Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RECONSTITUTION OF IRON-SUPEROXIDE DISMUTASE
Fumiyuki Yamakura
and
Koji Suzuki
Department of Chemistry, School of Medicine Juntendo University, Narashino, Chiba 275, JAPAN Received August 4,1976 Summary: Iron-free superoxide dismutase has been prepared by incubation with sodium carbonate buffer (pH ii.0) containing i0 mM dithiothreitol and 2 mM EDTA under anaerobic conditions and subsequent dialysis to return the pH to 7.8. Reconstituted enzyme was obtained from the apoenzyme by incubation under the same conditions, with addition of 12 mM ferrous ammonium sulfate instead of EDTA. The apoenzyme has no visible absorption and no significant amount of activity. The reconstituted enzyme had the same visible absorption and activity as the native enzyme. Four sulfhydryl groups per molecule of apoenzyme reacted rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) while holoenzyme reacted with the reagent very slowly.
Superoxide dismutase reaction
0~ + O~ + 2 H +
(EC 1.15.1.i), which catalyzes the > H202 + 02, is widely distributed
among oxygen-metabolizing organisms obes (2).
With a few exceptions
(i) and some obligate anaer-
(3,4), SOD was obtained as the
Mn or Fe-containing enzyme from procaryotes and mitochondria, whereas SOD from eytosoles of eukaryotes was found to contain Cu and Zn (i).
Since McCord and Fridovich (5) reported the prep-
aration of the apo- and reconstituted enzymes of Cu, Zn-SOD, many studies have been carried out on the properties of apo- and reconstituted
SOD (1,6,7).
Recently,
Ose and Fridovieh (8) have
extended the studies of the reversible removal of Mn atom from Mn-SOD of Escherichia eoli and the replacement of Mn with Co,Zn,
Abbreviation:
SOD, superoxide dismutase
Copyright © 1976 by Academic Press, Inc. All rights o] reproduction in any ]orm reserved.
1108
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or Ni,
but they failed
Te-SOD
of E s c h e r i c h i a
succeeded
and
from P s e u d o m o n a s is composed
crystalline hydryl
Fe-SOD clude
enzyme
groups
describe
coli using
the
removal
of Fe atom from
same technique
which
had
with the Mn-SOD.
Fe-SOD 40,000
in the r e v e r s i b l e
has a m o l e c u l a r
of two subunits
contains
of equal
i.i g atoms
per mol of enzyme
the p r e p a r a t i o n
(9).
ovalis.
Fe is e s s e n t i a l
Furthermore,
the results
4 sulfhydryl
groups
cule and/or
chelated
The results
of q u a n t i t a t i v e
of h o l o e n z y m e
might
of
(9).
of Fe and 4 mol
In this
for enzymatic
weight
size
The
of sulf-
eommunieation,
of apo- and r e c o n s t i t u t e d
from P s e u d o m o n a s that
ovalis
have
activity studies
enzyme
of
led us to conof Fe-SOD. indicate
be buried
we
that
in enzyme
mole-
to Fe atoms. MATERIALS
AND METHODS
Fe-SOD was i s o l a t e d from P s e u d o m o n a s ovalis as d e s c r i b e d p r e v i o u s l y (9). E n z y m a t i c a c t i v i t y was m e a s u r e d by the m e t h o d of McCord and F r i d o v i c h (5) with a few m o d i f i c a t i o n s (9). For the studies of the enzyme stability , the enzymatic a c t i v i t y was estimated by the m e t h o d of N i s h i k i m i et al. (10). Protein was determined by the m i c r o - b i u r e t method of Itzhaki and Gill (ii), using bovine serum a l b u m i n as standard. Iron was d e t e r m i n e d by the m e t h o d Of M a s s e y (12) except that b a t h o - p h e n a n t h r o l i n e was used as a colo r i m e t r i c reagent. P o l y a c r y l a m i d e gel e l e e t r o p h o r e s i s was p e r f o r m e d at 5 ° C in a c c o r d a n c e with the method of Davis (13). Mobilities of the apo- and r e c o n s t i t u t e d SOD on p o l y a c r y l a m i d e gels (6, 7.5, 9, and 10.5 %) were a n a l y z e d a c c o r d i n g to the method of H e d r i c k and Smith (14). SOD a c t i v i t y on p o l y a c r y l a m i d e gel was located by the p h o t o c h e m i c a l m e t h o d of B e a u c h a m p and F r i d o v i c h (15). Sulfhydryl groups were d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y using 5,5'dithiobis-(2-nitrobenzoic acid) as d e s c r i b e d by Ellman (16). Optical a b s o r p t i o n spectra were m e a s u r e d with a Hitachi Model 124 Spectrophotometer.
RESULTS Purified
Fe-SOD
min and the a c t i v i t y Fig.
i, the enzyme 30 mg of the
and c o n t a i n e d
1.12
AND DISCUSSION
was treated
at various
of the enzyme
was
completely
pH's
was measured. inactivated
Fe-SOD,
which
g atoms
of Fe per mol
As
shown
5
in
at pH 10.9.
had a specific
1109
at 30 ° C for
activity
of enzyme,
of 3,200
was dialyzed
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
I
>,o
-~ loo > u _J
25o if) w
I
o
I
9
I0
l
I
pH
Fig. i Effect of pH on the stability of SOD. The enzyme (34 ~g/ml) was i n c u b a t e d for 5 min at 30 ° C w i t h 0.i M s o d i u m c a r b o n a t e buffer, at various pH's as indicated. Residual a c t i v i t i e s were m e a s u r e d by the assay method of N i s h i k i m i et al. (i0).
against
distilled
water
carried
out under
anaerobic
The
enzyme
carbonate which
was
buffer
containing
against
(Whatman
DE-32)
eluted
by 50 mM of this
preparation
was
The a p o - S O D
lyzed
buffer instead
against
was
column
employed
of i N NaOH.
0.2 M sodium
for
buffer
carbonate
1110
The a p o - S O D to a DEAE-
(pH 7.8),
equili-
and then
i mM dithiothreitol. study.
25 min at 30 ° C with the
with a d d i t i o n Then
(pH 7.8)
previously
in the r e c o n s t i t u t i o n for
this
so-
(pH g.2)
buffer
and applied
buffer
This
5- 12 hr at 4 ° C,
phosphate
containing
incubated
of 2 mM EDTA.
substitution:
0.2 M s o d i u m
carbonate
water
were
i0 mM d i t h i o t h r e i t o l ,
(0,9 X 4 cm),
phosphate
buffer
as above,
N 2 gas
for 18 hr at 4 ° C.
ten fold with d i s t i l l e d
by 2.5 mM p o t a s s i u m
sulfate
0.2 M sodium
50 mM p o t a s s i u m
brated
alkaline
2 mM EDTA and
to pH ii by a d d i t i o n
against
procedures
25 min at 30 ° C with
0.5 mM d i t h i o t h r e i t o l
was diluted
This
using
i mM E D T A and i mM d i t h i o t h r e i t o l
subsequently
cellulose
The f o l l o w i n g
conditions
for
containing
was d i a l y z e d
containing and
incubated
pH was a d j u s t e d
lution
overnight.
of 12 mM ferrous enzyme
buffer
solution
(pH 9.2)
same
ammonium was dia-
containing
Vol. 72, No. 3, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 2 P o l y a c r y l a m i d e gel e ! e c t r o p h o r e s e s of native, apo-, and r e c o n s t i t u t e d SOD. Native SOD (a): 44 ~ g , a p o - S O D (b): 4 0 ~ g , r e c o n s t i t u t e d SOD (c): 3g ~ g , and r e c o n s t i t u t e d SOD (d): 4 ~ g were each subjected to p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s at pH 8.9 The gels (a,b,c) were stained for protein by Amido black i0 B; the achromatic zone indicates the enzymatic activity (d).
i mM d i t h i o t h r e i t o l at 4 ° C.
and I mM ferrous a m m o n i u m
A f t e r dialysis
of the r e c o n s t i t u t e d
changes
of 50 mM p o t a s s i u m phosphate
covered
by using a D E A E - c e l l u l o s e
buffer
sulfate for 5-12 hr enzyme against
(pH 7.8),
column as described
1111
two
it was reabove.
Vol. 72, No. 3, 1976
Fig.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2 shows
the e l e c t r o p h o r e t i c
reconstituted
SOD on p o l y a e r y l a m i d e
and one m i n o r
were
SOD.
The p r o t e i n
the zones
of the apo-
method
bands
isomers,
(Fig.
gels.
of the r e c o n s t i t u t e d activity,
described
2).
which
in the text. SOD have
It appeared
the
to the a n a l y t i c a l
method
and
protein
band
and r e c o n s t i t u t e d SOD c o i n c i d e d
were
located
Major
with
by the
protein
same m o b i l i t y
that the major
of the apo- and r e c o n s t i t u t e d
according
of the apo-
One major
in both the apo-
and r e c o n s t i t u t e d
enzyme
protein
bands
of the enzymatic
photochemical
native
observed
patterns
bands
as the
and minor
SOD were
"charge"
of H e d r i c k
and
Smith
(15).
0.8
LLI 0 Z
0.6 0.5
03 n- 0 . 4 0 (/') I:]O