CHEMICAL NATURE OF SPECIFIC OXYGEN CAPACITY IN HJEMOGLOBIN. BY RUDOLPH A. PETERS,
Scholar of Gonville and Gaius College, Cambridge.
(From the Physiological Laboratory, Cambridge.)
HISTORICAL AND CRITICAL.
Evidence for chemical combination of oxygen with ha?moglobin. Upon
general principles, it has been assumed by the majority of physiologists
that oxygen combines chemically with the iron of the molecule, and
that haemoglobin is essentially identioal for different species, at any rate
as regards the nature of its union with oxygen. In support of this view
of chemical combination, Huifner (), working with the spectroscope,
considered that he had demonstrated by his curves and by his extinction
coefficient relations (i.e. E1/E) that heemoglobin was identical in
different animals, existing as one substance throughout the higher
animals as regards its union with oxygen. Later, however, although
Gamgee () accepted Hiifn er's results, spectrophotometric methods
were to a certain extent discredited, owing to difficulty of accurate
observation, although Hiifner and his pupils extended the work later,
disproving the objections to spectrophotometric methods (i a). Meanwhile, indirect evidence accumulated in favour of chemical combination.
In 1904, Laidlaw3), extending the observations of Hoppe-Seyler,
showed that, whereas it was easy to split off the iron from reduced
haemoglobin, strong acids were required to remove it from oxyhemoglobin, that is to say, the oxygen had fixed the iron to the molecule,
probably by a chemical union. More recently, Barcroft working with
several observers has given considerable additional evidence; thus
Barcroft and Camis(4) showed that the differences in the dissociation
curves of different animals were due to variations in the salt content,
and that essentially the same curve could be obtained from dialysed
solutions of the haemoglobin of different animals, in this manner
indicating the probable existence of one heemoglobin; again, Barcroft
132
R. A. PETERS.
and Roberts(s) proved that a curve given by a completely dialysed
solution of heemoglobin approximated very closely to that deduced by
HIufner from theoretical considerations of a simple chemical mass
action; Barcroft and King(6) working upon the alteration of the
dissociation curve with temperature, gave strong evidence for the
reaction being of a chemical and not a physical nature, owing to the
enormous change produced by temperature; this was subsequently
confirmed by Barcroft and Hill (7) who were able to calculate a value
for the molecular weight of the haemoglobin molecule, approximating to
those obtained by chemical means from the heat of combination
observed. Again, Doug]as(s) showed that the relationship of oxygen
capacity of blood to its depth of colour was not materially disturbed
during regeneration following repeated heemorrhage in rabbits, thus
supporting the view that at least the hbmatin portion of htemoglobin is
the same for the same individual at different times. More recently,
Butterfield(24) and Masing and Siebeck(25) working with improved
spectrophotometric methods have given important evidence in favour
of a chemical relation of oxygen to iron in different animals and under
different pathological conditions, Butterfield by analogy with carbon
monoxide combination and the other observers by determining the
oxygen combined per gramme of hemoglobin estimated spectrophotometrically.
Evidence against chemical combination. Whereas the above facts
would seem to form overwhelming evidence in favour of a chemical
theory, those who have tried to obtain a value for the relation of oxygen
to iron in haemoglobin by direct methods have met with wide variations
in their results. Hoppe-Seyler(9) believed the evidence in favour of
the view that haemoglobins varied, and that there existed in the corpuscles
two substances, "arterin " in the arteries, and " phlebin " in the veins;
suibsequent evidence showed that his proofs were not tenable (see
Gamgee(10)). The question was investigated later by Bohr (i), who, so
far from establishing a definite relationship found the ratio extremnely
inconstant, and introduced the term " Specific Oxygen Capacity," which
he defined as "the ratio of comibined oxygen in cubic centimetres at
normal temperature and pressure to the iron in grams in a given amount
of a solution of haemoglobin formed from laked blood or a solution of
heomoglobin crystals, which had been saturated with air at 150 C."
Although Hufner had ascribed his variations to impurity of solutions
investigated, Bohr continued to believe in the accuracy of his results,
and held that the " Specific oxygen capacity varied in different animal
HAEMOGLOBIN.
133
species, in different animals of the same species, and in the same
individual at different times," a view which was supported subsequently
by Bornstein and Miillero2). In order to explain his experimental
results, Bohr believed in the existence of a series of oxyhaemoglobins
a, $, 'y, 8 with various specific oxygen capacities (throuigholut the
remainder of the paper specific oxygen capacity will be written
Sp.O.C.), the ratios obtained being due to various admixtures of these
substances. In addition, he and others working under him, believed
that the Sp.O.C. changed as the blood passed from the artery to the
vein; as their experiments did not show a constant variation, they were
forced to the conclusion that the variation was regulated by the needs
of the tissues. The view that the Sp.O.C. changed in the same animal
at different times they supported further by experiments upon bleeding,
by which a variation in Sp.O.C. was found upon regeneration following
a saline injection. The whole was considered by Bohr to be an
indication of the fact that the body could adjust its oxygen supply
according to the actual tissue needs. A considerable amount of
evidence is to be found in the pathological literature upon the subject,
which it is not possible to discuss in this paper (see Krauz, Kohler
and Schulz(26)).
Evidence for adsorption. Before giving the results of the direct
analyses made by these observers, mention must be made of Ostwald(1s)
who has suggested, and of Manchot(so) who considers, that be has
obtained proof of the view that oxygen is adsorbed by the hamoglobin
molecule, a view which can be fitted to the dissociation curves obtained
by the various observers.
Direct determinations. From theoretical considerations, if the combination is
Fe 02 in the reaction Hb + 02 ;±HbO2,
Fe + O,°2
then
22-394 1 =409
Sp. 0. = 02: Fe -1 1molecular
molecularvol.Fe02 =55-85
Therefore, if the Sp.o.0. is above the value 401, excluding errors
due to experiment, the hypothesis of simple chemical combination is
not tenable.
On the other hand, if the Sp.o.0. has a value below 401, excluding
experimental error, it may be concluded either that (a) haemoglobin is
not a simple substance, but a mixture (as Bohr believed), or (b) that the
1
This value is given by the most recent determinations of the density of oxygen.
R. A. PETERS.
134
combination is not chemical, or (c) that if chemical, the hemoglobin
under investigation is either (a) not pure (that is it contains small
quantities of substances such as inethaemoglobin, which would diminish
the 02 value) or (,8) not fully saturated.
The following are the values wbich have actually been obtained for
mammals:
Animal
Number of cases
Limits of ratio
...
...
...
Dog
22
17
32
...
Pig
Horse
Cat
328-468
378-429
301-391
(& 450)
284-401
379-426
372-403
Observer
...
Bohr(14)
Tobiesen P5) ...
Abrahamson (1")
it
Ox
Bohr(")
...
...
Bornstein and Miller(12)
5
9
5
Mean Sp.O.C.
375
388
351
341
411
401.21
(& 458 3)
Masing and Siebeck(25)
Man
Ox
Rabbit j
Ox
Butterfield (24)
,,
Average
3
(diseased) 11
Approx.
3972
389-395
3972
3912
3992
)Man (healthy)
385-409
(& 429)
Bohr(ii) also quotes a value for birds, 16 individuals, the blood of
which was divided into five parts and analysed. Sp.O.C. = 356-344,
mean 348. Again in one case upon Delphinus communis Sp.O.C.
= 413. For rabbits, he gives a value of 389.
The values for Sp.O.C. found directly by these observers seem at
first conclusive evidence against the chemical hypothesis, for Bohr,
Tobiesen, Abrahamson, and Muller have all obtained values 5 0/0
above the theoretical ratio 401. Therefore before passing to the results
of recent experiment, it is necessary to show that such an experimental
error was possible under the old methods of determination.
In the estimation of a ratio, errors may be cumulative or may divide
out in the calculation. Thus, supposing the ratio observed to be x, and
further supposing that there is a 1 0/o error + for x, and - for y, the
lOla, 100 101 /x\
resulting fraction would appear as °o-o x -9-Y 0-9 j) . That is the l ob
error has beea cunmulative, and appeared as 2 0/o. In this manner an
1 Born t ein and Muller 's results are for 380C. and not normal pressure (see p. 136).
These results have been calculated to correspond with above table. Butterfield' 8
are determined for CO and are therefore by analogy only.
2
HiEMOGLOBIN.
135
error of 3 0/0 upon each determination might appear as 6 0/0, that is with
an error of 3 0/0 in the iron and oxygen determinations respectively, the
ratio if theoretical might vary from 377-425.
That an error of this magnitude should have been present is not
improbable, wben the older methods of analysis are considered. The
method of oxygen estimation by the gas-pump was lengthy and admitted
of extremely few determinations, so that an error of 3 0/0 is probably a
low estimate. The difficulty of accurate determination of the absorption
coefficient for oxygen is also very great.
With regard to the iron estimations, it is noticeable that those
of Bohr and Tobiesen were performed by titration with potassium
perinanganate in the presence of chlorides, which, as it is known now,
interfere seriously with the titration. These observers did not make as
a rule duplicate determinations, except where they considered that the
results required confirmation; it is difficult also to obtain zinc absolutely
free from iron; and so taking into account the fact that the iron
estimations have to be performed upon between 30 and 50 milligrams,
and the method was not a good one, it is probable that the error in
the iron estimations was between 2 and 3 0/o at the least.
It is therefore possible to explain any value up to 425, and probably
up to 430, as due to experimental error. This however would not
explain those obtained above 430. By reference to the original papers
of Bohr(14) and Tobiesen(15), it can be seen that the former observer,
out of the 22 cases upon normal blood, has only 3 over 401 (viz. Sp. 0.
= 468, 437, 435), and in the whole paper including animtals under conditions of anaemia etc. between 80 and 90 estimations, Bohr only obtains
two more figures (namely Sp. 0. = 461, 426), which are above 420, except
for 549, which he marks with an asterisk. It therefore seems probable
that the figures 468, 437, 435 are due to errors, which were overlooked
possibly in the course of estimation; further, this is supported by evidence
from the experiments of Tobiesen, because he has obtained values within
much narrower limits (viz. Sp.O. 378-429 in 17 individuals, 429 being
the only value above 419). Therefore it seems probable that the few
high figures of Bohr may have been due to the difficulties of analysis
by the earliest methods.
The next observer Abrahamson (n) has only obtained one figure
450 over 391 in 32 cases, which would again indicate a possible experimental error. Bohr's figures for the horse would be within the
experimental error. Lastly, the work of Bornstein and Miillerwl
gives one value 458-3, which reduces to 402 when corrected for
136
R. A. PETERS.
temperature', so that there are no figures remaining as material
evidence against chemical combination.
Bornstein and Muller further conclude with Bohr that the
Sp.O.C. varies in the same individual at different times. They quote
experiments on two cats. In one case, the reduced value for the first
bleeding was 402, and for two subsequent bleedings 351, and 353; and
in the other, for a first 351 and a second 353; an interval of a fortnight
was allowed to elapse between the two times of drawing blood, and
the deficiency after the first was made up with isotonic solution. If
anything, there is a slight fall of Sp.O. but the difference is very small,
and would not seem to warrant the conclusion drawn from the observation. Bohr and Tobiesen have a number of experiments to establish
the same fact, some by bleeding and some by analysing the arterial and
venous blood separately. If the variations given by them are examined,
it will be seen that they are not constant in direction or amount, and
do not seem to warrant the views which Bohr held, chief among which
was the view that the Sp.O.C. changed according to the needs of the
animal. Further the inconstancy of the change was explained by the
varying tissue wants. Accordinig to the same observer, the result was
brought about by variation of the amount of a, /3, ty, 8 hsemoglobin
respectively. Arguing that a separation of these might to a certain
extent occur in the corpuscles, Haldane and Lorraine Smith(21)
determined the Sp.O.C. of centrifuged corpuscles, where they found
variations, but these again were not constant in direction.
With reference to the more recent work of Butterfield(d4) and
Masing and Siebeck (s), which has been carried out with all the
accuracy that improved spectrophotometric methods would permit, the
former observer has estimated the constancy of the chemical combination
of carbon monoxide with hoemoglobin from this arguing by analogy for
oxygen; the amount of haemoalobin was determined by spectrophotometric methods, and the CO absorbed, as reckoned per gm. of
haemoglobin, varies from 1P30-1-35 for the various animals; the iron
content varies from *0326-0-342 0/0; the final ratio CO/Fe varies from
386-428 in 11 cases of pathological condition, and although the average
399 is remarkable, a variation of 10°/o is large for the limits of the
average. Again Masing and Siebeck have investigated most carefully
the ratio of oxygen combined to iron content, but have obtained their
final result of Fe 02 by averaging the iron content per gm. of
1 As there was doubt as to whether the figures were calculated for 380 or 00, reference
was made to the writers themselves, who stated that the figures were calculated for 380 C.
HLEMOGLOBIN.
137
haemoglobin for a varying number of animals (ox, rabbit, goose, and also
for man) and taking the mean 0295 0/0 of (029-030 o/0) their last table,
whereas their total variation was 027-030 0/0.
The average 0295 0/0 is considerably lower than that of Butterfield
0335 0/0, and is explained as being due to difference in coefficients for
the spectrophotometer. Zino ffsky and others have obtained O034 0/0.
The iron average is compared with an average obtained from oxygen
determinations, which vary from 1i13-1-20 c.c., and hence the relation
02 to Fe is found to be that of chemical combination. Althouigh the
final result is close to the theoretical, approximately 397, the variations,
of which the average is taken, are considerable.
The absence of satisfactory direct determinations of 02 to Fe
in individual samples of haemoglobin solutions and the difficulty of
satisfactory correlation of spectrophotometric results make it desirable
that the work of Bohr and his followers should be done again with
methods of analysis of iron and oxygen, allowing a much higher degree
of accuracy. The method will be described.
METHODS.
The final technique of the method is described best by giving a full
account of the last experiment (Exp. 7), with notes as to the development of the means of estimation employed'.
Preparation of haimoglobin solution. Defibrinated sheep's blood was
collected and centrifuged to remove the serum without delay; the
corpuscles were reserved and washed twice with isotonic salt solution, in
order to obtain as pure a suspension of blood corpuscles as was possible2.
To the corpuscular suspension prepared in this manner was added twice
its volume of *5 o/0 NH. solution, so that 1 c.c. of corpuscles was laked
by 2 c.c. of NH, solution. This solution (A) was centrifuged again to
remove any unlaked elements or corpuscular debris, which might interfere
with the oxygen determination.
1 In every case the analysis was made by comparison of the results obtained by
estimating the amounts of oxygen and iron in different portions of the same homogeneous
hetmoglobin solution.
2 In the Exps. 1 and 2, .9 gm. of the corpuscular suspension was weighed into the
bottles of the Barcroft and Roberts differential apparatus, which were laked by making
up to 3 c.c.s with NH, solution; the suspension was similarly weighed, with the due
precaution of a thorough shaking, into the platinum crucible for iron estimation; in the
case of the cat, however, it was found impossible to obtain complete laking in this manner,
so that the method described below was adopted in the later experiments.
138
R. A. PETERS.
Oxygen determination. The determination of the oxygen was performed in the Barcroft and Roberts improved differential apparatusl6,
in which the amount of oxygen combined is estimated by removal with
potassium ferricyanide. A was kept in ice until the estimation upon
the following day when it was tboroughly shaken with air from' 10-15
minutes to obtain as full a saturation as was possible.
Owing to the fact that the apparatus constants were calculated for
3 c.c. respectively in each bottle, amounts of 3 c.c. were measured as
accurately as was possible with the dense solution into the apparatus
bottles from a 50 c.c. burette', the whole amount being drawn off without
levelling between each 3 c.c. In all 16 determinations were made
(-48 c.c.), two separate pairs for each apparatus. By this means the
error upon the whole 48 c.c. will be small, although that on each
estimation may be large for the following reason.
It was not found possible to read with certainty to more than 1 c.c.,
owing to density of solution and the difficulty of avoiding parallax, so
that the difference between individual amounts placed in the bottles
might be 2-9-3-1 c.c. or 6 0/0; an average of the whole 48 c.c. would
only show a difference of 48 1-47 9 c.c. or *5 0/0 as an error of measurement. The total error for the estimation in the cases where a large
number have been performed cannot be more than 1-5 0/02.
The following table gives the actual figures obtained in Exp. 7.
Temp. 15° C.
Barometer 748 mm.
(Owing to the fact that the amount of gas affected by temperature
is that in the differential tubes, the temperature taken was that of the
air surrounding the tubes, and not of the water in the bath in which the
bottles were immersed, as this stood at constant temperature throughout
the experiment.)
Columns I and II give the differences of pressure in millimetres
observed for the separate pairs of individual experiments. The bottles
were washed out between I and II, and II forms a completelv new set
of observations.
Column III gives the constant of the apparatus (C). This is
calculated according to the new formula(li), but in addition it has been
1 The solution was considered pure, if at this stage there was evidence of complete
laking, if the solution was of a good colour and showed no opacity. It was never allowed
to stand for more than 18 hours, in the cold, before determination of the blood gases.
2 This is shown clearly in Table A below. The separate estimations have been
grouped into amounts a, f, %y 8, and again into a + p, and y +B. In this manner the
differences between amounts of 12 c.c. and of 24 c.c. are not more than 1 0/0.
HEMOGLOBIN.
139
found necessary for greater accuracy to recalculate the constant for
each barometric pressure. (In an ordinary blood-gas experiment this
is not necessary as the values are not absolute, but comparative.)
TABLE A.
I
100
93
99 25
II
100
91
98
23
24
97 5
107-5
109-5
99 25
106
105-5
III
3667
3708
3509
3544
3327
3327
30
114-5
113-75
31
106-0
108-5
Bottlenumber
15
16
19
t20
a
IV
V
(.3667
( 3667
*3374
3449
j 3482
1 3455
*3576
-3527
*3640
3510
3177
'y *3639
3260
*3455
3614
.3537
*3439
3517
j3 1l3997
1-4188
2-8185
Average for 3 c.c.= 3534 c.c.
a 1-4053
y 1-4309
2-8362
a
Taking the constant of 15 as an example, the calculation for the barometric pressure
is performed as follows:
Formula for calculation of true amount of gas y, where y' is observed amount, is
y=y' x C, where C=-+A.
A refers to the diameter of the tubing and is unaffected so far as the experiment is
concerned. V however, the volume of the bottle and tubing, varies according to the value
of p, which is the pressure calculated in millimetres of clove oil (760 mm. Hg = 9774 mm.
clove oil)'.
In this case, with Bar .=748 mm. Hg, V=23&25, A=1-25 sq. mm.
V= 2-417.
p
C= (2-417 + 1-250) = 3667.
of
method
calculating the difference in practice is given below (p. 140).
A simpler
Columns IV and V give the true amount of gas calculated by
multiplying Column I by the constant to give Column IV, and
Column II by the constant to give Column V.
For the sake of comparison the Columns IV and V have been added
up in groups to show that the variation of 12 c.c. of A is very small.
Average for 3 c.c. = .3534 c.c.
Reducing to N.T.P. = -3298 c.c.
In 1 c.c. amount of oxygen united with haemoglobin
= 1099 c.c. 02.
1 Calculated by finding the specific gravity of the clove oil used in the experiments.
140
R. A. PETERS.
A simpler method of finding the difference due to atmospheric
Reduction FormulaI.
changes is as follows:
In the formula C= (p + A)() for a small change of P to P+ 8P, the change in C is
p~~~~
5C= p ap= -__ ap,
so that the new value of C is (p + A - js 5P), where P is any standard value, which here
is taken to be normal. In reducing the gas to normal pressure this has to be multiplied by
P+p P,ie (+p)
Hence multiplying and neglecting
comes to
(8p ),
which is of order of 1/1600, the product
8
-pA+AP
=
+
+A5P
p
p-C.......................
(1).
p is a constant for any given bottle, so that the small changes in the constant due to
a given change 8P in the barometric pressure can be allowed for by adding to the constant
a
value
7'60
,
where A
is the area of the tubing.
N.B. After this correction it is not necessary to oorrect the gas volume for pressure.
The only further correction needed is one for temperature.
Estimation of iron. After a trial of several methods, the new process
of estimating iron by titanium salts was adopted(is). An account of
the preparation of the standard solutions is unnecessary, as complete
details are given in New Methods of Volumetic Analysis(m7).
The following general equation represents the reaction
TiCI3 + FeCI3 = TiCl4 + FeCl2.
The method is of great value in the estimation of iron in ashed
corpuscles, because the end-point, determined by a saturated solution
of potassium sulphocyanide, is very clearly defined and is unaffected by
the presence of chlorides, which interfere with the permanganate
titration.
Exp. 7 was performed in duplicate in the following manner.
50 c.c. of A were evaporated carefully in a platinum crucible, and
cautiously ashed. In order to exclude the possibility of contamination
with extraneous iron, the evaporation and ashing were carried out
between two asbestos sheets, the lower of which had a circle cut out of
the hard asbestos, leaving three arms stretching from its circumference
to support the crucible. The ashing was carried out at a low temperature
until the fumes of nitrogenous matter were removed; at this stage the
ash, a hard black mass, was transferred to a muffle furnace in which
1 For the following calculation, I am indebted to Mr A. V. Hill.
IIHMOGLOBIN.
141
the carbon was burnt off in a current of air at the lowest possible
temperature'. It is this that constitutes the vital part of the process.
If the carbon is not completely burnt away, iron will still remain which
cannot be removed from the carbon by boiling with acids; whereas on
the other hand, if the ash is heated to a high temperature in removing
the carbon, the oxide of iron becomes insoluble. In both of these cases,
the result will be a loss of iron, and consequent raising of the value
obtained for the ratios.
In practice, it was found advisable to burn off the greater part of
the carbon at a low red heat, and then make a preliminary extract with
the strong HCI, filtering through an iron-free filter paper to keep back
unburnt carbon. This filter paper was then transferred to the crucible
with any remaining carbon to be reheated. By this means it was easier
to remove the whole of the carbon at a lower temperature.
When the ashing was carried out in this manner, the iron was easily
soluble in a small quantity of strong H Cl, and was transferred to a flask,
where it was neutralised with ammonia. In order to oxidise any iron
that may have become reduced in the process of ashing, one drop of a
solution of hydrogen peroxide was added at this stage, and the ferric
solution boiled for ten minutes to destroy any excess of peroxide. When
cold, the indicator, a saturated solution of potassium sulphocyanide, was
added in excess of the amount required to produce the deep-red colouration. The titration was carried out with the titanous chloride solution
until all the ferric iron was reduced to the ferrous condition, as indicated
by loss of colour.
In Exp. 7 the amounts of titanous chloride solution used were
(a) 7.75
ca
(b) 7-68
(Standardisation of the TiC13 was performed for each experiment by titrating against
10 c.c. of a standard solution of ferric iron, containing per c.c. solution *003571
gms. Fe.
Three such determinations gave for 10 c.c. of ferric solution, 19-2, 19-25, 19 2 c.c. TiC13.
Previous determinations had shown 19-2 c.c., which was therefore taken to equal *03571
gm. Fe.)
1 In this connection, I should like to express my thanks to Mr C. T. Heycock,
Director of the Metallurgical Department, Cambridge Chemical Laboratory. Not only
did he recommend to me the titanium process, but gave me the assistance of his valuable
advice throughout the experiments, finally placing the metallurgical muffle furnace at my
disposal.
2 It is interesting to note that before the use of the muffle furnace, the values obtained
were all high, viz. 429, 436, 404, 408, 422.
PH. XLIV.
10
R. A. PETERS.
142
Thus for
x 100-'O22gis
cotis775 x -03571
002882
(a) 1 c.c. Hb solution A contains
197 2 5x0
(b)
,,
(b),,
',,,
",,
Average of
7-68 x 03571 x 100 = 027gis
002857 gms
x50
~~~~19'2
}002882 = -002869 gms.
Difference under 1 0/0 of the figures.
Exp. VII. Determination of Ratio. In 1 c.c. of solution A, Oxygen= 1099 c.a.
Iron = -002869 gms.
*1099
Oxygen in c.c. =002869
Iron in gms.
RESULTS AND DISCUSSION.
For details of experiments, the reader is referred to the appendix.
The general results will be given here. The iron determinations were
always in duplicate.
Number
of exp.
Animal
I
II
Ox
Ox
III
IV
V
VI
VII
Cat
Sheep
Ox
Pig
Sheep
Number of oxygen
determinations
Average 02
reduced
Average
iron
Ratio
6
31-39 c.c.
*0798 gms.
393.3
5
400
32'15
'08025
(The experiments I and II were carried out by the original method of weighing
corpuscles (vide note on p. 137). The oxygen and iron are therefore per 100 c.c. solution.)
The remaining figures represent the amounts in 1 c.c. solution.
4
12
14
9
16
*0751 c.c.
*1073 (6)
*1102
*1146
*1101
'001814 gms.
*002784
'002767
-002869
*002962
393'8
385'7
398x2
387-0
883-1
Iron estimations. The differences in the two iron determinations
are, in I and II 2i°/oX in III '5°/0, IV under I°/o, V under 1/e, VI 1°/e,
VII under 1 /0.
Therefore in the last five experiments the iron estimation is probably
accurate to 1%/o. The two earlier experiments show a difference of 210/o,
so that as the highest is probably more correct, the value should be
slightly lower.
The experiments increased in accuracy from I to VII, the last
experiments being the most exact.
It has been previously shown that the error upon the oxygen
determinations, where as many as 10-16 have been made, is not more
than 1 e/o. In those in which fewer estimations were made, the error
may be slightly greater, but, even when that due to calculation of the
HLtXMOGLOBIN.
143
constant is taken into consideration, the total gas analysis error cannot
exceed 20/0 at the most. The iron estimation for the last five experiments has been shown to be within 1 0/o. At a high computation,
therefore, the total error in the ratio cannot exceed 3 0/0 in the last five
experiments, perhaps slightly more in the first two. Since this 30/0 has
to be taken upon either side of the ratio, the figures should not show
a difference of more than 6 0/0 between the lowest and highest, that is
should lie between 389-423, if the true Sp. 0.0. = 401, and if the
hemoglobin was pure and fully saturated.
An allowance must be made for oxygen dissolved in the solution
owing to the high pressures observed in the differential apparatus,
which has been interpreted as an addition of one to the value given for
the ratio.
Sp. 0. C. Tabulated according to the various animals.
Ox
393-3'
Cat
Sheep
400 -=397'2+1=398.
398-2
=393-8+1=395.
385-7 = 3844 + 1 = 385.
=387 +1=388.
Pig
Average of 7 animals=392+1=sea.
The values obtained for the Sp.O.C. of these four animals in seven
cases lie between 401-385, so that there is a difference of 40/, between
the lowest and highest figures, therefore it is justifiable to average them
in view of the experimental error of the analysis. Firstly, there are no
figures above the value required for the theory of chemical combination.
Secondly, the average 393 is about 20/o lower than 400 9 the theoretical
value. And thirdly, the experiments increased in accuracy, the last
three being the most satisfactory estimations performed, the last two of
which gave low results. This may be due either to want of complete
saturation', following the ordinary laws of balanced reactions, or to the
presence of breakdown products in the blood of the normal animal,
because the iron dissolved in the plasma is too small in amount to
affect the determination, if any exists. That rnethsemoglobin can exist
in the body during health as the result of various conditions is a wellknown fact, and it is probable that in this lie the results obtained by
1 Care was taken to shake thoroughly, so as to obtain complete saturation, which the
curves of B arc ro ft and King(6) show that it is possible to obtain at the temperature at
which the experiments were carried out.
10-2
144
R. A. PETERS.
observers upon regeneration. If the infusion of salt solutions into the
arteries and veins was not quite isotonic, there might be some decomposition products which had not disappeared before the following
bleeding. Apart from this conjecture, it must be assumed that the
discrepancies obtained by Bohr and others were due to length of
oxygen estimation, and to difficulty of completely ashing and estimating
iron. This explanation has been given by Aron(27), and the presence of
methmmoglobin bands in normal blood observed by Butterfield(%).
The evidence quoted above leaves no grounds for believing that the
Sp.O.C. varies in individuals of different species, different individuals
of the sanme species, or the same individual at different times. From
Bohr's view of the regulation of the oxygen supply to the tissue needs,
this is no longer necessary, because what he considered at first to be
effected by varying Sp.O.C. has been shown by Bohr, Hasselbach
and Krogh(23) and by Barcroft and Orbeli(22) to be accomplished
by means of acids. The former observers, by showing the effect of
carbon di-oxide, and the latter the effect of lactic acid upon the
dissociation curve of haemoglobin, have given direct evidence of a
means by which the tissues regulate their oxygen supply.
The results of the foregoing experiments justify the conclusion that
in the ox, cat, sheep and pig the haemoglobin is identical as regards its
iron-containing part. Further, since the experiments of Bohr(i on the
hsemoglobin of the horse, rabbit, birds and fishes, and of B u tterfield(24
and Masing and Siebeck(2) for men and other animals as has been
seen, give results closely corresponding with the theoretical, taking
into account the errors inherent in their methods, it may be concluded
that hamoglobin is identical as regards its iron-containing part throughout vertebrates. The fact that the three latter observers, in their
determinations of the extinction coefficient relations for a variety of
animals under diseased and healthy conditions, obtained variations only
between 1 55-1-63, when compared with the similarity of the spectroscopic characters of haemoglobin leads to the additional conclusion that
this identity applies to all the animals which contain the pigment
quoted by Gamgee(io).
Taken in conjunction with Laidlaw's(3) experiments, the fact that
the relation oxygen to iron is chemical would seem to indicate that
it is to the iron portion of the molecule that the oxygen is attached.
HLEMOGLOBIN.
140-
SUMMARY AND CONCLUSIONS.
The paper deals with a redetermination of the relation of oxygen
combined with hbemoglobin to the iron-containiiig part of the molecule,
known as "specific oxygen capacity." This capacity has been investigated
in hbamoglobin solutions formed by laking washed corpuscles.
New methods of analysis have been employed, the oxygen capacity
having been estimated by the differential apparatus, and the iron by the
titanium method.
The values obtained are independent of the concentration of haemoglobin and of a knowledge of the absorption coefficient for oxygen, and
therefore of spectrophotometric results.
The conclusions are as follows:
1. In a solution of haemoglobin of the ox, sheep, pig and cat, the
value obtained for the ratio oxygen to iron is essentially the same and
agrees within experimental error with the value required upon a hypoFe 02.
thesis of chemical combination Fe + 02
2. The ratio does not vary in the same individual at different
times.
3. The oxygen is attached to the iron-containing part of the
molecule as has been argued by Laidlaw and others.
4. The results obtained by previous observers combined with those
given in this paper may be taken as showing that haemoglobin, at any
rate as regards its iron-containing part, is identical throughout vertebrates.
This conclusion has been reached by Butterfield and Masing and
Siebeck working with different methods.
Besides the thanks which I have expressed already to Mr C. T.
Heycock, Goldsmith Reader in Metallurgy, I should like to express my
warmest gratitude to Mr Barcroft for his kind help throughout the
research, both for recommending the subject to me, and for help in the
oxygen estimation, several determinations of which he has done himself
in person. My thanks are also due to Dr Hopkins for much helpful
criticism.
REFERENCES.
(1) Hiifner, G. Arch. f. Anat. u. Physiol. p. 130. 1894.
(la) Hufner and Kuster. Arch. f. Anat. u. Physiol. Suppl. S. 387. 1904. See
also other paper by Huifner.
(2) Gamgee. ' OnHoemoglobin.' Schiafer's Text-book of Physiology, I. p. 185. 1898.
(3) Laidlaw. Journ. of Physiol. xxxi. p. 465. 1904.
(4) Barcroft and Camis. Ibid. xxxix. p. 374. 1909.
146
R. A. PETERS.
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
Barcroft and Roberts. Journ. of Physiol. XXIi. p. 143. 1909.
Barcroft and King. Ibid. xxxix. p. 374. 1909.
Bareroft and Hill. Ibid. xxxrx. p. 411. 1909.
Douglas. Ibid. xxxix. p. 453. 1909.
Hoppe-Seyler. Ztsch. f. physiol. Chem. xiiI. S. 477. 1889.
Gamgee. Schafer's Text-book, I. p. 186.
Bohr. For a full discussion vide Nagel's Handbuch, I. p. 93. 1909.
Bornstein and Miller. Arch. f. Anat. u. Physiol. p. 478. 1907.
Ostwald, W. Biochem. Ctrlb. p. 386. 1908.
Bohr. Skan. Arch. iII. p. 101. 1892.
Tobiesen. Skan. Arch. f. Physiol. vi. p. 273. 1895.
Bareroft and Roberts. Journ. of Physiol. xxxrx. p. 429. 1909.
Kneckt and Huppert. " New Methods of Volumetric Analysis."
F. Jahn. Ztsch. Physiol. Chemie, p. 308. 1911.
Barcroft and Higgins. Journ. of Physiol. XLII. p. 512. 1911.
W. Man chot. Liebig's Annalen, pp. 369, 370. 1909.
Haldane and Lorraine Smith. Journ. of Physiol. xvi. 1894.
Barcroft and Orbeli. Ibid. XLI. p. 355. 1910.
Bohr, Hasselbach and Krogh. Skan. Archiv. f. Physiol.
B utterfield. Ztsch. f. physiol. Chem. LXII. p. 173. 1909.
Masing. Deut. Arch. f. klin. Med. xcviii. p. 123. 1909.
Masing and Siebeck. Ibid. xcix. p. 130. 1910.
(26) Kraus, Kohler and Schulz. Arch. f. exp. Path. und Pharm. XLII.
(27) Aron, H. Biochem. Ztsch. III. 810.
APPENDIX.
In each case the blood was defibrinated and kept in the cold. It was centrifuged,
washed twice with isotonic salt solution, and either as in I and II the corpuscles kept and
weighed, or as in the remainder, the corpuscles laked with twice the volume of ammonia,
and then again centrifuged.
Exp. VII has been described in detail in the text.
Exp. I. Ox'8 blood. Defibrinated. Corpuscular suspension. Bar. 762-5. Temp. 16° C.
Oxygen estimaticDn.
Bottle
mm. o bserved
Constant
8
15
71L 5
6(D 5
[email protected]
3828
3859
3452
3485
3615
3656
19
20
15
16
Iron
Weight in bottle
In 100 gi ns.
*806 gm.
33-96 c.c.
-700 (6)
33.33
*671 (7)
32-12
-570 (8)
32-36
6,2)
*653 (7)
34-28
6) L5
*690
32-59
Reducing to N.T.P. 31-39 c.c. °2 in 100 gms. sol.
estimation. (-03571 gm. Fe= 19-3 c.c. TiC13.)
Average
33-11 c.c.
Iron in 100 c.c.
(a) 25-874 gms. ashed in Pt crucible required of TiC13 soln. 11 3 c.c.
,,
11-15 it
it
it
(P8) 26,178 gms. ,,
Average in 100 c.c. = *0798 gm. Fe.
. . Ratio = 393 3.
*0808 gm.
*0788 ,,
e/0 dff. 2J0/0.
HLEMOGLOBIN.
147
-Exp. II. Ox's blood. Corpuscular suspension weighed. Bar. 744. Temp. 15" C.
Oxygen estimation.
Bottle
19
20
15
16
8
-rved
mm. obse
Constant
Weight in bottle
In 1.00 gms.
3524
*5728 gm.
3 4'45
3558
*5522
3679
*6084
313'57
53 5
3721
*5691
14'98
64
3908
*6994
315'76
Reduced to N.T.P. 32'15 c.c. 02 in 100 gms.
56
53 15
55''5
14347
C
c.
Average
34'64 c.c.
C
Iron estimation. (-03571 gm. Fe=19'2 c.c. TiCl3.)
(a) 25'85 (8) gms. ashed in Pt crucible required of TiC13 11 0 c.c. In 100 gms. *0791 gm. Fe.
,,
,,
( 2581 (5)
,,
,,
,
11'3 ,,
08141
Diff. = 2 010.
Average *08025 gm. Fe per 100 gms. suspension.
Ratio = 400.
Exp. III. Cat. Corpuscular suspension laked with twice volume of ammonia, and
centrifuged again. Blood was taken from two cats which were pithed.
Oxygen estimation.
Bottle
19
20
15
16
Bar. 751-5 mm. Temp. 15° C.
mm. observed
Constant
Amount of soln.
In 3 c.c. soiln.
'2288
3 c.c.
3493
'2320
Average
3527
3'05
'2286 in 3 c.c.
3
*2264
3652
62
-3
*2271
61'5
3693
In 1 c.c. '07619 (6) c.c. Reduced for N.T.P. '07541 c.c. °2'
65'5
67
Iron estimation. ('03571 gm. Fe=19'2 c.c. TiCl3.)
Two amnounts of 45 c.c. ashed in separate crucibles, gave upon titration with TiCl3
'
4 '40
4°
c.c.
Whence Fe= 001810} 1 c.c. = *001814 gm. Fe. Difference = '5
l0o
Ratio =393'8.
This experiment was the last of three experiments upon cats. The first two were done
by the former method, and the corpuscles were not properly laked, the solution appearing
opaque; the values of 371 and 381 were obtained in these two experiments, but were discredited owing to insufficient laking, and the above method adopted for the third, which
was subsequently adopted for the remaining experiments.
148
R. A. PETERS.
ExP. IV. Sheep. Bar. 767. Temp. 170 C. By later method of laking in bulk with
ammonia.
Oxygen estimation. (Three separate sets of estimations.)
Bottle
C (a)
First
Second
Third
C (f)
(b)
(c)
(a)
15
99
98
98-75
-3564
*3539
-3528 + Average
3600
3651
16
92-25 91-5
92-5
*3334 for 3 c.c.
-3361
3643
3691
-3352
19
96-5
93
95-25
-3265 (-3196) l -3390 c.c.
-3318
3438
3492
20
96-5
98-5
97
3472
-3358
-3351
3526
-3420)
02.
Ns-
Differences of pressure in
mm. observed in different experiments.
_
,
Amounts of gas present in
3 c.c. calculated by multiplying readings by constants.
Constants. C (a)= constant calculated for 767, which multiplied by First and Second
gives (a) and (b) respectively.
C (B) = constant calculated for 753, which was height of barometer on the next day,
when the ' Third ' set of readings were taken. To obtain (c), the C (O) is multiplied by
Constants.
'Third' and the whole multiplied by 767 to obtain uniformity.
N.B. -3196 is disregarded.
Reduced for N.T.P. -3390 c.c.
-1073 (6) c.c. 02-
02
becomes -3221
c.c.
that is, in 1
02,
c.c.
there are
estimation. (-03571 gm. Fe= 19-2 c.c. TiCl2.)
Two parallel amounts of 70 c.c. were evaporated and ashed.
Required of TiCl soln. respectively 10-4 c.c. ) Diff.
10-5 c.c.
In 1 c.c. *002771
-002784 gm.
Iron
1
Average=
*0027981
Ratio
=
385-7.
Exp. V. As in text. Ox. Blood collected same day as preceding. Laked solution
analysed. Bar. 767. Temp. 170 C.
Oxygen estimation.
As in the preceding experiments separate sets of observations were carried out on
different days, but the whole have been reduced finally in the last columns to 767 mm.
pressure. The temp. was constant throughout, but on Nov. 15th Bar. = 753 mm. and on
16th= 750 mm. Hg.
a, ,3, -y, a = four separate sets of observations, a, # on Nov. 14th, 'a on 15th, and a on 16th.
Cag3, C'y, and C8=constants used for each set of readings respectively; to obtain
was
8' ';
results a',
750
753
-y
has also been
Readings
multiplied by 76-7 and
5'
by
~--.
C.C. 02 in 3 c.c. solution
a
I'
'y'
d'
Bottle
y
CapS Cy Cd a'
is
15
94
103
100-5 102-25 3600 3651 3660 -3384 -3708 -3531 -3659
-3547 -3438
92
3643 3691 3701
95
16
100-5 3438 3492 3502 -3421 -3344 -3602 -3441
19
99-5 97-25 103
3472 3526 3536 -3316 -3489 -3334 -3492
20
95-5 100-5 102-5 101
The average of these 14 results=-3479 c.c. Reduced to N.T.P. -3306 c.c. 02In 1 c.c. solution -1102 c.c. 02a
-
HfEJMOGLOBIN.
Iron estimation. ('03571 gm. Fe=19'2 c.c. TiCl3.)
Two parallel amounts of 70 c.c. were evaporated and ashed.
Required of TiCl3 respectively 10-33 c.c. l
10'41 c.c.
.1 c.c. solution contained 002752 gm I Fe respectively.
'002773 gm.)
Average for 1 c.c.= '002767 gms. Fe.
Ratio = 398'2.
149
Diff. under 1 0/0.
En'. VI. Pig. Blood collected 3 o'clock. Oxygen estimation performed on same day.
at 6 o'clock. Centrifuged after laking with NH3.
Oxygen estimation. Bar. 769. Temp. 17° C.
Bottle
Diff. in pressure
Constant
Oxygen in3 c.c. soln.
15
101 mm.
3601
'3645
A
16
3641
103'5
*3768
19
102
3436
'3505'
20
104'5
3470
'3625
Average for 3 c.c.
23
110'5
'3609
3267
3609 (6)
24
107'75
3267
'3520
25
107
3400
'3638
113'0
30
3118
'3524
31
114'25
3199
'3654
Average for 3 c.c. reduced (to N.T.P.) for temperature and pressure= '3439 c.c.
In 1 c.c. '1146 c.c. 02.
Iron estimation. ('03571 gm. Fe= 19'2 c.c.)
Two parallel amounts of 50 c.c. were evaporated and ashed.
They required respectively of TiCl8 8-08 c.c.
7'92 c.c. J
1 c.c. solution contains :002978} gin. Fe. Duff.=1 °/o.
Average for 1 c.c.-= 002962 gm. Fe.
Ratio= 387'0.
This is the second of two experiments performed upon the pig. The first has been
rejected, as it was omitted to centrifuge after laking, and a sediment was observed at the
conclusion of the experiment, the result working out 1 /0 lower than this last experiment
in which the solution was centrifuged as usual after laking.