1. Art and Diseño

Eur. J. Biochem. 194,713-720 (1990)
FEBS 1990
e
Human liver manganese superoxide dismutase
Purification and crystallization, subunit association and sulfhydryl reactivity
',
Yukihiko MATSUDA '%',
Shigeki HIGASHIYAMA', Yoshiyuki KIJIMA Keiichiro SUZUKI ', Kiyoshi KAWAN04,
Morio AKIYAMA5. Sumio KAWATA', Seiichiro TARUI *, Harold F. DEUTSCH ' and Naoyuki TANIGUCHI'
Department of Biochemistry, Osaka University Medical School, Japan
Second Department and First Department of Internal Medicine, Osaka University Medical School, Japan
Pathology Section, Osaka Rosai Hospital, Japan
Department of Physics, Sapporo Medical College, Japan
(Received July 27, 1990) - EJB 90 0906
Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver
(2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic
form.
Four of the eight cysteine residues in the tetramer reacted with 5,5'dithiobis(2-nitrobenzoic acid) or with
iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol
or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence
analysis of Nbs,-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of
Mn-SOD in 1O h SDS for 2 or 3 days at 25 "C or 5 min at 100"C gave material showing two prominent components
on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass
of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS
produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in
which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that
Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.
Superoxide dismutases (SOD) are found in all aerobic
organisms but not in obligate anaerobes [1, 21. The enzyme
catalyzes the dismutation of superoxide anion into molecular
oxygen and hydrogen peroxide [3]. The physiological function
of the enzyme appears to protect cells against reactive free
radicals by scavenging superoxide anions produced under oxidative conditions [I -31.
In mammalian tissue, three SOD isozymes are found: Cu,
Zn-SOD is predominantly located in the cytosol[4 - 71, extracellular SOD exists in the extracellular fluids [S] and Mn-SOD
appears to be localized mostly in the mitochondria1 matrix [9].
Cytosolic Cu,Zn-SOD is a well-characterized protein containing two identical subunits, each having an intrachain disulfide
and two sulfhydryls [lo, 111. The complete amino acids sequence of human Mn-SOD has been determined by Barra et
al. [12] and, except for minor differences, is in good agreement
with the structure deduced from its cDNA [13, 141. All MnSOD appear to be composed of four identical subunits [6,15,
161, although a recent study of a recombinant form suggests
a dimeric enzyme [17]. The gross structural properties of such
Correspondence to N. Taniguchi, Department of Biochemistry,
Osaka University Medical School, 2-2 Yamadaoka, Suita, Japan 565
Abbreviutions. SOD, superoxide dismutase; Nbs2, 5,5'-dithiobis(2-nitrobenzoic acid); LALLS, low-angle laser light scattering;
HPGC, high-performance gel chromatography.
Enzymes. Superoxide dismutase (EC 1.15.1.1); xanthine oxidase (EC 1.2.3.22); carboxypeptidase A (EC 3.4.12.2); carboxypeptidase B (EC 3.4.12.3); carboxypeptidase Y (EC 3.4.16.1); pepsin (EC
3.4.23.1); phosphorylase (EC 2.4.1.1); carbonic anhydrase (EC
4.2.1.1); ribonuclcase (EC 3.1.27.5).
proteins have received little study to date, although preliminary crystallographic study was performed using recombinant
Mn-SOD [18].
The present investigation details sulfhydryl reactivities and
the subunit association of human Mn-SOD.
EXPERIMENTAL PROCEDURES
Chemicals
Nitrobenzene tetrazolium, 4-amidinophenylmethylsulfonyl fluoride, guanidinium chloride, SDS and iodoacetamide
were purchased from Wako Chemicals, dithiothreitol, Nethylmaleimide 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2;
Ellman's reagent), urea and xanthine from Nacalai Tesque,
xanthine oxidase and carboxypeptidases A, B and Y from
Boehringer Mannheim. Pepsin and oxidized glutathione was
purchased from Sigma. DEAE-cellulose (DE-52) was obtained from Whatman Ltd, hydroxylapatite from Wako
Chemicals; Sephadex G-25 (PD-10 column), Sephacryl S-300,
Red Sepharose, Polybuffer Exchanger 94, activated thiolSepharose 4B and Polybuffer 96 from Pharmacia LKB.
Enzyme activity assay
Enzyme assays were carried out according to Beauchamp
and Fridovich [19]. Activity of Mn-SOD incubated with or
without denaturing agent and/or reducing agent was assayed
by a xanthinelxanthine oxidase/nitrobenzene tetrazolium system in sodium carbonate buffer at pH 10.2. Protein concentrations of samples were measured by the method of Bradford
[20] using bovine serum albumin as the standard.
714
Metal and EPR spectrometer analysis
The purified enzyme was extensively dialysed against
metal-free water (Milli Q"' quality) and subjected to the
analyzer. In order to determine manganese contents of the
protein, 10 pl of the enzyme (36 pg/ml) was subjected to a
polarized Zeeman atomic absorption spectrophotometer
(Hitachi, Z-8000). To identify Mn(I1) or Mn(II1) in the native
form, 1 ml enzyme (27 mg/ml) was employed in the EPR
spectrometer system (Varian, Type E-12).
Crystallization of Mn-SOD
Purified Mn-SOD (10 mg/ml in pH 8.2, 50 mM Tris/HCl
buffer) was crystallized by dialysis against 2.8 M ammonium
sulfate solution containing pH 8.2, ?ris/HCl buffer at 4°C.
A preliminary crystallographic study On Mn-SoD
be
published elsewhere.
Stability of Mn-SOD
To investigate the pH stability of Mn-SOD, the purified
enzyme (0.24 mg/ml) was incubated for up to 3 days at 4°C
in various buffers at pH 5.5-9.3. Buffers used were 0.1 M
sodium acetate, pH 5.5, sodium phosphate, pH 7.2, Tris/HCl,
pH 8.0, or monoethanolamine acetate, pH 9.3. Aliquots of
the mixture at appropriate intervals were removed and subjected to enzyme assay. This experiment was carried out in
duplicate.
To clarify stability of activity and subunit association of
Mn-SOD in denaturing solutions, the enzyme (0.24 mg/ml)
was incubated at 25°C in pH 8.0, 0.1 M Tris/HCl and 1 mM
EDTA in the presence of 1% SDS, 8 M urea or 6 M guanidinium chloride. The mixtures were adjusted to pH 8.0, if
necessary, with 0.1 M HC1. At appropriate time intervals,
aliquots were removed for enzyme assay and subjected to
SDS/PAGE.
SDS/ PAGE
Discontinuous polyacrylamide gel electrophoresis employing a SDS/Tris/glycine buffer was carried out according
to Laemmli et al. [21]. An aliquot of the non-heated protein
was mixed with an equal volume of the sample buffer containing 1% SDS and 50% glycerol, and left at room temperature
for 20 min prior to electrophoresis. The heated samples with
or without 5 mM 2-mercaptoethanol were cooled, mixed with
the sample buffer then electrophoresed. The Pharmacia molecular mass calibration kit was employed.
Low-angle laser light scattering (LALLS)
Molecular masses of the purified Mn-SOD and various
fractions were also determined by a LALLS technique in
combination with high-performance gel chromatography
(HPGC/LALLS) [22]. The 0.05 M sodium phosphate/O.l M
NaCl buffer, pH 7.5, employed was first passed through a
0.2-pm pore ultrafilter then through a degasser (ERC-3312,
Erma Optical Works). The buffer solution was supplied to
the 7.8 mm x 300 mm TSK-GEL G3000 SW-XL column
(TOSOH) at a constant flow rate of 0.5 ml/min. The column
temperature was maintained at 20°C by a thermostated water
supply (BL-51, Yamato Science Co. Ltd). Elution from the
column was monitored by three types of detectors connected
in series: an ultraviolet spectrophotometer (TOSOH, UV-
8000), a precision differential refractometer (TOSOH, RI8000) and a LALLS photometer (TOSOH, LS-8000). Bovine
serum albumin (66267 Da, E&'
= 0.670 mgiml), ovalbumin
(42750 Da, 0.735 mg/ml) and ribonuclease A (13700 Da,
0.706 mg/ml) standards were assayed three times. Native
Mn-SOD was diluted in the elution buffer to give concentrations of 0.243,0.486,1.217 and 2.434 mg/ml. 100 pl of each
enzyme sample or of the standard sample were employed. The
peak heights of the elution profile of the chromatogram were
used in the calculation of molecular masses and molecular
absorption coefficients of Mn-SOD by a standard method
1221.
ThioI grouv titrations
-
&
Thiol titrations with Nbs2 were carried out essentially by
the method ofEllman [23]. Mn-SOD (1 mg/ml) in 0.1 M Tris/
HCl buffer, pH 8.0, containing 1 mM EDTA was incubated
for 1 h at 25% or 5 min at 106°C in the presence or absence
of 1% SDS or 8 M urea. The samples were then mixed with
an equal volume of 10 mM Nbs2, kept at 25°C for 20 min,
and the increase in A412 was compared with that of a control
lacking Mn-SOD. A molar absorption coefficient of 14200
M- cm- for the 5-mercapto-2-nitrobenzoate anion was
used. To block free thiol groups of Mn-SOD, 0.75 mg/ml
native enzyme was incubated for 12 h in the dark at 37°C in
pH 8.0, 0.1 M Tris/HCl containing 50 mM iodoacetamide.
After desalting on a column of Sephadex G-25 (Pharmacia/
LKB, PD-10 column), the alkylated Mn-SOD was subjected
to thiol titration and SDS/PAGE. The modified enzyme was
heated in the presence of 8 M urea prior to electrophoresis.
'
Identification
of
a reactive sulfiydryl group
In order to identify the reactive site to sulfhydryl groups,
human liver Mn-SOD (100 nmol) was dissolved in buffer A
(0.2 M N-ethylmorpholine, pH 8.0 and 1 mM EDTA) containing 5 mM Nbs2 and incubated for 30 min at 25°C. The
mixture was then loaded on to a commercial column of
Sephadex G-25 (Pharmacia/LKB, PD-10) to remove low-molecular-mass reactants from the protein. The enzyme solution
obtained was then mixed with a 2-ml slurry of activated thiolSepharose 4B. After incubation at 37 "C for 30 min, the mixture was poured into a small column. Approximately 75% of
the Mn-SOD added was bound. After extensive washing of the
column with buffer B (formic acid/acetic acid/water, 1 :4: 45,
pH 1.9), the column contents were removed, suspended in the
2 ml of the above buffer B and digested with 80 pg pepsin for
12 h at 37°C. The level of pepsin used is approximately 0.8%
of the amount of Mn-SOD bound to the resin. The digest was
then poured into the original column, and washed successively
with 10 ml each of buffer B, buffer A containing 1 M NaC1,
and buffer A. The peptides bound to the column were then
eluted with buffer A containing 20 mM 2-mercaptoethanol.
The eluted peptide material was subjected to gas-phase amino
acid sequence analysis and the resulting amino acids labeled
with phenylhydantoin were analyzed by HPLC (Applied Biosystems, Model 120 A).
RESULTS
Isolation of Mn-SOD
Since liver and other tissues contain various forms of SOD,
the precise calculation of yields of Mn-SOD from the starting
715
Tdblc 1. Purificution table o f Mn-SOD
Step
Supernatant
DEAE-Cellulose
H ydroxylapatite
Sephacryl S-300
Chromatofocusing
2nd hydroxylapatite
Red Sepharose
Volume
Total protein
ml
9 000
20000
21 0
240
165
182
6.6
Total
activity
Specific
activity
Increase in
specific activity
mg
lo-" x units
units/mg
-fold
289000
67 700
27.2
8.93
-
-
-
-
5230
3 290
1280
454
320
1.91
2.49
2.10
2.33
1.96
369
757
1640
51 33
6100
1
2.1
4.5
14.0
16.6
extracts based on activity is not possible, even though the Cu,
Zn-SOD is largely inhibited by cyanide. For this reason, the
fractionation steps will be described without presentation of
activity/protein relationships for the crude extract and the
DEAE-cellulose chromatography eluent. After the hydroxylapatite chromatographic step, fractions were found to be free
from cyanide-sensitive proteins that affect the enzyme assay,
therefore the calculations were possible.
The following fractionation procedure was employed. Human normal liver was obtained at autopsy. Autopsy and research procedures were in accord with the ethical standards
of the Helsinki Declaration of 1975. The frozen liver (2 kg)
was thawed and homogenized with 4 vol. cold 10 mM sodium
phosphate buffer, pH 7.8, containing 1 mM benzamidine and
10 pM 4-amidinophenylmethylsulfonyl fluoride to inhibit
proteases. All purification procedures were performed at
about 4°C unless otherwise indicated.
The homogenate was centrifuged at 55000 x g for 60 min
and 9 1 supernatant were chromatographed by DE-52. 20 1
flow-through active fractions were applied directly on to a
hydroxylapatite column. Following washing of the column
with 100 mM sodium phosphate, pH 7.2, the enzyme was
eluted at about 25°C with 300 mM sodiumphosphate, pH 7.2
(Fig. IA). The active fractions were pooled and dialyzed
against 25 mM monoethanolamine acetate, pH 9.4, then concentrated to 50 ml with an Amicon PM-10 membrane. This
material was subjected to gel-exclusion chromatography on a
Sephacryl S-300 column which had been equilibrated with the
pH-9.4 buffer (Fig. 1B). The enzymatically active fractions
were pooled and the resulting 240 ml applied to a Polybuffer
Exchanger 94 chromatofocusing column. Following washing,
elution with tenfold diluted Polybuffer 96 acetate (pH 6.0)
was carried out. Mn-SOD was eluted at about pH 8.0
(Fig. 1C). The result indicates that while relatively small
amount of enzymatically inactive material were separated
in the hydroxylapatite and S-300 gel-filtration step, the
chromatofocusing experiment led to a significant loss of protein. The pooled fractions (165 ml) were applied to a second
hydroxylapatite column. Following washing, the protein was
eluted with a linear gradient made by adding 500 ml400 mM
sodium phosphate, pH 7.2, to the same volume of 100 mM
sodium phosphate, pH 7.2. Inspection of Fig. 1D indicates
that this second hydroxylapatite chromatographic step led to
the loss of a considerable amount of inert protein. The 182-ml
volume of the active fractions was extensively dialyzed against
20 mM sodium phosphate buffer, pH 7.2, and appled to a Red
Sepharose column. The enzyme was eluted with a linear salt
gradient made by adding 300 ml 20 mM phosphate buffer,
pH 7.2, containing 1 M NaC1, to 300 ml of the same buffer
without NaCI. It can be seen from Fig. 1 E that the enzymatic
activity elutes as a single symmetrical component.
The specific activity of this purified Mn-SOD showed an
increase of 17-fold from the protein eluted from the first
hydroxylapatite column (Fig. 1A). The cyanide-resistant SOD
activity in the starting homogenate supernatant comprises
about 40% of the total reported in Table 1. If one assumes
that this represents only Mn-SOD the overall recovery at the
final stage of the Red Sepharose chromatography (Fig. 1E) is
about 18%. This may be compared with the recovery of protein. The homogenate supernatant from 2 kg of liver showed
the presence of 1.73 g Mn-SOD when assayed by an ELISA
method employing a monoclonal antibody [9, 241. When this
was compared with 320 mg purified enzyme recovered, there
was an overall yield of 18.5%. Thus, the recoveries based on
enzymatic and immunochemical methods are in good agreement.
The isolated Mn-SOD was homogeneous on SDS/PAGE.
Its specific activity was 6100 units/mg which is a higher value
than that reported previously [16]. The enzyme contained
0.314% (by mass) manganese, as judged by atomic absorption
spectrophotometry. This corresponds to 1.04 Mn/subunit,
equivalent to 4 Mn/mol native tetramer. In order to identify
Mn(I1) or Mn(II1) in the native enzyme, EPR spectrum was
recorded. However, no signals were obtained, indicating that
the native Mn-SOD has Mn(II1) because in the native Mn
containing enzymes, only Mn(I1) can be detected by EPR as
reported by Sugiura et al. [25].
The purified Mn-SOD was concentrated with an Amicon
PM-10 membrane to 45 mg/ml then stored at -35°C in the
presence of 50% glycerol until use. A photomicrograph of the
crystallized Mn-SOD is shown in Fig. 2.
Determination of molecular mass by HPGCiLALLS
The elution pattern of Mn-SOD (48.6 pg) when subjected
to the HPGCiLALLS method is shown in Fig. 3. The MnSOD was eluted after 18 min as a single peak. The glycerol is
particularly prominent in the refractometer assay and is seen
to elute after 26 min. The apparent molecular mass of native
Mn-SOD was calculated to be 88.6 f 2 kDa (mean f SD).
Its molecular absorption coefficient at 280 nm was determined
as 1.926 x lo5 M-' cm-'. A monomeric form of the enzyme
prepared by treatment with 1 % SDS, as will be described later,
exhibited a single component with an apparent molecular
mass of 21.3 f 4 kDa. This indicates that Mn-SOD is a noncovalently linked tetrameric enzyme. Amino acid composition
and partial sequence data have indicated that its subunits are
identical (data not shown), as reported in [13, 141.
716
I
1
A
A
Fraction
number
Fraction
number
.
Fraction
Fraction
number
to
l5Ic
-0
number
50
100
150
Fraction
200
250
300
350
400
number
Fig. 1 . Chromatographic patternsfor purification of human Mn-SOD.
The size of each column and volume of each fraction collected are
given in parentheses. (A) Hydroxylapatite column (7 cm x 42 cm)
chromatography( I8 mi). (B) Sephacryl S-300column ( 5 cm x 125 cm)
chromatography (10 ml). (C) Chromatofocusing with Polybuffer Exchanger 94 column (1.8 cm x 55 cm) chromatography ( 5 ml). (D) The
second hydroxylapatite column (2.5 cm x 30 cm) chromatography
( 5 ml). (E) Red Sepharose column (2.2 cm x 23 cm) chromatography
(3.6 ml). Each eluate fraction in A -E was assayed for cyanide-insensitive SOD activity (0)and protein (--- -). For experimental details,
see Results
p H stubility
Stability in denaturing solutions
As shown in Fig. 4, Mn-SOD was most stable in the buffer
at pH 7.2 where 98% of the original activity was retained for
1 day at 4 C. At pH 8.0 or 9.3, 80-90Y0, and at pH 5.5,65%
activity was retained. The enzyme lost up to 30% of its activity
after 3 days incubation at pH 7.2, 8.0 or 9.3, but about 50%
of initial activity at pH 5.5.
Mn-SOD loses about 20% of its activity in pH 8.0, 0.1 M
Tris/HCl after 3 days at 25"C, retaining its tetrameric structure as shown in Fig. 5 . In the presence of 1% SDS, all activity
is lost within 6 h. The enzyme, however, retained its molecular
mass of 88.6 kDa within a day, then gradually dissociated to
a monomeric form; finally, most of the enzyme appeared to be
717
m
2
m
0
1
r h l
3
Day
Fig. 2. Crystals of human liver Mn-SOD. Magnification, x 400
A B C
n
D
E
F
G H I
4
88kDa
25kDa
*23kDa
I
/
0
/
/
Day 1
/
20
10
Retention t i m e (rnin)
Fig. 3. The elution patterns ofhuman Mn-SODfrom a TSK-GEL G3000
SW-YL ( TOSOH, 7.8 mm x 300 mm) f o r molecular mass determination by HPGCILA LLS. The concentration gradients were obtained
photometrically (LS), refractometrically (RI) and spectrophotometrically (UV). The elution patterns in the figure are for 48.6 wg
enzyme. The gain settings of the detectors were 32 for the photometric,
64 for the refractometric, and A = 0.32 for the spectrophotometric
measurements
0
1
Day 2
30
2
3
Day 3
B
Fig. 5 . The labile nature of Mn-SOD at 25°C in 0.1 M TrislHCI,
p H 8.0, under varying conditions ( A ) and SDSjPAGE ( B ) . (A) ( 0 )
Control; (0)8 M urea; ( A ) 6 M guanidinium chloride; (U ) 1YoSDS.
An aliquot of each sample was removed at the indicated timcs for
activity measurements. Each point presents the mean of a duplicate
experiment. (B) Sequential analysis was carried out for changes of
molecular structure of the enzyme on SDSjPAGE as described in the
text. Lanes A - C, D - F and G -I represent the samples incubated for
1,2 and 3 days, respectively. Three samples under different conditions
were electrophoresed and demonstrated as follows: lanes A, D and
G, control; lanes B, E and H, 8 M urea; lanes C, F and I, 1% SDS
in the monomeric form after 3 days. This indicates that MnSOD is a non-covalently linked tetrameric enzyme. Two
monomeric components corresponding to molecular masses
of 23 kDa and 25 kDa were distinguished. After 2 days, the
amount of the 23-kDa and 25-kDa species appeared to be
equivalent, but after 3 days, the amount of the 23-kDa form
seemed to be greater than that the 25-kDa form. This indicates
that the 25-kDa species appeared to convert to the 23-kDa
species. In 6 M guanidinium chloride, about 50% of the activity was lost in 3 days. In 8 M urea, the enzyme remains
active and a tetrameric form was observed which was essentially similar to the control form, even though minor monomeric components were observed.
Incubationtime ( day )
Fig. 4. The stability of Mn-SOD at 4°C in the following 0.1-M buffers
ut various pH. ( 0 )Sodium acetate buffer, pH 5.5; (0)phosphate
buffer, pH 7.2; ( A ) Tris/HCl buffer, pH 8.0; (El) monoethanolamine
acetate buffer, pH 9.3. At the denoted time, an aliquot of the solution
was removed for enzyme assays and calculated in duplicate
Thiol titration of Mn-SOD
Under non-denaturing conditions, 4 thiols/mol(88.6 kDa)
enzyme (Table 2) were derivatized. In the presence of 1YOSDS
or 8 M urea solution for 1 h at 25°C a similar value was
71 8
-Fable 2. ThioI t i r i a t i o n of liiiniun Mn-SOD under various conditions
Thiol titrations mere curried out with 5 mM Nbsz at pH 8.0, as
described in the text. All mixtures contained 0.1 M Tris/HCl and
1 inM EDTA, pH 8.0. Thiol titration was carried out after removing
dithiothreitol by gel filtration. Alkylation of Mn-SOD is described in
the text. Each d u e reprcsenls the mean of a duplicate or a triplicate
experiment
Incubation conditions
Sulfhydryl group/Mn-SOD
25 c
for 1 h
100 c
for 5 min
niol/mol
Natlvc Mn-SOD
Alkylated Mn-SOI>
Native Mn-SOD 1 % S D S
N a t ~ v cMil-SOLI 1 % SDS
I 0 m M dithiothreitol
Ndtive Mil-SOD 8M urea
Native Mil-SOD XM uiea
10 mM dithiothieilol
kDa
A
B
C
D
-
41
03
39
40
24
40
41
19
33
38
82
E
F
sulfhydryl residues reactive. This indicates that the unreactive
thiol groups are exposed by these treatments. However, in the
absence of dithiothreitol, these residues apparently combine
with the reactive sulfhydryls to form oxidative intrachain disulfides, even in the presence of 5 mM EDTA.
Amino acid analyses of the carboxylamidomethylated
native protein showed that almost 4 mol sulfhydryl groups
were alkylated (data not shown). Ellman titration of the alkylated protein gave a value of only 0.3 thiols/mol. However, in
8 M urea, followed by heating at 100°C after carboxylamidomethylation, the titration showed 4.0 sulfhydryls. Thus
4 out of the 8 cysteine residues in the tetramers are reactive with the Ellman's reagent as well as to alkylation by
iodoacetamide, whereas the other residues are not, unless the
protein is completely denatured in the presence of reducing
reagent.
Effect
of thiol seagents on the enzjwze nctivitj
The enzymatic activity was not changed by treatment with
iodoacetamide, iodoacetic acid, Nbs2, N-ethylmaleimide,
oxidized glutathione, 2-mercaptoethanol or dithiothreitol
under conditions where the 4 reactive thiols noted could be
derivatized. This indicates that sulfhydryl modification of MnSOD did not affect the enzyme activity.
C
94
67
SDSIPAGE
43
30
4
25kDa
20.1
14.4
Fig. 6. Rrsu1t.s of .SDS,:PAGE ufter incubation of M n - S O D under
wrying condition.c in 0.1 M Tris!HCI, p H 8.0. The samples, which had
all been heated at 100 C for 5 niin in 1 % SDS, were treated as
follows: (A) standards; (B) control (10 wg); (C) reduced with 5 mM
2-mercaptoethanol (20 pg): (D) control (2 pg); (E) reduced with
5 mM 2-mercaptoethanol (2 pg); (F) alkylated with iodoacetamide
(2 pg); ( G ) alkylatcd with iodoacetamide and reduced with 5 m M
2-mercaptoethanol ( 2 pg)
obtained. This probably suggests incomplete denaturation,
since the tetraineric molecule has eight non-covalently crosslinked cysteine residues. However, heat treatment (1OO'C for
5 min) in the presence of SDS or urea gave relatively low
values, suggesting that some of the thiol groups were oxidized
to form disulfides. Incubation of the enzyme with SDS for
3 days at 25 C also gave low values. The conversion of the
tetramer to monomers accompanied by the loss of reactive
sulfhydryls means that some intrasubunit disulfide bonds
are readily formed rather than the oxidative formation of
disulfide-bridged oligomers.
Following reduction of Mn-SOD with 10 mM dithiothreitol in 8 M urea or in lo/" SDS at 25°C for 1 h, only 4
sulfhydryl groups were found. This indicates that the other 4
unreactive cysteine residues have no mixed disulfide bonds on
the outer sphere and are probably buried in the molecule.
Mn-SOD treated with 10 mM dithiothreitol in 1% SDS
or in 8 M urea and heated at 100 'C for 5 min showed almost 8
As shown in Fig.6, after heating at 100 C for 5 min in
pH 8.0, 0.1 M TrisjHCl containing 1% SDS or 1% SDS and
5 mM 2-mercaptoethanol, the Mn-SOD was subjected to
SDS/PAGE. When heated in SDS alone, a major component
corresponding to 23 kDa and a minor one of 25 kDa were
observed. Another two faint bands were observed, with molecular masses of 43 kDa and 54 kDa. However, in the presence
of 5 mM 2-mercaptoethanol, a single band corresponding to
a monomer of 25 kDa was observed. This indicates that
the 43-kDa and 54-kDa molecules are probably proteins
oligomerized by oxidative disulfide formation which convert
to 25-kDa species by 2-mercaptoethanol. The enzyme alkylated with iodoacetamide followed by heating in the presence
of SDS or SDS plus 2-mercaptoethanol, revealed the same
result on SDS/PAGE, where a major molecule of 25 kDa and
a minor one of 27 kDa were observed, but neither 43-kDa nor
54-kDa molecules. A minor band of 27 kDa is probably a
byproduct of the carboxyamidomethylation reaction to
certain amino acid residues due to long incubation time. We
conclude that when the heat treatment of Mn-SOD in SDS
is carried out in the absence of 2-mercaptoethanol or
dithiothreitol, the likely formation of intrasubunit disulfide
bonds occurred, and this is associated with conformational
changes that give an apparent molecular mass of 23 kDa in
SDSjPAGE experiments. These results are consistent with
those obtained by thiol titrations. One might speculate that a
proteolytic enzyme contaminant was activated by heat treatment and converted the 25-kDa species to one of 23 kDa.
However, this was unlikely since alkylation or treatment with
2-mercaptoethanol after the heat treatment produced the
25-kDa species. This indicates a reversible conversion from
the 23-kDa to the 25-kDa species.
Identification of the reactive suif;hq'dsyl group of Mn-SOD
The two cysteine residues known to be present in each
subunit are Cys140 and Cys196. As described above, only
719
one cysteine residue is alkylated or titrated at 25°C under
denaturing conditions. In order to identify this residue, the
Mn-SOD was incubated with Nbs2, then subjected to thiolSepharose chromatography. The Nbs2-reactive cysteine was
expected to bind to the thiol-Sepharose column through a SH/
SS exchange reaction. After peptic digestion of the Mn-SOD
material bound to the column, it was eluted with 2-mercaptoethanol. Amino acid sequence analysis indicated the presence
of two peptides in this eluate. Both were found to correspond
to the C-terminal region, one comprising residues 182- 198
and the other 194 - 198 (Met-Ala-Xaa-Lys-Lys). The cysteine
residues were not detected. These peptides are formed by
cleavages on the carboxyl side of Trpl81 and Tyr193, respectively, and are in keeping with the high specificity of pepsin
for cleavage at such residues. The binding of these peptides to
the thiol-Sepharose column clearly indicate that Cys196 was
the reactive sulfhydryl and is most probably located on the
outer surface of the enzyme. In a separate experiment, the
Mn-SOD which had been treated with iodoacetic acid or
iodoacetamide was digested with a mixture of carboxypeptidases Y or A and carboxypeptidase B, and the amino
acids released were analyzed. However, 2 mol lysine (Lys197
and Lys198) were quantitatively released from the C-terminus
of Mn-SOD, but none of the cysteine that has been alkylated
with iodoacetic acid or iodoacetamide was detected. The alkylated cysteine residue apparently was not susceptible to cleavage by either carboxypeptidase A or Y.
calculated from the amino acid composition data [13,14]. The
subunit molecular mass on SDSjPAGE are somewhat higher
than the value reported previously for other Mn-SOD isolates
[6,15, 161. However, the N-terminal analysis of the 30 residues
was identical with those reported by Barra et al. [12]. This
indicates that the our isolated human Mn-SOD does not occur
in a precursor form [17, 321. The Stokes' radius of the native
enzyme, obtained as subdata from the HPGCILALLS system,
was estimated to be 3.8 nm which corresponds to a molecular
mass of 75 kDa for a globular protein. This indicates that
native Mn-SOD is a non-globular tetrameric enzyme.
As anticipated from the sequence data [12] Mn-SOD was
found to contain 8 mol cysteine. In chicken liver Mn-SOD, the
8 cysteines appear to be involved in disulfide bond formation
within each subunit [6]. In the present study, we found that
human liver Mn-SOD contains four readily reactive cysteine
residues/mol tetramer under both denaturing and nondenaturing conditions. Heating in the presence of 1% SDS
showed no reactive sulfhydryls and predominantly converted
the native tetramer (88.6 kDa) into monomers (23 kDa) containing an intimubunit disulfide bond.
The presence of a Mn-SOD monomer of 23 kDa was noted
in the SDSjPAGE experiments following heat treatment in
urea or SDS solution. It seems reasonable to assume that
formation of this material involves intrasubunit disulfide bond
formation with an associated conformational change to a
more compact type of molecule. On the other hand, we have
no conclusive data to explain the appearance of 43 kDa
and 54 kDa species. These molecules are probably oligomers
of the 25 kDa monomer with interchain disulfide, such as
DISCUSSION
dimer or trimer. Such an assumption appears to be reasonable,
Our isolation method did not employ carboxymethyl- since reduction with 2-mercaptoethanol diminished the
cellulose chromatography at pH 5.5 since its use appeared to amount of these species and generated the 25 kDa form. As
inactivate the enzyme as described [15]. As shown in Fig. 4, shown in Fig. 5, the structural changes responsible for generpH stability test of purified human Mn-SOD also supported ating the 23-kDa species from the 25-kDa species appear to
result from conversion of a subunit containing free sulfhydryl
our method.
Human Mn-SOD is a relatively stable enzyme and retains groups to one having an intrachain disulfide.
This result again suggests that no intrasubunit disulfide
most of its activity and tetrameric structure in pH 8.0, 0.1 M
Tris/HCl containing 8 M urea at 25°C. At acidic pH in the bonds are present in the native enzyme. The reactive cysteine
presence of 8 M urea or 6M guanidinium chloride, the enzyme residue was found to be Cys196. One might speculate that
rapidly loses activity concomitant with loss of manganese as nascent polypeptide chains of Mn-SOD are folded so as to
described [26, 271. In a 1% SDS solution at 25"C, as shown prevent intrasubunit disulfide formation. In fact most of proin Fig. 5, human Mn-SOD rapidly loses activity [28] and teins are likely to start folding in order just after translation
gradually dissociates to monomers. This indicates that the from mRNA, but some protein requires C-terminal peptides
Mn-SOD was inactivated prior to dissociation in SDS and to complete its structure [33, 341. Mn-SOD synthesized in
finally converts a tetramer to a monomer, some via the 25-kDa cytosol must be transported into mitochondria where it posform. Following repeated freezing and thawing, the Mn-SOD sibly becomes the active tetrameric form. This transport
underwent inactivation and dissociation in SDS, even at 25 "C system and subsequent folding and oligomerization mechanfor a 20-min incubation (data not shown). Heating in SDS or ism in mitochondria is still unclear. To clarify this point,
urea also converts the native enzyme to monomers, forming including the role of the C-terminal peptides of the enzyme,
predominantly intrachain disulfide bonds. The likely forma- further study will be required.
The result of the present study indicates that human Mntion of intrasubunit rather than intersubunit disulfide linkages
may be explained by the distance between two cysteine resi- SOD is a tetrameric enzyme consisting of four identical subdues [30]; that is, actual distance between two intrachain units, in which one cysteine (Cys196) is readily reactive, and
cysteines would be very close compared to those of intersub- the other (Cysl40) is apparently buried in the molecule. Under
unit cysteines. Crystallographic studies of human Mn-SOD physiological conditions, Cys196 of Mn-SOD may be highly
are necessary, but to date this data has not been completely reactive to intracellular sulfhydryl compounds such as
reported 1181. A structure deduced from X-ray analysis of glutathione. It is well known that various proteins form a
mixed disulfide with glutathione and undergo functional or
bacterial Mn-SOD [31] seems to support our results.
The HPGCiLALLS is a method for molecular mass deter- structural changes (for review see [35]). Whether the mixed
mination based on hydrodynamics. Utilizing this we have disulfide formation of Mn-SOD with glutathione occurs in
obtained an apparent molecular mass of 88.6 kDa, a subunit the tissues is an interesting problem. In fact we found that in
molecular mass of 21.3 kDa and a molecular absorption coef- liver tissues two forms of Mn-SOD exist, and one of them has
ficient of 1.926 x lo5 M-' cm-l. These values are close to a an acidic isoelectric point [36]. We are now studying whether
subunit molecular mass of 22.2 kDa and an E of 18.1 x lo4 or not this heterogeneity is due to a mixed disulfide formation.
720
The authors are indebted to Mr Y. Sakamoto and Mr N. Koike
of Central Laboratory for Research and Education, Osaka University
Medical School for performing the amino acid and sequence analysis.
This work was supported by a grants-in-aid for Cancer Research from
the Ministry of Education, Science and Culture, and Welfare of Japan,
and of the Yasuda Memorial Foundation.
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