Vol. 70, No. 4, 1976
BIOCHEMICAL
FERROCYANIDE:
AND BIOPHYSICAL
AN INHIBITOR
OF CYTOCHROME OXIDASE
C. A. Yu and Linda Laboratory
of Bioenergetics,
Received
April
Yu
State
Albany,
RESEARCH COMMUNICATIONS
University
New York
of New York
12222
5,1976
SUMMARY--Ferrocyanide inhibits cytochrome oxidase activity with an inhibition constant of.0.8 mM and a binding rate constant of 0.0033 set-1. Treatment of the enzyme with ferrocyanide caused the reduction of cytochrome 5 but not 3, as judged by the Soret to a absorption ratio, which was established using phopholipid depleted oxidase in the absence of inhibitor. The spectral characteristics of ferrocyanide treated oxidase are presented and the possible inhibition site is discussed. In spite
of the many important
and the contradictions
among the
of inhibitors,
with
wealth
coupled
of information
of fact, chrome
long
Detailed
fluoride, effect
formate,
used widely
reported
before.
cyanide
was first
present constant
inhibitors
reported
of cytochrome
was mentioned.
inhibitory
behavior, on both
In this
The inhibitory this
oxidase,
a but not 3
chemical has not by ferro-
but no inhibitory
kinetics
"lipid-sufficient"
been
effect
we wish and the
to
dissociation
and "lipid-depleted"
oxidase.
Cytochrome
Copyright AN rights
of cyto-
sulfide,
communication
the binding
MATERIALS
depleted,
5).
et al . (6)
a
enzyme was established
although
of cytochrome
the use
As a matter
studies
such as azide,
ferrocyanide,
by Jacobs
(l),
has provided
the
of this
(4,
unanswered
oxidase.
into
available
in the studies
of ferrocyanide
cytochrome
on other
The reduction
activity the
existence
common reagent,
has been
technique,
introduced
are also
remaining
of cytochrome
the
etc.
of a very
on oxidase
(2) were
still
of investigators
spectroscopic
before
studies
findings
the studies
CO and cyanide oxidase
(3).
in
questions
oxidase
containing
preparation,
9 and 12 nmoles
0 1976 by Academic Press, Inc. in any form reserved.
of reproduction
AND METHODS both
phospholipid
sufficient
heme a per mg protein,
1115
and
respectively,
BlOCHEMlCAL
Vol. 70, No. 4,1976
were
prepared
according
to the method
The enzyme preparation containing used
was made in
1% sodium
to ensure
AND BIOPHYSKAL
cholate
reported
RESEARCH COMMUNlCATlONS
in this
50 mM phosphate
laboratory
buffer,
pH 7.4, The latter
and 0.1 M ammonium sulfate.
the complete
clearness
(7).
of the enzyme solution
was
and caused
no interference. Cytochrome ferrocyanide
c (type
were
III)
was obtained
the products
twice
from water.
Asolectin
carbon
monoxide
from Metheson
cially
at the highest Activity
determined
(7)
was determined
of Merck
Co.
Other
and heme 5 content
model
(8)
described
14,
Sigma.
Ferricyanide
The latter
and
was recrystallized
from
Concentrate
chemicals
were
Associates
obtained
and
commer-
available.
by the biuret
done in a Cary,
Co.
was purchased
purity
by the methods
from
of cytochrome
previously.
method
(9).
Protein
All
spectrophotometer
oxidase
were
concentration
spectral
measurements
were
at room temperature.
RESULTS AND DISCUSSION z
inhibitory
-on cytochrome chrome
depleted This
3 mM.
type
42 umole
increasing
could
30 nmole
was first
and cytochrome
reaction
was started
incomplete
inhibition
inhibition
constant
75 nmole
with
0.7 mM, was observed
volume for
to the mixture
was found when heart
The reaction in terms
pH 7.4,
of heme
and
of 1.5 ml.
Cytochrome
4 minutes
in the
reaction
one minute
before
the
In spite
even at higher
concentration,
muscle
1116
and lipid
of ascorbate.
to be 0.8 mM.
of
the same behavior.
oxidase
buffer,
ferrocyanide
the addition
cyto-
concentration
experimentally.
2, 0.2 nmole
by ferrocyanide, (Ki)
inhibited
showed
of phosphate
-c was added with
asolectin,
in a final
ferrocyanide
lipid-sufficient
be demonstrated
cytochrome
of
ferrocyanide
80% at an inhibitor
with
of ferrocyanide incubated
that
constant
preparations,
by replenishing
vessel
viz.
Both oxidase
of ascorbate, amount
oxidase
up to about
of inhibition contained
and the dissociation was observed
activity
followed
mixture 2,
oxidase--It
oxidase
approximately
effects
A slightly
preparation
of the
lower
was tested.
the Ki,
BIOCHEMICAL
Vol. 70, No. 4, 1976
In contrast chloride
(6) did
cytochrome
to the reported
observation
not
inhibitory
reverse
--for
to that
introduced
similar
uptake
rate
into
the assay
also
determined.
on mitochondria, effect
magnesium
of ferrocyanide
time
straight
line
of plot
slope
simple.
--et al.
after
of log
(10)
on
rate
of ferrocyanide of 0.0033
versus
the behavior
is monophasic,
the
6.7 mM ferrocyanide
constant
that
a technique
and following
constant
(act.t/act.o)
indicated
It
rate
order
using
introducing
the binding
The first the
-of ferrocyanide--By
by Nicholls
mixture,
from
rather
the binding
against
obtained
is
the
RESEARCH COMMUNICATIONS
oxidase.
Rate constant
oxygen
AND BIOPHYSICAL
set
time
was
-1
was
plot.
The
of ferrocyanide
and more like
sulfide
binding
than
cyanide
binding. Spectral
properties
phospholipid
is removed
the enzyme becomes found
between
oxidase N,N,N'
a and g3
not
The block
ascorbate
a prolonged
was obtained.
due to the
traces
presence
anaerobic
spectra
versus
a33'
cytochromes
form minus
s and -3, a than
a3+a33+, ratio
respectively.
those
Table
I summarizes
ratio
and u absorption
was incubated
a complete
versus
spectra
anaerobically reduction
aerobic
oxidase
prepara-
condition
and
spectra
respectively.
From these
These
(11)
of cytochrome
of a
2+ 3
spectra,
to a to be 3.1 and 19 for results
the percent
of
case was presumably
the delipidated
of Soret
1117
c and a was
the difference
of 4.1 and 18.5
of inhibitor. of Soret
showed
cytochrome
cytochrome
of a3 in this in
has been
of cytochrome
the same conditions,
present
the absorption
values
oxidase
when
preparation,
1 shows the difference
of the anerobic
oxidized
and a2'a32'
we calculated
of Fig.
that
transfer
when delipidated
The reduction
of activity
Difference
tion.
under
oxidase
(TMPD) only
line
known
of electron
in the
When delipidated
period
is
cytochrome
Therefore,
The solid
a . -i+ a .
minus
a and 5
better
with
the active (7).
(7).
2 and a--It
,N' ,-tetramethyl-p-phenylendiamine
of a2+
minus
from
inactive
was reduced
reduced,
for
-of cytochrome
should
obtained
in the
of contributions a and a . 3
be considered presence and the
Vol. 70, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
i ,i
Fig.
1.
TABLE.
, 450
I I 500 550 WA'iELENGTH,nm
I 600
6
Difference spectra of reduced minus oxidized form of cytochromes 5 and 53. One ml of phospholipid depleted cytochrome oxidase solution containing 20 pM heme 5 and one ml of freshly prepared reducing solution containing 30 mM ascorbate, 10 uM cytochrome c and 200 PM TMPD were pipetted separately into two double sector cuvettes with 0.5 cm lightpath in each sector. After measuring the base line in a Cary 14, the cuvette in sample beam was then mixed throughly and the spectrumzas recorded which gives difference spectra of cytochrome a versus a3+ The mixed cuvette was then covered under argon and (-1. placed again in sample beam and absorption at 445 nm was recorded versus time until no further increase was observed. The difference spectra were then taken again, which give a2+*as2+ versus a3+*as3+ (---). The difference spectra of cytochrome as2+ versus a3 3+ were obtained by the prolonged incubated mixture versus the immediately mixed sample (- - - -).
Absorption
Ratio
of
a
and Soret
of Cytochromes
a and g3
% contribution Soret/a Components
a; 605 nm (Red-Ox)
Soret;
445 nm (Red-Ox)
Cytochrome
2
80
41
Cytochrome
g3
20
59
Cytochrome
a+
100
100
g3
1118
3.1 19 6.6
BIOCHEMICAL
Vol. 70, No. A,1976
Fig.
2.
Ferrocyanide induced absorption spectra change of cytochrome oxidase. Active cytochrome oxidase, heme a 17.5 JIM, in 50 mM phosphate buffer, pH 7.4, containing 1% sodium cholate and 0.1 M ammonium sulfate was used. Solid (--), dash (- - -), dotted ("""') and broken (-a- *-) lines represent oxidized, dithionite reduced, ferrocyanide treated and ferrocyanide plus ascorbate treated samples, correspondingly. Soret region was measured in a 0.5 cm light path cuvettes.
Effect --
of ferrocyanide
--on the
oxidase--Addition
of ferrocyanide
partial
of cytochrome
reduction
2 reduction spectra
depended
between
and absence
ferrocyanide
ferrocyanide
monoxide treated
cyanide
was maximized
solution.
The action
keeping redox only
showed
oxidase. when
low
ascorbate
itself
Figure
2 shows
and untreated to that
amount
enough
to reduce
reduced the absolute
ratio all
cytochrome spectra
1119
used.
The difference in
Soret
the presence
properties
was present
the cytochrome
It
of
a by ferro-
ferricyanide, high.
spectra
to a absorption
of cytochrome
was to reduce
to ferricyanide
of cytochrome
of the difference
of ascorbate
a
extent
on the spectral
The reduction
caused
samples
by the low
no effect
of ascorbate
preparation
of ferrocyanide
identical
a small
-of cytochrome
not 55 and the
a3+ as judged
the ferrocyanide potential
amount
were
properties
to the oxidase
treated
a2+ versus
Carbon
spectral
a but
upon the
of ascorbate
of cytochrome ratio.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
thus
in
thereby provided
-a rather
a
than
2. of the
Soret
the
and a regions
of
Vol. 70, No. 4, 1976
ferrocyanide reduced
BIOCHEMICAL
treated
forms
cytochrome
increase
Soret
absorption
could
cause
shifted
in absorption
to the when
the oxidase
when
caused
treated
was first
of dithionite.
the Soret
the ferrocyanide
treated
Similar
a blue sample
absorption directly
then
shift
shift
CO
results
by dithionite
no spectral
in
the dithionite
at 445 nm is
added.
However,
s3 (because
decrease
reduced caused
The increase
with
in
to
sample,
as compared
of ferrocyanide
of
a absorption -3
of cytochrome
of decrease
Addition
the reduction
cytochrome
However,
Ferrocyanide
in the absence
comparison.
to the ferrocyanide
The extent
amount
ferrocyanide.
observed
not only
a significant
at 445 nm was observed.
with
for
at 445 nm was observed.
shift).
oxidase,
and dithionite
included
was due to the reduction
untreated
observed
The oxidized
the oxidized
was added
a spectral
proportional
are
preparation
When dithionite
a further
reduced
also
oxidase.
spectra
to oxidase
a but
430 nm.
cytochrome
of untreated
of ferrocyanide
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in
treated the a region
of about
was further
were
1 nm was
reduced
by
dithionite. The possibility contamination was ruled
of the inhibition
of cyanide out
for
two reasons.
the same inhibition spectra
were
not
In fact,
upon prolonged directly
suggested
that
located
reduction
incubation.
ascorbate
andis
oxidase
a complete
without the
action
between
of untreated
ferrocyanide
reagent.
oxidase
while
increased
upon
site
cytochrome
in
a and a . -3
the
the absorption
the prolonged
significantly.
in the
latter
of s3 was presumably
of ferrocyanide
1120
Secondly,
445 nm absorption
cytochrome
showed
the same conditions,
of a and s3 was reached
involving
solution
in the presence
change
under
The reduction
due to the
ferrocyanide
enzyme was observed of oxygen,
being
recrystallized
No significant
treated
inhibited
in the
inhibited
identical.
in the absence
ferrocyanide
as that
and ferrocyanide
at 445 nm of cyanide incubation
inhibitors First,
constant
of cyanide
of ascorbate
or other
of ferrocyanide
2. is
These
by
results
different
In addition,
case
from cyanide in
the
a region,
BIOCHEMICAL
Vol. 70, No. 4,1976
more blue ferrocyanide
shift
was observed inhibited
ACKNOWLEDGEMENTS--This Institute discussion
of Health. with
Dr.
in
sample research
AND BIOPHYSICAL RESEARCH COMMUNlCAl’lONS
cyanide
upon
the
treated addition
was supported
We acknowledge
oxidase
than
that
of
of dithionite. by the grants
the encouragement
from
from
National
and valuable
T. E. King. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Lemberg, R., Physiol. Rev. (1969) 69, 48-121. Warburg, 0. (1926) Biochem. Z. 177, 471. Keilin, D., Hartree, E. F. (1939) Proc. Roy. Sot (London) M, 167. Nicholls, P., and Chance, B. (1974) In Molecular Mechanism Oxygen Activation (ed. 0. Hayaishi), PP. 479-534, Acad. Press, N. Y. Lemberg, R., and Barrett, J. (1973) Cytochromes pp. 17-57, Acad. Press. N. Y. Jacobs, G. G., Andrews, E. C., and Crane, F. C. (1964) in Oxidases and Related Redox Systems (King, T. E., Mason, H. S., and Morrison, M eds.) Vol. 2, pp. 784-812, Wiley, N. Y. Y;: C. A., Yu, L., and King, T. E. (1975) J. Biol. Chem. m, 13831392. Kuboyama, M., Yong, F. C., and King, T. E. (1972) J. Biol. Chem. 247, 6375-6383. Gornall, A. G., Bardawell, C. J., and David, M. M. (1949) J. Biol. Chem. 177, 751-766. Nicholls, P., Van Buuren, K. J. H., and Van Gelder, B. F. (1972) Biochim. Biophys. Acta 275, 279-287. Horie, S., and Morrison, M. (1964) J. Biol. Chem. 239, 1438-1441.
1121
BIOCHEMICAL
FERROCYANIDE:
AND BIOPHYSICAL
AN INHIBITOR
OF CYTOCHROME OXIDASE
C. A. Yu and Linda Laboratory
of Bioenergetics,
Received
April
Yu
State
Albany,
RESEARCH COMMUNICATIONS
University
New York
of New York
12222
5,1976
SUMMARY--Ferrocyanide inhibits cytochrome oxidase activity with an inhibition constant of.0.8 mM and a binding rate constant of 0.0033 set-1. Treatment of the enzyme with ferrocyanide caused the reduction of cytochrome 5 but not 3, as judged by the Soret to a absorption ratio, which was established using phopholipid depleted oxidase in the absence of inhibitor. The spectral characteristics of ferrocyanide treated oxidase are presented and the possible inhibition site is discussed. In spite
of the many important
and the contradictions
among the
of inhibitors,
with
wealth
coupled
of information
of fact, chrome
long
Detailed
fluoride, effect
formate,
used widely
reported
before.
cyanide
was first
present constant
inhibitors
reported
of cytochrome
was mentioned.
inhibitory
behavior, on both
In this
The inhibitory this
oxidase,
a but not 3
chemical has not by ferro-
but no inhibitory
kinetics
"lipid-sufficient"
been
effect
we wish and the
to
dissociation
and "lipid-depleted"
oxidase.
Cytochrome
Copyright AN rights
of cyto-
sulfide,
communication
the binding
MATERIALS
depleted,
5).
et al . (6)
a
enzyme was established
although
of cytochrome
the use
As a matter
studies
such as azide,
ferrocyanide,
by Jacobs
(l),
has provided
the
of this
(4,
unanswered
oxidase.
into
available
in the studies
of ferrocyanide
cytochrome
on other
The reduction
activity the
existence
common reagent,
has been
technique,
introduced
are also
remaining
of cytochrome
the
etc.
of a very
on oxidase
(2) were
still
of investigators
spectroscopic
before
studies
findings
the studies
CO and cyanide oxidase
(3).
in
questions
oxidase
containing
preparation,
9 and 12 nmoles
0 1976 by Academic Press, Inc. in any form reserved.
of reproduction
AND METHODS both
phospholipid
sufficient
heme a per mg protein,
1115
and
respectively,
BlOCHEMlCAL
Vol. 70, No. 4,1976
were
prepared
according
to the method
The enzyme preparation containing used
was made in
1% sodium
to ensure
AND BIOPHYSKAL
cholate
reported
RESEARCH COMMUNlCATlONS
in this
50 mM phosphate
laboratory
buffer,
pH 7.4, The latter
and 0.1 M ammonium sulfate.
the complete
clearness
(7).
of the enzyme solution
was
and caused
no interference. Cytochrome ferrocyanide
c (type
were
III)
was obtained
the products
twice
from water.
Asolectin
carbon
monoxide
from Metheson
cially
at the highest Activity
determined
(7)
was determined
of Merck
Co.
Other
and heme 5 content
model
(8)
described
14,
Sigma.
Ferricyanide
The latter
and
was recrystallized
from
Concentrate
chemicals
were
Associates
obtained
and
commer-
available.
by the biuret
done in a Cary,
Co.
was purchased
purity
by the methods
from
of cytochrome
previously.
method
(9).
Protein
All
spectrophotometer
oxidase
were
concentration
spectral
measurements
were
at room temperature.
RESULTS AND DISCUSSION z
inhibitory
-on cytochrome chrome
depleted This
3 mM.
type
42 umole
increasing
could
30 nmole
was first
and cytochrome
reaction
was started
incomplete
inhibition
inhibition
constant
75 nmole
with
0.7 mM, was observed
volume for
to the mixture
was found when heart
The reaction in terms
pH 7.4,
of heme
and
of 1.5 ml.
Cytochrome
4 minutes
in the
reaction
one minute
before
the
In spite
even at higher
concentration,
muscle
1116
and lipid
of ascorbate.
to be 0.8 mM.
of
the same behavior.
oxidase
buffer,
ferrocyanide
the addition
cyto-
concentration
experimentally.
2, 0.2 nmole
by ferrocyanide, (Ki)
inhibited
showed
of phosphate
-c was added with
asolectin,
in a final
ferrocyanide
lipid-sufficient
be demonstrated
cytochrome
of
ferrocyanide
80% at an inhibitor
with
of ferrocyanide incubated
that
constant
preparations,
by replenishing
vessel
viz.
Both oxidase
of ascorbate, amount
oxidase
up to about
of inhibition contained
and the dissociation was observed
activity
followed
mixture 2,
oxidase--It
oxidase
approximately
effects
A slightly
preparation
of the
lower
was tested.
the Ki,
BIOCHEMICAL
Vol. 70, No. 4, 1976
In contrast chloride
(6) did
cytochrome
to the reported
observation
not
inhibitory
reverse
--for
to that
introduced
similar
uptake
rate
into
the assay
also
determined.
on mitochondria, effect
magnesium
of ferrocyanide
time
straight
line
of plot
slope
simple.
--et al.
after
of log
(10)
on
rate
of ferrocyanide of 0.0033
versus
the behavior
is monophasic,
the
6.7 mM ferrocyanide
constant
that
a technique
and following
constant
(act.t/act.o)
indicated
It
rate
order
using
introducing
the binding
The first the
-of ferrocyanide--By
by Nicholls
mixture,
from
rather
the binding
against
obtained
is
the
RESEARCH COMMUNICATIONS
oxidase.
Rate constant
oxygen
AND BIOPHYSICAL
set
time
was
-1
was
plot.
The
of ferrocyanide
and more like
sulfide
binding
than
cyanide
binding. Spectral
properties
phospholipid
is removed
the enzyme becomes found
between
oxidase N,N,N'
a and g3
not
The block
ascorbate
a prolonged
was obtained.
due to the
traces
presence
anaerobic
spectra
versus
a33'
cytochromes
form minus
s and -3, a than
a3+a33+, ratio
respectively.
those
Table
I summarizes
ratio
and u absorption
was incubated
a complete
versus
spectra
anaerobically reduction
aerobic
oxidase
prepara-
condition
and
spectra
respectively.
From these
These
(11)
of cytochrome
of a
2+ 3
spectra,
to a to be 3.1 and 19 for results
the percent
of
case was presumably
the delipidated
of Soret
1117
c and a was
the difference
of 4.1 and 18.5
of inhibitor. of Soret
showed
cytochrome
cytochrome
of a3 in this in
has been
of cytochrome
the same conditions,
present
the absorption
values
oxidase
when
preparation,
1 shows the difference
of the anerobic
oxidized
and a2'a32'
we calculated
of Fig.
that
transfer
when delipidated
The reduction
of activity
Difference
tion.
under
oxidase
(TMPD) only
line
known
of electron
in the
When delipidated
period
is
cytochrome
Therefore,
The solid
a . -i+ a .
minus
a and 5
better
with
the active (7).
(7).
2 and a--It
,N' ,-tetramethyl-p-phenylendiamine
of a2+
minus
from
inactive
was reduced
reduced,
for
-of cytochrome
should
obtained
in the
of contributions a and a . 3
be considered presence and the
Vol. 70, No. 4, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
i ,i
Fig.
1.
TABLE.
, 450
I I 500 550 WA'iELENGTH,nm
I 600
6
Difference spectra of reduced minus oxidized form of cytochromes 5 and 53. One ml of phospholipid depleted cytochrome oxidase solution containing 20 pM heme 5 and one ml of freshly prepared reducing solution containing 30 mM ascorbate, 10 uM cytochrome c and 200 PM TMPD were pipetted separately into two double sector cuvettes with 0.5 cm lightpath in each sector. After measuring the base line in a Cary 14, the cuvette in sample beam was then mixed throughly and the spectrumzas recorded which gives difference spectra of cytochrome a versus a3+ The mixed cuvette was then covered under argon and (-1. placed again in sample beam and absorption at 445 nm was recorded versus time until no further increase was observed. The difference spectra were then taken again, which give a2+*as2+ versus a3+*as3+ (---). The difference spectra of cytochrome as2+ versus a3 3+ were obtained by the prolonged incubated mixture versus the immediately mixed sample (- - - -).
Absorption
Ratio
of
a
and Soret
of Cytochromes
a and g3
% contribution Soret/a Components
a; 605 nm (Red-Ox)
Soret;
445 nm (Red-Ox)
Cytochrome
2
80
41
Cytochrome
g3
20
59
Cytochrome
a+
100
100
g3
1118
3.1 19 6.6
BIOCHEMICAL
Vol. 70, No. A,1976
Fig.
2.
Ferrocyanide induced absorption spectra change of cytochrome oxidase. Active cytochrome oxidase, heme a 17.5 JIM, in 50 mM phosphate buffer, pH 7.4, containing 1% sodium cholate and 0.1 M ammonium sulfate was used. Solid (--), dash (- - -), dotted ("""') and broken (-a- *-) lines represent oxidized, dithionite reduced, ferrocyanide treated and ferrocyanide plus ascorbate treated samples, correspondingly. Soret region was measured in a 0.5 cm light path cuvettes.
Effect --
of ferrocyanide
--on the
oxidase--Addition
of ferrocyanide
partial
of cytochrome
reduction
2 reduction spectra
depended
between
and absence
ferrocyanide
ferrocyanide
monoxide treated
cyanide
was maximized
solution.
The action
keeping redox only
showed
oxidase. when
low
ascorbate
itself
Figure
2 shows
and untreated to that
amount
enough
to reduce
reduced the absolute
ratio all
cytochrome spectra
1119
used.
The difference in
Soret
the presence
properties
was present
the cytochrome
It
of
a by ferro-
ferricyanide, high.
spectra
to a absorption
of cytochrome
was to reduce
to ferricyanide
of cytochrome
of the difference
of ascorbate
a
extent
on the spectral
The reduction
caused
samples
by the low
no effect
of ascorbate
preparation
of ferrocyanide
identical
a small
-of cytochrome
not 55 and the
a3+ as judged
the ferrocyanide potential
amount
were
properties
to the oxidase
treated
a2+ versus
Carbon
spectral
a but
upon the
of ascorbate
of cytochrome ratio.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
thus
in
thereby provided
-a rather
a
than
2. of the
Soret
the
and a regions
of
Vol. 70, No. 4, 1976
ferrocyanide reduced
BIOCHEMICAL
treated
forms
cytochrome
increase
Soret
absorption
could
cause
shifted
in absorption
to the when
the oxidase
when
caused
treated
was first
of dithionite.
the Soret
the ferrocyanide
treated
Similar
a blue sample
absorption directly
then
shift
shift
CO
results
by dithionite
no spectral
in
the dithionite
at 445 nm is
added.
However,
s3 (because
decrease
reduced caused
The increase
with
in
to
sample,
as compared
of ferrocyanide
of
a absorption -3
of cytochrome
of decrease
Addition
the reduction
cytochrome
However,
Ferrocyanide
in the absence
comparison.
to the ferrocyanide
The extent
amount
ferrocyanide.
observed
not only
a significant
at 445 nm was observed.
with
for
at 445 nm was observed.
shift).
oxidase,
and dithionite
included
was due to the reduction
untreated
observed
The oxidized
the oxidized
was added
a spectral
proportional
are
preparation
When dithionite
a further
reduced
also
oxidase.
spectra
to oxidase
a but
430 nm.
cytochrome
of untreated
of ferrocyanide
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in
treated the a region
of about
was further
were
1 nm was
reduced
by
dithionite. The possibility contamination was ruled
of the inhibition
of cyanide out
for
two reasons.
the same inhibition spectra
were
not
In fact,
upon prolonged directly
suggested
that
located
reduction
incubation.
ascorbate
andis
oxidase
a complete
without the
action
between
of untreated
ferrocyanide
reagent.
oxidase
while
increased
upon
site
cytochrome
in
a and a . -3
the
the absorption
the prolonged
significantly.
in the
latter
of s3 was presumably
of ferrocyanide
1120
Secondly,
445 nm absorption
cytochrome
showed
the same conditions,
of a and s3 was reached
involving
solution
in the presence
change
under
The reduction
due to the
ferrocyanide
enzyme was observed of oxygen,
being
recrystallized
No significant
treated
inhibited
in the
inhibited
identical.
in the absence
ferrocyanide
as that
and ferrocyanide
at 445 nm of cyanide incubation
inhibitors First,
constant
of cyanide
of ascorbate
or other
of ferrocyanide
2. is
These
by
results
different
In addition,
case
from cyanide in
the
a region,
BIOCHEMICAL
Vol. 70, No. 4,1976
more blue ferrocyanide
shift
was observed inhibited
ACKNOWLEDGEMENTS--This Institute discussion
of Health. with
Dr.
in
sample research
AND BIOPHYSICAL RESEARCH COMMUNlCAl’lONS
cyanide
upon
the
treated addition
was supported
We acknowledge
oxidase
than
that
of
of dithionite. by the grants
the encouragement
from
from
National
and valuable
T. E. King. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Lemberg, R., Physiol. Rev. (1969) 69, 48-121. Warburg, 0. (1926) Biochem. Z. 177, 471. Keilin, D., Hartree, E. F. (1939) Proc. Roy. Sot (London) M, 167. Nicholls, P., and Chance, B. (1974) In Molecular Mechanism Oxygen Activation (ed. 0. Hayaishi), PP. 479-534, Acad. Press, N. Y. Lemberg, R., and Barrett, J. (1973) Cytochromes pp. 17-57, Acad. Press. N. Y. Jacobs, G. G., Andrews, E. C., and Crane, F. C. (1964) in Oxidases and Related Redox Systems (King, T. E., Mason, H. S., and Morrison, M eds.) Vol. 2, pp. 784-812, Wiley, N. Y. Y;: C. A., Yu, L., and King, T. E. (1975) J. Biol. Chem. m, 13831392. Kuboyama, M., Yong, F. C., and King, T. E. (1972) J. Biol. Chem. 247, 6375-6383. Gornall, A. G., Bardawell, C. J., and David, M. M. (1949) J. Biol. Chem. 177, 751-766. Nicholls, P., Van Buuren, K. J. H., and Van Gelder, B. F. (1972) Biochim. Biophys. Acta 275, 279-287. Horie, S., and Morrison, M. (1964) J. Biol. Chem. 239, 1438-1441.
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