Vol.
64,
BIOCHEMICAL
No. 4, 1975
SELECTIVE SOLUTI
tiESTRUCTION ONS
OF
OF
SUPEROXI
F.
Bossa,
.;t D.
It
B.
+++
di di
Institute
+
SM
April
E.
Martin
Biologica, Molecol
a
Cancer
Bag,
and
Calabrese”
and
hi
gh
:
D.S.1
Lower
1 to
10
(for
(1).
It
see
2
radicals KBr yield
0
are
KCNS
H
atoms
a
recent
is
wel
of
the however,
enzyme
i nact the
of
Zeal
Nuclear
Sciences,
and.
produced i nto OH
f
radicals
in
that
loss
secondary that i on
secondary
basi to
s of data Br- was e x2 ent of
the
deposition
the
approximate
of
enzymes
activity
(2).
When
OH
radicals
are
converted
these
2
secondary
under
certai radi
cat
CNS2
only
these
solutes
can
such to
an
It
has
also
react
with
as
equal been
cause
(5-8).
circumstances can
to
(3,4).
radicals n
s
or
ratio chemistry
of
Br-
saturated
radiation
exposure
of
solutions
aqueous
radicals
obtai ned observed ami no
pH and presence or abtosses with residual enof some vital residues.
after aqueous
of
known
on
f ute
review
present,
i vat
di
bovine superoxi de few types of residues confirmed the indicaacids. Furthermore de-
on the exposed Different
depending of these identification
and
in are
shown,
solution,
at i on
results or
Department,
Institute
New
of irradiated that only a radicals and in free ami no
radicals
radi
N
of Rome, and Rome, Italy.
22,1975
reactive
with
Rotilio i$‘$
Physics
.R.
Hutt,
Ami no acid analyses solutions showed by H and Br-
energy
G.
University del C.N.R.,
are
Research,
losses were observed of copper. Correlation activity permitted
The
IRRADIATED
Fielden
ti ons obtai ned from work struction of lysi ne - unexpected in free solutions of ami no acids in the copper-containing protein. acid sence zymic
IN
U.K.
address
Private
SummaryI dismutase are destroyed
RESIDUES
COMMUNICATIONS
-;+
5PX,
2
Permanent
Received
ACID
RESEARCH
Dl SKUTASE,
l.,
Chimica Biologi of
Sutton
AMI NO
DE
-2 +L+
Roberts
Istituto Centro
BIOPHYSICAL
z‘t
Barra,
Peter
AND
In four
free of
Vol.
64,
No. 4, t 975
the
common
tophan, be
BIOCHEMICAL
amino tyrosi
acids n :,
by
tra
radicals
at
free
completion
solution
can
be
possible,
me
with
in
methods
human
can
be
show Iectivel te
y
loss
ami
mutase
solutions.
and
acid
in
no
the
ami
residues
Samples
that aci
the protein. Al i quots
should
of
an
enzy-
of
ami
no
modi
ficat
ion
in
using
and
enzymes
d
has
dues
concerned
in
i n
protei
of
one
bovi has
i ndi
active
and
of
carrels the
se-
i on
we
superoxi
de
played
the
cate
site
to se-
to
cat
already of
method
deeilable
more
ne
bovine
radicals
ns or
cata-
this
is
communi
studies
the
to
before
present
enzyme
known
ied
it
irradiated
the
I
i nduced
chemistry
of
appl
systems,
mechanistic es
been
wel
However,
I n the of
with
radiation
resi
achieved
a
enzyme that
hi
di
s-
major
(11-14) sti
di
ne
(15-17).
METHODS of
of
it
types
been
destruction
native
bovine
red by the method of McCord complete apo-protei ns were et al e (19). Co60 irradiation lutions in 1 mM phosphate carried out tions contained Analar grade. give unreactive the OH radical
in
systems
chemical
already
enzyme
analysis
assay
AND
spec-
results
enzyme
(10).
for
no
studi
MATERIALS
smutase
Radiation
be
the
product
specific
method
residues.
acid
may
can
with
these
to
trypacids
inactivation
in
the
with
biochemical
amino
their
If
extension
analysis
modified
report
and
upon
has
valid
activity
lectively
role
and di
attack
of
pH
complex
technique
as no
namely
reactivity
correlate
success
(5-8)
ami
to
an
superoxide
by
i ed
attack
radical
.regarded
their
reaction,
to
of
residues
and
the
(9), These
of
COMMUNICATIONS
studies.
measure
secondary
lytic
of
provide
enzyme
Some
of
principle,
and
for
response function
RESEARCH
proteins
histidine.
a
appl
in
and
radical
residue
the
be
secondary
acid
the as
the
BIOPHYSICAL
contained
cysteine
differentiated
secondary
AND
as
superoxide
dismutase
and fridovich obtained by the of approximately or pyrophosphate
(18). methods buffer
were
prepa-
Copper-free and of Roti I io 20 JJM enzyme sounder N20 was
outlined by Ewing (20) and Roberts (7). The either 50 mM t-butanol or 10 mM KBr, both OH radicals were scavenged In the former case, alcohol radi cal s and i n both cases H atoms concentration) were avai I able for react ion
irradiated
holoprotei
n
1304
and
an
uni
rradi
ated
sol2 of
control
to
(l/10 with
Vol.
44, No. 4, 1975
were
di
BIOCHEMICAL
I uted
M ethanol
to
pulse stituted phosphate,
The
method 7
and
remaining
RESULTS
AND The
vities
Dl SCUSSI
results
are
superoxide
dismutase
data
in
H
and
previously
modification was
only
i ndi
the
by
rectly
on
ami
no
exposed
of case
loss
of
free
lysi
in lysi
the ne
the
the
the A more
on
ne
of
lysine
various
the
ami of
no
acid
a either
smal
or
an
I a
ne
is
to
Br;
the
no
gh
on cate
active
21,
ned
free
H
the
the
results
th,at
the
of
copper-free
i n the
lows
be
ruled
is
a
presence conforma-
better
a more
leads
there residues
protein al
atoms
solutions
only
data
reactions
affected
with
here
1305
from
in
cannot
the
atom
indicates
However,
of
chemical
of
the
occurs
observed
H
Br;
22).
and
metal,
reactivity
the
expected
that
that
efficiency
(9)
obtained
indication (9)
in
The
destroyed
the
in
of
(10). are
used
and
study
acids
of
radicals.
inspection
fuller
confirm
not
Data
actidoses
eesidue
(7,
obtai
Residual
elsewhere
be
hi
data
i ndi
the
a
selectivity
dostructi
destruction led
the
identification
simply
to
detai
assay present.
system,
1.
ami
enzymes
Br;. nc
presence
lysine
with
presence out with
Table
and
to
by
of
to
may
on
pyrophos-
for assay reference
for
of
free
f ar
lysi
basis
residues
copper
work
the
lysi
This
mk!
analyzed
with
types
enzyme
certain
to
that
copper.
in
this
supported of
proteins
show
few
So
limitations
enzyme
a
proteins.
in
4
The al so
published
of
be
tion,
from
exposing
standard
n was reconin 5 mhl pyro-
nst
column
agreement
in
the
acids
shown
selective
may
single
a
controls. EDTA was
in the carried
under
only
inactivation
found
using a system).
sufficiently
However,
and
HCI were
inactivation
radicals
are
I i ted 6N
irradiation
obtained
radicals
lyophi
are
that
Bri
agai
0.08
by
protei CuSO
~~r\l
s
0.1
were withdrawn calculated
unirradiated that lo-4F.l
reasonable
demonstrate
alysi
f
ON
after
conditions
di
with analyses
obtained
measured
9.2
Copper-free nst 100
al iquots activity
except
COMMUNICATIONS
containing
pH
agai
column
RESEARCH
pyrophosphate
14).
in
was
i nstrument, (2
2 ml.! resultant
of
enzyme
BIOPHYSICAL
further
after hydrolysis of phenol. The BC200 instrument
of
then
activity recovered for holoprotei n
Hi o-Cal Optics
of
i n
the
(11, dialysis
overnight pH
content crystal
in
at
After reconstitution, the percent recovery
to the was as
by
1 PH
assayed
radiolysis by
ph;ate. with
about
and
AND
exposure
direct out,
rather
effect in
special to
further
which case. consi
-
in
Significant
Reactive
#
*
No
radicals
and
were
residues H atoms
in
t-butanol,
than
H
above
13.5
was
not atoms
+
Bri
60 33
25
60
KBr
51
325
11.0
5.3
8
KBr
KBr
4.0
9
405
t-but
in
found.
4.1
5.5
1.2
0.3
75
30
KBr
0.5
60
7.8
6
560
KBr
47
3.6
9
790
t-but
t-but
3.1
76
the
His
49
ng
KBr
$
KBr.
modified
superoxide enzyme gives
Residues
bovine
0
7.4
present
( KDGis)
Activity remai ni
ation of the untreated
85
other
irradi of
present.
on
47
t-but
in
*
are
content composition
I
t-but
K Br
t-butanoi
7.4
11.0
7.4
PH
t i ons
residues
ami noacid amino acid
Condi
Th e tryptophan
destruction
activity
conditions. (23).
M.W.
Cu)
no
33.000
Copper-free enzyme (2 Zn atoms/
oenzyme
Lys
different and 22
HOI
#
Changes
TABLE
2
2
0
0
0
0
4.3
0.9
0
11.6
3.0
3.0
0
LYS
M.W.
7.0
33.000
in 2 Tyr
2.0
1.4
0.9
1.5
2
1.5
0.7
0
Tyr
per
dismutase His,
16
Vol.
64, No.
4,1975
derations. on
BIOCHEMICAL
The
susceptibi
conditions
we
or
have
reactivity
upon
of
(hi
pH
tested;
at
gni
ficant
losses
other
gh
about
were
by
to
row
and
I ates the
usion 3
with two
hi
di fal
number
of
or
lysi
Is
at
both
respondi
ng may
protein
regions
impair
the
be
of
ons
tested,
the
di
cattri
ne
resi,dues.
buted
By
with
dues.
not far
by copper
it
only the
active appears
ficati
the
of correof
dose
and
i nacti
vati
on
di
of
a
ffe-
in
cor-
the
cor-
destroyed
but
site
centre,
the
strict
the
active
on
dose,the
and
rice
of
figures
the
that
active
brought
increase
si
can
which
in may
mechanisms.
e
64,
BIOCHEMICAL
No. 4, 1975
SELECTIVE SOLUTI
tiESTRUCTION ONS
OF
OF
SUPEROXI
F.
Bossa,
.;t D.
It
B.
+++
di di
Institute
+
SM
April
E.
Martin
Biologica, Molecol
a
Cancer
Bag,
and
Calabrese”
and
hi
gh
:
D.S.1
Lower
1 to
10
(for
(1).
It
see
2
radicals KBr yield
0
are
KCNS
H
atoms
a
recent
is
wel
of
the however,
enzyme
i nact the
of
Zeal
Nuclear
Sciences,
and.
produced i nto OH
f
radicals
in
that
loss
secondary that i on
secondary
basi to
s of data Br- was e x2 ent of
the
deposition
the
approximate
of
enzymes
activity
(2).
When
OH
radicals
are
converted
these
2
secondary
under
certai radi
cat
CNS2
only
these
solutes
can
such to
an
It
has
also
react
with
as
equal been
cause
(5-8).
circumstances can
to
(3,4).
radicals n
s
or
ratio chemistry
of
Br-
saturated
radiation
exposure
of
solutions
aqueous
radicals
obtai ned observed ami no
pH and presence or abtosses with residual enof some vital residues.
after aqueous
of
known
on
f ute
review
present,
i vat
di
bovine superoxi de few types of residues confirmed the indicaacids. Furthermore de-
on the exposed Different
depending of these identification
and
in are
shown,
solution,
at i on
results or
Department,
Institute
New
of irradiated that only a radicals and in free ami no
radicals
radi
N
of Rome, and Rome, Italy.
22,1975
reactive
with
Rotilio i$‘$
Physics
.R.
Hutt,
Ami no acid analyses solutions showed by H and Br-
energy
G.
University del C.N.R.,
are
Research,
losses were observed of copper. Correlation activity permitted
The
IRRADIATED
Fielden
ti ons obtai ned from work struction of lysi ne - unexpected in free solutions of ami no acids in the copper-containing protein. acid sence zymic
IN
U.K.
address
Private
SummaryI dismutase are destroyed
RESIDUES
COMMUNICATIONS
-;+
5PX,
2
Permanent
Received
ACID
RESEARCH
Dl SKUTASE,
l.,
Chimica Biologi of
Sutton
AMI NO
DE
-2 +L+
Roberts
Istituto Centro
BIOPHYSICAL
z‘t
Barra,
Peter
AND
In four
free of
Vol.
64,
No. 4, t 975
the
common
tophan, be
BIOCHEMICAL
amino tyrosi
acids n :,
by
tra
radicals
at
free
completion
solution
can
be
possible,
me
with
in
methods
human
can
be
show Iectivel te
y
loss
ami
mutase
solutions.
and
acid
in
no
the
ami
residues
Samples
that aci
the protein. Al i quots
should
of
an
enzy-
of
ami
no
modi
ficat
ion
in
using
and
enzymes
d
has
dues
concerned
in
i n
protei
of
one
bovi has
i ndi
active
and
of
carrels the
se-
i on
we
superoxi
de
played
the
cate
site
to se-
to
cat
already of
method
deeilable
more
ne
bovine
radicals
ns or
cata-
this
is
communi
studies
the
to
before
present
enzyme
known
ied
it
irradiated
the
I
i nduced
chemistry
of
appl
systems,
mechanistic es
been
wel
However,
I n the of
with
radiation
resi
achieved
a
enzyme that
hi
di
s-
major
(11-14) sti
di
ne
(15-17).
METHODS of
of
it
types
been
destruction
native
bovine
red by the method of McCord complete apo-protei ns were et al e (19). Co60 irradiation lutions in 1 mM phosphate carried out tions contained Analar grade. give unreactive the OH radical
in
systems
chemical
already
enzyme
analysis
assay
AND
spec-
results
enzyme
(10).
for
no
studi
MATERIALS
smutase
Radiation
be
the
product
specific
method
residues.
acid
may
can
with
these
to
trypacids
inactivation
in
the
with
biochemical
amino
their
If
extension
analysis
modified
report
and
upon
has
valid
activity
lectively
role
and di
attack
of
pH
complex
technique
as no
namely
reactivity
correlate
success
(5-8)
ami
to
an
superoxide
by
i ed
attack
radical
.regarded
their
reaction,
to
of
residues
and
the
(9), These
of
COMMUNICATIONS
studies.
measure
secondary
lytic
of
provide
enzyme
Some
of
principle,
and
for
response function
RESEARCH
proteins
histidine.
a
appl
in
and
radical
residue
the
be
secondary
acid
the as
the
BIOPHYSICAL
contained
cysteine
differentiated
secondary
AND
as
superoxide
dismutase
and fridovich obtained by the of approximately or pyrophosphate
(18). methods buffer
were
prepa-
Copper-free and of Roti I io 20 JJM enzyme sounder N20 was
outlined by Ewing (20) and Roberts (7). The either 50 mM t-butanol or 10 mM KBr, both OH radicals were scavenged In the former case, alcohol radi cal s and i n both cases H atoms concentration) were avai I able for react ion
irradiated
holoprotei
n
1304
and
an
uni
rradi
ated
sol2 of
control
to
(l/10 with
Vol.
44, No. 4, 1975
were
di
BIOCHEMICAL
I uted
M ethanol
to
pulse stituted phosphate,
The
method 7
and
remaining
RESULTS
AND The
vities
Dl SCUSSI
results
are
superoxide
dismutase
data
in
H
and
previously
modification was
only
i ndi
the
by
rectly
on
ami
no
exposed
of case
loss
of
free
lysi
in lysi
the ne
the
the
the A more
on
ne
of
lysine
various
the
ami of
no
acid
a either
smal
or
an
I a
ne
is
to
Br;
the
no
gh
on cate
active
21,
ned
free
H
the
the
results
th,at
the
of
copper-free
i n the
lows
be
ruled
is
a
presence conforma-
better
a more
leads
there residues
protein al
atoms
solutions
only
data
reactions
affected
with
here
1305
from
in
cannot
the
atom
indicates
However,
of
chemical
of
the
occurs
observed
H
Br;
22).
and
metal,
reactivity
the
expected
that
that
efficiency
(9)
obtained
indication (9)
in
The
destroyed
the
in
of
(10). are
used
and
study
acids
of
radicals.
inspection
fuller
confirm
not
Data
actidoses
eesidue
(7,
obtai
Residual
elsewhere
be
hi
data
i ndi
the
a
selectivity
dostructi
destruction led
the
identification
simply
to
detai
assay present.
system,
1.
ami
enzymes
Br;. nc
presence
lysine
with
presence out with
Table
and
to
by
of
to
may
on
pyrophos-
for assay reference
for
of
free
f ar
lysi
basis
residues
copper
work
the
lysi
This
mk!
analyzed
with
types
enzyme
certain
to
that
copper.
in
this
supported of
proteins
show
few
So
limitations
enzyme
a
proteins.
in
4
The al so
published
of
be
tion,
from
exposing
standard
n was reconin 5 mhl pyro-
nst
column
agreement
in
the
acids
shown
selective
may
single
a
controls. EDTA was
in the carried
under
only
inactivation
found
using a system).
sufficiently
However,
and
HCI were
inactivation
radicals
are
I i ted 6N
irradiation
obtained
radicals
lyophi
are
that
Bri
agai
0.08
by
protei CuSO
~~r\l
s
0.1
were withdrawn calculated
unirradiated that lo-4F.l
reasonable
demonstrate
alysi
f
ON
after
conditions
di
with analyses
obtained
measured
9.2
Copper-free nst 100
al iquots activity
except
COMMUNICATIONS
containing
pH
agai
column
RESEARCH
pyrophosphate
14).
in
was
i nstrument, (2
2 ml.! resultant
of
enzyme
BIOPHYSICAL
further
after hydrolysis of phenol. The BC200 instrument
of
then
activity recovered for holoprotei n
Hi o-Cal Optics
of
i n
the
(11, dialysis
overnight pH
content crystal
in
at
After reconstitution, the percent recovery
to the was as
by
1 PH
assayed
radiolysis by
ph;ate. with
about
and
AND
exposure
direct out,
rather
effect in
special to
further
which case. consi
-
in
Significant
Reactive
#
*
No
radicals
and
were
residues H atoms
in
t-butanol,
than
H
above
13.5
was
not atoms
+
Bri
60 33
25
60
KBr
51
325
11.0
5.3
8
KBr
KBr
4.0
9
405
t-but
in
found.
4.1
5.5
1.2
0.3
75
30
KBr
0.5
60
7.8
6
560
KBr
47
3.6
9
790
t-but
t-but
3.1
76
the
His
49
ng
KBr
$
KBr.
modified
superoxide enzyme gives
Residues
bovine
0
7.4
present
( KDGis)
Activity remai ni
ation of the untreated
85
other
irradi of
present.
on
47
t-but
in
*
are
content composition
I
t-but
K Br
t-butanoi
7.4
11.0
7.4
PH
t i ons
residues
ami noacid amino acid
Condi
Th e tryptophan
destruction
activity
conditions. (23).
M.W.
Cu)
no
33.000
Copper-free enzyme (2 Zn atoms/
oenzyme
Lys
different and 22
HOI
#
Changes
TABLE
2
2
0
0
0
0
4.3
0.9
0
11.6
3.0
3.0
0
LYS
M.W.
7.0
33.000
in 2 Tyr
2.0
1.4
0.9
1.5
2
1.5
0.7
0
Tyr
per
dismutase His,
16
Vol.
64, No.
4,1975
derations. on
BIOCHEMICAL
The
susceptibi
conditions
we
or
have
reactivity
upon
of
(hi
pH
tested;
at
gni
ficant
losses
other
gh
about
were
by
to
row
and
I ates the
usion 3
with two
hi
di fal
number
of
or
lysi
Is
at
both
respondi
ng may
protein
regions
impair
the
be
of
ons
tested,
the
di
cattri
ne
resi,dues.
buted
By
with
dues.
not far
by copper
it
only the
active appears
ficati
the
of correof
dose
and
i nacti
vati
on
di
of
a
ffe-
in
cor-
the
cor-
destroyed
but
site
centre,
the
strict
the
active
on
dose,the
and
rice
of
figures
the
that
active
brought
increase
si
can
which
in may
mechanisms.
e
