HEMOGLOBIN OETSTALS OP BODENTS' BLOOD
181
On the Haemoglobin Crystals of Rodents' Blood.
By
W. I>. Halliburton, IMC.D., B . S c ,
Assistant Professor of Physiology, University College, London.
(From the Physiological Laboratory, University College; London.)
THE crystals of haemoglobin since their first discovery have
been described by various observers as occurring in^ no less
than five out of the six crystallographic .systems. Subsequent
investigators have reduced this number: to two, namely, the
rhombic system, in which the haemoglobin from.the blood of
most animals crystallises; and the hexagonal system,:in which
that from the blood of certain rodents is said to crystallise.
This research was undertaken at Professor Lankester's suggestion, in order, first, to ascertain whether these six-sided
crystals really belonged to the hexagonal system ; and, secondly^.
to find, if possible, an explanation of the difference of crystalline
form that haemoglobin presents in different animals, while in
its other chief properties haemoglobin is universally the same.
It will be convenient to take the Bubject under the following
1. Historical.
,.
2. Hexagonal blood-crystals.
3. Influence of the other constituents of the blood on the
crystalline form of haemoglobin crystals.
4. The crystalline forms of haemoglobin obtained by mixirigthe blood from different animals.
5. Can squirrel's haemoglobin be obtained in any form other
than hexagonal crystals ?
6. Conclusions and remarks.
182
W. D. HALLIBURTON.
1. Historical.
Oxyhsemoglobin crystals were first described by Eeichert1 as
occurring in the uterus of a pregnant guinea-pig; by Leydig8
as occurring in the alimentary canal of the leech; and by
Kolliker,3 obtained from the blood of the dog, python, and
other animals. Kolliker considered the crystals to be composed of a more or less modified haematin. Funke4 was, however, the first to make complete observations upon them, and
to recognise their true nature. Kunde,5 working at the same
time, made extensive observations from a comparative point of
view, and was the discoverer of the exceptional form of the
crystals in the guinea-pig and squirrel. Since then many
investigators have worked at the subject, notably Lehmann,8
Rollett,7 von Lang,8 and Preyer,9 in whose exhaustive treatise
a complete bibliography of the subject up to 187] is given.
Our present knowledge of the crystalline form that haemoglobin assumes may now be summarised as follows:
a. In the great majority of animals10 in which haemoglobin
1
Reichert, ' Miiller's Arohiv,' 1849, p. 197Leydig, • Zeitsch. f. wiss. Zool.,' Bd. i, 1849, p. 116.
Kolliker, ' Zeitsch. f. wiss. Zool.,' Bd. i, 1849, p. 266.
4
Funcke, ' Zeitsch. f. nat. Med.,' N. P., Bd. i, 1851, p. 184; Bd. ii,
1852, p. 204 and p. 288. "De sanguine venaj lievates," 'Diss. Lipsise,"
1851.
6
Kunde, ' Zeitsch. f. nat. Med.,' N. F., Bd. ii, 1852, p. 276.
6
Lehmann, ' Ber. d. k. Sach's Ges. d. Wissen.,' 1852, p. 22.
7
Rollett, ' Sitzungsber. d. Wien. Akad.,' Bd. xlvi, 1862, p. 65.
8
Lang, ibid.
9
Preyer, ' Die Blutkrystalle,' Jena, 1871.
10
To the animals falling under this rule I can add several, the crystalline
form of the hemoglobin of which have not been hitherto recorded. I am
much indebted for specimens of the blood of these animals to my friend
Mr. F. E. Beddard, of the Zoological Gardens.
Opossum (Didelphys cancrivora).—Very large and dark red crystals,
can be readily obtained. They belong to the rhombic system.
Kangaroo (Macropus giganteus).—Crystals are more soluble, and
so less readily obtained. They are rhombic prisms, slenderer than in the
opossum.
s
3
HEMOGLOBIN CRYSTALS OF RODENTS* BLOOD.
183
occurs, vertebrate and invertebrate, crystals of it can be
obtained in the form of prisms and plates belonging to the
rhombic system.
b. The exceptions to this rule hitherto noted are the
following:
i. Guinea-pig. Haemoglobin crystals from the blood of this
animal are tetrahedra, once supposed to belong to the regular
system, but now shown by von Lang to be in reality rhombic.
ii. Lehmann mentions that similar tetrahedra may be obtained from the blood of the mouse and rat. This has not
since been confirmed.
iii. In several birds the crystals obtained are also tetrahedra.
iv. In three animals—the squirrel, the hamster, and the
mouse—six-sided plates have been described.
v. In one of these, the hamster, rhombohedra are described
as occurring also.
2. Hexagonal Blood-crystals.
We will take the three animals in which the haemoglobin is
said to crystallise in the hexagonal form one by one.
a. Squirrel.—The discovery of the fact that haemoglobin
crystals from this animal are six-sided plates was made by
Kunde (1852). Writing in the same year, Lehmann asserts
that though these crystals are six-sided they do not belong to
the hexagonal system. He gives, however, no reasons for this
assertion. Lang and Preyer arrived at the opposite conclusion
i. e. that they do belong to the hexagonal system, from the study
of their optical properties.
Belideus breviceps (a marsupial).—Crystals similar to those of the
opossum.
Seal (Phoea vitulina).—Rhombic prisms, many of them very short
and simulating hexagons. Easily obtained.
Bear (Ursus syriaous).—Bunches of rhombic needles, easily obtained.
They are slenderer than those obtained from dog's blood as a rule, some being
almost silken in appearance.
Hydromys leucogaster (white-bellied beaver rat).—Rhombic prisms.
Sus leucomystax (white-whiskered swine).—Rhombic prisms.
Water-vole (Arvicola aquatica).—Crystals are obtained easily by
adding water to the blood. They are of the usual rhombic shape.
184
W. D. HALLIBURTON.
My own observations are as follows:—The crystals can be
obtained with the greatest ease by simply adding a drop of
water to a drop of defibrinated blood on a slide, and covering
it; in less than a minute crystals appear. I have also prepared
them by other methods; l but in all cases the crystalline form
is the same. When first formed the crystals are six-sided
plates, many equilateral, but many not. After recrystallisation,
however, the crystals are then all but perfectly regular. The
quetions then arises, Do they belong to the hexagonal system or
not ? To this question one of the three following answers must
be the correct one.
• 1. They do belong to the hexagonal system.
2. They do not belong to the hexagonal system, but are
rhombic crystals, having a so-called "hexagonal habit." In
mineralogy instances are known of such occurrences. This is
the case with copper-glance, some of whose crystals so closely
resemble hexagonal ones that several mineralogists believed
that there were two kinds, one being hexagonal. Again, mica
is an instance of a monoclinic crystal with "hexagonal
habit."
FIG.
1. \
/
FIG.
2.
Fio. 3.
Suppose A B c D (fig. 1) to be the basal plane of a rhombic
plate, and the angle A B C to be approximately 120°, the lines
1
The method that I have found best for the preparation of blood-crystals
iri most animals is to add to defibrinated blood a sixteenth of its volume of
ether, and then to shake for two or three minutes until the liquid becomes of
a clear lake colour; in the course of time, varying from five minutes to three
days, crystals form in abundance (' Gamgee's Physiological Chemistry,' p. 87)
HAEMOGLOBIN OEYSTALS OF EODENTS* BLOOD.
185
joining A C, B D being the axes. Then if the angles D A B ,
D c B be replaced, as shown by the dotted lines, a hexagon
will be produced differing but little from a regular hexagon.
3. The third alternative is that they may belong to the
rhombic system by being twins, consisting, of three parallelograms or six triangles, as is shown in figs. 2 and 3. Twins
are, however, rare in the rhombic system.
In order to settle this question it is necessary to examine
the optical properties of the crystals.
Crystals may be divided, according to their optical properties, into three classes:
1. Isotropic.—Those in which there is no distinction of
different directions as regards optical properties. This includes
crystals belonging to the r e g u l a r system. They have but
one refractive index, i. e. refract light like amorphous bodies
do, singly.
%. Uniaxal.—Those in which the optical properties are
the same for all directions equally inclined to one particular
direction, called the optic axis, but vary according to this inclination. This class includes crystals belonging to the
dimetric system (crystals with three rectangular axes, two of
them being equal) and the hexagonal system. The optic
axis corresponds with the principal crystallographic axis.
In the direction of this axis a ray of light is refracted singly,
and in other directions doubly.
3. Biaxal.—This includes the remaining three systems
of crystals, the trimetric or rhombic (three rectangular axes
all unequal), the monoclinic, and the t r i c h i n i c . In these
there are always two directions along which a ray is singly
refracted.
The best test, as to whether a substance is doubly refractive
or not, is this : If between crossed nicols, which consequently
appear dark, a substance be interposed that make's the darkness give place to illumination, however feeble, that substance
is doubly refractive. This action is termed the depolarisation
of the ray.
The crystals of squirrel's haemoglobin I submitted to this
186
W. D. HALLIBURTON.
test, with the result that no depolarisation of the light can be
detected, when they are examined with the apparent basal
plane perpendicular to the axis of the instrument and rotated;
nor when a quartz plate is inserted do they produce any modification of the tint, as the stage is turned. The instrument
used was a Zeiss polarising microscope.
Hence the presumption is that they belong to the hexagonal
system, as rhombic crystals with hexagonal habit or rhombic
twins would produce some double refraction examined in this
way.
I submitted the question as to whether this was conclusive
to Professor Lewis, of Cambridge, and he kindly wrote to me
in answer as follows:
" The observation under the microscope between crossed
nicols, so far as it goes, is rather in favour of t h e ' crystals
being hexagonal, that is, presupposing that the field remains
dark when the crystal is rotated in the field of view. However,
this is not quite conclusive, and in such cases greater certainty
would be obtained if the crystals were placed under a Bertrand's polarising microscope, to see the shape of the interference rings and cross."
It should be here stated that uniaxal crystals in the direction
of their optic axis exhibit a symmetrical cross and circular
rings; in biaxal crystals the rings are oval, or at any rate not
circular, and the cross is not symmetrical. This is the case,
because the resistance to displacement in the three cardinal
directions called the axes of elasticity are all unequal in biaxal
crystals. This is true, not only for the crystalline substance
itself, but also for the luminiferous ether that pervades it.1
Acting on Professor Lewis's advice, I submitted the crystals
to Professor Judd, who with Mr. Fletcher's co-operation
examined them, aud gave me the following report, for which I
am much indebted to him :—"I have every reason to believe
the crystals belong to the hexagonal system from their form,
and their extinction between crossed nicols. I regret, however,
1
The cardinal directions are, however, believed not to be the same for the
ether as for the material of the crystal.
HEMOGLOBIN CRYSTALS OP EODBNTS* BLOOD.
187
to find that their minute size, and especially their extreme
tenuity, prevents our applying the crucial test of the interference figures seen in convergent polarised light.
"Bertrand devised a form of microscope which enables these
interference figures to be studied in the minute crystals seen
in their rock sections, and von Lasaulx has improved this
apparatus. We have what I believe to be the best form of the
Bertrand-Lasaulx apparatus constructed by Nachet; but even
employing an immersion objective magnifying 650 diameters,
the crystals are still so small as to give neither rings, nor cross,
nor brushes.
" I greatly regret that we have not been able to apply this
test. I fear that no instrument exists which will accomplish
what you desire; and Mr. Fletcher, on theoretical grounds,
doubts whether it would be possible under any conditions to
apply the test to such minute crystals."
The largest crystals of squirrel's haemoglobin that I have
obtained were those formed by the addition of water to the
defibrinated blood; they varied in size from 4001 to "005 m. in
breadth.
Since receiving Professor Judd's report, I have tried to
obtain larger crystals by Gscheidlen's1 method. He seals
defibrinated blood in narrow glass tubes, which are then kept
at a temperature of 37° C. for several days. On opening these
tubes and emptying their contents into a watch glass, crystals
of great size are formed from dog's blood after evaporation has
occurred.
With squirrels' blood, however, I have not obtained larger
crystals by this method than by the first. The reason for this
seems to be the extreme readiness with which squirrels' haemoglobin crystallises. It is a well-known fact that bodies that
crystalise rapidly crystallise in small and numerous crystals.
If some method could be devised for retarding, but not preventing, the crystallisation of squirrel's haemoglobin, we might
then be able to obtain crystals of it large enough to which to
apply this crucial test.
1
' Physiologische Methodik,' p. 361,
188
W; D. HALLIBURTON.
'
The matter must therefore be left incomplete up to this
point for the present. The probability, however, is greatly in
favour of the crystals being-true hexagons.
We have seen that in order to have a rhombic plate with
hexagonal habit, it is necessary that one of its angles be
approximately 120°; I measured the angles in the rhombic
plates found in the rat, and found that they averaged 129°.
I shall also presently show that it is possible by the intermixture of the blood of different animals to obtain crystals
closely resembling hexagons, but which are not so, as is shown
by their optical properties.
b. Mouse.—Kunde was the first to describe the haemoglobin
crystals of this animal. He made eighteen observations, and
the crystals he found were fine needles and prisms.
Bqjanowski1 was the next to make observations on these
crystals. He describes and figures them as six-sided plates
resembling in form those from squirrel's blood, of a flesh
colour, and very soluble in water. He prepared them by the
addition of a mixture of equal parts of alcohol and ether to
the blood. No description of their optical properties is given.
He remarks, " I have not been able to observe the fine needles
described by Kunde."
Preyer repeated these experiments, and confirmed the observations of Kunde, not those of Bojanowski. He obtained
small prismatic crystals.
I have myself experimented with the blood of eighteen
mice, and the result has been again to confirm Kunde's observations. The crystals are exceedingly difficult to obtain, and
in some cases 1 have had to repeat the process of freezing and
thawing many times after the addition of alcohol, before succeeding in obtaining them. They are very soluble in water.
The crystals are exceedingly small rhombic prisms. They are
nearly colourless, and it is only when they are heaped together
that any red tinge at all can be perceived in them. In one
case in which by the addition of ether to the blood I obtained
crystals of fair size after allowing the mixture to stand for five
1
Bojanowski, 'Zeitscli. f. wiss. Zool.,' Bd. xii, 1863, p. 333.
HEMOGLOBIN CRYSTALS OF EODENTS BLOOD.
189
days, the crystals still showed this same peculiarity, namely, in
being nearly colourless. I have successfully employed Bojanowski's method for the preparation of the crystals, namely,
the addition of a mixture of alcohol and ether to the blood; but
in no case did hexagonal crystals form. Mouse's haemoglobin
also differs from squirrel's in being very soluble in water ; this
is admitted by Bojanowski; one would therefore expect a
p r i o r i that its crystalline form would be different.
c. H a m s t e r (Cricetus vulgaris).—My remarks under
this heading will be only historical. I have not myself been
successful in obtaining one of these animals. The crystalline
form of the haemoglobin was first described by Lehmann, who
found rhombohedra and six-sided plates. His experiments
were repeated by Preyer,! whose observations on the subject are
very complete. He fornd both crystalline forms, viz. sixsided plates, and rhombohedra. This is interesting since the
rhombohedron belongs to the hexagonal system. By examination between crossed nicols he found that the six-sided plates
had no action in " depolarising " the ray, and he therefore concludes that they, like squirrel's haemoglobin crystals, are true
hexagons.
d. Conclusions.—The presumption in favour of the
haemoglobin crystals of the squirrel and hamster being true
hexagons is exceedingly great. In the case of the mouse, it
seems to be almost equally certain that the crystals are not as
a rule hexagonal. I should not like, however, to deny that
haemoglobin may sometimes in the case of the mouse crystallise
in this way, because of some observations I have made on the
haemoglobin crystals of the rat.
Crystals are obtained from the blood of this animal with
great ease; mere addition of water to the blood causes almost
immediately an abundant crop of crystals. On this account
the blood of this animal is used by the students in the practical
classes at University College for the preparation of haemoglobin
crystals. Professor Schafer told me that on looking over the
students' preparations he had occasionally seen hexagons to1
' Die Blutkrystalle,' p. 262.
190
W. D. HALLIBURTOlT.
gether with the ordinary rhombic prisms and plates. In
order to verify this, I have made numerous specimens of the
crystals from the blood of about fifteen rats. As a rule, no
hexagons were present; but on three occasions I have detected
hexagonal plates—very few in number, perhaps not more than
one or two on the slide—among the rhombic crystals. There
appeared to be nothing special either about the animal used
or the method employed in these cases. The diameter of
these crystals averaged about the same as in squirrel's blood
(003—"003 m.). Between crossed nicols they also behaved
the same as squirrels' haemoglobin crystals, viz.-remained dark
in all positions.
In addition to this, if crystallisation be watched under the
microscope, a single corpuscle will often be observed to set
into a minute hexagon. This is what Preyer calls intraglobular
crystallisation. He describes it as occurring in the blood of
the hamster. It can also be observed in the blood of the
rat. The crystals apparently so formed last but a few seconds,
the corpuscles then becoming shrunken, or irregular, and very
often under the subsequent action of water, globular. It is
therefore possibly a stage in the crenation of the corpuscle.
But, apart from this, it is undoubtedly the fact that hexagonal
crystals are occasionally found in the blood of the rat.1 It
1
Since writing the above, I have received the following in a letter from Mr.
Sheridan Lea, of Cambridge. He says :—" When I was showing a class how
to put up permanent specimens of haemoglobin crystals from rat's blood, we
obtained uniformly hexagons, instead of prisms. This I have neither ever
noticed or heard of before, and I thought it might be of interest to you.
The method employed was that of Stein (' Centralb. f. d. med. Wiss.,' 1884,
No. 23, and ' Virchow's Archiv,' 97, 483)." I had myself occasionally used
Stein's method of preparing crystals from rat's blood, but had always obtained
the usual rhombic prisms. On receiving Mr. Lea's letter I made a large
number of preparations of haemoglobin crystals by this method. The method
consists in simply mounting a drop of deiibrinated blood in a drop of Canada
balsam. In the case of some animals, among which were man and the mouse,
I was not able to get any crystals at all. In the commoner mammals, dog
and cat, the crystals obtained were very fine specimens of rhombic prisms.
In the guinea-pig and squirrel they presented the usual tetrahedral and
hexagonal shapes respectively. With rat's blood, however, the results were
HEMOGLOBIN CRYSTALS OP EODUNTS* BLOOD.
191
would therefore be possible that such crystals occasionally may
occur in the blood of other animals, such as the mouse, the
usual form of whose blood-crystals is, however, rhombic.
The rats employed in the above experiments were the
common house rat, and also tame rats.
3. Influence of the other Constituents of the Blood on the
Crystalline form of Haemoglobin Crystals.
These experiments, as well as those in the next section of
this paper, were undertaken at the suggestion of Professor
Schafer.
The blood-crystals of an animal have the same form whether
they be obtained from the fresh blood, or from the blood from
which the fibrin has been removed. Fibrin, or its precursor
in the blood-plasma fibrinogen, has then no influence on the
form of the blood-crystals.
The following experiments were undertaken to ascertain
whether the other constituents of the blood-plasma, which are
all contained in the serum, have any effect in influencing the
form of the crystals.
The method of experimentation was as follows:—Defibrinated
blood is taken in a tube and centrifugalised for about half an
very strange. In the majority of cases the usual rhombic needles were formed ;
but in a few cases I confirmed Mr. Lea's observations, and obtained perfectly
regular hexagons; in some oases the hexagons would occupy one part of the
slide only, while the remainder was filled with the ordinary prisms. Hexagons
seemed to form where the proportion of blood to balsam was small, and they
were formed especially at the edges of a preparation where the drop of blood
had probably had time to dry somewhat before being covered with Canada
balsam. These hexagons remained dark in the dark field of the polarising
microscope. After a day or two they cracked in a peculiar way, and seemed
then to be made up of minute needles radiating from a centre. This may or
may not indicate the way in which they are formed. The fact that they
occurred most in parts of the field where there was leaBt water seems, however, to confirm the theory advanced later in the paper, viz. that the difference
of crystalline forms in haemoglobin is due to different amounts of water of
crystallisation.
192
W. D. HALLIBURTON.
hour; the corpuscles settle at the bottom of the tube, and
the supernatant serum is pipetted off. To the corpuscles the
blood-serum of some other animal is added, the mixture shaken,
and the mixture again centrifugalised; the serum is again
pipetted off, and more added. After repeating this process
several times, the corpuscles of one animal are obtained in the
serum of another animal without any of the serum of the first
animal being in the mixture. Haemoglobin crystals are then
prepared from this mixture. In some cases the foreign serum
dissolves the haemoglobin and disintegrates the corpuscles.
This was first pointed out by Landois.1
Mere addition of the blood-serum of one animal does not
as a rule cause the formation of blood-crystals. It does so,
however, sometimes.2 This is explicable on the assumption
that the blood-serum used is very watery, and the haemoglobin
of the other animal crystallises very readily. I have myself
come across no case in which it occurred.
My results may be best given in the form of the following
table. I have given not only the effect of the foreign serum on the
crystalline form of haemoglobin, but also the effect on the corpuscles themselves, as to whether they are disintegrated or not.
Corpuscles of
In Serum of
Squirrel
Rut
Dog
Rat
Guinea-pig
Guinea-pig Cat
Guinea-pig Dog
Cat
Mouse
Rat
Squirrel
Squirrel
Effect on the Corpuscles.
Effect on thD Crystalline Form
of the Hemoglobin.
Much dissolved
Very little dissolved
Very little dissolved
Little if any dissolved
Nearly entirely dissolved
Much dissolved
Little dissolved
Nil.
Nil.
Nil.
Nil.
Nil.
Nil.
Nil.
The result of these experiments is to show that the serum of
one animal has no influence in causing a change of the haemoglobin crystals of another animal.
I next examined in a qualitative manner the serum of certain
1
' Die Transfusion des Blutes,' Leipzig, 1874.
2
An instance of such action is recorded by Professor Sctaafer (" Blood
Transfusion," ' Trans. Obst. Soc. London,' 1879, p. 317).
HEMOGLOBIN CRYSTALS OF KODENTS' BLOOD.
193
rodents with regard to the proteids or albuminous substances
contained in it. I obtained similar results in all animals,
results which show, too, that the serum proteids of rodents
agree with those in other mammalian animals which I had
previously investigated.1 The proteids, the most important
bodies in the blood-plasma, being similar, the serum would
not on a priori grounds be suspected of influencing the
crystalline form of haemoglobin. The results I have obtained
with regard to the heat-coagulation temperatures of these
bodies is shown in the following table.
Tem p e r a t u r e s of Coagulation of the P r o t e i d s in the
Blood of c e r t a i n R o d e n t s .
"Same of Proteiil.
Guinea-pig. |
Squirrel.
C.
c.
Globulins—
C. i C.
C.
56° : 56° ' 56°
Fibrinogeu. .
0
'75°
75°
7i>
Serum globulins .
C.
75°
75"
Albumins—
y
!72°-3'
73° , 70°'•70°-l°|72°
76° '• 77° • 78° i77°
:77°(smallinamount)77°'
84° i 84° , 84°
87° (trace)84° (very abundant) " "
The stromata of the red blood-corpuscles might, however,
possibly be supposed to have some influence on the crystalline
form of the haemoglobin. We have seen that crystallising the
haemoglobin of one animal from the serum of another yielded
negative results; squirrel's haemoglobin remained hexagonal,
rat's and guinea-pig's rhombic pi-isms and tetrahedra respectively, whatever the serum in which they had been dissolved.
A similar result followed crystallisation from a fluid consisting
of serum plus the dissolved stromata of the corpuscles of some
other animal. This was obtained by adding to the blood one
sixteenth of its volume of ether, and letting it stand; the
crystals of haemoglobin which formed were filtered off, and the
ether evaporated from the filtrate which consisted of the serum
with the stromata of the corpuscles dissolved in it.
1
Halliburton, "Periods of Serum," 'Journal of Physiology,' vol. v, p. 152.
VOL. XXVIII, PART 1.
NEW SER.
N
191
W. D.
HALLIBURTON.
So far then these experiments seem to show that the difference of crystalline form is due to some inherent quality of the
haemoglobin itself, and not due to any agency in the blood
external to the hsemoglobin.
4. The Crystalline forms of Haemoglobin obtained by
mixing the Blood from different Animals.
By mixing the defibrinated blood from two animals, whose
haemoglobin crystallises differently, and then preparing crystals,
I thought I might obtain some new forms resulting from the
mixture. Here my experiments have yielded mostly negative
results, but the one positive result I have obtained from such
experiments warrants me in recording the whole. The blood
of two animals were mixed in about equal proportions, shaken
thoroughly, and then haemoglobin crystals prepared by the
ether method.
It will be convenient here again to give my results a tabular
arrangement.
Form of Hremoglobiii Crystals prepared from the Mixture.
Rat
Rat
Squirrel
Dog
Dos
Squirrel
Both rbombic prisms and hexagous present.
Guiuea-pig No rhombic prisms of the shape usually seen in rats'
blood present. No tetrahedra. Crystals are all
rhombic prisms with hexagonal habit.
Guinea-pig Hexagonal phtes and tetrahedra both present. Many
tetrahedra imperfect. The tetrahedra were all reduced to about half the size of those prepared from
the unmixed blood of the same guinea-pigs.
Squirrel
Fine rhombic needles and hexagonal plates both present in abundance.
Guinea-pig The greater number of the crystals formed are very
small tetrahedra, about a quarter the size of those
prepared from the blood of the same guiuea-pigs.
The optical properties are, however, the same.
Rhombic prisms very slender, like those of dog's
blood, also seen.
The second case, that of mixing blood from the rat and
guinea-pig, is interesting, and demands further description.
It shows that it is possible to obtain a new form of haemoglobin
HEMOGLOBIN CRYSTALS OF EODRNTS* BLOOD.
195
by mixing that from two animals in which the crystalline form
is different. It also shows that rhombic haemoglobin crystals
may assume a hexagonal type (fig. 4). These crystals are not,
however, perfect or equilateral hexagons, two of the sides
being longer than the other four.
E
The side A B = E D = -0019 m. (average).
The sides B C = C D = E F = F A = '00125 m. (average).
This irregularity is possibly to be accounted for by the fact
that, in rats' hEemoglobin crystals, the angles corresponding to
B C D , A F E, are 51°. In order to obtain perfect hexagons
of a rhombic type it is necessary, as before stated, that this
angle be 60°.
Under crossed nicols these crystals appear perfectly bright,
so contrasting with the true hexagons obtained from the blood
of the squirrel and hamster.
This result was not, however, always obtained; in one or two
cases I obtained as a result of mixing the blood of these two
animals a mixture of crystals; that is prisms and tetrahedra.
5. Can Squirrel's Haemoglobin be obtained in any form other
than Hexagonal Crystals ?
Another set of experiments was performed with the object
of breaking down the hexagonal constitution of the haemoglobin of squirrels' blood. The first method tried was that of
driving off the water of crystallisation, and of then adding
water to the dehydrated hsemaglobin.
The hsemaglobin was obtained in a state of purity and dried
over sulphuric acid until it lost no more weight. Then it was
examined, and found to have its normal spectroscopic proper-
196
W. B. HALLIBURTON.
ties. It was heated to 100° C. in a water oven, and again
examined. It had lost but a slight amount of weight. It was
rather more insoluble in warm water than previously, but the
spectroscopic properties, and the form of the crystals obtained
from the solution, remained as before. This confirms the observation previously made by Hoppe-Seyler that dry haemoglobin
is not decomposed by a temperature of 100° C. It was again
heated in the water oven at 100° C. until there was no further
loss of weight. I t was then heated to ]20° C. in an air-bath,
and again examined. It was found to have lost considerably
in weight, to have lost its crystalline lustre, to he brown in
colour (hjematin) and to be insoluble in water. That is, it
parts with its water of crystallisation at a temperature which
decomposes it, with the formation of heematin, the proteid
matter becoming at the same time coagulated and insoluble.
Experiments were then tried with the object of ascertaining
whether a lower temperature will remove the water of crystallisation in a Torricellian vacuum. This I did by means of a
Pfliiger's mercurial air-pump. The action of the vacuum alone
converted the dried haemoglobin, at any rate partially, into the
conditions of methaemoglobin. The water of crystallisation
seemed to be completely lost at a temperature of 50°—60° C ,
as subsequent heating to 120° C. produced no further loss of
weight. But this temperature was also sufficiently high to
decompose the haemoglobin in such a way as to render it
insoluble, or almost so, in water, and therefore no crystals could
be subsequently obtained from it.
The next method adopted was to convert the haemoglobin by
various reagents into methsemoglobin ; then by reducing agents
to form once more hsemoglobin, and then obtain crystals of
this. But the reducing agents used were found to hinder the
formation of crystals.
The third and simplest method was to repeatedly recrystallise
the haemoglobin, when it was found after three or four recrystallisations that no six-sided crystals were obtained, but a
mixture of rhombic needles and tetrahedra, and in some cases
the latter were absent. This is interesting in connection with
HEMOGLOBIN CRYSTALS OF BODENTS* BLOOD.
197
the reverse experiment already related, in which crystals simulating hexagons were obtained by mixing together the blood of
the rat and guinea-pig, and in which the same result was
obtained from a mixture of the solutions of the pure hsemoglobin of the same animals.
6. Conclusions and Remarks.
What the difference between the various forms of, haemoglobin may be, it cannot be a very deep or essential one. The
difference in crystalline form is associated with a difference of
solubility in water and other reagents; but the spectroscopic
characters, the decomposition products, the compounds it forms,
of which hsemin is a readily obtained example, are universally
the same. Not only so, but Hoppe-Seyler has shown1 that iu
various animals dried haemoglobin has the same or nearly the
same elementary composition.
Have we then to deal •with a case of polymorphism? The
terms dimorphism and polymorphism cannot be applied to any
substance which crystallises in two or more forms, unless the
composition of that substance be exactly the same in all cases.
Instances of dimorphism in the mineral world are carbon and
sulphur among the elements, and sal ammoniac, potassium
iodide, cuprous oxide, &c, among compounds. The conditions
on -which dimorphism depend are two: first, temperature,
secondly, the solvent from which the substance crystallises.
If, as in the case of many mineral salts, the compounds are
united with different proportions of water of crystallization, we
have to deal with different hydrates, and the case is not one of
true dimorphism ; an instance of this is sulphate of soda.
The case seems to me to narrow itself down to this in the
case of haemoglobin; either we have here a case of polymorphism, or the crystalline forms are due to the combination with varying proportions of water of crystallisation. In
the absence of a rational formula for haemoglobin it would
be unsafe to affirm the former of these two alternatives. Moreover, the conditions that are known to produce dimorphism in
1
'Pliysiologisclie Chemie,' p. 377.
198
W. D. HALLIBURTON.
minerals, namely, differences of temperature and of solvent, have
in the case of haemoglobin no influence.
If we then fall back on the latter alternative, the question
which arises is whether there are any facts to support it. The
explanation that the varying form of oxyhsemoglobin is due to
varying quantities of water of crystallisation may be otherwise expressed by saying that we have to deal with different
hydrates of oxyhsemoglobin. This would account for the
varying solubilities of these substances in water and other
reagents, and at the same time is not such an essential difference as to prevent the chief properties of haemoglobin from
being universally the same.
Turning to Hoppe-Seyler's researches on this subject of water
of crystallisation, it is seen that its amount varies considerably. The following is his table i1
Per<ventage of Water of Crystnllisot
Dog's hsemoglobin .
Guinea-pig's ,,
Squirrel's
„
Goose's
3 to 4
7
9-4
9'4
In an earlier paper,2 the same author gives rather different
percentages, viz. for guinea-pig's haemoglobin 6, for goose's
hsemoglobin 7, and for squirrel's haemoglobin 9. Dr. Christian
Bohr 3 has more recently made observations on the water of
crystallisation of dog's hsemoglobin, and as the result of
thirteen experiments he finds that its amount varies from 6*3
to 1"2 per cent. It is thus seen that great variations occur in
the numbers obtained by these experiments. The reason for
this variation seems to me to be the great difficulty of obtaining htemoglobin in a pure state, and also possibly because the
method adopted, which is the same as that carried out in
similar investigations on inorganic salts, is not applicable to
such a complex and much less stable organic compound as
1
' Pliysiologische Chemie,' p. 377.
'Med. Chem. TJntersuckungen/ Heft iii, 1S68, p. 370.
' ' Experimental TJntersuchungen iiber die Sauerstoffaufnahme des Blutfarbstoffes,' Kopenhagen (Olsen and Co.), 1885.
3
HAEMOGLOBIN CRYSTALS OF RODENTS* BLOOD.
199
haemoglobin; in other words, the temperature necessary to
drive off the water of crystallisation is also sufficient to cause
certain decomposition changes in the pigment.
My experiments have shown that squirrel's haemoglobin will
under certain circumstances crystallise in forms other than the
usual hexagonal form. A crucial experiment in order to see
whether this is due to uniou with different amounts of water of
crystallisation would have been first to ascertain the amount
of this water in the hexagonal crystals, and then in the
rhombic crystals obtained by recrystallisation. I have performed three such experiments, but the results obtained are so
conflicting, and exhibit variations as great as in Bohr's experiments, that it is impossible to draw any conclusions from
them, except the negative one that we cannot by our present
methods of research make any definite statement with regard
to the water of crystallisation of haemoglobin.
Even if it be found ultimately that the difference in crystalline form is dependent on varying amounts of water of crystallisation, the difficulty is only explained up to a certain point.
What is left unexplained is the nature of the agency that
causes the haemoglobin of some animals to unite with a certain
amount of water of crystallisation, and that of other animals
with a different amount. That some such substance or agency
does exist would seem to be the inevitable result of the recrystallisation experiments which have been related.