E. A. (Edward Albert) Sharpey-Schäfer.

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In a series of researches, which extended from 1867 to 1872, Lankester
investigated the distribution of haemoglobin throughout the animal kingdom,
and comparatively few facts have since been added to those which he
published in 1872."

The following are among the jDrincipal facts hitherto ascertained in rela-
tion to the distribution of haemoglobin.^

Haemoglobin occurs : —

1. In special corpuscles —

(a) In the blood of all vertebrates, excepting Le2:)toeex>lialus and Amjpliioxus.

^ Felix Hopj^e iu Tubingen, "Ueber das Verhalten des Blutfarbstoffes im Spectrum
des Sonnenlichtes," Virchow's Archiv, 1862, Bd. xxiii. S. 446-449; " Ueber die chenii-
sclien u. optischen Eigenscliafteu des Blutfarbstotfs," Virchow's Archiv, 1864, Bd. xxix.
S. 233-245.

^ " Ueber den Farbstoff der Muskeln," Virchow's Archiv, 1865, Bd. xxxiii. S. 79;
Kiihne, " Lehrbuch d, phys. Chemie," 1868, S. 288.

^ " Das Bhit der Ptegenwiirmer, " Journ. f. frakt. Chcm., Leipzig, 1839, Bd. xvi. S. 152.

■* " Ziir Kenntniss der Verbreitung des Haematins," Sitzungsh. d. k. Akad. d. JFissensch.,
Wien, 1861, Bd. xliv. S. 615-630.

^"Observations with the Spectroscope," Journ. Anat. and Physiol., London, 1867,
S. 114.

** " Optisclie Eigenschaften des Bhitfarbstolfs," Centralhl. f. d. mcd. JFissensch., Berlin,
1867, S. 196.

' "A Contribution to the Knowledge of Hsemoglobin, " Fi-oc. Boy. S'oc. London, 1872,
vol. xxi. pp. 70-81.

^ The student is advised to read the interesting chapter, entitled "The Blood of
Invertebrate Animals," in Halliburton's Text-Book, see pp. 316-330.


(In AmpMoxus Lankester failed to obtain spectroscopic evidence of the pre-
sence of lisemoglobin, though Wilhelm Mliller of Jena had described the
corpuscles of this vertebrate as of a pale red colour.) :

(&) In the perivisceral fluid of some species of the vermian genera,
Glycera, Capitilla, and Phoronis :

(c) In the lamellibranchiate molluscs Solen and Area.

2. Diffused in a vascular or ambient liquid —

(a) In the peculiar vascular system of the chaetopodous annelids, very
generally, but with apparently arbitrary exceptions :

(h) In the vascular system (which represents a reduced perivisceral cavity)
of certain leeches [Nephelis, Hirudo), but not of all :

(c) In the vascular system of certain turbellarians, as in PoUa sanguirubra :

(d) In a special vascular system (distinct from the general blood system)
of a marine parasitic crustacean (undescribed), observed by Professor Edouard
van Beneden :

(e) In the general blood system of the larva of the dipterous insect
Chironomus ; and in Musca domestica : ^

if) In the general blood system of the pulmonate mollusc PlanorMs. Mr.
H. C. Sorby expressed the opinion that probably the colouring matter found in
the blood of Planorhis is not identical Avitli haemoglobin. I have shown, how-
ever, that the position of the absorption-bands of the colouring matter of the
blood of Plcmorbis coincides exactly with that of the haemoglobin bands : ^

(g) In the general blood system of the crustaceans Daphnia and
Cheirocephalus (Lankester) ; also in Apus and Gypris.^

3. Diffused in the substance of muscular tissue —

(a) In the voluntary muscles generally of Mammalia, and probably of
birds, and in some muscles of reptiles :

(&) In the muscles of the dorsal fin of the fish Hippocampus, being
generally absent from the voluntary muscular tissue of fish :

(c) In the muscular tissue of the heart of Vertebrata generally :

(cZ) In the unstriped muscular tissue of the rectum of man, being absent
from the unstriped muscular tissue of the alimentary canal generally :

(e) In the muscles of the pharynx and odontophore of the gastropodous
molluscs (observed in Lymnwus, Paludina, Littorina, Patella, Chiton,
Aplysia), and of the pharyngeal gizzard of Aplysia, being entirely absent from
the rest of the muscular and other tissues and the blood of these molluscs :

(/) In the muscular tissue of the pharyngeal tube of Aphrodite aculeata
(Lankester), being absent from the rest of the muscular tissue, and from the
blood in this animal, and absent from the muscular tissue generally in all
other annelids, as far as yet examined.

4. Diffused in the substance of nervous tissue —

(a) In the chain of nerve ganglia of Aphrodite aculeata (Lankester). In
this annelid the chain of nerve ganglia possesses a bright crimson colour. The
colour is most intense in the supra-oesophageal ganglion, which has as intense
a colour as a drop of fresh human blood. The colour impregnates the nerve
itself, and is not contained in a liquid bathing the tissue :

(&) An exactly similar observation has been made by Hubrecht, who found
haemoglobin in the red-coloured cerebral ganglia of certain Nemertine worms,
which possess no coloured blood corpuscles.*

1 MacMimn, "Animal Cliromatology," Proc. Birmingham Phil. Soc, vol. iii. p.
130 (quoted at second-hand).

2 Gamgee, "A Text-Book of the Physiological Chemistry of the Animal Body,"vol. i. p. 131.
^ Regnard et Blanchard, "ISTote sur la presence de rhemoglobine dans le sang des

crustac^s branchiopodes," Compt. rend. Soc. de biol., Paris, 1883, pp. 197-200.

* A. A. W. Hubrecht, " Untersuch. ueber Nemertinen aus dem Golf von Neapel,"
Niederland. Arch. f. Zoologie, 1876, Heft 3, Abstract in Jahresh. ii. d. Fortschr. d. Thier-
Chem., Wiesbaden, Bd. vi. S. 92.


The Peobable Eelations of the Blood-Colouking Matter to
THE other Constituents of the Coloured Corpuscles.

Without encroaching upon the domain of histology, reference must
be made to the two principal views which have been advanced in
reference to the structure of the coloured corpuscles.

According to the first/ which dates from the time of Bidloo,^ Wells,^
and Hewson/ and which was strongly advocated by Schwann, the
coloured corpuscles of the blood are vesicular bodies, possessing an
external envelope enclosing fluid contents.

This view has been revived and strongly insisted upon by Schafer,^
who briefly describes the structure of the red corpuscle in the following
sentence : — " Each red corpuscle is formed of two parts, a coloured and
a colourless, the former being a solution of ho&moglohin ; the latter, the
so-called stroma, which is in by far the smaller quantity, being composed
of various substances, chief among these being lecithin and cholesterin,
together with a small amount of cell globulin." ^

According to the second view, which, in its present form, we owe to
EoUett '^ and Brlicke,^ and which for many years found general favour, the
coloured blood corpuscle is not considered as vesicular, but as a viscous
solid mass composed of a colourless, highly elastic framework, tlu stroma
(EoUett) denser at the periphery than at the centre, in the interstices
or trabeculte of which haemoglobin and the other constituents of the
corpuscles are contained.

Without attempting to decide which of these views, if either, is the
correct one, it is expedient to consider some questions bearing upon
them, and towards the solution of which we possess important facts.

Making for the moment the assumption which, as will be shown in
the sequel, is denied by Hoppe-Seyler, that oxyhaemoglobin exists as
such in the coloured blood corpuscles, the question arises, in what
physical state does it occur ? Is it simply dissolved in the liquid
contents of the corpuscles, or is it dissolved in virtue of its being in com-
bination with other constituents ? Is it in a soHd condition ? and if so,
is there any evidence as to whether its structure is crystalline or
amorphous ?

That the colour of the blood does not depend upon a simple aqueous
solution of haemoglobin, is evident when we consider that the blood
corpuscles are among the soft parts of the body which contain the least
water ; ^ and that not only is the water which the coloured corpuscles
contain altogether insufficient to hold the hEemoglobin in solution, but in
some animals, the haimoglobin of which is more sparingly soluble than

^ a reference to and discussion of the earlier literature relating to this view will be
found in Gamgee's "Physiological Chemistry," vol. i. p. 72.

^ " Anatomia humani corporis, 1685," quoted by Milne-Edwards, "Lecoiis, etc.,"
tome i. ]). 66.

3 "On the Colour of the Blood," Phil. Trans., London, 1797, p. 429.

* Hewson's Works, Syd. Soc.

^ " Quain's Anatomy," 1891, vol. i. pt. 2, p. 210.

^ Halliburton and Friend ; since shown to be a nucleo-proteid. — Editor.

' Sitzungsh. d. k. Akad. d. JFissensch., Wien, 1862, Bd. xlvi. Abth. 2, S. 73.

** Brllcke apjilied the term Oekoid to the stroma, ibid., Wien, 1867, Bd. Ivi. Abth. 2,
S. 79.

® According to Bunge, "Zur quantitativen Analyse des Blutes," Ztschr. f. Biol.,
Mlinchen, 1876, Bd. xii. S. 191, the blood cor]niscles contain 36"7 parts of solids, and 63"3
parts of water ; muscular tissue contains about 25 per cent, of solids, and 75 per cent, of
water ; nerves contain about 22 per cent, of solids, and 78 per cent, of water.


is generally the case, the whole of the water contained in the blood would
not suffice to dissolve the hcemoglobin stored up in the coloured

That the haemoglobin is not contained in the blood corpuscles in the
form of infinitely minute crystals, is proved by examining the corpuscles
between crossed Nicols, when they are found not to be doubly refract-
ing ; whilst crystals of hsemoglobin, even when reduced to a state of most
minute subdivision, are so.^

Furthermore, no crystalline or granular structure can be discovered
when the coloured corpuscles are examined with the highest powers of
the microscope.

The assumption was made by Preyer,- that hcemoglobin exists in the
corpuscles in combination with potassium, alkaline solutions jDossessing the
property of dissolving much larger quantities of hcemoglobin than pure water,
and potassium being the most abundant of the mineral constituents of the
coloured corpuscles of man, though by no means of all animals.^ But, as a
matter of fact, the coloured blood corpuscles do not behave as if they contained
free haemoglobin in a solid condition, or in solution, or a solution of an alkaline
compound of hsemoglobin. Only one proof of this statement need be given in
this place, others being adduced when discussing the remarkable, and, as it
appears to me, untenable proposition of Hoppe-Seyler, that the blood-colouring
matter, as it exists in the living corpuscles, differs remarkably in properties from
hsemoglobin, so that it should be distinguished by a separate name or names.
The one proof to which reference is made is furnished by the fact that the
colouring matter of the red corpuscles is not extracted from them by the plasma,
or serum, or by fairly concentrated solution of neutral salts, as would be the
case if they contained free hsemoglobin, or an alkaline compound of that
substance.'^ To explain the fact that hsemoglobin is retained by the corpuscles,
Hoppe-Seyler advanced the plausible hypothesis, that it exists in them in
combination with some constituent of the stroma, and he expressed the opinion
that this constituent is lecithin. There are absolutely no grounds for the
latter assumption ; and it has indeed yet to be proved that the phosphorus-con-
taining principle of the stroma of the coloured corpuscles is lecithin and not
protagon, as had been very positively asserted by Hermann.^

Without attempting to speculate beyond the facts which we possess,
it may, however, be assumed that haemoglobin exists in the blood,
corpuscles in the form of a compound with a yet unknown constituent
of the corpuscle. This compound, the existence of which we are forced
to assume, is characterised by remarkable instability, for it is decomposed,
setting free the haemoglobin, which then passes into solution — (1) when
the blood plasma or serum, in which the corpuscles are suspended, is
diluted ; (2) when certain substances act upon the corpuscles (ether,
chloroform, salts of the bile acids, certain products of putrefaction);
(3) by the action of heat ; by alternate freezing and thawing ; by induction
shocks, etc.

1 W. Preyer, "Die Blutkrystalle," Jena, 1871, S. 28.

'Ibid., S. .30-33.

^ G. Bunge, " Zur quantitativeii Analyse des Blutes," Ztschr. f. Biol., Miinchen 1876
Bd. xii. S. 191.

"* This is no real proof that hsemoglobin is not in solution ; it is merely a statement of
the fact that it is indiffusible through the unaltered envelope of the corpuscle. It is,
moreover, capable of proof that the contents of the red corpuscles are completely fluid
during life. Cf. p. 193, lines 9 to 12.— Editor.

5 Arch./. Anat. u. Physiol., Leipzig, 1866, S. 33.



Hypothesis of Hoppe-Seyler, that the coloured substance of the
corpuscles possesses properties which differ from those of hsemo-
globin— Arterin (?), and Phlebin (?). — It has been shown that we are
forced to assume the existence in the coloured corpuscles of a very unstable
compound of haemoglobin. Hoppe-Seyler, as far back as 1877/ expressed the
opinion that whilst the compound or compounds of haemoglobin existing in the
blood corpuscles absorb the rays of the spectrum precisely as solutions of
haemoglobin, in other respects very remarkable differences can be detected,
certain of these differences being, in his opinion, of great physiological

Subsequently,^ Hoppe-Seyler, returning to this subject, endeavoured to
prove by a variety of arguments that such are the differences between the
properties of the colouring matter as it exists in the coloured corpuscles and
pure haemoglobin, that we cannot logically assert that they are identical. He
examined in detail the differences in behaviour which had been observed by
himself and by others, between the blood-colouring matter as it exists in the
corpuscles and solutions of pure oxy- or reduced haemoglobin. He referred to
the undoubted fact that the colouring matter, as it exists in the coloured
corpuscles, is not dissolved out by serum, liquor sanguinis, or saline solutions,
of a certain strength. It does not, he alleged, crystallise, nor readily yield
its dissociable oxygen when heated in vacuo ; it readily decomposes peroxide
of hydrogen (H.,0.,), setting free ordinary inactive oxygen, and is not oxidised
during the process ; a solution of potassium ferricyanide does not for a long
time attack the blood corpuscles, or convert their colouring matter into

On the other hand, a solution of oxyhaemoglobin (or, as Hoppe-Seyler
preferred to express it, of the oxyluemoglohins, so as to recall the fact of the
minor differences presented by the haemoglobin of different species of animals)
is soluble iu serum or in blood plasma, or in solutions of the neutral salts ;
it crystallises with greater or less facility, according to the animal whence
the blood is obtained. When thoroughly pure, it has scarcely any power
of decomposing HgO.,, but under the influence of this body it is readily

Solutions of crystallised oxyhaemoglobin, Hoppe-Seyler maintained, give
up their dissociable oxygen with difficulty and incompletely, when heated
in vacuo. When blood is saturated with CO, this gas can subsequently be
entirely removed, by passing a stream of hydrogen gas through it for some
hours, or by long-continued boiling in vacuo. On the other hand, when a
solution of oxyhaemoglobin is saturated with CO, and the solution is heated
in vacuo, the poisonous gas is, Hoppe-Seyler stated, given off with great
difficulty and incompletely.

Lastly, highly dilute solutions of potassium ferricyanide readily convert the
oxyhaemoglobins into methaemoglobin.

The evidence by which Hoppe-Seyler endeavoured to prove that the
properties of the blood-colouring matter, as it exists in the corpuscles, differ so
greatly from those of haemoglobin, that we cannot with truth say that this
body exists in them, is, on every single point, of so unsatisfactory a character
as not to stand a moment's investigation, and would lead us to reject his
hypothesis, even if we had not been placed in possession of some remarkable
facts bearing on this subject, which have been ascertained by the method of
spectrophotometry. The non-crystallisation of the colouring matter as it
exists in the coloured corpuscles might, were it really true, well be explained
by the fact that haemoglobin does not exist in a free state, but is combined

1 " Physiologische Chemie," Berlin, 1877, Th. 1, S. 381.

" "Beitriise '^ur Kenntniss dcs Blutfarbstoffes, " Zlschr. f. physxol. Clicm., Strassburg,
1889, Bd. xiii. S. 477.

''ARTERIN" (?) AND ''FBLEBIN" (.?)• 191

with another constituent of the corpuscle ; but the statement itself, as made
by Hoppe-Seyler, is incorrect. Although the fact has been denied by some
writers, there can be no question whatever, on the evidence of so eminent
an observer as Kiihne,^ as well as of Funke,'^ Brisegger and Bruch,^ Bdttcher,
KoUiker, L. Beale,^ Owsjannikow,-^ Richardson,*^ and Klebs, that what Preyer
terms " intraglobular crystallisation " can and does occur, i.e. a single crystal
forms in the interior of a coloured blood corpuscle. The process is most easily
followed in the blood corpuscles of certain fishes,'^ though it has also been
observed in those of the dog (Kiihne) and of the rat.^ The most remarkable
fact with regard to intraglobular crystallisation is, that when water is added
to a preparation exhibiting it, the crystal at once disappears, and the cor-
puscles resume their original appearance.'^

Again, at first sight, the difference in behaviour of the blood corpuscles
and of hsemoglobin towards peroxide of hydrogen appears thoroughly in
favour of Hoppe-Seyler's hypothesis. It was, however, shown by Bergengruen,
who first discovered the facts in reference to H.^Og, that the decomposing
action exerted by the blood corpuscles on H2O2 depends upon their stroma.
Solutions of perfectly pure crystals of oxyhsemoglobin have no action what-
ever on peroxide of hydrogen, whilst the stroma of the coloured blood
corpuscles exerts an intense action.^ All forms of protoplasm (splenic
cells, colourless corpuscles, yeast cells), decompose H^O,, though the stroma
of the coloured corpuscles acts most powerfully. The fact of the decom-
posing action being exerted by the stroma, and the stroma only, explains
why the blood corpuscles are not oxidised whilst oxyhsemoglobin is so, the
colouring matter in the corpuscles not coming in contact with the unde-
composed HgOg.

The greater readiness, as compared with pure htemoglobin, with which,
according to Hoppe-Seyler, the blood corpuscles give up either the oxygen or
the carbonic oxide which may be combined with their colouring matter (if the
facts were true, which we are not prepared to admit), would be much more
probably due to a katalytic action, exerted by some other constituent of the
corpuscle, than to any radical difference between the colouring matter of the
corpuscles and haemoglobin.

The one point of difference between the colouring matter of the corpuscles
and oxyhsemoglobin, which at first sight appears most difficult to explain, is
the action of solution of potassium f erricyanide. As von Mering ^ showed, if
fresh defibrinated blood be mixed with solutions containing 2|^, 5, and 10 per
cent, of the ferricyanide, the mixture assumes a scarlet colour, and even after
twenty-four hours contains the blood-colouring matter unaltered. On adding,
however, the same solution of the ferricyanide, in the same proportions, to
solutions of pure oxyhsemoglobin, they assume almost insiantaneously the
colour, and exhibit the spectrum of methsemoglobin.

^ W. Kiiline, Vircliow's ArcJdv, Bd. xxxiv. S. 423.

2 "Ueber Blutkrystallisation, " Ztschr. f. rat. Med., 1852, N.F., Bd. i. S. 288-292.

^ " Blutkrystalle, und organisclie Krystalle neberhaujjt" (15tli Sept. and loth Oct.
1852), Ferhandl. d. naturf. Gesellsch. in Basel, 1854-1857, Bd. i. S. 173-185.

* "Observations upon the Nature of tlie Red Blood Corpuscles." Quart. Journ. Micr.
Sc, London, 1864, pp. 32-43.

^ "Zur Histologic der Blutkorperchem," Bidl. Acad. d. se. de St. Pdershourg, vol. viii.
pp. 561-572 (describes intraglobular crystallisation in Osmerus e2JerIanits).

^ "Structure of the Red Blood Corpuscles," Philadelphia, 1870 (describes intraglobular
crystallisation in Menohranclius).

'' Kilhne, see W. Preyer, "Die Blutkrystalle, " Jena, 1871, S. 2 and 3. See also Funke's
"Atlas of Physiological Chemistry" (London, printed for the Cavendish Society, 1858),
Plate X. fig. 5, and the description at p. 17 of the "Description of the Plates."

^ Paul Bergengruen, "Ueber die "Wechselwirkung zwischen Wasserstoffsuperoxyd und
verschiedenen Protoplasmaformen," Inaug. Diss., Dorpat, 1888.

^ "Ueber die "Wirkung des ferricyan. Kalium auf Blut, " Ztschr. f. physiol. Chem.,
Strassburg, 1883, Bd. viii. S. 186, 189.



The extraordinary difference in the result predisposes one at first to
conclude that it must be due to a radical difference betwen the colouring
matter of the corpuscles and oxyhsemoglobin, such as Hoppe-Seyler believed to
exist. If, however, instead of the solutions mentioned above, solutions ten
times more dilute (containing 0"25, 0'5 and 1 per cent.) be mixed with
defibrinated blood in the same proportions as before, the mixture assumes
instantly the colour, and exhibits the spectrum of methsemoglobin. In this
case the dilute solution extracts, in the first instance, the blood-colouring
matter from the corpuscle, and then the ferricyanide acts upon the solution,
exactly as it does when brought in contact with a solution of crystals of
oxyhsemoglobin. The fact that the strong solution of potassium ferricyanide
does not act upon the colouring matter of the blood corpuscles, is due to its
incapacity to reach, in the first instance, the oxyhsemoglobin of the corpuscles.
In this case also, it appears that the difference (supposed) between the colour-
ing matter, as it exists m the intact blood corpuscles and solutions of haemo-
globin, is only an apparent one.

Though closely connected with the subject which has been discussed in
this section, the views of Bohr ^ (avIio believes that he has succeeded in
establishing, in addition to the already known oxyhaemoglobin, the existence of
at least three additional compounds of oxygen with hsemoglobin, all possessing
the spectrum of oxyhaemoglobin, but differing in elementary composition and
in their capacity to combine with oxygen), will be referred to under the heading
of "Oxyhaemoglobin." There can be no question, however, that these views
have been completely disproved by Hiifner," the supposed individual oxy haemo-
globins of Bohr being mechanical mixtures of pure oxyhaemoglobin with
products of its decomposition, — the necessary results of the methods of pre-
paration followed by the Scandinavian observer.

We have shown that even admitting, for the sake of argument, the
correctness of all Hoppe-Seyler's statements, these when carefully analysed
aff'ord no evidence whatever in support of his bold hypothesis. Whilst such
is the case, the splendid investigations of Hufner ^ have conclusively proved
that, in respect of its power of combining with oxygen, the blood-colouring
matter, as it exists in the coloured blood corpuscles, behaves precisely as a
solution of pure haemoglobin of the same concentration. Further, by the
method of spectrophotometry, Hiifner has shown, as could be done by no
other method, that the colouring matter of the blood is one — haemoglobin —
and that in every specimen of living blood, this colouring matter exists, partly
as oxyhaemoglobin and partly as reduced haemoglobin.

The discussion which has preceded Avill have prepared the reader for the
conclusion, which appears to be the only one which can legitimately be based
upon the facts in our possession — to wit, that whilst oxyhemoglobin and
reduced haemoglobin exist in the coloured blood corpuscles in the form of
loose or unstable combinations with some other constituent of the corpuscle,
evidence is altogether wanting in support of Hoppe-Seyler's contention that
the blood- colouring matter, as it exists in the corpuscles, possesses properties
so different from those of oxyhaemoglobin and of reduced haemoglobin, as to
warrant its being looked upon as a distinct substance, to be distinguished by
a different name. Hoppe-Seyler suggested,^ indeed, that the colouring matter
of arterial blood should be called Arterin, to distinguish it from oxyhaemo-

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 27 of 147)