solution, presumably be- increased at low pH. At pH 9.6, a saturat-
cause its protonated form does not react ing concentration of superoxide dismutase
with metal ion. The pK value for the (47 nM) inhibited the reduction by 98%, in
dissociation (NH30H+ C$NH,OH + H+) is contrast to 85% at pH 10.2.
5.9 (25). Although the pK value for the Participation of H,O, in the Autoxidu-
dissociation (NH,OH e NH,O- + H+) has 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
PH
assure the involvement of H,O, in the
FIG. 4. Effect of pH on the initial rate of NBT autoxidation, the effect of Na,S,O,, which
reduction. The reaction mixture consisted of 24 PM decomposes H202, was examined because,
NBT, 0.1 mre EDTA, 1 mM hydroxylamine, and 0.03%
unfortunately, catalase is inhibited by hy-
(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 OFpH 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 NBT (mM)
Tris-HCl, pH 9.2 1.5
FIG. 5. Effect of the concentration of NBT on its
Tris-HCl, pH 9.6 2.0
reduction induced with autoxidation of hydroxyla-
Sodium carbonate, pH 9.6 1.9
mine. The reaction mixture contained, in a total
Sodium carbonate, pH 10.2 25.5
volume of 1.0 ml, 50 mM sodium carbonate, pH
D The reaction mixture contained, in a total vol- 10.2, 0.1 mM EDTA, 0.1 mM hydroxylamine, 0.03%
ume of 1.0 ml, 1 mM hydroxylamine and 0.1 rnre (v/v) Triton X-100, and the indicated concentration
EDTA in the indicated buffered solutions. of NBT.
 HYDROXYLAMINE AUTOXIDATION AND OS- 193
droxylamine. Na.&O, at 5 mM inhibited ion and/or metal complex such as Cu2+
the rates of NBT reduction and nitrite and EDTA-Fe2+ because autoxidation is
formation by 50 and 60% respectively, in enhanced. The observations described
the case of 24 PM NBT. However, when above are inferred by the following se-
the concentration of NBT was high enough quences of reactions.
to saturate (1.2 mM, cf. Fig. 51, where all NH,OH + Me” + NH,0 . + H+ + Me”-’ (a)
of the 02- formed during autoxidation of NH,0 . + 0, + NO- + Ob- + 2H+ (b)
hydroxylamine reduced NBT and did not Men-1 + O9 + Men + Ox- (c)
produce HzOz, the inhibition by Na&O, 20,- + 2H+ + Hz02 + 02 Cd)
was not detected. Na,S,O, up to 100 mM NH,O. + H,O, + NO- + H+ + H,O (4
did not affect the NBT reduction by 02- 2NO- + Op + 2NO,- U-J
generated by the FMN-photochemical sys- A metal (Me”) oxidizes hydroxylamine
tem at pH 7.8 (23). As summarized in to the hydroxylamine radical [reaction
Table III, superoxide dismutase inhibited
the H,O,-induced reduction of NBT, indi- (a)]. 02- is generated by the univalent
cating the participation of H,Oz and gen- reduction of 0, by a hydroxylamine radical
eration of O,- in the autoxidation of hy- [reaction (b)l and by a reduced metal ion
droxylamine. [reaction (c)l. O,- dismutates sponta-
neously or catalytically with superoxide
Mechanism dismutase [reaction (d)]. H202 participates
in the autoxidation [reaction (e)] because
The reduction of NBT and the inhibition the scavenging H202 by thiosulfate in-
by superoxide dismutase indicate the gen- hibits the autoxidation. O,- formed in re-
eration of O,- during autoxidation ofahy- actions (b) and (c) reduces NBT and this
droxylamine. The reaction is a free radical reduction was inhibited by superoxide dis-
chain reaction initiated by a free metal mutase. However, a minor part of the
NBT reduction might be induced by the
TABLE III hydroxylamine radical since about 14% of
EFFWTS OF BENZOATE,MANNITOL, FORMATE, the reduction is insensitive to superoxide
THIOBULFATE, CYANIDE, HYDR~CXN PEROXIDE, AND dismutase. At neutral pH, hydroxylamine
SUPEROXIDEDISMUTABEON AU~XIDATION OF is oxidized by 02- to nitrite according to
HYDROXYLAMINE” reaction (g>, as confirmed by Elstner et al.
Additions Relative NBT Relative ni- (10).
reduction rat.45 trite forma-
tion rate
NH,OH + 20,- + H+ + NO,- + H,O, + H,O (g)
Control* 100 100
Benzoate, 0.2 mM 100 108 Superoxide dismutase inhibited the nitrite
Mannitol, 0.2 mM 100 84 production by only 12%, indicating that
Formate, 0.2 mM 96 94 reaction (g) is not a major reaction of
Na&03 autoxidation at pH 10.2. Hughes and Nick-
1rnM 86 - lin have proposed that an intermediate
5rnM 50 39 (NO-) is first formed by attack of an oxy-
10 rnM 52 gen atom to hydroxylamine (31).
HzOz, 0.1 mM 158 178 K,[Ni(CN),I, which is known to react with
H,O,, 0.1 mM, + SOD, 18 NO- to form K,[Ni(CN),NO] (32), slightly
47 nM
SOD, 47 nM 14 88
inhibited the autoxidation of hydroxyla-
Anaerobic 0.08 0.04 mine, indicating that the species NO-
KCN, 2 mM 95 would be formed during the reaction.
Hydroxylamine is a powerful mutagenic
0 Reaction conditions were the same as in Table
I, except for the addition of the compounds indicated
and phage inactivating agent that specifi-
and of 0.1 mM EDTA. Anaerobic conditions were cally attacks cytosine. Its mechanism has
obtained by bubbling nitrogen gas through the re- been explained by the addition of NHOH-
action mixture. to the 6-position of the double bond (33).
* Control rates were 0.0086 AA,,, Jmin and 20.7 The data mentioned above suggest that
nmol of nitrite formed/20 min. the radicals formed during the autoxida-
194 YASUHISA KONO

mo- o/O-O
-
tion involve these biochemical effects by
hydroxylamine. v-----l
Assay of Super-oxide Dismutase Activity
by Means of Inhibition of NBT
Reduction with Hydroxylamine
A&oxidation 2Q /
0
=‘O- s
/ 0’ 4
Most measurements of superoxide dis- ;40- O s /

/fym:
mutase activity are based on its ability to ts
:*
inhibit the O,--dependent reaction. When ii /
‘I
the concentration of a compound (A), rt*
which reacts with 02-, is the saturation OO
SOD ‘“(“w lO
level to prevent a spontaneous dismuta- Oo IO 20 40 20
tion of 02- in the absence of superoxide S~oxlda tiZLY.d”“,
dismutase, the following relationship FIG. 6. Effect of superoxide dismutase on the
holds (21, 34, 35): initial reduction rate of NBT induced with autoxi-
dation of hydroxylamine. The reaction mixture con-
V/v = 1 + k’[SODl, tained, in a total volume of 1.0 ml, 50 mM sodium
where v and V are the reaction rates of carbonate, pH 10.2, 0.1 mM EDTA, 1.2 mM NBT,
(A) in the presence and absence of super- 0.1 mre hydroxylamine, 0.03% (v/v) Triton X-100,
and superoxide dismutase as indicated. V and v are
oxide dismutase, respectively, and k’ is
the at&oxidation rates in the absence and presence
k,/K, [Al (k, and k, are the second-order of superoxide dismutase. The rate in the absence of
rate constants of the reaction between O,- superoxide dismutase was 0.0070 AA%,,,,,/min.
and A and between 02- and superoxide
dismutase, respectively). In the present
system, (A) is NBT. Figure 5 shows the III). This method has the advantage of
initial rate of NBT reduction with 0.1 mM simplicity and has found favor in labora-
hydroxylamine as a function of the concen- tories for situations in which multiple as-
tration of NBT. The rate was saturated at says must be performed, as in monitoring
1.2 mM and is 0.0070 min-‘. The inhibition column eluates for super-oxide dismutase
of NBT reduction by superoxide dismutase activity.
in Fig. 6 was measured at the saturating
ACKNOWLEDGMENTS
level of NBT. The maximum inhibition
was 85%. A replot of these data shows I would like to thank Professor A. Takagi for hia
that V/v is proportional to the concentra- encouragement during the course of this work. I
would also like to express my sincere thanks to Dr.
tion of superoxide dismutase. The enzy- K. Asada of Kyoto University for his valuable
matic unit of superoxide dismutase has discussions and suggestions during the preparation
generally been defined as the amount of of this manuscript.
the enzyme required to inhibit the reduc-
tion by 50%. In the present system, one REFERENCES
unit corresponds to 5.1 nM spinach Cu,Zn- 1. KONO, M., AND TANIGUCHI, S. (1960) Bbchim.
superoxide dismutase, in a toal volume of Biophys. Actu 43, 419-430.
1.0 ml. This method is as sensitive as the 2. WALKER, G. C., AND NICHOLAS, D. J. D. (1961)
method based on cytochrome c reduction B&him. Bbphys. Acta 49, 361-368.
by xanthine-xanthine oxidase for Cu,Zn- 3. HOFMAN, T., AND LEE, H. (1953) B&hem. J.
superoxide dismutase (21). Cu,Zn-super- 54, 579-563.
oxide dismutase is efficiently inhibited by 4. FALCONE, A. B., SHUG, A. L., AND NICHOLM,
D. J. D. (1963) B&him. Biophys. Actu 77,
cyanide (361, whereas Mn (37)- and Fe 199-208.
(38)-enzymes are unaffected. Thus, cya- 5. ANDERBON, J. H. (1964)B&hem. J. 91, 6-17.
nide may be used as a means of distin- 6. MOEWE, P. C., AND AUDRIETH, L. F. (1959) J.
guishing between different types of super- Inorg. Nucl. Chem. 11, 242-246.
oxide dismutase. Potassium cyanide did 7. ANDEIWON, J. H. (1964) Analyst 89, 357-362.
not afikct the rate of NBT reduction (Table a. HUGHES, M. N., AND NICKLIN, H. G. (1970) J.
 HYDROXYLAMINE AUTOXIDATION AND O,- 195
Ckm. Sot. A, 925-928. 24. MCCORD, J. M., AND FIUDOVICH, I. (1969) J.
9. EL~TNER, E. F., S~o~wa, C., AND HEUPEL, A. BioZ. Ckm. 244, 6056-6063.
(1975) 2. Naturforsch. 3Oc, 53-56. 25. ERLENMEYEB, H., FLIEBL, C., AND SIGEL, H.
10. EL~TNE~, E. F., AND HEUPEL, A. (1976) Anal. (1968) Chimiu 22, 433-434.
Biockm. 70, 616-620. 26. CWPP, L. B. (1967) in The Chemistry of the OH
11. MICRA, H. I?., AND FRIDOVICH, I. (1971) J. Bid. Group, p. 43, Prentice-Hall, Englewood
Ckm. 246, 6886-6890. Cliffs, New Jersey.
12. Mrsar, H. P., AND FBIDOVICH, I. (1972) J. Btil. 27. NETA, P., AND DOWMAN, L. M. (1968) Advan.
Ckm. 247, 188-192. Ckm. Ser. 81, 222-230.
13. MIS~A, H. P., AND FRIDOVICH, I. (1972) J. BioZ. 28.. MATHEBON, M. S., MULAC, W. A., WEEKS, J. L.,
Chem. 247, 3170-3175. AND RABANI, J. (1966) J. Phys. Ckm. 70,
14. MILLER, R. W., AND RUP, u. (1973) J. Biol. 2092-2099.
Ckm. 248, 6084-6090. 29. SCHWAZ,H. A. (1964) Radiat. Res. (Suppl.) 4,
15. COHEN, G., AND HEIKKILA, R. E. (1974) J. Bb!. 89-113.
Chem. 249, 2447-2452. 30. HARBER, F., AND WEISS, J. (1934) Proc. Roy.
16. NIBHIKIMI, M. (1975) A&. B&km. Biophys. Sot. Ser. A. 147, 332-351.
166, 273-279. 31. HUGHES, M. N., AND NICKLIN, H. G. (1968) J.
17. MISRA, H. P. (1974) J. Biol. Chem. 249, 2151- Chem. Sot. A, 450-452.
2155. 32. NAST, R., NYAL, K., AND GI~IZIWOK, E. (1952)Z.
18. MARKLUND, S., AND MABKLIJND, G. (1974) Eur. Anorg. AZZg. Ckm. 267, 304-314.
J. Biockm. 47, 469-474. 33. LAWLEY, P. D. (1967) J. Mol. BioZ. 24, 75-81.
19. MIEIU, H. P., AND FRIDOVICH, I. (1976) Biockm- 34. NIBHIKIMI, M. (1975) B&km. Biophys. Res.
ktry 15, 681-687. Commun. 63, 463-468.
20. ASADA, K., URANO, M., AND TAKAHA~HI, M. 35. SAWADA, Y., AND YAMAZAKI, I. (1973) B&him.
(1973) Eur. J. B&hem. 36, 257-266. Biophys. Acta 327, 257-265.
21. A~ADA, K., TAKAHABHI, M., AND NACATE, M. 36. ROTILIO, G., BRAY, R. C., AND FIELDEN, E. M.
(1974) Agric. Biol. Chem. 38, 471-473. (1972) B&him. Biophys. Acta 268.605-609.
22. ENDRES, G., AND KAUFMAN, L. (1937) Liebigs 37. WEISIGER, R. A., AND FIIIDOVICH, I. (1973) J.
Ann. 530, 184-194. BioZ. Ckm. 248, 3582-3592.
23. KONO, Y., TAKAHASHI, M., AND ASADA, K. (1976) 38. YOST, F. J., AND FIIIDOVICH, I. (1974) Arch.
Arch. Biockm. Biophys. 174, 454-462. Biockm. Biophys. 161, 395-401.