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A new method for the determination of ligand dissociation rate constant of carboxyhemoglobin
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A NEW METHOD FOR THE DETERMINATION
OF LIGAND DISSOCIATION
RATE CONSTANT OF CARBOXYHEMOGLOBIN
V.S.
Sharma;
H.M. Ranney*
J.F. Geibel and T.G. Traylor Departments of Medicine* and Chemistry University of California at San Diego La Jolla, California 92037
Received
August
21,
1975
Microperoxidase binds CO faster and more strongly than does hemoSUMMARY: We have used this observation to determine the overall CO-dissociaglobin. tion rate constants, e, of carbo hemoglobin. The results of our studies P is not very different indicate that the rate constant from the statistically corrected &,, suggesting that, compared to Hb02, cooperativity in the dissociation rate constants of carboxyhemoglobin is greatly reduced. INTRODUCTION Although played
reactions
an important
of gaseous
ligands,
of carbon role
in enhancing
thus
far
(CO>4Hb4 has been studied rate
constants
(CO-Hb) lack Since
and the
have
remained
of a suitable the
rate
the breaking
step-wise constants. the
step:
beyond
that in
of the metal-ligand information
as the reaction
rate
reach binds ligand
regarding
constants,
of the three
rate of existing
CO faster
first
step-wise
constant
mainly
rates
is
and to a lesser
reflected degree,
due to the than
is most
provide
and electronic
of successive
from
dissociation
methods,
dissociation
proceeds
ligand
and more strongly
steric
mechanism
of carboxyhemoglobin
reactions
the
(Hb) have
reaction
dissociation
environment reaction
hemoglobin of the
the dissociation
bond,
The changing ligation
our knowledge
dissociation the
step
(CO) with
The remaining
overall
reagent
the heme pocket. Hb tetramer
only
(1).
limiting
mate and direct
monoxide
Hb. likely
more intienvironment
heme pockets
of the
in the values in the
of
of
overall
Suchxinformation is not available from the kinetic parameters of JL Hb Lv 4 4x4 Hb4L3 + L (= CO, O2 or NO). In fact, the values of x4 and
Vol. 66, No. 4,1975
xi
BIOCHEMICAL
are similar
to the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
corresponding
values
of noncooperative
species
such as
ar and B chains. In this
communication
we present
a method
for
the overall
dissociation
rate
constant
of CO-Hb.
the
dissociation
rate
constant
e
corrected
value
overall
statistically MATERIALS
of
direct
determination
The results
is not
very
of
indicate
different
that
from
the
c,.
ANLI METHODS
CO-dissociation rates were studied by mixing reduced MP with CO-W and following the optical density changes occurring during the reaction: were made on human hemoglobin Hb4(CO) + 4MP+4MPCO + Hb . All studies dialyse 3 overnight against 0. 4 M phosphate buffer of pH 7. The kinetic studies were made at 200C with a Durrum stopped-flow apparatus using a Lun light path cuvette and a tungston light source as described previously (2). CO-Hemoglobin solutions were prepared by mixing calculated amounts of deoxy Hb, and CO solutions in a known volume of deoxygenated buffer. Partially saturated CO-Hb solutions were prepared similarly. The last traces of oxygen were removed by adding a few crystals of dithionite to the syringe containing the CO-Hb solution. Microperoxidase (Sigma MP-11) solutions were prepared by dissolving a weighed amount of MP in buffer and deaerating it thoroughly by bubbling nitroThe reagent was reduced by adding a few crystals of dithionite to the gen. deaerated solution. RESULTS Our preliminary than
deoxy
the
of Hb.
suitability The method
CO-dissociation with
rate
3@l solution
.0175
set-1
for
ml,
m-1:
see discussion
the
results
cent
tested
The reaction
The first
of the reaction
time
plot
time
course.
CO
(ml/m-l
studying
An eight
constant,
and co-workers, with
100 times
fold
kinetic
excess plot
process,
shown
in Figure
set -l
1302
for
which
agreement (3).
Table
of with 1 shows
of NP concentra-
covers
the
of MP was mixed
as can be seen from 1, which
reac-
gave a value
CO-Hb as a function
order
= 2 x lo'),
(Mb),
in excellent .017
faster
CO dissociation
on CO-myoglobin
order
rate
experiments is a first
vs.
for
is known.
by Antonini
of similar
- O.D.,)
was first
the dissociation
MP binds
and more strongly reagent
of CO-Mb.
reported
(O.D.po
that
of the
constant
the value
tion.
indicated
Hb (ml = 2 x 107M-%ec-l)
suggesting tions
studies
the
In
5 to 90 per
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1
CO-dissociation
rate
constants
for
5%
Bb A at 20°,
Rate Constant
O.lM
phosphate
(See-')
buffer,
bm>
3
3
3
.009
416
3
3
6
.Ol
416
3
3
9
.008
416
3
3
25
.008
416
3
3
30
.009
416
1
1
6
.009
423,
80
80
110
.009
556
80
40
110
.009
556
80
20
110
.009
556
Average
1
All
1
The rate was studied, slit
point
(O.lmm
trapolating
slit
was independent
of the
MP-MPCO),
At slit on the
of heme before
- l.Omm) more
423 nm (steep for
inPM
constant
dependence
HbCO),
.017 + .OOl
but was dependent
width
width
are
width width, the kvs.
portion
than
doubled
rates
and were slit
width.
portion
band),
in good agreement
with
the values
1303
The slit
of the Soret not
to zero
slit
for
band).
significantly
width,
in the
416 nm (isobestic
were
curve
reaction
increase
constant.
rates
width
the
the
same at 419 nm (maxima
absorption
so steep
at which
A ten fold
the rate
was the
of the Soret
and 440 nm (not
419
of the wavelength slit
O.lunn and lower,
440
mixing.
on the
reaction
419,
= .009 + .OOl
3-25
concentrations
pH 7.0
dependent
obtained
or the
limiting
by ex-
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t(sec.1
Figure
1:
value
order kinetic plot for the reaction of IQ with KHdT = EOlT = phosphate buffer, pH 7.0. = 30pM.
of ,e obtained
filters
by reducing
(.OOY See-l)It
tion
First O.lM [WI,
should
with
in the
CO.
reaction
(Kodak
the
Gelatin
intensity
filter
be pointed
out
Therefore,
at each pH value
time
that
light
of interest
is
neutral
density
No. 96, ODl,
OD2 and OD3).
pH values
Mp shows a slow
at higher
there
using
CO-Hb at 20°, 3.0 @I;
it
is necessary
no reaction
reac-
to ascertain
between
that
CO and MP.
DISCUSSION In the overall
reaction
of MP with
CO-Hb,
the
following
reactions
ought
to be considered: Hb + CO MP
+
“I?!?
co
-d[HbCOldt I
=
If we make study-state of the reaction,
it
(1)
MPG0
1 [-CO-j
-
approximation
e'[HbCO]
for
can be shown that:
1304
free
CO after
the initial
stages
BIOCHEMICAL
Vol. 66, No. 4, 1975
. . .
the
equation
(III)
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
becomes:
-d [HbCO]
=
1 [HbCOj
- h[Hb]
l
S
09
'dt For CO-dissociation II,
the second
first
from HbCO to be the
term
term.
Under
and independent is
excellent
linearity
jl
than expected
are
of the
on the other
stant
is
only
tion
Roughton
hand,
order
(4)
plots
values
higher =
was independent
the value
are not work
CO-Hb is
greatly
reduced.
rate
constants
dissociation very
different
on the effects
from
those
for for
time constants
suggesting
of the
is
liganded
of 2,3-diphosphoglycerate,
reaction
much higher
of the present rate
large
shift
saturated the
consistent
interin dimer-
saturated Hb.
level with
These of cooperthe
liganded
CO-Hb (6).
Further
and pH on e is
ACKNOWLDGMENT
National
*cooperativity
work
was supported
Institutes
by grants
AM-18348-2
of Health.
in dissociation
rate
constants
1305
idea
CO-Hb
in progress. This
con-
the CO-dissocia-
Partially
temperature
the
no heme-heme
range,
CO from partially
fully
The
3, a value
for
as fully
are
section.
results
expected
view
conditions
are much
to oxyhemoglobin, This
in HbCO
1 4 or they
nM Hb concentration
as compared
that,
to the
dissociation
of Hb concentration.
indicate
I and
order
CO-dissociation
In spite
observations
the
of the
(0.25),
the overall
at the same rate
that
last
The kinetic
CO-Hb (50% and 20%) reacted
ativity*in
in the
Q/y
alone
c (=.009). to 40.0
these
to
that
that
than
That
most
statistically
reactions
be first
of step-wise
estimated
indicate
over
in
as compared
will
given
interpretation.
in 0.5
constant
reaction
considerations
second
step
be negligible
by the data
related
04/4(..008)
equilibria
rate
either
limiting
concentration.
the numerical
slightly
(1,5):
tetramer
the
first
on statistical
study
action
of the
f4.
correctness
conditions
supported
that
!2 and
greater than
strongly
indicates
13,
these
(V) should
of microperoxidase
satisfied
course
of equation
rate
and HL-13581
from
the
Vol. 66, No. 4,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6.
Antonini, E. and Gibson, Q.H. (1960) Biochem. J. 76, 534-538, Sharma, V.S., Noble, R.W. and Ranney, H.M. (1974) J. Mol. Biol. 82, 139-149. Antonini, E, and Brunori, M. (1971) Hemoglobin and Myoglobin in Their Reactions with Ligands (Amsterdam: North Holland Publishing Co.), p. 266, Roughton, F.J.W. (1954) J. Physiol. 126, 359-383. Gibson, Q.H. (1959) Progr. Biophys. Biophys. Chem. 2, 2-53. Brunori, M., Bonaventura, .J., Bonaventura, C., Antonini, E. and Wyman, J. (1972) Proc. Nat. Acad. Sci. USA 69, 868-871.
1306
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A NEW METHOD FOR THE DETERMINATION
OF LIGAND DISSOCIATION
RATE CONSTANT OF CARBOXYHEMOGLOBIN
V.S.
Sharma;
H.M. Ranney*
J.F. Geibel and T.G. Traylor Departments of Medicine* and Chemistry University of California at San Diego La Jolla, California 92037
Received
August
21,
1975
Microperoxidase binds CO faster and more strongly than does hemoSUMMARY: We have used this observation to determine the overall CO-dissociaglobin. tion rate constants, e, of carbo hemoglobin. The results of our studies P is not very different indicate that the rate constant from the statistically corrected &,, suggesting that, compared to Hb02, cooperativity in the dissociation rate constants of carboxyhemoglobin is greatly reduced. INTRODUCTION Although played
reactions
an important
of gaseous
ligands,
of carbon role
in enhancing
thus
far
(CO>4Hb4 has been studied rate
constants
(CO-Hb) lack Since
and the
have
remained
of a suitable the
rate
the breaking
step-wise constants. the
step:
beyond
that in
of the metal-ligand information
as the reaction
rate
reach binds ligand
regarding
constants,
of the three
rate of existing
CO faster
first
step-wise
constant
mainly
rates
is
and to a lesser
reflected degree,
due to the than
is most
provide
and electronic
of successive
from
dissociation
methods,
dissociation
proceeds
ligand
and more strongly
steric
mechanism
of carboxyhemoglobin
reactions
the
(Hb) have
reaction
dissociation
environment reaction
hemoglobin of the
the dissociation
bond,
The changing ligation
our knowledge
dissociation the
step
(CO) with
The remaining
overall
reagent
the heme pocket. Hb tetramer
only
(1).
limiting
mate and direct
monoxide
Hb. likely
more intienvironment
heme pockets
of the
in the values in the
of
of
overall
Suchxinformation is not available from the kinetic parameters of JL Hb Lv 4 4x4 Hb4L3 + L (= CO, O2 or NO). In fact, the values of x4 and
Vol. 66, No. 4,1975
xi
BIOCHEMICAL
are similar
to the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
corresponding
values
of noncooperative
species
such as
ar and B chains. In this
communication
we present
a method
for
the overall
dissociation
rate
constant
of CO-Hb.
the
dissociation
rate
constant
e
corrected
value
overall
statistically MATERIALS
of
direct
determination
The results
is not
very
of
indicate
different
that
from
the
c,.
ANLI METHODS
CO-dissociation rates were studied by mixing reduced MP with CO-W and following the optical density changes occurring during the reaction: were made on human hemoglobin Hb4(CO) + 4MP+4MPCO + Hb . All studies dialyse 3 overnight against 0. 4 M phosphate buffer of pH 7. The kinetic studies were made at 200C with a Durrum stopped-flow apparatus using a Lun light path cuvette and a tungston light source as described previously (2). CO-Hemoglobin solutions were prepared by mixing calculated amounts of deoxy Hb, and CO solutions in a known volume of deoxygenated buffer. Partially saturated CO-Hb solutions were prepared similarly. The last traces of oxygen were removed by adding a few crystals of dithionite to the syringe containing the CO-Hb solution. Microperoxidase (Sigma MP-11) solutions were prepared by dissolving a weighed amount of MP in buffer and deaerating it thoroughly by bubbling nitroThe reagent was reduced by adding a few crystals of dithionite to the gen. deaerated solution. RESULTS Our preliminary than
deoxy
the
of Hb.
suitability The method
CO-dissociation with
rate
3@l solution
.0175
set-1
for
ml,
m-1:
see discussion
the
results
cent
tested
The reaction
The first
of the reaction
time
plot
time
course.
CO
(ml/m-l
studying
An eight
constant,
and co-workers, with
100 times
fold
kinetic
excess plot
process,
shown
in Figure
set -l
1302
for
which
agreement (3).
Table
of with 1 shows
of NP concentra-
covers
the
of MP was mixed
as can be seen from 1, which
reac-
gave a value
CO-Hb as a function
order
= 2 x lo'),
(Mb),
in excellent .017
faster
CO dissociation
on CO-myoglobin
order
rate
experiments is a first
vs.
for
is known.
by Antonini
of similar
- O.D.,)
was first
the dissociation
MP binds
and more strongly reagent
of CO-Mb.
reported
(O.D.po
that
of the
constant
the value
tion.
indicated
Hb (ml = 2 x 107M-%ec-l)
suggesting tions
studies
the
In
5 to 90 per
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1
CO-dissociation
rate
constants
for
5%
Bb A at 20°,
Rate Constant
O.lM
phosphate
(See-')
buffer,
bm>
3
3
3
.009
416
3
3
6
.Ol
416
3
3
9
.008
416
3
3
25
.008
416
3
3
30
.009
416
1
1
6
.009
423,
80
80
110
.009
556
80
40
110
.009
556
80
20
110
.009
556
Average
1
All
1
The rate was studied, slit
point
(O.lmm
trapolating
slit
was independent
of the
MP-MPCO),
At slit on the
of heme before
- l.Omm) more
423 nm (steep for
inPM
constant
dependence
HbCO),
.017 + .OOl
but was dependent
width
width
are
width width, the kvs.
portion
than
doubled
rates
and were slit
width.
portion
band),
in good agreement
with
the values
1303
The slit
of the Soret not
to zero
slit
for
band).
significantly
width,
in the
416 nm (isobestic
were
curve
reaction
increase
constant.
rates
width
the
the
same at 419 nm (maxima
absorption
so steep
at which
A ten fold
the rate
was the
of the Soret
and 440 nm (not
419
of the wavelength slit
O.lunn and lower,
440
mixing.
on the
reaction
419,
= .009 + .OOl
3-25
concentrations
pH 7.0
dependent
obtained
or the
limiting
by ex-
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t(sec.1
Figure
1:
value
order kinetic plot for the reaction of IQ with KHdT = EOlT = phosphate buffer, pH 7.0. = 30pM.
of ,e obtained
filters
by reducing
(.OOY See-l)It
tion
First O.lM [WI,
should
with
in the
CO.
reaction
(Kodak
the
Gelatin
intensity
filter
be pointed
out
Therefore,
at each pH value
time
that
light
of interest
is
neutral
density
No. 96, ODl,
OD2 and OD3).
pH values
Mp shows a slow
at higher
there
using
CO-Hb at 20°, 3.0 @I;
it
is necessary
no reaction
reac-
to ascertain
between
that
CO and MP.
DISCUSSION In the overall
reaction
of MP with
CO-Hb,
the
following
reactions
ought
to be considered: Hb + CO MP
+
“I?!?
co
-d[HbCOldt I
=
If we make study-state of the reaction,
it
(1)
MPG0
1 [-CO-j
-
approximation
e'[HbCO]
for
can be shown that:
1304
free
CO after
the initial
stages
BIOCHEMICAL
Vol. 66, No. 4, 1975
. . .
the
equation
(III)
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
becomes:
-d [HbCO]
=
1 [HbCOj
- h[Hb]
l
S
09
'dt For CO-dissociation II,
the second
first
from HbCO to be the
term
term.
Under
and independent is
excellent
linearity
jl
than expected
are
of the
on the other
stant
is
only
tion
Roughton
hand,
order
(4)
plots
values
higher =
was independent
the value
are not work
CO-Hb is
greatly
reduced.
rate
constants
dissociation very
different
on the effects
from
those
for for
time constants
suggesting
of the
is
liganded
of 2,3-diphosphoglycerate,
reaction
much higher
of the present rate
large
shift
saturated the
consistent
interin dimer-
saturated Hb.
level with
These of cooperthe
liganded
CO-Hb (6).
Further
and pH on e is
ACKNOWLDGMENT
National
*cooperativity
work
was supported
Institutes
by grants
AM-18348-2
of Health.
in dissociation
rate
constants
1305
idea
CO-Hb
in progress. This
con-
the CO-dissocia-
Partially
temperature
the
no heme-heme
range,
CO from partially
fully
The
3, a value
for
as fully
are
section.
results
expected
view
conditions
are much
to oxyhemoglobin, This
in HbCO
1 4 or they
nM Hb concentration
as compared
that,
to the
dissociation
of Hb concentration.
indicate
I and
order
CO-dissociation
In spite
observations
the
of the
(0.25),
the overall
at the same rate
that
last
The kinetic
CO-Hb (50% and 20%) reacted
ativity*in
in the
Q/y
alone
c (=.009). to 40.0
these
to
that
that
than
That
most
statistically
reactions
be first
of step-wise
estimated
indicate
over
in
as compared
will
given
interpretation.
in 0.5
constant
reaction
considerations
second
step
be negligible
by the data
related
04/4(..008)
equilibria
rate
either
limiting
concentration.
the numerical
slightly
(1,5):
tetramer
the
first
on statistical
study
action
of the
f4.
correctness
conditions
supported
that
!2 and
greater than
strongly
indicates
13,
these
(V) should
of microperoxidase
satisfied
course
of equation
rate
and HL-13581
from
the
Vol. 66, No. 4,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6.
Antonini, E. and Gibson, Q.H. (1960) Biochem. J. 76, 534-538, Sharma, V.S., Noble, R.W. and Ranney, H.M. (1974) J. Mol. Biol. 82, 139-149. Antonini, E, and Brunori, M. (1971) Hemoglobin and Myoglobin in Their Reactions with Ligands (Amsterdam: North Holland Publishing Co.), p. 266, Roughton, F.J.W. (1954) J. Physiol. 126, 359-383. Gibson, Q.H. (1959) Progr. Biophys. Biophys. Chem. 2, 2-53. Brunori, M., Bonaventura, .J., Bonaventura, C., Antonini, E. and Wyman, J. (1972) Proc. Nat. Acad. Sci. USA 69, 868-871.
1306