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globin, whilst that contained in venous blood should be named Phlehin, to

1 " Ueber die Verbindungen des Hamoglobins mit Sauerstoff," "Ueber die specifische
SaiierstofTinenge des Bhites iind die Bedeutimg derselben fiir den resinratorischen StofF-
wechsel," CentralU. f. Physiol., Leipzig u. Wien, 1890, 15d. iv. S. 242, 254.

- In this place it is only necessar}' to refer to one of Htifner"s papers. See G. Hufner,
"Neue Versiiche zur Bestiminung der Sauerstoffcapacitat des Bhitfarbstoffs," Arch. /.
Physiol., Leipzig, 1894, S. 130, 176. Refer particularly to pp. 130, 134, 175, 176.

'^ Ztschr.f. physlol. Chem., Strassburg, 1889, Bd. xiii. S. 495.



ox YH^MO GL OB IN. 1 93

distinguish it from reduced haemoglobin ! The only distinction which Hoppe-
Seyler found to exist between " arterin " and " phlebin " consisted in the alleged
greater ease with which the hypothetical constituent of arterial blood yielded
its dissociable oxygen, when boiled in vacuo., as compared with the hypo-
thetical constituent of venous blood. To establish this alleged difference
between arterial and venous blood would require a body of experimental facts,
such as does not exist. Even were the difference shown to be a real one, it
would in no way support the hypothesis of a radical difference between the
colouring matter of arterial and venous blood. But the investigations of
Hiifner, which have proved with mathematical accuracy that the colouring
matter of the blood behaves, both in so far as its optical characters and
its relations to oxygen are concerned, precisely as a solution of haemoglobin,
and is tJie only coloured constituent of the corpiiscles, complete the demonstra-
tion of the erroneous nature of the hypothesis advanced by Hoppe-Seyler
on this subject.

OXYH^MOaLOBIN.

Methods of Pkeparation.

Introductory remarks. — It has already been stated that the blood-
colourmg matter of different species of animals is not, in all particulars,
absolutely identical. Although behaving in the same manner in refer-
ence to the gases w^ith which it can combine to form more or less
easily dissociated compounds, and whilst possessing identical powers of
absorbing the rays of the spectrum, the haemoglobin of different animals
exhibits differences (1) in crystalline form, (2) in solubihty, (.3) in the
quantity of water of crystallisation, (4) in percentage composition.
These differences will be carefully examined in the sequel, but attention
is drawn to them in this place, in relation to another point of difference,
namely, the variation in the facility of separating haemoglobin in a
crystalhne form. From the blood of certain animals, crystals of hemo-
globin can most readily be prepared, whilst in other cases the task
is one of very considerable difficulty. Among the conditions which
influence the result, the degree of solubility of the blood-colouring
matter is the chief. Thus the blood of the rat, the guinea-pig, and
the squirrel, which contains the least soluble hasmoglobin, yields crystals
with great facility ; whilst the blood of man, and that of the domestic
herbivorous animals, which possess liEemoglobin of remarkable solu-
bility, yields crystals with extraordinary difficulty. It is impossible
to state with accuracy the relative facility of crystalhsation of the
haemoglobin of different animals, but the following statements are pro-
bably correct. The blood of the rat, the guinea-pig, and the squirrel
crystalhses most readily ; next comes the blood of the cat, the dog, and
the horse ; the blood of man and the pig follow, whilst that of the
rabbit, the sheep, the ox, and the frog crystallise with the greatest
difficulty.

The principle upon which the majority of the methods for the
separation of haemoglobin in a crystalline form are based is the
following : — To effect the solution of the haemoglobin of the coloured
corpuscles in the serum, or in water, added to the previously separated
corpuscles ; and thereafter, by the addition of alcohol, or of ether, or
by the agency of cold, or of both cold and alcohol or ether conjointly,
sometimes aided by the process of evaporation, to cause the haemoglobin,
VOL. I. — 13



194



HAEMOGLOBIN.



which is sparingly sohible in dilute alcohol, especially at low tempera-
tures, to crystallise. In the case of animals, the hasmoglobin of whose
blood is very sparingly soluble, the addition of alcohol or ether is often
dispensed with. We shall, in the first place, describe those methods
which readily furnish haemoglobin crystals for the purposes of micro-
scopical research, and then the methods which are employed for the
preparation and purification of large quantities of haemoglobin.

Methods employed in preparing small quantities of haemoglobin
for microscopic examination. — 1. Funkcs method} — From the blood of
those animals whose blood crystallises readily, but especially in the
case of the rat, oxyhtemogldbin can be obtained for microscopic exami-
nation in three or four minutes, by receiving a drop of blood on a
glass slide, adding a drop of distilled water, mixing the two hquids
by means of a needle, and spreading the mixture over the central part
of the slide. When the edges of the liquid commence to dry, cover
with a microscopic covering glass. Crystals of haemoglobin form at
once.

2. Bollett's method? — A platinum capsule is placed in a freezing
mixture, and freshly defibrinated blood is poured into it, so as to convert
it into a lump of red ice. After being in the freezing mixture for half
an hour the blood is allowed to thaw gradually, and the contents of the
capsule are poured into a glass vessel of such dimensions that the
bottom is covered by the lake-coloured blood to a depth of 15 mm. ;
the glass vessel is then set aside in a cool place. In a short time,
the blood of rats, of guinea-pigs, and of squirrels, treated by this method,
furnishes well-formed crystals.

3. Gscheidlens method.^ — Defibrinated blood, which has been exposed
to the air for a period of twenty-four hours, is sealed in narrow glass
tubes (vaccine tubes answer well), and these tubes are then placed in
the incubator and kept a temperature of about 37° C. for some days.
On opening the tubes and emptying their contents into a watch glass or
on a glass slide, and allowing some time for evaporation to take place,
crystals of extraordinary size are obtained.

4. Max Sehultzes method.'^ — Defibrinated blood is heated (on a warm
stage, in the case of a microscopic preparation) to a temperature of 60° F.,
when the corpuscles dissolve and the blood becomes lake-coloured ; it
is then allowed slowly to cool and to evaporate. This method may be
employed with large quantities of blood, and Preyer ^ found that by no
other method did he obtain as fine and as large crystals from horse's
blood.

In addition to the four methods which have been above described as
most conveniently yielding crystals of oxyhaemogiobin, when these are
desired on a small scale, there are many others which have been employed,
and which occasionally give good results.

Thus Piollett ^ found that when induction shocks were passed through
blood, it became lake-coloured and yielded crystals of haemoglobin, and

1 Ztschr.f. rat. Med., 1851, S. 185.

2 "Versuche und Beobaclitungen am Blute," Sitzungsh. cl. h. Akad. d. JVisscnsch.,
Wien, 1863, Bd. xlvi. S. 77.

2 Arch.f. d. yes. Physiol., Bonn, 1878, Bd. xvi. S. 421.

* " Eiu lieitzbarer Objecttiscli und seine Verwendung Lei Untersuclimigcii des Blutes,"
Arch.f. mikr. Anat., Bonn, 1865, Bd. i. S. 31.

5 "Die Blutkrystalle," S. 23.

* Sitzunrjsb. d. k. Akad. d. Wissensch., Wien, 1852, Bd. xlvi. S. 75.



PREPARATION OF OXYHEMOGLOBIN. 195

A. Schmidt ^ made the same observation in reference to the blood near
the positive pole, when this liquid is subjected to the action of a
constant current.

Without undergoing any other treatment, the blood of asphyxiated
animals often crystallises. Blood which has been deprived of its gases,
by boiling in vacuo, often crystallises. Indeed, by this method, Preyer ^
succeeded in the very difficult task of crystallising sheep's blood.

Methods of preparing considerable quantities of oxyhsemo-
globin. — Certain of the methods already recommended for the prepara-
tion of hajmoglobin, when small quantities only are needed for the
purposes of microscopic investigation, might be employed in the pre-
jjaration of larger quantities. Other processes, however, are to be
preferred, and of these some which are specially to be recommended
are given below. Of these processes, the first, or Hiifner's modification
of it, should, by preference, be employed, especially if a preparation, as
free as possible from products of decomposition, be desired.^

Hoppe-Seylers method.'^ — Defibrinated blood is mixed with ten times
its volume of a solution of sodium chloride^ (made by diluting one
volume of a saturated solution of NaCl with nine volumes of water),
and the mixture is poured into shallow basins, which are set aside in a
cool place, so as to allow the greater part of the blood corpuscles to
settle.^ The supernatant liquid is decanted, and the magma of corpuscles,
mixed with a small quantity of water, is poured into a stoppered
separating funnel. The contents of this funnel are treated with an
equal volume of ether. After repeated, but not too violent, agitation,
the deep red aqueous solution is separated from the supernatant ether,
and filtered as quickly as possible. The clear red filtrate cooled to 0° C.
is then mixed with one-fourth of its volume of absolute alcohol, likewise
cooled to 0° C. The mixture is then maintained for a couple of days
(and, if crystallisation has not occurred, even longer), at a temperature of
— 5° C. to - 10° C. In a period varying between twenty-four and
forty-eight hours crystallisation has usually occurred, and, unless the
solution of haemoglobin was too dilute, the whole of the liquid has set into
a mass of crystals. The crystals are now collected on a filter (the process
of filtration being carried on at as low a temperature as possible, in any
case below 0° C.) and washed several times with a previously cooled
mixture, composed of one volume of absolute alcohol and four volumes
of distilled water. The filter with its contents is now placed between
sheets of filtering paper, and as much as possible of the adhering mother-
liquor is removed by gentle pressure. The oxyhgemoglobin thus

^ "Zuv Krystallisation des Blutes," Firchoiv^s Archiv, 1864, Bd. xxix. S. 29.

- "Die Blutkrystalle," S. 19 and 20.

^ The blood of the dog, and especially of the horse, are to be preferred for the preparation
of large quantities of oxyhfemoglobin. As the success of the various operations depends
upon their being conducted at a low temperature, the preparation of hfemoglobin for purposes
of research should only be attempted in the depth of winter.

■* " Beitriige zur Kenutniss des Blutes des Menschen und der Wirbelthiere," Med.
Chem. Uoitersuch., Berlin, 1866, S. 170, 180-185; " Handbuch d. physiologisch. chem.
Analyse," Berlin, 1893, Aufl. 6, S. 274.

^ In the preparation of haemoglobin from the blood of birds, amphibia, and fish, sodiuna
sulphate is to be employed in the place of sodium chloride. In the case of mammalian
blood, it presents no advantages over sodium chloride.

^ Instead of allowing the corpuscles to separate, as described, it is preferable to employ a
centrifugal machine. The separation of the corpuscles from the mixture of serum and salt
solution is not only very much more rapid, but also much more complete, and therefore the
obtaining of pure oxy haemoglobin is facilitated.



196 HAEMOGLOBIN.

obtained may now be purified by being recrystallised. With this object
the moist crystals are removed by means of a spatula from the filter,
and placed in a flask or beaker, and about three times their volume
of distilled water is added. The mixture is heated to 55° C, the solu-
tion filtered ; the filtrate is cooled to 0° C, and to every four volumes
one volume of absolute alcohol, cooled to 0° C, is added. The mixture
is then cooled to - 5° C. or - 10° C.

When the oxyheemoglobin separates again, this process of crystallisa-
tion may be repeated five or even six times, providing the temperature
at which the various operations are conducted be a very low one. The
recrystallised hfemoglobin obtained by these processes may be employed
to make standard solutions of the body, or it may be dried. It is very
questionable, however, whether the recrystallising of oxyhcemoglobin is
advisable, for reasons to be stated below, it being probably better to purify
the crystals by repeated washings with ice-cold water. Hoppe-Seyler
states that oxyhaemoglobin can only be dried, without decomposition, in
vacuo, at a temperature under 0° C. If dried at a higher temperature it
assumes a dark colour, and ceases to be entirel}^ soluble in distilled water.

Zinoffsky,^ who worked with oxyhemoglobin prepared from the
blood of the horse, found that, when spread out in very thin layers, it
could be dried in vacuo in eight hours, without undergoing decomposi-
tion, at a temperature of 10° C. to 20° C. He found that the oxy-
haemoglobin thus prepared was entii'ely soluble in distilled water, and
that the solution was not precipitated by lead acetate ; proving that no
methffimoglobin had been formed.

Hfemoglobin which has been dried in vacuo, over sulphuric acid or
phosphoric anhydride, at a temperature of 0° C, may be heated to 110° C.
or 115° C, without undergoing any decomposition.

Modifications of Hoppc-Scylers method. — (a) Among numerous modi-
fications may be mentioned one employed by Hiifner,^ and which may
with advantage be adopted in laboratories provided with centrifugal
machines. The blood is not treated with salt solution, but the corpuscles
are separated by the action of the centrifuge alone. Crystals thus
obtained are treated with ice-cold water, separated by the centrifuge,
and this process repeated several times. Mnally, the crystals are dried
on porous plates made of cellulose, or solutions are made of the yet
moist crystals, and the percentage of hemoglobin in them determined.

(h) The defibrinated blood of the dog is mixed with its own volume
of distilled water, and the diluted fluid is treated with one-fourth its
volume of alcohol. The mixture is kept for twenty-four hours, at a
temperature which must be lower than 0° C. The crystals which
separate are dissolved in about three tunes their bulk of distilled water,
at a temperature of 30° C, and the solution being cooled to 0° C, a
fourth of its volume of absolute alcohol at 0° C. is added. The fluid
should be kept in a freezing mixture at a temperature of — 10° C. to
- 20° C. for twenty-four hours. The whole fluid then becomes con-
verted into a magma of crystals. The process of recrystalhsation may
be several times repeated.



^ "Ueber die GriJsse des Haniogloljin-moleculs," ^to/ir. f. physiol. Clicm., Strassburg,
1886, Bd. X. S. 15-34. See "Darstelhmg des Ilamoglobiiis," S. 18-24.

'^ " Beitvag zur Leiire von Blutfarbstoffe," Beitr. z. Physiol. 0. Luchvig z. s. 70 Gchnrtst.
etc., Leipzig, 1887, S. 74-81 ; and "Neiie Versuche, u.s.w.," ^rc/i. /. Physiol., Leipzig,



PURIFICATION OF HEMOGLOBIN. 197

(c) Defibrinated blood is treated with about one-sixteenth its
vokime of ether (say 31 c.c. of ether to 500 c.c. blood), and the mixture
shaken for some minutes. It is then set aside in a cool jjlace. After a
period, varying from twenty-four hours to three days, the liquid has
been converted into a thick magma of crystals. These may be separated
by placing in tubes and using the centrifugal apparatus. The cakes of
crystals are treated with a mixture of one part of absolute alcohol and
four parts of distilled water, and again centrifugalised. By repeating
this process the crystals are ultimately obtained free from serum
albumin. The crystals may be dissolved in water and recrystallised, as
described in Hoppe-Seyler's method.

In addition to the methods described, many others have been
suggested, and to these only a passing reference need be made.

Thus Kiihne devised a method based upon the fact that the stroma
of the coloured corpuscles is dissolved by the addition of a watery
solution of crystallised bile (a mixture of sodium glycocholate and
taurocholate).^ Hiifner ^ and his pupil Otto employed a 1 per cent,
alcoholic solution of chinoline, or a watery solution of the hydrochlorate
of the same base, to prepare oxyhaemoglobin from pig's blood, though
Otto afterwards found ^ that, by taking special precautions, Hoppe-Seyler's
method is available, even in the case of pig's blood, and indeed preferable
to all others.

Remarks on the puriflcation of hsemoglobin. — It has, until lately,
been assiuned that in the preparation of pure oxyhae.niogiobin the body should
be recrystaUised as frequently as possible, with the object of getting rid of all
traces of adherent albuminous and saline impurities derived from the plasma
or serum. Since spectrophotometry has supplied us with a method of deter-
mining, with an accuracy previously unattainable, the purity of a colouring
matter, it has been found that although oxyhaemoglobin which has been
recrystallised, when examined in the ordinary manner, exhibits a spectrum
which appears identical with that of the colouring matter which has been only
once crystallised, its spectrophotometric constants have changed ; in other
words, when oxyheemogiobin is recrystallised it undergoes a change, possibly
only affecting its physical, but more probably affecting its chemical constitution
also. The knowledge of these facts has caused Hiifner in his recent researches
to employ hsemoglobin which has not been recrystallised.

If precautions are taken in the first instance to separate (by the most perfect
filtration, followed by prolonged centrifugalising) all formed elements and acci-
dental solid impurities from the solution of blood corpuscles which is to be
crystallised, and if the crystalline mass of oxyhaemoglobin obtained be repeatedly,
say five or six times, treated with ice-cold water, the resulting solution being
each time separated from the midissolved crystals by very rapid and very
prolonged centrifugalising, the portion of the original crystals still left undis-
solved will be found, on chemical, microscopical, and spectrophotometric
investigation, to furnish evidence of being a pure substance.

The new method is more easily and much more expeditiously carried out
than the old.

Elementary composition of oxyhaemoglobin dried at 110°-115° C.

— Before describing either the physical or chemical properties of the

1 Centralhl. f. cl. mccl. Wif<senscTi. , Berlin, S. 833.

" The account of Hlifuer's discovery of this method is contained in a paper by his pupil,
F, Otto, "Ueber das Oxyharaoglobin des Schweines," Ztschr. f. physiol, Ghem., Strassburg,
1882-83, Bd. vii. S. 57.



198 HEMOGLOBIN.

blood-colouring matter, it is advisable to consider its elementary com-
position, and to ascertain how the results of chemical analysis bear on
the question as to htemoglolnn being a definite chemical individual, its
composition being invariable.

Heemoglobin is a compound of carbon, hydrogen, nitrogen, sulphur,
iron, and oxygen. The crystals of haemoglobin contain water of crystal-
lisation, which varies considerably in amount in the haemoglobin of
different animals. When ignited, pure haemoglobin obtained from
mammahan blood yields an ash composed entirely of ferric oxide ; the
haemoglobin of birds and fishes, and probably of all animals with
nucleated corpuscles, yields on ignition an ash which, in addition to
FejOg, contains phosphoric anhydride (P2O5), derived in all probability
from nuclein contained in the corpuscles.^

The earlier analyses of oxyhaemoglobin made by C. Schmidt ^ and
by Hoppe-Seyler 2 exhibited results which appeared to indicate that
crystallised oxyhaemoglobin is a body of constant composition. From
the analyses of these two observers, and his own determinations of the
iron and sulphur in crystallised oxyhaemoglobin, Preyer deduced the
following as the mean percentage composition of oxyhaemoglobin : —





In 100 Parts.




c




54-00


H




7-25


^




16-25


Fe .




0-42


S




0-63







21-45



100-00

On the assumption, which a large number of facts have since shown
to be almost certainly correct, that the molecule of haemoglobin contains
one atom of iron, Preyer assigned to it the empirical formula CeooHggo
ISTig^FeSgOi^o, the molecular weight being 13,332.



Analysis of Oxyhcemoglohin dried at a temperature above 100° CS' (H


oppe-Seyler).


Oxyhasmoglobin of


Water of Crystallisa-
tion in the "Crystals
which had been
dried in vacuo.


C.


H.


N.


0.


s.


Fe.


P2O5.


Dog .


3-4 per cent.


•53-85


7-32


16-17


21-84


0-39


0-43




Goose .




54-26


7-10


16-21


20-69


0-54


0-43


0-77


Guinea-pig .


6 „ „


54-12


7 '36


16-78


20-68


0-58


0-48




Squirrel


9 ,, ,.


54-09


7-39


16-09


21-44


0-40


0-59





^ Y. Inoko, " Einige Bemerkungen ueber phospliorhaltige Blutfarbstoffe," Ztschr. f.
physiol. C'hcm., Strassburg, 1894, Bd. xviii. S. 57.

^"Analyse der Blutlvrystalle," in Bottcher's monogvapli, "Ueber Blutkrystalle,"
Dorpat, 1862.

^ "Beitriige zur Kcnntniss des Blutes des Menschen und der Wirbelthiere ; Zusammen-
setzung der Farbstofflcrystalle des Meerschweinchen- und de.s Plunde-blutes," Med. Chem,
Untersitch., Berlin, 1868, S. 186 et seq.



ELEMENTARY COMPOSITION OF OXYHEMOGLOBIN 199



The subsequent researches of Hoppe-Seyler soon demonstrated,
however, that the blood crystals obtained from the blood of different
animals did not possess an identical composition, though the differences
brought out by Hoppe-Seyler's analyses were not very great. His
results are shown in the table on p. 198.^

The very numerous analyses of oxyhsemoglobin of different animals,
made in recent years by Kossel,^ Otto,^ Zinoffsky,'* Hufner,^ Jaquet,^
and others exhibit, however, such extraordinary discrepancies in the
results of ultimate organic analysis as to preclude a precise answer
being given to such simple questions as the following : —

Is haemoglobin a body, having a constant composition in animals
of the same species ?

Does the hemoglobin of different animals vary in chemical com-
position, and if so, within what limits ?

Results of the more recent Analyses of Oxyhcemoglohin (1878-1890).



Oxyhgemoglobin of


0.


H.


N.


S.


Fe.


0.


P2O5.




Dog .


53-85


7-32


16-17


0-89


0-43


21-84




Hoppe-Seyler. '^


,, .






53-91


6-62


15-98


0-540


0-333


22-62




Jaquet.^


))






54-57


7-22


16-38


0-568


0-336


20-93




Jaquet. ^


Horse






54-87


6-97


17-31


0-650


0-47


19-73




Kossel.i*'


))






54-76


7-03


17-28


0-67


0-45


19-81




Otto."


))






54-40


7-07


17-40


0-66


0-45


19-74




Blicheler.^"


>j






51-15


6-76


17-94


0-39


0-335


23-43




Zmoffsky.13


Ox .






54-66


7-25


17-70


0-447


0-400
0-336


19-54




Hilfner."
Hlifner.15


Pig ;






54-17


7 '-38


16-23


0-660


0-430


21-360




Otto. 16








54-71


7-38


17-43


0-479


0-399


19-602




Hufner."


Hen!






52-47


7-19


16-45


0-857


0-335


22-500


0-197


Jaquet. 1^



1 Med. Chem. UntersucJi. , Berlin, 1868, Heft 3, S. 370.

" The results of the analyses made by Dr. Kossel were published in a paper by Hoppe-
Seyler, entitled "Weitere Mittheilungen ueber die Eigenschaften des Blutfarbstoffs — Das
Oxyhamoglobin des Pferdeblutes, " Ztschr. f. physiol. Chem., Strassburg, 1878-79, Bd. ii.
S. 149-155.

^ "Ueber das Oxyhamoglobin des Schweines," ibid., 1882-83, Bd. vii. S. 57-68.

* "Ueber die Grosse des Hamoglobinmoleculs," ibid., 1886, Bd. x. S. 16-34.

^ " Ueber das Oxyhamoglobin des Pferdes," Ztschr. f. physiol. Chon., Strassburg,
1883-84, Bd. viii. S. 358-365; " Beitriige zur Lehre vom Blutfarbstoffe," in Beitr. z.
Physiol. G. Ludioig z. s. Gebtirtst. etc., Leipzig, 1887, S. 74-81 ; "Neue Yersuche zur
Bestimmung des Sauerstoffscapacitat des Blutfarbstoffs," Arch. f. Pliysiol., Leipzig, 1894,
S. 130-176. See especially S. 174-176.

" " Elementaranalyse des Hundeblut-Hamoglobins," Ztschr. f.physiol. Chem., Strass-
burg, 1888, Bd. xii. S. 285-288 ; Beitrage zur Kenntniss des Blutfarbstoffes, " ibid.,
1890, Bd. xiv. S. 289-296.



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