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BY
(From
JAMES
the Chemical
Laboratory
LOUIS
AND
of Harvard
for publication,
F. FIESER.
University,
October
Cambridge.)
28, 1924.)
(Received
B. CONANT
Methemoglobin
one hydrogen equivalent is required
Na&04
+ 2MHb
-+
in the reaction:
2Hb
+ 2NaHSOa
The experimental
procedure
for the electrometric
titration
was similar
to that
described
in our earlier
paper
except
that a general
method
of
operating
was developed
which
could
be employed
with
solutions
containing
mixtures
of reduced
hemoglobin
and methemoglobin.
The titration vessel of 50 cc. capacity
was provided
with a mechanical
stirrer,
a
nitrogen
inlet and water-sealed
outlet,
a bright
platinum
and platinized
platinum
electrode,
a siphon
to a saturated
calomel
electrode,
and a
burette-tip
inlet.
Pure atmospheric
nitrogen
was obtained
by passing
commercial
compressed
gas through
three
Friedrich
wash
bottles
containing
alkaline
anthrahydroquinone
sulfonic
acid-sodium
hyposulfite
solution.
A rapid
stream
of gas so purified
was without
effect
on the
Comparison
iF;0
0s
$*
z&
z-4
cc.
8.88
5.31
5.31
3.495
3.497
0.13
5.1
5.1
5.80
5.65
2.96
2.88
13.79
3 77
10.02
6.00
6.00
0.494
0.480
0.12
{ 0.10
4.55
7.14
7.26
6.45
4.50
4 48
2.94
3.21
3.26
13.00
13.78
3.58
1.27
1.28
5.64
5.70
5.70
0.521
0.545
0.554
0.13
10.58
13.10
3.27
2.65
3.46
3.47
13.78
2.59
11.19
6.70
6.70
0.516
0.517
0.15
11.99
12.11
3.44
3.37
4.13
4.08
13.77
1.33
12.45
7.45
7.45
0.554
0.549
0.08
i 0.10
7.39
8.27
8.31
4.10
4.25
4.14
3.03
3.52
3.44
14.11
14.19
14.11
4.18
3.76
3.70
9.93
10.43
10.41
5.94
6.25
6.25
0.485
0.564
0.550
0.10
8.87
8.70
4.07
4.02
3.61
3.50
14.12
3.16
10.96
6.56
6.56
0.550
0.533
0.10
9.02
10.01
10.22
3.64
4.23
4.07
3.28
4.23
4.16
14.12
14.12
3.64
1.21
1.32
10.48
12.91
12.90
6.34
7.74
7.74
0.518
0.546
0.539
G-2
1
G-2
{ 0.13
0.13
10.61
3.69
Using anthrahydroquinone
.instead
of Na&04;
cordingly.
0.10
8.3
4.30
7.98
4.13
/m. per
100ce.
1.91
G-l
G-2
m. per
00 cc.
13.79
G-2
!z
rm. pm
zoocc.
2.68
2.64
G-2
G-2
nols
q ::
s
6.10
5.85
F-l
G-2
G-2
ix
e .
k
-2
4.4
4.5
G-l
cc.
$
a
,ms
0.13
15.64
2.57
9.42
9 51
9.50
3.92
14.19
1.25
12.94
7.75
0.506
disulfonate
as the reducing
agent
headings
of columns
to be read
ac-
3.57
3.30
14.12
2.80
11.32
6.80
6.80
0.525
0.485
4.03
14.12
1.56
12.56
7.52
0.535
(0.10
$G
F-.,0
'rn
-22
az
F4
its Deter-
;;
3
r!
5
5
X
F-3
G-l
I.
of the Electrometric
Titrations
of Methemoglobin
and
mination
EY the Method
of Van Slyke and Stadie.
a
.$
F-l
597
Methemoglobin
Since
in
these
experiments
we
were
not
interested
in
the
poten-
but only the change of E.M.F. corresponding to the end-point, it was found best to add small increments of
reducing agent (about 0.2 cc.) rather rapidly, noting the potential
tials
during
after
each
the
titration
addition
and
continuing
the
addition
of
hyposulfite
potential
of a mixture
of indigo
disulfonate
and its reduction
product
or
upon
the color
of the completely
reduced
solution.
The solutions
of
hemoglobin
employed
were prepared
by laking
the corpuscular
cream
of
horse blood
with
about
twice
the volume
of water
and centrifuging
to
remove
the small
amount
of sediment;
different
samples
of. corpuscular
cream are denoted
by letters,
and successive
lakings,
by numbers,
throughout this paper.
In the experiments
listed
in Table
I, varying
proportions
of the total
hemoglobin
were
converted
to methemoglobin
by adding
small
amounts
of saturated
ferricyanide
solution
to 30 cc. of the hemoglobin
solution
contained
in a tonometer.
After
evacuation
and shaking
for 5 minutes
to insure complete
reaction,
a sample
of known
volume
was
withdrawn,
equilibrated
with the oxygen
of the air by shaking
in a tonometer for 15 minutes,
and the oxygen
capacity
then determined
in a Van
Slyke
apparatus
(2).
The remainder
of the solution
was then completely
freed from oxygen
by evacuating
the tonometer
and filling
with nitrogen
and repeating
this process
eight to ten times.
A 10 cc. sample
was then
pipetted
from the tip of the tonometer
and quickly
transferred
to the titration vessel which
contained
10 cc. of a phosphate
buffer
solution
(pH 6.8)
and had previously
been swept
out with a rapid
stream
of nitrogen
for
10 minutes.
The stream
of nitrogen
was continued
for 5 to 10 minutes
more
and the electrometric
titration
with
sodium
hyposulfite
was then
commenced.
The sodium
hyposulfite
solution
(about
0.01 N) was prepared
before
a determination
by dissolving
0.2 gm. of the solid in 100 cc.
of water,
containing
0.5 gm. of sodium
carbonate.
It was protected
in
the burette
with
a layer
of xylene
and it was standardized
by rapidly
titrating
electrometrically
a 10 cc. sample
of 0.01 N KaFe(CN)B
solution
immediately
after each determination.
Since a standardization
could
be
completed
in 10 minutes
after
the hemoglobin
titration,
an error
due to
the deterioration
of the solution
was not serious,
although
it was found
that the reducing
equivalent
of the hyposulfite
solution
so prepared
fell
off about
3 per cent per hour.
J. B. Conant
and L. F. Fieser
599
determination
of methemoglobin a not very satisfactory
analytical
procedure as compared with Stadies method and the one given in
the following paper.
It should be mentioned in this connection
that the electrometric titration of reduced hemoglobin with potassium ferricyanide (cf. previous paper (1) ) is even more difficult to
carry out with accuracy and is therefore unsuitable as an analytical method.
II.
Oxidation-Reduction
was prepared
in the usual
manner
(7).
horse
blood,
thrice
washed
with
hyperwas laked with the least possible
amount
Methemoglobin
600
The concentration
of the methemoglobin
solution was determined by a method which is given in detail in the following paper
and the validity of which has been carefully demonstrated.
The
solution employed contained 6.15 (~kO.02) per cent of methemoglobin. Carefully measured amounts of this solution and a 10.66
(-10.05) per cent solution of freshly prepared hemoglobin solution (from the same horse blood) were mixed in such proportions
that three solutions resulted, having the following composition.
Solution.
Hemoglobin
cont,ent.
mJZ8 per 1.
A
B
1.06
2.12
4.24
x 10-z
x 10-z
X 1O-3
Methemoglobin
content.
mozs per 2.
3.07
2.45
1.23
x
X
IO-3
lo+
10-s
of water.
To 500 cc. of the warm
concentrated
solution,
1.5 gm. of potassium ferricyanide
were added and the solution
was stirred
until
the reaction had become
complete.
The dark
solution
was cooled
to 0 and
0.25 volume
of 95 per cent alcohol,
also cooled
to O, was added
with
vigorous
stirring.
After
standing
for 2 days in a well packed
salt-ice
mixture,
a copious
crop of minute
crystals
had appeared.
These
were
separated
from the mother
liquor
by centrifuging
and immediately
dissolved
in distilled
water
at 37.5.
The crystallization
was repeated
and
the thick
paste
separated
by the centrifuge
was dissolved
in water
without
further
removal
of mother
liquor
by filtration.
The solution
so
obtained
(400 cc.) was clear reddish
brown
in color and no solid residue
was detected
on filtering.
It undoubtedly
contained
small amounts
of
ferricyanide
or its reduction
product
and of alcohol;
these
impurities
had no noticeable
influence,
however,
and presumably
would
not influence
our results.
601
KH,PO,
HaBOa
HaBOa
+ NaHP04
..................
+ borax .......................
+ NaOH
.....................
6.42
8.44
9.45
2.5
0.833
1.306
Added
to
30 cc. of hemoglobin
solution.
cc.
1.25
4.20
2.30
In carrying
out an experiment
30 cc. of a given mixture
(A or B or C)
were measured
into a tonometer,
the buffer
solution
was added,
and the
tonometer
tightly
sealed with
a rubber
stopper,
coated
with
collodion.
After
evacuating
and filling
with pure nitrogen
ten times,
the tip of the
tonometer
was connected
with a tube projecting
through
the stopper
of
a 4 oz. bottle
which served
as the electrode
vessel.
It was equipped
with
a sealed nitrogen
outlet,
a bright
and a gold-plated
platinum
electrode,
and a siphon
tube containing
saturated
potassium
chloride
solution.
The
side tube of the tonometer
was connected
with
the nitrogen
supply
and
the stop-cock
turned
so as to connect
the side tube with the tip and allow
nitrogen
to enter the electrode
vessel through
the tube which extended
to
the bottom
of the bottle.
A second
similarly
equipped
vessel was connected
in series with the first.
After
sweeping
out the two bottles
with
a rapid stream
of nitrogen
for 4 to 1 hour, half of the solution
was run into
the first vessel under
a pressure
of nitrogen,
which
could be regenerated
by placing
the vessel again in connection
with the nitrogen
supply.
The
inlet
and outlet
tubes were then closed with
pinch-cocks,
the tonometer
was removed
and carefully
connected
with
the second
vessel
without
introducing
any air.
After
a few minutes
additional
sweeping,
this bottle
was filled with solution.
In a few cases nitrogen
was allowed
to bubble
through
the solution
in one of the bottles
for 10 to 15 minutes
but no
difference
in the potentials
due to this additional
precaution
could
be
detected.
Measurements
were made at 25 f 0.05 against
the saturated
calomel
electrode.
The potentials
on both
the bright
platinum
and the goldplated
electrodes
were read every
5 to 10 minutes
for periods
varying
from
4 to 2 hours.
In this period
of time a fair degree
of constancy
had been
reached
in each case.
The first few readings
were usually
erratic
and
were discarded.
After
that
the potential
in some cases became
quite
PH
Methemoglobin
602
Time.
a (Gold).
9r (Pt).
Remarks.
min.
-0.157
-0.155
-0.158
-0.167
Shaken.
10
-0.150
-0.153
-0.155
-0.158
Quiet.
Shaken.
20
-0.145
-0.148
-0.156
-0.159
Quiet.
Shaken.
30
-0.139
-0.144
-0.156
-0.163
Quiet.
Shaken.
40
-0.143
-0.149
-0.150
-0.156
Quiet.
Shaken.
50
-0.139
-0.144
-0.155
-0.155
Quiet.
Shaken.
constant,
the readings
on both electrodes
agreed,
and were
not altered
by vigorous
shaking;
in other
cases the constancy
and agreement
were
not so good
and shaking
altered
the potential.
In the latter
event,
however,
it was perfectly
apparent
when
the initial
drift,
due, presumably,
to the establishment
of equilibrium
at the liquid
junction
and
to the attainment
of the temperature
of the thermostat,
had ceased
and
when no further
change was taking
place.
Consequently,
from five to ten
readings
on each electrode
for the quiet and the shaken
solution
were recorded at regular
intervals
during
the period
of this state of relative
constancy
and the average
of these was taken
as the potential
of the cell.
Thus,
for example,
the following
cell potentials
were recorded
in Experiment
18.
603
[MHbl
(1)
%-observed
rfl
o.osg
I%
,Hbl
Oxidation-Reduction
II.
Potentials
and MethemoT
-0.059
1%
PH
Electrode
potentials
referred
to the normal
hydrogen
electrode.
LHbl
pn
(Column
Column
7+
4)
ro%
(oxygenated).
~volts
23
24
11
12
17
18
A
A
B
B
c
c
6.4{
volts
volts
volts
-0.027+0.146+0.151+0.148
-0.027 +0.145+0.150/+0.147
-0.0041+0.119
+0.146+0.134
,
'
-0.004+0.115
+0.139 +0.130
+0.032+0.107
+0.135+0.120
+0.032+0.086+0.110,+0.098
Average....................................]
25
26
19
20
7
8
13
14
' -0.027+0.048
+0.049+0.048
A
,4
-0.027 +0.048 +0.051+0.049
-0.027,+0.049
+0.061+0.053
A
-0.027+0.040+0.059
+0.050
A
8.41f
-0.004+0.068+0.086+0.077
B
-0.004.t0.051f0.063
$0.060
B
+0.032 +0.012+0.038+0.024
c
c
,+0.032+0.008+0.022
$0.015
Average ....................................
uo1t.s
volts
$0.121
$0.120
+0.130
+0.126
+0.152
+0.130
+0.233
t-O.231
-to.209
+0.196
+0.227
+0.209
+0.130
+0.217
$0.021'1
+0.020
$0.026
+0.023
+0.073
+0.056
+0.056
+0.047
+0.204
f0.046
(?)
g:;;$
(?)
+O. 196I
+o. 192
+0.187
+0.189
+0.193
+0.193
+0.184
$0.164
$0.174
to.165
+0.176
+0.156
Average ....................................
+0.006
+0.164
globin
of Mixtures
of Hemoglobin
at Various
pH Values
(SW).
Methemoglobin
604
+0.500
K31
t
3
g
---_
--S_
7e (CN),-KJe
&
EN&
----.
+0.300
8
$ +0200
s
d
F to.100
s
3
3
ox 0.000
-0.100
pH+
10
11
12
FIG.
1. Oxidation-reduction
potentials
of K3Fe(CN)s-K4Fe(CN)e
and
Hb-MHb.
The curves
showing
the changes
of normal
oxidation-reduction potential
with hydrogen
ion concentration
are as marked;
the crosses
represent
experimental
points
given
in this paper,
the circles,
previous
determinations
by the titration
method.
The broken
line AB is the
estimated
true Hb-MHb
potential
curve
at 735 mm. of 02; the curve
rG2
gives
the observed
potentials
of Hb-MHb
mixtures
saturated
with
oxygen.
+0.400
IConant
(l),
p.
411.
with the values given in the first paper (1) which were obtained by
the titration
method.
This is illustrated by Fig. 1 in which the
present determinations
and the earlier ones are both plotted.
In
one respect it is necessary to modify the earlier views; the sharp
drop in potential between pH 8.5 and 9.8, which was indicated by
the first measurements,
has not been confirmed.
Instead, the
change of potential with change in pH seems to extend gradually
over the whole range. Not too much significance can be attached
to the exact magnitude of this change, however, since the activity
of the hemoglobin and methemoglobin
is undoubtedly
greatly
affected by the salt concentration
as well as by the hydrogen ion
concentration
and in our present work no data are available to
enable us to distinguish between these two effects of the buffer
solution.
Thus, while we believe that the oxidation-reduction
potential of methemoglobin
in any solution of definite pH is
probably fairly close to the value corresponding
to the curve in
Fig. 1, the exact slope of. this particular
curve is probably not
very significant.
The general downward
trend indicates that
methemoglobin
is a weaker acid than hemoglobin, but instead of
only 1 acidic hydrogen atom being involved as first suggested1
there are probably several acid groups functioning in this range.
Passing oxygen or carbon monoxide into a mixture of hemoglobin and methemoglobin was shown in the previous paper (1) to
increase the oxidation-reduction
potential.
The reason is evident
from a consideration of equation (1) ; if the term [Hbl is greatly
diminished by combination of the hemoglobin with the gas (formation of oxy- or carboxyhemoglobin)
and [MHb] stays unchanged,
the potential will increase.
Hemoglobin
combines with either
oxygen or carbon monoxide, methemoglobin
does not; hence,
saturating the solution with one of these gases changes the potential greatly.
The values given in the last column of Table II
(ao,) were obtained by passing oxygen into the electrode vessel
after the determinations
in the absence of oxygen were complete.
The vessels were shaken and the flow of oxygen was continued
until a constant potential was reached which is recorded in the
table.
The position
of these potentials
is indicated
in Fig. 1.
Undoubtedly these values are much lower than the true potential
606
Methemoglobin
III.
Action of Ferricyanide
on Hemoglobin
Xolutions.
Initial
Hb
concentration.
gm. per 100 CC.
14.16
13.78 I Average
14.10
14.01
14.01 i
(By extrapolation,
Average*..................
III.
of Hemoglobin
by Varying
Amounts
Pres&ce
of 6, (755 Mm.)
(t = 25 * 3).
Final
Hb
concentration.
&Fe(CN)e
equivalents 9 m.
g m per 100 cc
0.138
0.200
0.276
0.300
0.368
0.368
0 552
1.104
4.1.
. ...
0.50
0.73
1.00
1.09
1.33
1.33
2 00
4.00
of KsFe(CN)s
in
Fraction
(
1Sb CO~YBI
into MHI
(RI
0.365
0.554
0.754
0.792
0.837
0.826
0.875
0.892
0.64
0.26
0.11
0.10
0.12
0.09
0.11
0.27
per100ee.
8.89
6.25
3.44
2.91
2.30
2.46
1.77
1.51
13.5for
...............
..............................
The Hb content
was determined
in the usual
way
capacity
as measured
in the Van Slyke
apparatus;
the
every
case equilibrated
with
oxygen
and the apparent
corrected
for dissolved
oxygen
at 735 mm. of pressure.
* Omitting
the first value.
1.00)
0.15
from
the oxygen
solutions
were in
oxygen
capacity
TABLE
Extent
608
Methemoglobin
Hemoglobin
+ K3Fe(CN)e
methemoglobin
+ K4Fe(CN)e
= [K,Fe(CN)&
[K,Fe(CN)d
= [MHb]
- [MHbl
[MHb] = R [Hbli
(3)
K = (E - R) (1 - RI F
R2
,,=g=
(E-R)
F
609
potential
of K8Fe(CN)6)
= 0.059
log
lFe(CN)~llll [Hblt,t,t
[MHbl
= 0.059log
K = 0.372
Methemoglobin
(A B), parallel to the observed potentials of the oxygenated hemoglobin mixtures, has been drawn.
This curve, we believe, represents approximately
the true oxidation-reduction potential at 73.5
mm. of pressure of oxygen of the hemoglobin-methemoglobinsystem,
and its position determinesthe behavior of such a systemwhen treated
with oxidixing agents.
The downward trend of this curve with increasing alkalinity is
of importance and is verified by a few experiments we have performed in buffered solutions which are recorded in Table IV. In
each experiment the hemoglobin solution employed was prepared
by hemolysis of twice washed fresh corpuscular cream (horse).
The requisite amount of concentrated buffer solution (previously
described) to give a resulting 0.1 M solution was diluted with the
hemoglobin solution to 50 cc. After determining the oxygen
capacity of this buffered solution, a 10 cc. sample was treated with
a definite amount of solid potassium ferricyanide and shaken in
vacua. The solution was then equilibrated with oxygen at atmospheric pressure and the oxygen capacity again determined. It
will be noted that the amount of oxidation per equivalent of
ferricyanide at pH 6.42 is about the same as in the experiments
with the unbuffered solution given in Table III.
In the more
alkaline buffers, however, much more complete oxidation takes
place. This would be predicted from a potential curve corresponding to Al? in Fig. 1; the more nlka.line the solution the
greater the difference in potential between the ferricyanide potential (which is essentially independent of the pH) and the hemoglobin potential (7&, and the farther the reaction proceeds.
The facts represented graphically in Fig. 1 explain the success
of the Haldane method of determining the oxygen capacity of
hemoglobin. Although the amount of ferricyanide employed in
this analytical procedure usually corresponds to only about two
equivalents, the reaction liberates practically all the oxygen
because it is carried out either at a very low oxygen pressure
(Van Slyke method) or in alkaline solution. Thus in the original
Haldane procedure (lo), sodium carbonate was added, and
ammonia has often been similarly employed; in the Van Slyke
procedure, sodium hydroxide is often added to absorb the carbon
dioxide. The addition of any of these substances would make the
solution at least as alkaline as pH 9.5, and under these conditions
J. B. Conant
and L. F. Fieser
611
TABLE
Oxidation
of Hemoglobin
Experiment No.
Solution.
IV.
by Ferricyanide
in Buffer
Solutions
of Oxygen
at 755 mm. (25).
PH
Percentage
Initial.
in the Presence
Hb.
P&er adding
1GFe(CN)a.
equivalents
per cent
2
3
4
5
J-l
J-l
J-2
K-l
K-l
6.42
6.42
6.42
6.42
6.42
11.25
11.25
9.04
10.87
10.87
2.49
0.77
1.99
3.27
2.49
0.992
4.71
1.0
1.0
2.0
77.8
91.2
77.9
65.8
77.2
6
7
8
K-l
K-l
K-l
8.44
8.44
8.44
11.10
11.47
11.47
0.98
0.53
0.34
2.0
1.17
1.55
91.3
95.4
97.2
K-l
9.45
11.27
0.14
2.0
99.0
612
Methemoglobin
613
From equation
[Hb] = F [total
[MHb]
n-735= Tn + 0.059 log F ,total Hbl
Hb],
we have,
Since by definition
[MHb]
= [total Hb]
(5)
Substituting
- 0.059 log F
log F = -4.5
F = 3 X
1O-6
Hills equation,
(6)
and, therefore,
(7)
Y
log 1-Y
= log KI
+ n log z
when y = 0.5
logK1
--nlogz
614
Methemoglobin
blood2
one can estimate
that
x = 8 mm. when
y = 50 per cent for unbuffered
horse hemoglobin
at 25.
If n = 2.5 (a value
Hill has found
to
hold for a number
of cases), log K1 = 2.25.
Of course,
this is only a rough
approximation,
but will serve to show whether
our results
are of the right
order
of magnitude
as compared
with
the actual
measurements
of the
oxygen
dissociation
curves
of horse oxyhemoglobin.
It is evident
that
since F = 1 - y and, for very
small values
of F, 1 - F may be taken
as
equal to I without
serious
error,
equation
(6) becomes
(8)
log+=
-logF=logKi+nlogz
log
(9)
where
x1 = pressure
at 50 per cent saturation
and ~2 = pressure
at which
the fraction
uncombined.
Solving
this with
the data
just
given
(log F = -4.5 from our measurements,
x2 = 8 mm., estimated
from
Ferrys
results),
we find n = 2.28.
The agreement
between
this and the
values Hill has found for n (from 2.2 to 2.5) is merely
another
way of showing the entire
agreement
between
these apparently
very distantly
related
observations.
F represents
and
Hill
(17),
Fig.
4.
Substituting
-2.25
for log K,, n = 2.5, and x = 735, we find log F = 4.93:
or F is practically
10M5. The agreement
with the value
calculated
from
our measurements
(3 X lo-&) is all that could
be expected
and is gratifying
evidence
of the correctness
of our interpretations.
In connection
with
the validity
of Hills
equation
over
a very
wide
range
of oxygen
pressures,
it is important
to note that if n = 1, the divergence
between
F calculated
from
the dissociation
curves
and our
Y
measurements
would
be enormous.
If the simple
formula
= K1
1-Y
were a correct
expression
of the behavior
of the oxyhemoglobin-hemoglobin-oxygen
system,
our
results
would
be quite
inexplicable
since
1~1~~ would
then be expected
to be about
0.1 volt below the value we have
found;
the ferricyanide
reaction
then should
go to practical
completion,
even in a neutral
solution
saturated
with
oxygen.
It is worth
while
pointing
out, perhaps,
that
measurements
like ours of the equilibrium
between
an oxidizing
agent
(such as ferricyanide)
and hemoglobin
under
definite
conditions
enables
one to calculate
n directly.
Combining
equations
(7) and (S), we have
615
IV.
Structure
The relationship
been expressed
of Methemoglobin
between
by
the
methemoglobin
Hb /O
\d
or its
and hemoglobin
has
Hb /O
\J)
is the reduced hemoglobin molecule
molecule.
Although for the past
investigators
(in particular
Kiister
the formula HbOH, most texts stil!
following
and Hemoglobin.
equivalent
different
and very
symbols:
recently
Roaf
and
616
Methemoglobin
617
(NH,) Ht
G]
(Pr)
Hemoglobin
.,Fe (OOC)
(ferro
H.[
(02)
(Pr)rFe(OOC)
compound).
q
-G
Oxyhemoglobin.
(NH,)
Hz
[ (Pr)4Fe(OOC)
Methemoglobin
(ferri
compound).
The symbol (Pr) stands for the four pyrrole residues found in
hematin minus 4 hydrogen atoms (i.e. 4 pyrrole ions) and G for
the globin molecule of which one carboxyl group and one amino
group are indicated.
The negative valence of the complex ion
would be three in the ferro compound, hemoglobin, as 5 negative
ions (4 pyrrole and 1 carboxyl ion) are involved in the complex.
Besides the acidic hydrogen atoms which are thus pictured as
being necessary consequences of the formation
of the complex
ion, there are, of course, others in the large globin molecule and
probably one or two joined to the pyrrole nuclei. These and the
free amino groups of the protein are not represented.
In very
alkaline solution the value of x: in Na,(Hb)
would be the sum of
the 3 hydrogen atoms depicted above and the others just mentioned; at the isoelectric point the 3 hydrogen atoms would be
undissociated
and probably would be taken up by three amino
groups in the protein molecule; isoelectric hemoglobin, from this
point of view, would be represented
rather by an inner salt
formula than by that given above. Such a formula might be
written:
NH2
(Pr),Fe(OOC)
NHs+
/
G-NHa+
\NH$
Methemoglobin
618
Acid
Hemoglobin..
Oxyhemoglobin.
Methemoglobin.
... . .
.
.
.
..
.. . .
solution.
(HsHbH)
(H,HbO,H)
(H,MHbH)
+
+
+
Isoelectric.
Alkaline.
Hs(Hb)
Ha(Hb0.J
H,(MHb)
Na,(Hb)
NasHbOz
NazMHb
610
620
Methemoglobin
(a) 2H,(HbOz)+2H,(MHb)
+ 02 + (HzOd))
(b) 2H,(Hb)
+ Oz+2H,(MHb)
+ (HzOz
(?))
(c) BH,(Hb)
+ 2H3(HbOa)+iHz(MHb)
+ 2(H202
(?))
We hope to publish later some results concerning the various factors affecting the decomposition
of hemoglobin solutions in the
presence of oxygen.
We may state here in advance that the evidence is very clear that in many cases of the formation of methemoglobin, equation (a) does not fit the observed facts and the choice
lies between (b) and (c). There is every reason to believe this to
be true in general and, therefore, strictly speaking, the problem is
not the decomposition
of oxyhemoglobin,
but the oxidation of
hemoglobin by oxygen or by oxyhemoglobin.
Roaf and Smarts
and Quagliariellos
results, therefore, must be interpreted
on this
basis. We have suggested above a probable explanation in terms
of equation (b) ; a similar scheme, if oxyhemoglobin
is the oxidizing agent, is represented by (c).
We are indebted to the Cyrus M. Warren Fund of the American
Academy of Arts and Sciences for a grant which has helped defray
the expenses of this investigation.
SUMMARY.
1. The electrometric
titration
of methemoglobin
with sodium
hyposulfite has been compared with the Van Slyke and Stadie
methods
of determining
methemoglobin.
The results
afford
additional evidence that one hydrogen equivalent is involved in
the change from methemoglobin to hemoglobin.
BIBLIOGRAPHY.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Conant,
J. B., J. Biol. Chem.,
1923, lvii, 401.
Van Slyke,
D. D., and Neill,
J. M., J. Biol.
Chem.,
1924, lxi, 523.
Van Slyke,
D. D., Proc. Nat. Acad. SC., 1921, vii, 229.
Stadie,
W. C., J. Biol.
Chem.,
1920, xii, 237.
Van Slyke,
D. D., and
Stadie,
W. C., J. Biol.
Chem.,
1921, xlix, 32.
Peters,
R. A., J. Physiol.,
1912, xliv,
131.
Conant,
J. B., and Lutz,
R. E., J. Am. Chem. SOL, 1923, XIV, 1047;
1924, xlvi,
1254.
Htifner,
G., and Otto,
J., Z. physiol.
Chem.,
1882-83,
vii, 65. Jiiderholm,
A., 2. Biol.,
1884, xx, 419.
Henderson,
L. J., J. BioZ. Chem.,
1920, xii, 401.
Van Slyke,
D. D.,
Hastings,
A. B., Heidelberger,
M., and Neill,
J. M., J. Biol. Chem.,
1922, liv, 505.
Bancroft,
J., and Camis,
M., J. Physiol.,
1909-10,
xxxix,
118.
Kolthoff,
I. M., 2. anorg.
u. allg. Chem.,
1920, cx, 143.
Haldane,
J., J. Phykol.,
1897-98,
xxii,
298.
von Zeynek,
R., Arch.
Physiol.,
1899, 460.
von Reinbold,
B., 2. physiol.
Ciiem.,
1913, lxxxv,
250.
Wertheimer,
R., Biochem.
Z., 1920, cvi, 12.
2. The oxidation-reduction
potentials of mixtures of hemoglobin
and methemoglobin
have been measured.
The results are in
general agreement with those predicted from the electrochemical
equations and the values obtained by the titration method.
3. The reaction between potassium ferricyanide and hemoglobin
solutions has been shown to be reversible, proceeding to a definite
end-point dependent on the hydrogen ion concentration
and the
In alkaline solutions or at
oxygen pressure above the solution.
low oxygen pressures the reaction between equimolecular amounts
is practically complete; in neutral solutions saturated with oxygen
it is only about 80 per cent complete.
The results are in accord
with our electrochemical formulation of the hemoglobin-methemoglobin system and Hills equation for the oxyhemoglobin-hemoglobin-oxygen
system; such results afford a drastic test for an
equation of this type.
4. It is shown that the behavior of hemoglobin, methemoglobin,
and oxyhemoglobin
is in accord with a complex ion formula previously suggested.
The discrepancies
between our electrometric
titrations and the results of two recent investigations
on the formation of methemoglobin
by acids are discussed and a probable
explanation of the apparent contradiction
is given.
622
14.
15.
16.
17.
18.
19.
20.
21.
22.
Methemoglobin
ARTICLE:
METHEMOGLOBIN
James B. Conant and Louis F. Fieser
J. Biol. Chem. 1925, 62:595-622.