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"^ This analysis, which is adduced for purposes of comparison, does not fall within the
dates given above, having been published in 1868.

^ Ztschr. f. 2}hysiol. Chem., Strassburg, 1888, Bd. xii. S. 285.

^ Ibid., 1890, Bd. xiv. S. 289.
" Ibid., 1878-79, Bd. ii. S. 149. Refer to Note 2.
" Arch. f. d. ges. Physiol, Bonn, 1884, Bd. xxxi. S. 240.

12 Hiifner, Ztschr. f. ]}hysiol. Chem., Strassburg, 1883-84, Bd. viii. S. 358. This paper
contains the results of Biicheler's researches, which have been carried out under Hlifner's
direction, and had appeared as a Tiibingen Dissertation in 1883.

13 Ibid., Strassburg, 1886, Bd. x. S. 16.

-'^ Beitr. z. Physiol. C. Ludivig z. s. Geburtst. etc., Leipzig, 1887, S. 74.

'^^Arch.f. Physiol., Leipzig, 1894, S. 174.

^^ Ztschr. f.iohysiol. Chem., Strassburg, 1882-83, Bd. vii. S. 57.

" Beitr. z. Physiol. C. Ludwig z. s. Geburtst. etc. , Leipzig, 1887, S. 74.

^^ Ztschr. f. physiol. Chem., Strassburg, Bd. xiv. S. 289.



200 HAEMOGLOBIN.

Were we to admit the accuracy of the work of all the observers,
whose results are exhibited on the table on p. 199, we should be forced
to the conclusion that haemoglobin is a body which does not only vary
considerably in composition in different animals, but does not possess a
constant composition even in different individuals of the same species.
Thus, whilst Kossel found the percentage of carbon in the oxyheemoglobin
of the horse to be 54"87, and the mean of a large number of analyses
by Kossel, Otto, and Blicheler gave 54"68, Zinofisky, as a result of his
analyses (only two in number, so far as the carbon and hydrogen are
concerned !), found the percentage of carbon in the haemoglobin of the
horse to be 51"15 (!!). A body in which the carbon differs by 3'72 per
cent, in different specimens cannot, it will be argued, be a chemical indivi-
dual. But to draw this conclusion in reference to htemoglobin from the
facts in our possession would certainly be an error. The discrepancies
between the results of the analyses of the heemoglobin of the same
animals are doubtless due to differences in the purity of the substance
analysed, and to errors of analysis. The preparation of perfectly pure
oxyhemoglobin, entirely free from contamination with other con-
stituents of the blood corpuscles and from products of decomposition, is
much more difficult than has, until very recently, been supposed. In
the attempt to purify the substance by crystallising it as frequently as
practicable, nearly all observers have in all probability decomposed it,
and have afterwards analysed a mixture of oxyheemoglobin and products
of its decomposition. How far this is the source of the above discrep-
ancies must now, in the light of recent spectrophotometric work, be
carefully enquired into. Moreover, assuming that perfectly pure crystal-
lised oxyhsemoglobin is at the disposal of the analyst, the task of drying
without decomposing it is one of peculiar difficulty, concerning the
method of execution of which the chemists who have carried out the
researches under discussion have been by no means agreed. Thus, whilst
some (following Hoppe-Seyler's directions) have dried the oxyhsemoglobin
intended for analysis, in the first instance in vacuo at 0° C, and only
afterwards at higher temperatures, others (Zinoffsky, Hlifner, Jaquet)
have dried the substance in vacuo at ordinary temperatures (15° to
18° C), and subsequently at 110° to 115° C.

It is conceivable, nay probable, that some of the differences in the results
of different observers may have depended upon the above-mentioned differ-
ence in the treatment of the substance analysed. But, unquestionably, some
of the best marked differences must dej)end upon differences in the
method of analysis employed {e.g. where one observer determines the JST in
oxyhsemoglobin by Will and Varrentrapp's method, whilst another employs
Dumas' method), and upon accidental errors of analysis, which can easily be
rendered obvious, by making a considerable number of analyses.

For instance, it appears to me that the percentage of carbon given by
Zinoffsky,^ as representing the proportion of this element in the hcemoglobin
of the horse, must be due to imperfect combustion. Whilst this observer
carried out the determinations of iron and sulphur in the hcemoglobin of the
horse in the most elaborate and perfect manner, making many analyses of
each of three separately prepared specimens of crystallised haemoglobin, he
rested satisfied with only two determinations of carbon and hydrogen, and
two determinations of nitrogen (the latter by the method of Will and

^ "Ueber die Grbsse des HamoglobinmolectUs," -^<sc7w. /. phys'wl, Chem., Stiassburg
18S6, Bd. X,



ELEMENTAR V COMPOSITION OF OX YHyEMO GI OB IN. 2 o i



Varrentrapp), though the results which he obtained differed in a remarkable
manner from all those of previous observers. It is clear that whilst very great
value must be attached to the determination of the iron and sulphur contained in
haemoglobin, made by Zinoffsky, his conclusions as to the percentage of carbon
and hydrogen must be rejected, as being based upon an insufficient number
of analyses, and as being in all probability incorrect. This opinion is supported
by the remarkable discrepancy between his results and those of other observers
— a discrepancy which cannot be accounted for by differences in purity of the
bodies analysed.

While it is almost inconceivable, and against the weight of evidence, that
haemoglobin derived from animals of the same species should not have a
constant composition, the differences in centesimal composition which certainly
do exist between the haemoglobin of certain animals and that of others
cannot surprise us when we reflect that haemoglobin does exhibit marked
physical differences in different animals — that it exhibits variations in
crystalline form, in the amount of water of crystallisation, and in solubility.

The study of the general results of the ultimate analyses of oxy-
haemoglobin made of recent years forces us assuredly to the conclusion
that new and still more precise investigations are needed before we can
lay claim even to so limited a knowledge as that of its precise centesimal
composition. Nevertheless, it would be wrong to leave the study of
the more recent researches without drawing attention to certain of the
numerical results obtained, which are more deserving of confidence
than others.

The most characteristic and the most important of the elements
which enter into the composition of hfemoglobin is its iron. Iron is
the typical element in a molecular group which exists and possesses
identical chemical and physical properties in all the varieties of
haemoglobin with which we are acquainted. Besides furnishing us
with data by which the molecular weight of hcemogiobin may be
calculated, the amount of iron appears to bear a definite relation to
the quantity of the dissociable oxygen and carbonic oxide which
haemoglobin combines with. For these reasons, an extremely accurate
determination of the iron in hcemoglobin, carried out with all the
precision which the present state of science permits of, has been a great
desideratum. Such determinations have been carried out by Zinoffsky,
Jaquet, and Hiifner (see p. 199).



Hsemoglobin of


Fe per cent.


Authority.


Dog
Horse
Ox .
Hen


0-336
0-335
0-336
0-336


Jaquet.
Zinoffsky.
Hiifner.
Jaquet.



These observers have determined the proportion of iron in the
oxyhsemoglobin of the dog, the horse, the ox, the pig, and the hen.
They have shown : First, that the amount of iron in the blood-colouring
matter of these animals is decidedly smaller than had been assumed
on the basis of the older analyses. Secondly, that in the animals
mentioned the percentage of iron in the hemoglobin is identical, so
that we may conclude that in these very different animals, in spite of



202 HAEMOGLOBIN.

the discrepancies between the results of the ultimate organic analyses
yet made, the oxyhsemoglobin possesses the same molecular weight.
The concordance between the more recent determinations of the u'on
of oxyhsemogiobin is well shown in the table given on the previous
page.

On the assumption that one molecule of haemoglobin contains one
atom of u"on, the molecular weight of the haemoglobin of the dog, horse,
ox, and hen would be 16,669, and this result is borne out, as will be
afterwards shown, by the volume of oxygen or of carbonic oxide which
enters into combination with the blood-colouring matter.

In addition to the estimation of the iron in haemoglobin, that of
the sulphur has been carried out with remarkable care by Hiifner,^
Zinoffsky, and Jaquet ; and their results, whilst establishing that the
centesimal composition of the blood-colouring matter of all animals is
not identical, show that in haemoglobin the sulphur stands to the iron
in deiinite relations.

Thus Zinoffsky's analyses appear to estabhsh that in the haemoglobin
of the horse the sulphur is to the iron in the relation of two atoms of
the former to one of the latter element, and Hiifner has shown that
exactly the same relation obtains in the case of the haemoglobin of the
ox and the pig. On the other hand, Jaquet's analyses of the haemo-
globin of the dog indicate that in it three atoms of sulphur correspond
to one atom of iron. When, in a subsequent section, we shall examine
the products of decomposition of haemoglobin, we shall show that, under
the influence of acids and alkalies, the blood-colouring matter breaks
up into an iron-containing body (of which the composition and the
properties vary according to the presence or absence of oxygen during
the decomposition) and into an albuminous body or bodies. The
sulphur of haemoglobin belongs to the albuminous part of the molecule,
and the difference in the relation of S to Fe, brought out by the
researches of Hiifner, Zinoffsky, and Jaquet indicates that the albuminous
moiety of the haemoglobin molecule varies in different animals, and that
among the points of difference is the difference in the proportion of
sulphur. This point will be certainly cleared up by future researches
specially directed to its elucidation ; it may be remarked, however,
that the proportion of sulphur in different albuminous bodies does
exhibit great variations.

It appears to me, moreover, that we must not lose sight of the possi-
Inlity (even when there is no evidence afforded by ultimate organic
analysis of there being a difference in the percentage composition of the
alljuminous part of the haemoglobin moiety), and indeed probability, that
hcemoglohins varying in certain ijhysical 'pro'perties may he formed by the
linldng of the iron-containing molecule to various iwlymeric comhioiations of
the same albuminous molecule.

Although it is highly probable that the molecular weight of the
haemoglobin of the dog and of the ox (16,669), as determined by the
iron determinations of Jaquet and Hiifner, and by determinations by
Hiifner of the volumes of and CO with which haemoglobin combines,
has been ascertained with correctness, or nearly so, the discrepancies in
the results of the detenninations of C, H, and N, made by different
observers, are too great to warrant our placing confidence in the
empirical formulae which have been assigned to haemoglobin. Of these

^ " Bestimmung d. Sauerstoffscapacitat d. Blutfarbstoffs," S. 76.



CRYSTALLINE FORM OF IL^MOGLOBIN. 203

empirical formultie, that calculated by Jaquet for the luemoglobin of the
dog is probably the nearest the truth, namely —

^758-*^ 1203-^ 196^3-^ 6^218

Why should ha3moglobin possess so enormously high a molecular
weight ? The question suggested itself to the acute mind of Bunge, who
has furnished us with one reason which is • eminently suggestive : " The
enormous size of the hsemoglobin molecule," says this writer, " finds a
teleological explanation, if we consider that iron is eight times as heavy
as water. A compound of iron, which would float easily along with the
blood current through the vessels, could only be secured by the iron
being taken up by so large an organic molecule." ^

When discussing the compounds and products of decomposition of
oxyhaemoglobin and hsemoglobin, we shall have again to revert to and
further examine certain of the facts which have found a place in this
section.

The crystalline form, the amount of water of crystallisation, the
solubility, and the diffusibility of oxyhsemoglobin. — Although, as has
already been stated, the oxyhsemoglobin of different animals varies con-
siderably in the facility with which it crystallises, we now know that the
haemoglobin of all animals, without exception, may, by suitable treat-
ment, be obtained in the crystalline form.'^ Great differences exist in
the solubility of the blood-colouring matter obtained from different
animals, and, as might have been anticipated, the blood of these
animals whose hsemoglobin is least soluble (as the rat, the guinea-pig,
and the squirrel) yields crystals of oxyhsemoglobin most readily ; whilst
the converse is also true, i.e. the oxyhsemoglobin of man, of the rabbit,
the sheep, and the ox, all of which are exceedingly soluble, yield crystals
with considerable difficulty. It was, indeed, long supposed to be impos-
sible to obtain large quantities of oxyhsemoglobin from the blood of
certain of these animals.

As a rule, crystals of oxyhsemoglobin are of such a size that their
form, and even their crystalline nature, cannot be made out by the
naked eye. The blood of certain animals, however, as the dog, and
particularly the horse, yields under favourable circumstances rhombic
prisms of macroscopic size. From horse's blood Hoppe-Seyler frequently
obtained prisms over 5 mm. in length and 4 mm. in thickness. The
colour of crystals of oxyhsemoglobin appears different, according to
their size or the number aggregated together.'^ Thus the finest needles
or prisms of oxyhsemoglobin, when seen singly under the microscope,
appear almost colourless, or possess the yellowish tint characteristic of
the coloured corpuscles. On the other hand, large crystals, or consider-

1 From the results of Hilfner's analyses of the hsemoglobin of the ox, but substituting
his most recent determinations (1894) of the iron for the older ones, published in 1887, I
have calculated for the hsemoglobin of this animal the formula —

2 G. Bunge, " Text-Book of Physiological and Pathological Chemistry." Translated by
L. G. Wooldridge : London, 1890, p. 24.

^ It was Dr. Otto Funke who first asserted, as the result of his own researches, " that
all blood is capable of crystallisation, whatever animal or organ it may be taken from." — ■
"Explanation of the Plates" of his "Atlas of Physiological Chemistry," p. 15 (see p. 205,
note 1).

■^ F. Hoppe-Seyler, "Das Oxyh'amoglobin des Pferdeblutes, " Ztschr. f. ■physiol, Cliein.,
Strassburg, 1878-79, Bd, ii, S. X49.



2 04 HEMOGLOBIN.

able aggregations of the smaller crystals, exhibit, like aggregations of
blood corpuscles, the red colour characteristic of the blood.

We shall now examine successively the most important facts con-
nected with the (1) form, (2) quantity of water of crystallisation,
(3) solubility, presented by crystallised oxyhai'mogiobin, (4) dih'usibility.

1. Form. — (a) The blood of man and of the immense majority of
animals yields oxyheemoglobin which crystalhses in rhombic prisms or
needles belonging to the rhombic system.

(&) The oxyha^moglobin of the guinea-pig presents crystals which
were described by Lehmann as regular octohedra. They were, however,
shown by the eminent crystallographer v. Lang^ to be tetrahedra
belonging to the rhombic system.

The blood of certain birds,^ and occasionally apparently of the
rat ^, ^, ^, yields crystals of the same form.

(c) The oxyheemoglobin of the squirrel crystalhses normally in the
form of six-sided plates belonging, as proved by v. Lang, to the hexagonal
system. These crystals had been first described by Lehmann and Kunde.
Thie blood of the hamster {Cricetus vulgaris) contains oxyhaimoglobin
which crystalhses, as Lehmann showed, in rhombohedra and six-sided
plates belonging to the hexagonal system. Halliburton,® who has studied
the crystallography of oxyhemoglobin with great care, has made the
interesting observation that " after recrystallising squirrel's haemoglobin
several times the hexagonal constitution of the crystals is broken down,
and the crystals obtained are either rhombic prisms or a mixture of
these with rhomlDic tetrahedra."

Eollett,'^ taking for granted that oxyhsemoglobin, from whatever source
obtained, possessed the same chemical composition, argued, from the fact of
its crystallising generally in the rhombic, but in the case of the squirrel in the
hexagonal system, that oxyhaemoglobin should be looked upon as dimorphous.

Halliburton, however, with perfect correctness, hesitates to admit this
view, which could only be held if we were certain that the haemoglobins whose
crystals belong to different systems possess identical composition, and suggests
that perhaps the difference in the crystalline form, as well as the difference in
solubility of the haemoglobins which crystallise differently, depends upon
varying quantities of water of crystallisation— that, in fact, the haemoglobins
which crystallise in different systems represent " different hydrates of oxy-
haemoglobin." ^ This may be the case, though it appears to me that the cause
of the difference lies deeper.

It has been previously stated — and the grounds for the statement will be
given in a subsequent section — ^that, notwithstanding the perplexingly dis-
cordant results of the analyses of oxyhaemoglobin, there is, in the ha3moglobin
of all animals, absolute identity of the essential iron-containing nucleus, i.e.

1 111 a paper by A. Rollett, entitled "Versuche u. Beobachtungen am Blut, iiebst
kiystallographiscli. u. optiscb. Mittbeilungen iieber die Blutkiystalle von v. Lang,"
SUzungsb. d. Ic. Akad. d. JVissensch., Wien, 1862, Bd. xlvi. S. 66-98.

- Halliburton, "Text Book of Cliemical Pbysiology," London, 1891, S. 270.

■", •* Knnde, Lebniann, see Preyer, "Die Blutkry.stalle," S. 38.

■' Hoppe-Seylcr, "Ueber die Krystallformcn der Blutkrystalle," 3fed. Chem. Untersuclt.,
Berlin, 1868, S. 195.

''"Preliminary Coniimmication on tbe Hiemoglol)in Cry.stals of Rodents," Journ.
ricyslol., Cambridge and London, 1886, vol. vii. p. 2 ; Quart. Journ. Micr. Sc, London,
vol. xxviii. p. 181.

'' Loc cit.

^ Halliburton, "Text IJook of Chemical Physiology," section on tlie " Crystal! ogra])liy of
Oxyluemoglobin," pp. 270-274. The student is recommended to read this interesting and
suggestive section.



THE WATER OF CRYSTALLISATION.



205



of that moiety of the molecule on which its colour and its pliy.siological func-
tion depends. At the same time, there is such a dilfereuce in the ratio of
S:re in the haemoglobin of certain animals as renders it highly probable, or
rather certain, that, in the haemoglobin of different animal groups, the albu-
minous moiety of the complex molecule differs.' Such being the case, it is not
surprising that certain of the physical characters of hsemogiobin, such as
crystalline form and solubility, should exhibit variations.^ IS'or can Ave lose
sight of the possibility, to which I have already drawn attention, that the
differences in the hsemoglobins of certain animals may be due to their being
formed by the linking of the iron-containing molecule with different polymers
of the same albuminous group. The existence of hsemoglobins varying some-
what in their percentage of iron renders this view highly probable.

2. Quantity of water of crystallisation. — Eemarkable difticulties
encounter the observer in his attempts to determine the amount of
water of crystalUsation of oxyhaemoglobin, and considerable discrepancies
are to be noticed in the results obtained by different processes.

In order to make the determination, pure oxyh&emoglobin is dried
in vacuo at 0° C, and after ceasing to lose weight under these conditions
it is heated to a temperature of 115° C.

The following are some of the principal and most rehable results
obtained : —



OxyhiBinoglobin.


Water of

Crystallisation

per cent.


Authority.


Dog

Horse ....

Pig

Guinea-pig

Squirrel ....


3-4

3-94

5-9

6

9


Hoppe-Seyler.

Htifner.

Otto.

Hoppe-Seyler.

Hoppe-Seyler.



According to Bohr,^ the water of crystallisation of oxyhsemoglobin may
vary in amount between 1"2 and 6 '3 per cent., but these results, like others
obtained by the same author, and to which reference has been made (see p.
192), are explicable by the fact that his preparations of haemoglobin did not
represent the pure substance, and contained products of decomposition.

Without taking Bohr's results into consideration, there can be no
doubt that crystals of oxyhaimoglobin of different animals exhibit
differences in the amount of water of crystallisation. Assuming the
above results to be correct, the highly soluble oxyha?moglobin of the
pig, which crystallises in rhombic prisms, possesses the same amount of
water of crystallisation as the very sparingly soluble oxyhemoglobin of
the guinea-pig, separating in the form of tetrahedra.

3. Solubility. — The difticulties which encounter the observer in

^ The reader is referred to an admirable account of all the researches on the Crystal-
lography of Hsenioglobin, up to the date of its publication (1871), to the chapter entitled
"Krystallformen des Blutroths," in Preyer's work, "Die Blutkrystalle." Very fine
coloured engravings of the hpenioglobin crystals of various animals — amongst others, of
man, the guinea-pig, and the squirrel — are to be seen in Punke's "Atlas of Physiological
Chemistry," being a Supplement to Lehmanu's "Physiological Chemistry," London,
printed for the Cavendish Society, 1853. See plate x. and pp. 15-17 of the appended
letterpress.

^ "Exp. Untersuchungen ti. die Sauerstoffaufnahme des Blutfarbstotfes," Copenhagen,
1885.



2o6 HyEMOGLOBIN.

determining the water of crystallisation of the blood-colouring matter
are surpassed by those attending the estimation of its solubility. It is
doubtless in some measure due to the difficulty, almost the impossibility,
of eliminating every trace of certain of the reagents (especially the
alcohol), employed in the preparation of the body, that any attempts
to determine with precision the solubility of oxyheemoglobin have
failed. The chief cause of the discrepancies between the observations
of different observers is, however, probably that they were unaware
of the physical, and perhaps also chemical, changes which hsemoglobin
undergoes in the process of recrystallisation.

The oxyhemoglobin of all birds, of the ox, of the pig, and of man
is distinguished by its great solubility, the relative solubility increasing
in the above order. Next in order of solubility comes the haemoglobin
of the horse, dog, squirrel, guinea-pig, and rat, the latter being certainly
the least soluble.

According to C. Schmidt, 100 grms. of water at 18° C. dissolve 15"59 grms.
of the crystallised oxyhfemoglobin of the dog. From the fact that the oxy-
haemoglobin analysed by C. Schmidt when ignited yielded on an average 0'91
per cent, of P.jOg, we are in a position to state that the body he experimented
with was very impure, and consequently that his estimate of its sohibility in
water possesses no value. Hof)pe-SeyIer found that 100 c.c. of water at
5° C. dissolved 2 grms. of the dry oxyhsemogiobin of the dog.

Lehmann found that one part of the dry crystallised oxyhaemoglobin of
the guinea-pig required 597 parts of water to dissolve it, but the temperature
at which the determination was made is not stated ; ^ moreover, it is more
than doubtful whether the substance experimented with was pure.

The present state of our knowledge permits us, therefore, to state
that the oxyhemoglobin of different animals differs in no property so
remarkably as in its solubility in water. It appears, further, that oxy-
hemoglobin — which, according to the more recent researches, contains
the same percentage of iron (that of the horse, ox, dog, and pig), and
therefore presumably possesses the same molecular weight, and which,
further, crystalUses in the same manner — exhibits marked differences in
solubility. As the oxyhemoglobins of the horse and of the dog seem,
in so far as the water of crystallisation is concerned, to be identical,
and as the researches of Hlifner and his school have proved the identity



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 29 of 147)