Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE USE OF A C E T Y L A T E D OF S U P E R O X I D E
Angelo
FERRICYTOCHROME
RADICALS
AZZI,
Cesare
C F O R THE D E T E C T I O N
PRODUCED
IN B I O L O G I C A L
MEMBRANES
MONTECUCCO
and C h r i s t o p h
RICHTER
C.N.R. Unit for the Study of P h y s i o l o g y of M i t o c h o n d r i a , and I n s t i t u t e of G e n e r a l Pathology, U n i v e r s i t y of Padova, Italy. Received
May
15,1975
SUM}~_ARY A c e t y l a t i o n of 60% of lysine r e s i d u e s of horse heart f e r r i c y t o c h r o m e c r e s u l t s in m o r e than 95% d e c r e a s e of its a b i l i t y to be r e d u c e d by m i t o c h o n d r i a l and m i c r o s o m a l r e d u c t a ses and to b e c o m e o x i d i z e d (after c h e m i c a l reduction) by m i t o c h o n d r i a l oxidase. The a b i l i t y of a c e t y l a t e d f e r r i c y t o chrome c to be r e d u c e d by 02 - r a d i c a l s is m a i n t a i n e d , m a k i n g this d e r i v a t i v e u s e f u l for the d e t e c t i o n of 02 - r a d i c a l s in b i o l o g i c a l systems c o n t a i n i n g c y t o c h r o m e c r e d u c t a s e s or oxidases. M i t o c h o n d r i a l m e m b r a n e s can reduce a c e t y l a t e d ferric y t o c h r o m e c at a rate of 0.5 n m o l e s . m i n - l . m g -I. Such a reaction is 82% i n h i b i t e d by 2.8 x I0-8M s u p e r o x i d e dismutase. INTRODUCTION The t e c h n i q u e s superoxide
currently
radicals
adrenochrome
(I), the r e d u c t i o n
or f e r r i c y t o c h r o m e
c
The choice
cated
does
not p e r m i t
radicals
(7,8).
auto-oxidation
(6). The p r e s e n c e
in m i t o c h o n d r i a ,
presence
are also
in m e m b r a n e s
of c y t o c h r o m e that
Copyright © 19 75 by Academic Press, Inc. All rights o f reproduction in any form reserved.
597
of some
and nuclei
of s u p e r o x i d e
cytochrome
(mitochondria,
c reductases
acetylation
can lead of a nitro-
microsomes
found w i t h
complireac-
for example,
the use of this dye as an i n d i c a t o r
and nuclei) It is k n o w n
but also
Complications
due to a w i d e s p r e a d somes
of i n t e r f e r i n q
has the d i s a d v a n t a g e ,
of a d r e n o c h r o m e reductase
in a c o m p l e x
is p a r t i c u l a r l y
of a n u m b e r
that not only c o - o x i d a t i o n ,
bluetetrazolium
(2)
of t e t r a n i t r o m e t h a n e
to be e m p l o y e d
such as m e m b r a n e s ,
The use of e p i n e p h r i n e
to the f o r m a t i o n
of to
(5).
due to the p r e s e n c e
tions.
of e p i n e p h r i n e
of n i t r o b l u e t e t r a z o l i u m
of the t e c h n i q u e
system,
for the d e t e c t i o n
(3), the r e d u c t i o n
(4) and p o l a r o g r a p h y
biological
employed
are the c o - o x i d a t i o n
micro-
and oxidases.
lysyl
residues
c,
of
Vol. 65, No. 2, 1975
ferricytochrome reduction
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
c destroyes
and o x i d a t i o n
able to be reduced has almost chrome
by chemical
negligible
c derivative
were
it w o u l d
detection
of 02 - species,
cient.and
a relatively
MATERIALS
agents
still
since
enzymatic is still
such as d i t h i o n i t e
(9).
able
be of large use
to u n d e r g o
the h e m o p r o t e i n
autooxidation
radicals,
02 - r a d i c a l s
its a b i l i t y
(9), w h i l e
If a c e t y l a t e d
to be r e d u c e d in b i o l o g i c a l
cyto-
by s u p e r o x i d e systems
it has a h i g h e x t i n c t i o n
h i g h rate c o n s t a n t
and
for coeffi-
(106 M -I sec -I) w i t h
(10).
AND M E T H O D S
F e r r i c y t o c h r o m e c was a c e t y l a t e d a c c o r d i n g to the p r o c e d u r e of M i n a k a m i et al. (9). At O°C, 50 mg of c y t o c h r o m e c were d i s s o l ved in 5 ml of a h a l f - s a t u r a t e d s o l u t i o n of sodium acetate. U n d e r stirring ten times excess acetic a n h y d r i d e w i t h r e s p e c t to the lysine groups was added, and the r e a c t i o n w a s c a r r i e d out for 30 minutes. The s o l u t i o n was d i a l y z e d in a 5/8 inch Thomas dialyzing tubing at O°C for 12 hours against 11 of d i s t i l l e d w a t e r w i t h four c h a n g e s of water. A c e t y l a t e d c y t o c h r o m e c was stored at - 20°C. The extent of a c e t y l a t i o n was d e t e r m i n e d by the n i n h y d r i n m e t h o d (11). The p e r c e n t m o d i f i c a t i o n is given by % acetylation
= 1OO
(I -
S acetylated S native
)
w h e r e S is the slope of the p l o t of a b s o r b a n c e at 570 nm versus the c o n c e n t r a t i o n of native or a c e t y l a t e d c y t o c h r o m e c. The c o n c e n t r a t i o n of c y t o c h r o m e c was d e t e r m i n e d in the reduced form at 550 nm using the e x t i n c t i o n c o e f f i c i e n t emM = 27.7. F e r r o e y t o c h r o m e c was o b t a i n e d by r e d u c t i o n w i t h a fe~f grains of d i t h i o n i t e f o l l o w e d by a gel f i l t r a t i o n on S e p h a d e x G-25 M. The c y t o c h r o m e was eluted w i t h d e g a s s e d d e s t i l l e d w a t e r and collected under N 2. S u c c i n a t e - o x i d a s e was m e a s u r e d p o l a r o g r a p h i c a l l y , with a C l a r k - t y p e electrode. C y t o c h r o m e c d e p l e t e d m i t o c h o n d r i a (1.5 mg/ml) w e r e i n c u b a t e d in a b u f f e r c o n t a i n i n g 0.25 M sucrose, IO m M Tris-HCI pH 7.4 and I ~M rotenone. The c o n c e n t r a t i o n of n a t i v e or a c e t y l a t e d c y t o c h r o m e c was 5 ~M. The r e a c t i o n w a s i n i t i a t e d by the a d d i t i o n of 5 m M succinate, and the a c t i v i t y e x p r e s s e d as nmoles O~ c o n s u m e d . m i n - l . m g -I. z Succinate-cytochrome c r e d u c t a s e a c t i v i t y was m e a s u r e d in a H i t a c h i - P e r k i n Elmer s p e c t r o p h o t o m e t e r in 250 ~ M Sucrose, 10 ~ M T r i s - H C l pH 7.4, I mM KCN, and I0.7 ~M c y t o c h r o m e c. The r e a c t i o n was started by adding 3 mM suecinate. P r o t e i n c o n c e n t r a t i o n was 180 ~g/ml. F e r r o c y t o c h r o m e c o x i d a s e was tested by adding 210 ~g/ml of a s u s p e n s i o n of c y t o c h r o m e c d e p l e t e d rat liver m i t o c h o n d r i a to b o t h sample and r e f e r e n c e c u v e t t e s c o n t a i n i n g 250 r~4 sucrose, 10 m M T r i s - H C l pH 7.4. 8.8 UM f e r r o c y t o e h r o m e c w e r e also present in the sample cuvette. The d e c r e a s e in a b s o r b a n c e at 550 nm
598
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
was p l o t t e d on s e m i l o g a r i t h m i c p a p e r as a f u n c t i o n of time and the first order v e l o c i t y c o n s t a n t c a l c u l a t e d from the slope of the plot. N A D P H - c y t o c h r o m e c r e d u c t a s e was a s s a y e d in a m e d i u m like that for s u c c i n a t e - c y t o c h r o m e c r e d u c t a s e (without KCN) and the r e a c t i o n was s t a r t e d by a d d i n g I m M NADPH. The p r o t e i n c o n c e n t r a tion of m i c r o s o m e s was 235 ~g/ml. Rat liver m i t o c h o n d r i a (13), beef heart m i t o c h o n d r i a l fragm e n t s (14) and m i c r o s o m e s (15) w e r e p r e p a r e d by s t a n d a r d p r o c e dures. C y t o c h r o m e c (type VI, f r o m horse heart) was from Sigma C h e m i c a l Co. acetic a n h y d r i d e f r o m Merck, Darmstadt, x a n t h i n e o x i d a s e from Boehringer. All o t h e r c h e m i c a l s w e r e c o m m e r c i a l l y a v a i l a b l e r e a g e n t p u r e products. RESULTS
AND DISCUSSION
Reduction oxide
of n a t i v e
and a c e t y l a t e d
ferricytochrome
c by super-
radicals. Superoxide
se ~ [ s t e m
radicals,
(16) w e r e
the rate of r e d u c t i o n cytochrome
generated
able to r e d u c e being
c concentration
50 ~M c y t o c h r o m e
c. W h e n
by the x a n t i n e - x a n t i n e native
progressively until
the
cytochrome higher
a maximum
c
I),
at i n c r e a s i n g
was r e a c h e d
same e x p e r i m e n t
oxida-
(Fig.
at about
was c a r r i e d
out w i t h
native
5
~2
sb
1~o Cytochrome c, pM
Fig.
1 - -eduction radicals.
of n a t i v e
and a c e t y l a t e d
cytochrome
c by 02-
E x p e r i m e n t a l conditions: c v t o c h r o m e c (native Or acetylated) was d i s s o l v e d in 50 m M p h o s p h a t e b u f f e r c o n t a i ning 10 m M KCI and 200 UM xantine, s a t u r a t e d w i t h oxygen, pH 7.4. R e d u c t i o n of c y t o c h r o m e c (at 550 nm, in a H i t a c h i - P e r k i n Elmer s p e c t r o p h o t o m e t e r Mod. 124) was started by the a d d i t i o n of 17 Hg/ml x a n t i n e oxidase.
599
Vol. 65, No. 2, 1975
acetylated
cytochrome
ry to attain native
slightly
decreased,
when
However
limited
Thus,
02
dismutase,
Enzymatic
reduction
The a b i l i t y
its e f f i c i e n c y cytochrome
chain
succinate less than
3.3 % of the rate
in
obtained
under
dismutase.
cytochrome
tested
oxidase
(Table
c.
electrons
by a s s a y i n g activity
of
I). The rate of o x y g e n
mitochondria,
of excess
being
to s u p e r o x i d e
c to t r a n s f e r was
succinate
c extracted
is
of b o t h native
beeing
of a c e t y l a t e d
mitochondria
c
to be c o m p e t e n t
superoxide
cvtochrome
in r e c o n s t i t u t i n g
c was
production.
sensitive
of m i t o c h o n d r i a
in the p r e s e n c e
cytochrome
of
is o n l y
of the zero order,
of the r e d u c t i o n
of c y t o c h r o m e
radicals
The r e d u c t i o n
and o x i d a t i o n
to that of
of c y t o c h r o m e
c appears
at 2.5 x IO-8M
c extracted
consumption
became
was n e c e s s a -
that the a b i l i t y
by 02
acetylated
c was h i g h l y
of a c e t y l a t e d
in the r e s p i r a t o r y
supplemented
acetylated
in the p r e s e n c e
cytochrome
of native
c
cyto-
c.
The s u c c i n a t e - c y t o c h r o m e the zero order chrome
indicates reduced
formation.
92% i n h i b i t i o n conditions
chrome
if excess
cvtochrome
the above
cytochrome identical
the c o n c e n t r a t i o n
cytochrome
radicals
and a c e t y l a t e d
as m u c h
by the rate of radicals
acetylated
detecting
was
finding
c to b e c o m e
the rate of its r e d u c t i o n
presumably
with
twice
rate of reduction,
c. This
cytochrome
limiting.
present
c about
the m a x i m u m
cytochrome
acetylated
rate
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
c
rate c o n s t a n t
(at 550 nm)
the p r e s e n c e
The oxidase using
acetylated
order
reaction
of 4% w i t h
cytochrome
with
respect
7.7% w i t h
acetylated
c. A c e t y l a t i o n enzymatic chondria
reduction
Acetylation
c with
c appears
and o x i d a t i o n without
by s u p e r o x i d e
in
for native
c depleted
mitochondria (a first
c), had a rate c o n s t a n t
hemoprotein.
c reductase
cytochrome
and m i c r o s o m e s ,
reducibility
to c y t o c h r o m e
of c y t o c h r o m e
higher
c as the substrate
to the native
NADPH-ferricytochrome
ferricyto-
mitochondria,
30 folds
by
c.
of c y t o c h r o m e
ferrocytochrome
respect
than
measured
of excess
c extracted
was m o r e
activity
activity,
of the r e d u c t i o n
by c y t o c h r o m e
of succinate,
than for a c e t v l a t e d
c reductase
of rat liver m i c r o s o m e s respect
to native
to depress
of c y t o c h r o m e substantial
cytochrome
drastically
c both
was
the
in m i t o -
modifications
of its
radicals.
of c y t o c h r o m e
c is thus
600
a simple
and v e r y useful
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
ENZYMATIC
REDUCTION
I
AND O X I D A T I O N
OF N A T I V E A N D A C E T Y L A T E D
CYTOCHROME
C
E x p e r i m e n t a l c o n d i t i o n s : d e t a i l s c o n c e r n i n g the p r e p a r a t i o n s u s e d and the e n z y m a t i c tests are r e p o r t e d in the s e c t i o n of Methods.
cytochrome native rate
%
rate
Enzyme system succinate-cyt c r e d u c t a s e +
6.4
100
Ferrocytochrome c o x i d a s e (210 ~g/ml) ++
0.046
100
NADPH-cyt c reductase +
10.4
100
succinate
60
1OO
oxidase +
+ ++
expressed
in n m o l e s
expressed
as first o r d e r
modification radicals
even
these membranes
are t e s t e d
radicals,
one
reduction
or o x i d a t i o n
of s u p e r o x i d e superoxide
rate c o n s t a n t ,
in the p r e s e n c e
should,
for t h e i r
permits
which
ability
be a w a r e
of c y t o c h r o m e
dismutase,
radicals,
which
of m i t o c h o n d r i a
however,
0.2
3.1
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE USE OF A C E T Y L A T E D OF S U P E R O X I D E
Angelo
FERRICYTOCHROME
RADICALS
AZZI,
Cesare
C F O R THE D E T E C T I O N
PRODUCED
IN B I O L O G I C A L
MEMBRANES
MONTECUCCO
and C h r i s t o p h
RICHTER
C.N.R. Unit for the Study of P h y s i o l o g y of M i t o c h o n d r i a , and I n s t i t u t e of G e n e r a l Pathology, U n i v e r s i t y of Padova, Italy. Received
May
15,1975
SUM}~_ARY A c e t y l a t i o n of 60% of lysine r e s i d u e s of horse heart f e r r i c y t o c h r o m e c r e s u l t s in m o r e than 95% d e c r e a s e of its a b i l i t y to be r e d u c e d by m i t o c h o n d r i a l and m i c r o s o m a l r e d u c t a ses and to b e c o m e o x i d i z e d (after c h e m i c a l reduction) by m i t o c h o n d r i a l oxidase. The a b i l i t y of a c e t y l a t e d f e r r i c y t o chrome c to be r e d u c e d by 02 - r a d i c a l s is m a i n t a i n e d , m a k i n g this d e r i v a t i v e u s e f u l for the d e t e c t i o n of 02 - r a d i c a l s in b i o l o g i c a l systems c o n t a i n i n g c y t o c h r o m e c r e d u c t a s e s or oxidases. M i t o c h o n d r i a l m e m b r a n e s can reduce a c e t y l a t e d ferric y t o c h r o m e c at a rate of 0.5 n m o l e s . m i n - l . m g -I. Such a reaction is 82% i n h i b i t e d by 2.8 x I0-8M s u p e r o x i d e dismutase. INTRODUCTION The t e c h n i q u e s superoxide
currently
radicals
adrenochrome
(I), the r e d u c t i o n
or f e r r i c y t o c h r o m e
c
The choice
cated
does
not p e r m i t
radicals
(7,8).
auto-oxidation
(6). The p r e s e n c e
in m i t o c h o n d r i a ,
presence
are also
in m e m b r a n e s
of c y t o c h r o m e that
Copyright © 19 75 by Academic Press, Inc. All rights o f reproduction in any form reserved.
597
of some
and nuclei
of s u p e r o x i d e
cytochrome
(mitochondria,
c reductases
acetylation
can lead of a nitro-
microsomes
found w i t h
complireac-
for example,
the use of this dye as an i n d i c a t o r
and nuclei) It is k n o w n
but also
Complications
due to a w i d e s p r e a d somes
of i n t e r f e r i n q
has the d i s a d v a n t a g e ,
of a d r e n o c h r o m e reductase
in a c o m p l e x
is p a r t i c u l a r l y
of a n u m b e r
that not only c o - o x i d a t i o n ,
bluetetrazolium
(2)
of t e t r a n i t r o m e t h a n e
to be e m p l o y e d
such as m e m b r a n e s ,
The use of e p i n e p h r i n e
to the f o r m a t i o n
of to
(5).
due to the p r e s e n c e
tions.
of e p i n e p h r i n e
of n i t r o b l u e t e t r a z o l i u m
of the t e c h n i q u e
system,
for the d e t e c t i o n
(3), the r e d u c t i o n
(4) and p o l a r o g r a p h y
biological
employed
are the c o - o x i d a t i o n
micro-
and oxidases.
lysyl
residues
c,
of
Vol. 65, No. 2, 1975
ferricytochrome reduction
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
c destroyes
and o x i d a t i o n
able to be reduced has almost chrome
by chemical
negligible
c derivative
were
it w o u l d
detection
of 02 - species,
cient.and
a relatively
MATERIALS
agents
still
since
enzymatic is still
such as d i t h i o n i t e
(9).
able
be of large use
to u n d e r g o
the h e m o p r o t e i n
autooxidation
radicals,
02 - r a d i c a l s
its a b i l i t y
(9), w h i l e
If a c e t y l a t e d
to be r e d u c e d in b i o l o g i c a l
cyto-
by s u p e r o x i d e systems
it has a h i g h e x t i n c t i o n
h i g h rate c o n s t a n t
and
for coeffi-
(106 M -I sec -I) w i t h
(10).
AND M E T H O D S
F e r r i c y t o c h r o m e c was a c e t y l a t e d a c c o r d i n g to the p r o c e d u r e of M i n a k a m i et al. (9). At O°C, 50 mg of c y t o c h r o m e c were d i s s o l ved in 5 ml of a h a l f - s a t u r a t e d s o l u t i o n of sodium acetate. U n d e r stirring ten times excess acetic a n h y d r i d e w i t h r e s p e c t to the lysine groups was added, and the r e a c t i o n w a s c a r r i e d out for 30 minutes. The s o l u t i o n was d i a l y z e d in a 5/8 inch Thomas dialyzing tubing at O°C for 12 hours against 11 of d i s t i l l e d w a t e r w i t h four c h a n g e s of water. A c e t y l a t e d c y t o c h r o m e c was stored at - 20°C. The extent of a c e t y l a t i o n was d e t e r m i n e d by the n i n h y d r i n m e t h o d (11). The p e r c e n t m o d i f i c a t i o n is given by % acetylation
= 1OO
(I -
S acetylated S native
)
w h e r e S is the slope of the p l o t of a b s o r b a n c e at 570 nm versus the c o n c e n t r a t i o n of native or a c e t y l a t e d c y t o c h r o m e c. The c o n c e n t r a t i o n of c y t o c h r o m e c was d e t e r m i n e d in the reduced form at 550 nm using the e x t i n c t i o n c o e f f i c i e n t emM = 27.7. F e r r o e y t o c h r o m e c was o b t a i n e d by r e d u c t i o n w i t h a fe~f grains of d i t h i o n i t e f o l l o w e d by a gel f i l t r a t i o n on S e p h a d e x G-25 M. The c y t o c h r o m e was eluted w i t h d e g a s s e d d e s t i l l e d w a t e r and collected under N 2. S u c c i n a t e - o x i d a s e was m e a s u r e d p o l a r o g r a p h i c a l l y , with a C l a r k - t y p e electrode. C y t o c h r o m e c d e p l e t e d m i t o c h o n d r i a (1.5 mg/ml) w e r e i n c u b a t e d in a b u f f e r c o n t a i n i n g 0.25 M sucrose, IO m M Tris-HCI pH 7.4 and I ~M rotenone. The c o n c e n t r a t i o n of n a t i v e or a c e t y l a t e d c y t o c h r o m e c was 5 ~M. The r e a c t i o n w a s i n i t i a t e d by the a d d i t i o n of 5 m M succinate, and the a c t i v i t y e x p r e s s e d as nmoles O~ c o n s u m e d . m i n - l . m g -I. z Succinate-cytochrome c r e d u c t a s e a c t i v i t y was m e a s u r e d in a H i t a c h i - P e r k i n Elmer s p e c t r o p h o t o m e t e r in 250 ~ M Sucrose, 10 ~ M T r i s - H C l pH 7.4, I mM KCN, and I0.7 ~M c y t o c h r o m e c. The r e a c t i o n was started by adding 3 mM suecinate. P r o t e i n c o n c e n t r a t i o n was 180 ~g/ml. F e r r o c y t o c h r o m e c o x i d a s e was tested by adding 210 ~g/ml of a s u s p e n s i o n of c y t o c h r o m e c d e p l e t e d rat liver m i t o c h o n d r i a to b o t h sample and r e f e r e n c e c u v e t t e s c o n t a i n i n g 250 r~4 sucrose, 10 m M T r i s - H C l pH 7.4. 8.8 UM f e r r o c y t o e h r o m e c w e r e also present in the sample cuvette. The d e c r e a s e in a b s o r b a n c e at 550 nm
598
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
was p l o t t e d on s e m i l o g a r i t h m i c p a p e r as a f u n c t i o n of time and the first order v e l o c i t y c o n s t a n t c a l c u l a t e d from the slope of the plot. N A D P H - c y t o c h r o m e c r e d u c t a s e was a s s a y e d in a m e d i u m like that for s u c c i n a t e - c y t o c h r o m e c r e d u c t a s e (without KCN) and the r e a c t i o n was s t a r t e d by a d d i n g I m M NADPH. The p r o t e i n c o n c e n t r a tion of m i c r o s o m e s was 235 ~g/ml. Rat liver m i t o c h o n d r i a (13), beef heart m i t o c h o n d r i a l fragm e n t s (14) and m i c r o s o m e s (15) w e r e p r e p a r e d by s t a n d a r d p r o c e dures. C y t o c h r o m e c (type VI, f r o m horse heart) was from Sigma C h e m i c a l Co. acetic a n h y d r i d e f r o m Merck, Darmstadt, x a n t h i n e o x i d a s e from Boehringer. All o t h e r c h e m i c a l s w e r e c o m m e r c i a l l y a v a i l a b l e r e a g e n t p u r e products. RESULTS
AND DISCUSSION
Reduction oxide
of n a t i v e
and a c e t y l a t e d
ferricytochrome
c by super-
radicals. Superoxide
se ~ [ s t e m
radicals,
(16) w e r e
the rate of r e d u c t i o n cytochrome
generated
able to r e d u c e being
c concentration
50 ~M c y t o c h r o m e
c. W h e n
by the x a n t i n e - x a n t i n e native
progressively until
the
cytochrome higher
a maximum
c
I),
at i n c r e a s i n g
was r e a c h e d
same e x p e r i m e n t
oxida-
(Fig.
at about
was c a r r i e d
out w i t h
native
5
~2
sb
1~o Cytochrome c, pM
Fig.
1 - -eduction radicals.
of n a t i v e
and a c e t y l a t e d
cytochrome
c by 02-
E x p e r i m e n t a l conditions: c v t o c h r o m e c (native Or acetylated) was d i s s o l v e d in 50 m M p h o s p h a t e b u f f e r c o n t a i ning 10 m M KCI and 200 UM xantine, s a t u r a t e d w i t h oxygen, pH 7.4. R e d u c t i o n of c y t o c h r o m e c (at 550 nm, in a H i t a c h i - P e r k i n Elmer s p e c t r o p h o t o m e t e r Mod. 124) was started by the a d d i t i o n of 17 Hg/ml x a n t i n e oxidase.
599
Vol. 65, No. 2, 1975
acetylated
cytochrome
ry to attain native
slightly
decreased,
when
However
limited
Thus,
02
dismutase,
Enzymatic
reduction
The a b i l i t y
its e f f i c i e n c y cytochrome
chain
succinate less than
3.3 % of the rate
in
obtained
under
dismutase.
cytochrome
tested
oxidase
(Table
c.
electrons
by a s s a y i n g activity
of
I). The rate of o x y g e n
mitochondria,
of excess
being
to s u p e r o x i d e
c to t r a n s f e r was
succinate
c extracted
is
of b o t h native
beeing
of a c e t y l a t e d
mitochondria
c
to be c o m p e t e n t
superoxide
cvtochrome
in r e c o n s t i t u t i n g
c was
production.
sensitive
of m i t o c h o n d r i a
in the p r e s e n c e
cytochrome
of
is o n l y
of the zero order,
of the r e d u c t i o n
of c y t o c h r o m e
radicals
The r e d u c t i o n
and o x i d a t i o n
to that of
of c y t o c h r o m e
c appears
at 2.5 x IO-8M
c extracted
consumption
became
was n e c e s s a -
that the a b i l i t y
by 02
acetylated
c was h i g h l y
of a c e t y l a t e d
in the r e s p i r a t o r y
supplemented
acetylated
in the p r e s e n c e
cytochrome
of native
c
cyto-
c.
The s u c c i n a t e - c y t o c h r o m e the zero order chrome
indicates reduced
formation.
92% i n h i b i t i o n conditions
chrome
if excess
cvtochrome
the above
cytochrome identical
the c o n c e n t r a t i o n
cytochrome
radicals
and a c e t y l a t e d
as m u c h
by the rate of radicals
acetylated
detecting
was
finding
c to b e c o m e
the rate of its r e d u c t i o n
presumably
with
twice
rate of reduction,
c. This
cytochrome
limiting.
present
c about
the m a x i m u m
cytochrome
acetylated
rate
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
c
rate c o n s t a n t
(at 550 nm)
the p r e s e n c e
The oxidase using
acetylated
order
reaction
of 4% w i t h
cytochrome
with
respect
7.7% w i t h
acetylated
c. A c e t y l a t i o n enzymatic chondria
reduction
Acetylation
c with
c appears
and o x i d a t i o n without
by s u p e r o x i d e
in
for native
c depleted
mitochondria (a first
c), had a rate c o n s t a n t
hemoprotein.
c reductase
cytochrome
and m i c r o s o m e s ,
reducibility
to c y t o c h r o m e
of c y t o c h r o m e
higher
c as the substrate
to the native
NADPH-ferricytochrome
ferricyto-
mitochondria,
30 folds
by
c.
of c y t o c h r o m e
ferrocytochrome
respect
than
measured
of excess
c extracted
was m o r e
activity
activity,
of the r e d u c t i o n
by c y t o c h r o m e
of succinate,
than for a c e t v l a t e d
c reductase
of rat liver m i c r o s o m e s respect
to native
to depress
of c y t o c h r o m e substantial
cytochrome
drastically
c both
was
the
in m i t o -
modifications
of its
radicals.
of c y t o c h r o m e
c is thus
600
a simple
and v e r y useful
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
ENZYMATIC
REDUCTION
I
AND O X I D A T I O N
OF N A T I V E A N D A C E T Y L A T E D
CYTOCHROME
C
E x p e r i m e n t a l c o n d i t i o n s : d e t a i l s c o n c e r n i n g the p r e p a r a t i o n s u s e d and the e n z y m a t i c tests are r e p o r t e d in the s e c t i o n of Methods.
cytochrome native rate
%
rate
Enzyme system succinate-cyt c r e d u c t a s e +
6.4
100
Ferrocytochrome c o x i d a s e (210 ~g/ml) ++
0.046
100
NADPH-cyt c reductase +
10.4
100
succinate
60
1OO
oxidase +
+ ++
expressed
in n m o l e s
expressed
as first o r d e r
modification radicals
even
these membranes
are t e s t e d
radicals,
one
reduction
or o x i d a t i o n
of s u p e r o x i d e superoxide
rate c o n s t a n t ,
in the p r e s e n c e
should,
for t h e i r
permits
which
ability
be a w a r e
of c y t o c h r o m e
dismutase,
radicals,
which
of m i t o c h o n d r i a
however,
0.2
3.1
