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

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by the linking of one molecule of oxygen to a molecule of the highly
complex, iron-containing, crystalline colouring matter, " heemoglobin,"
and I have subsequently shown that this conception has received con-
firmation through the fine researches of Hiifner on the molecular
weight of haemoglobin and on the volume of oxygen with which it
can combine. In the present section, reference must be made to ad-
ditional facts which, besides possessing an interest of their own, throw
fresh light on the nature of oxyhsemoglobin, and, in a measure, on the
function subserved by it, although this subject will be more fully dis-
cussed under the heading of " Eespiration."

It had been noticed independently by Claude Bernard ^ and by Hoppe,"^
that blood which had been treated with carbonic oxide, or the blood of
men and animals asphyxiated by charcoal fumes, presents an intensely bright
arterial colour, but that, unlike arterial blood, it does not in a few hours change
to a venous hue, but retains its vermilion tint for long periods of time. The
idea forced itself on the minds of both Claude Bernard and Hopjje, that through
the action of CO the power which the coloured corpuscles possess of acting as
oxygen-carriers had in some way been interfered with. Claude Bernard has,
however, the merit of being the first to show that, when brought in contact
with the blood, CO is absorbed and displaces oxygen ; and he afterwards based
upon these facts a method for the quantitative determination of the oxygen of
the blood.

At the same time as, and independently of, Bernard, Lothar Meyer ^

^ "Ueber Hamoglobin,"^rc7i./. Physiol., Leipzig, 1890, Phys. Abth., S. 385.

2 " Besthnmimg der Sauerstoffcapacitat des Blutfarbstoffs, " ibid., 1894, Plivs. Abth.
S. 140 and 175.

^ "Lemons sur les effets des substances toxiques et m^dicamenteuses," Paris, 1857,
p. 158 ; also " Propriety des liq. de I'organisme, " Paris, 1859, tome i. p. 355.

■* "Ueber die Einwirivung des Kolilenoxydgases auf das Haniatoglobulin, " VircJiow's
Archiv, 1867, Bd. xi. S. 288.

^ "Die Gase des Bhites," Gottingen, 1857; and Ztsclir. f. rat. Med., 1858, S. 256;
"De sanguine oxydo carbonico infecto," Diss. Inaug., Vratislavice, 1858.


showed — (1) that the absorption of oxygen and carbonic oxide by blood does
not proceed according to Dalton and Henry's law — a proof, amongst many
others, that these gases are chemically combined with some constituent of
the blood and not held in a state of simple solution ; (2) that blood which
has been deprived of its gases by boiling in vacuo, combines with the same
volume of carbonic oxide as of oxygen — in other words, that when carbonic
oxide replaces the oxygen of the blood, one molecule of the former takes the
place of one molecule of the latter {i.e. CO replaces Oo). Lothar Meyer
further showed that hsematin could not be the body with which 0^ and CO
entered into combination, and expressed the surmise that it might prove
to be the same as constituted the red blood crystals described by Lehmann.^
The truth of the surmise was soon proved beyond the possibility of doubt,
it being shown that the O^- and CO-compounds of the blood-colouring matter
are isomorphous, that they are characterised by a similarity in their power of
absorbing light, but that the CO-compound is distinguished by not being
decomposed by reducing agents (Hoppe-Seyler).

Hermann - subsequently showed that just as CO possesses the power
of displacing the oxygen of oxyhcemoglobin, nitric oxide (NO) in its
turn is capable of displacing CO, one molecule of the former replacing
one molecule of the latter, the NO -compound being, like the CO-com-
pound, absolutely irreducible.

The three compounds of hsemoglobin were shown to be isomorphous,
to be characterised by a highly florid colour, only slightly differing in
tint one from the other ; their visible spectrum was found to be distin-
o-uished by two absorption-bands between D and E, at first sight appear-
ino- identical in the three cases, though careful measurement revealed a
very slight shifting of the bands towards the more refrangible end of the
spectrum in the case of the CO-compound.

They were all three found to be free from pleochromatism —
a character in which they differ strikingly from reduced haemo-
globin. Whilst the CO - compound is much more stable than the
Og-compound, the NO-compound is again more stable than the CO-

It was at first believed that the CO-compound, unlike oxyhsemo-
globin, could not be dissociated. I was the first to show that by the
long-continued passage of neutral gases through solutions of CO-hsemo-
globin, the CO is gradually driven out, and reduced haemoglobin is
obtained.^ Bonders,^ to whom the discovery of the fact is always
ascribed, drew attention to it in a highly interesting theoretical paper.
Zuntz ^ immediately afterwards showed, in contradiction of Nawrocki,^
that blood saturated with carbonic oxide, when boiled in vacuo, gives up
its carbonic oxide and that it manifests the absorption-band of reduced

1 " Consideranti enim quse his in rebus din versatus Lehmann de rubris illis sanguinis
crystallis nuper publicavit, plus quam verisimile videbitur, hac cum substantia et oxygenium
et oxydum cai-bonicum conjunctionem chymicam posse inire"; Lothar Meyer, "De
sanguine oxydo carbonico infecto," p. 12.

'^ " Ueber die Wirkungen des Stickstoffoxydgases auf das Blut," Arch. f. Physiol.,
Leipzig, 1865, S. 469.

3 A. Gamgee, "On Poisoning by Carbonic Oxide Gas, and by Charcoal Fumes,"
Journ. Anat. and Physiol., London, 1867, vol. i. pp. .339-346.

^ Donders, " Der Chemismus der Athmung, ein Dissociations-process," Arch. f. d. ges.
Physiol., Bonn, 1872, Bd. v. S. 20-26.

"^ " Lst Kohlenoxydhamoglobin eine feste Verbindung ? " Arch. f. d. ges. Physiol.,
Bonn, 1872, Bd. v. S. 584-588.

" " De Claudii Bernardi metliodo oxygenii copiam in sanguine determinandi," Inaug.
Diss., Vratislavicc, 1863.



hsemoglobin, and Podolinski ^ succeeded in dissociating blood saturated
with nitric oxide, by passing a stream of hydrogen through it for an
hour and a half; at the end of which time the blood presented the
absorption-band of reduced hsemoglobin.

Having passed in review the chief facts which exhibit the relation-
ship existing between the different compounds of haemoglobin, and which
illustrate the nature of the combination of haemoglobin with gases, some
of the characters and properties of CO-hsemoglobin and NO -haemoglobin,
but particularly of the former, must be systematically though briefly

Carbonic Oxide Hemoglobin (Co-Haemoglobin).

Mode of 'preparation. — A current of pure carbon monoxide is passed
through a saturated solution of oxyhsemoglobin. The solution acquires a
carmine-like tint in contrast to the scarlet colour of oxyhsemoglobin. This
solution is then cooled to 0" C, and, after being treated with one-fourth of
its volume of alcohol previously cooled to 0° C, is set aside at a tempera-
ture which must not rise above 0° C, but which should be as low as possible.
After some hours or days, the CO-compound, which is more sparingly soluble
than Og-haenioglobin, separates in crystals, of which the forms are identical
with those of that body.

The absorption of light by CO-haemoglobin. — (a) The visible
spectrum. —Solutions of this body possess more of a bluish-red tint
than the Og-compound. If solutions of equal concentration of the
oxygen and carbonic-oxide compounds be compared, it will be found, on
spectroscopic examination, that the CO-compound absorbs the blue rays
of the spectrum to a less degree than oxyhsemoglobin.

Between D and E are seen two absorption-bands which, unless very
closely studied, appear absolutely identical with those of oxyhsemo-
globin (see Plate I., Spectrum 6). On careful measurement, however, it
is seen that both the bands are very slightly shifted in the direction
of E; that is to say, towards the violet end. This is best seen by
noticing the interval between D and the adjacent border of the first
absorption-band ; in the case of the CO-compound this interval is broader
than in that of the 02-compound.

The spectrophotometric constants of CO-hsemog-lobin. — These
constants were re-determined by Hlifner in 1894, at the same time as
those of oxy- and reduced haemoglobin, and for the same spectral
regions, with the results exhibited below.^ The coefficients of extinction
in the case of CO -haemoglobin are designated for the region X 554-X 565,
e„ and for the region X 531'5-X 542-5 e'^, whilst the corresponding
absorptive relations are designated A^ and Ac.



X 554-A 565

X 531 -S-A. 542-5



^ " Ueber die Austreibbarkeit des CO- und NO- aiis dem Blute," ArcJi.f. d. ges. Physiol.
Bonn, 1872, Bd. vi. S. 553-555.

" Hiifner, op. cit,, S, 141 and 142.



(h) The photographic spectrum of CO-hsemoglobin. — In Fig. 35
are shown reproductions of the photographic spectrum of this com-
pound, contrasted with that of the oxygen compound. The band of
Soret is just as well marked in the one as in the other, but in the
case of the CO-hamoglobin there is a decided shifting of the band
in the extreme violet towards the red, which is somewhat curious,
considering that the bands in the visible spectrum are, though to a
much less extent, shifted in the opposite direction. I have shown that
there is absolute identity in the position of the absorption-band in the
extreme violet, in the case of the CO- and NO- compounds of hfemoglobin.^

The principal characteristic reactions of CO-heemoglobin.
1. When treated with Stokes' reagent, solutions of ammonium sulphide,
and the like, no change whatever occurs, either in the colour or the
spectrum of blood saturated with carbonic oxide, or in solutions of
pure CO-hsemoglobin.



Fig. 35. — The photographic spectrum of oxyhemoglobin and of CO-hremoglobin.

2. The blood of men or animals asphyxiated by carbonic oxide, or
by a gas containing it (charcoal fumes, coal gas), if pretty fully saturated,
possesses and retains for a long time a florid arterial colour, and when
diluted is found to be partially or completely irreducible. Hoppe-
Seyler found that if such blood is sealed in glass tubes, it may retain for
some years its characteristic spectroscopic properties, and even admit
of CO being boiled out, with the aid of tlie mercurial pump, and
identified by chemical analysis.

3. The addition of a concentrated solution of sodium hydrate (density
1'3) to blood, saturated with CO in the proportion of about two parts
of the former to one of the latter, causes the blood to assume a fine
scarlet colour, and to deposit a cinnabar-red precipitate. The same
coloration and precipitate is produced with solutions of pure CO-
haemoglobin. According to Hoppe-Seyler, the precipitate is composed
of CO-ha;moglobin, rapidly passing into CO-htemochromogen. When
normal blood is treated in the same way with sodium hydrate, it is

' Oamgee, Proc. Roy. Soc. London, 1896, voL lix. p, 276,


converted into a black shining mass, which when spread in thin layers
over porcelain appears of a greenish-brown colour.

4. Aqueous neutral solutions of pure CO-hsemogloljin, when heated
to boiling point, furnish a bright red precipitate, composed of coagulated
albuminous substances and CO-hsemochromogen (Hoppe-Seyler).

5. Solutions of carbonic oxide hcemoglobin, treated with NO in the
absence of oxygen, are at once decomposed, and liberate GO (Hiifner).

NiTEic Oxide Haemoglobin (NO-H^moglobin).

Mode of preparation. — So great is the affinity of nitric oxide for oxygen,
that, when it comes in contact with it, deep red fumes of nitrogen peroxide, NO.,,
is formed. When this gas comes in contact with Avater, the decomposition
indicated in the following equation occurs : —

3N0, + H^O = 2HNO3 + NO.

But as all free acids decompose the colouring matter of the blood, before
causing nitric oxide to act upon blood certain precautions must he taken ;
for even if atmospheric oxygen be eliminated and nitric oxide caused to act
upon oxyhaemoglobin, nitrous oxide would be formed at the expense of the
oxygen of that body ; and next, by the action of water, nitric acid, which
would immediately decompose the haemoglobin.

Two methods of proceeding are open to us — (a) To add to the solution of
oxyhaemoglobin which is to be subjected to the action of nitric oxide, sufficient
alkali to neutralise the nitric acid which will be formed. Such a solution
must be placed in a flask, permitting of the whole of the air above the solution
being driven out and replaced by a neutral gas, before allowing access to the
nitric oxide. After the latter has exerted its action, care must be taken again
to pass a neutral gas through the apparatus and solution, so as to remove all
traces of free nitric oxide.

(b) The solution of oxyhsemogiobin is subjected to the long-continued
action of carbonic oxide, so as to form COdisemoglobin and to expel all traces
of dissolved oxygen. Otherwise, the process is constructed as described
under {a). This process would be certainly preferred, if it were desired to
crystallise the NO-compound.

Physical and chemical characters. — Blood saturated with nitric oxide
possesses almost as florid a colour as CO-blood, though Hermann says
that it does not present the slight bluish shade of the latter. It exhibits
no dichroism. Solutions of NO-htemoglobin, or diluted NO-blood,
exhibit a visible spectrum in which, as I have convinced myself, the
bands occupy precisely the position of the two oxyhsemoglobin bands.
In the photographic spectrum, however, the band in the extreme violet
exhibits absolute coincidence with that of CO-hsemoglobin.

NO -haemoglobin can be crystallised, and, as Hermann showed, the
crystals are identical with those of oxyhsemoglobin and CO-hfemoglobin.

Alleged (but Pkoblematical) Compounds of Hjsmoglobin

with G-ases.

1. With hydrocyanic acid.— The most discrepant statements have
been made in reference to the very simple question — whether hydrocyanic
acid added to, or passed through, blood affects the characters of its absorption-
spectrum. In spite of these, it may be definitely stated that, at ordinary
temperatures, and when acting for moderate periods, hydrocyanic acid leads
VOL. I.- — 16


to no change in the physical characters of the blood, of which the spectrum
remains unchanged, and of which the property of being reduced by suitable
agents remains unaffected.

Upon what appears to me to be altogether insufficient evidence, Hoppe-
Seyler,^ however, came to the conclusion that hydrocyanic acid forms an easily
decomposed compound with haemoglobin. If hydrocyanic acid be added to
a solution of oxyhsemoglobin, on crystallising out the latter it retains some of
the acid. These crystals may be repeatedly crystallised, and when dried in
vacuo over sulphuric acid they are found to contain hydrocyanic acid. The
supposed compound of hj^drocyanic acid with oxyhsemoglobin presents an
absorption-spectrum absolutely identical with that of oxyhsemoglobin, and is
reduced just as easily by such agents as ammonium sulphide or Stokes's re-
agent. On the other hand, blood to which hydrocyanic acid has been added
shows the bands of oxyhsemoglobin for a much longer time than normal blood.

It appears to me that no proof whatever has been advanced of the
existence of a chemical compound of oxyhsemoglobin with HCN.

That some hydrocyanic acid should adhere to hsemoglobin, as it crystallises
out of the mother liquor which contains the acid, is quite in accordance with
a number of experiences of a similar kind, and can by itself afford no evidence
of an actual compound existing. The resistance of blood to which hydro-
cyanic acid has been added, to decomposition, when confined in a sealed or
closed vessel, can, on the other hand, be easily explained by the unquestion-
able arrest or slowing of the process of putrefaction in the presence of hydro-
cyanic acid. It is, undoubtedly, the jiroducts of putrefaction which are the
causes of the ap2Jarently spontaneous reduction of the oxyhsemoglobin of blood
confined in a receptacle to which air has no access ; so that an agent which
does inhibit putrefaction — as hydrocyanic acid unquestionably and admittedly
does — and, at the same time, does not, at ordinary temperatures, decompose
oxyhsemoglobin, would be expected to act as hydrocyanic acid has been found
to do in furthering the persistence of the oxyhsemoglobin bands.

What I have just stated in reference to the probable non-existence of a
compound of HCJS" with oxyhsemoglobin, does not imply my disbelief in the
existence of an interesting compound of hydrocyanic acid with methsemo-
globin, described by Kobert, which will be discussed after the latter body
has been described.

2. With cyanogen. — Ray Lankester - believed that cyanogen formed a
compound with hsemoglobin, probably analogous to the CO- and NO-com-
pounds, and characterised by an absorption-band, resembling that of, but
obviously not due to, reduced hsemoglobin. Many discordant statements have
been published on this matter. It appears that by the prolonged action of
cyanogen, as by the prolonged action of HON, there is produced Kobert's
cy^nogenmethsemoglobin (see p. 248).

3. With acetylene (CoHo). — Bistrow and Liebreich ^ surmised that acety-
lene forms a very unstable compound with hasmoglobin, easily reducible by
sulphide of ammonium and similar agents. On the evidence at present at our
disposal, the existence of this compound must be considered as more than

4. With carbon dioxide. — According to Bohr, hsemoglobin forms a series
of compounds with carbon dioxide, which possess spectra identical with those
of reduced hfemoglobin. He states, further, that if a solution of hsemoglobin
be brought in contact with a mixture of oxygen and carbon dioxide, the

1 " Cyanwasserstofiliseinoglobinverbinduiigen," Med. -cliem. Untersueh. , Berlin, 1868, S.

^ " Ueber den Einfluss des Cyangases auf Hanioglobin nach spectroscopischen Beobach-
tungen," Arch. f. d. ges. Physiol., Bonn, 1869, Bd. ii. S. 491-49.3.

*" Ueber die Wiikimg des Acetylens auf das Blut," Ber. d. deutsch. chem. Gesellsch.,
Berlin. 1868, Bd. i. S. 220.


amount of either of these gases which is absorbed is independent of the

A careful study of the whole of Bohr's researches on this subject, as well
as those on the various hypothetical compounds of heemoglobin Avith oxygen,
has convinced me that his work is pervaded by fallacies, which spring in part
from erroneous methods of work, in part from a non-appreciation of physical
principles of which the exactitude is beyond dispute ; the discussion of Bohr's
statements in a text-book would be, under these circumstances, altogether out
of the question.


Introductory observations. — It has already been stated, that when
the blood-colouring matter is subjected to the action of strong alkalies
and of acids, or even of salts possessing an acid reaction, or to the
action of heat, of alcohol, and of many other chemical agents, it under-
goes a decomposition of which the chief products are an albuminous
substance or substances, and a colouring matter which contains the whole
of the iron originally present in the oxyheemoglobin or hsemoglobin

Under ordinary circumstances, when oxyhsemoglobin is decomposed in
the presence of air, the coloured product of decomposition is the body
we know as hcematin, the amount of which produced corresponds theo-
retically to 3 '8 per cent, of the oxyhsemoglobin. Traces of organic
acids are said to result from the decomposition, the main product of
which is, however, composed of the albuminous residue of the blood-
colouring matter {vide infra). If, however, instead of decomposing
oxyhtemoglobin, we employ reduced haemoglobin and carry out the
process in the complete absence of oxygen, we obtain, not htematin, but
a body of which some of the optical characters were first described
by Stokes, and which he named reduced hcematin, to indicate that it
may be obtained by the action of reducing agents on hsematin. Instead
of employing this term, it is better to adopt that of hsemochromogen,
introduced by Hoppe-Seyler, to whom we owe nearly all the knowledge
we possess with regard to it. According to Hoppe-Seyler, heemochro-
mogen constitutes the coloured radicle of the blood-colouring matter,
upon which its essential optical properties and its property of com-
bining with oxygen, carbonic oxide, and nitric oxide depend.

Under the influence of carbonic acid, and very dilute acids acting
for comparatively short periods of time, oxyhsemoglobin, long before the
complete splitting up into hsematin, undergoes a change which is doubt-
less of the nature of a decomposition ; this change is identical with that
which is also brought about by a variety of oxidising agents, typically
by ozone, nitrites, and potassium ferricyanide ; to the body which
results, the name of methsemoglobin has been given. It will be con-
sidered first amongst the decomposition products of oxyhtTemoglobin.
We shall show it to be a substance which is formed in the living body,
under the influence of certain poisonous agents, and is occasionally
found in old blood extravasations ; it possesses the power of forming
molecular compounds with certain bodies, such as nitrites, hydrocyanic
acid, and cyanogen.


The Albuminous Eesidue of the Blood-Coloueing Matter.

An unfortunate error has become popular, and has, indeed, been
propagated by a large number of text-books, namely, that when oxy-
hcemoglobm is decomposed, it splits up into hgematin and a definite
albuminous matter belonging to the group of globulins, and designated
gldbin. There is absolutely no ground for such a statement. The term
globin was, it is true, assigned by Preyer to an albuminous substance,
which he obtained as a product of the spontaneous decomposition of
solutions of oxyhsemoglobin, but this body did not possess the character-
istic properties of the globulins, and there is no ground for considering
it as representing the albuminous body which, by linking to itself a
coloured iron-containing radicle, forms crystalline haemoglobin.

Our knowledge on this matter is indeed of the most unsatisfactory
character. We know, and have shown (see p. 207), that solutions of oxy-
hsemoglobin in the presence of many of the reagents for albumin (so long
as these do not decompose the blood-colouring matter) behave quite differ-
ently from solutions of the native albumins, globulins, etc. Thus copper
sulphate, mercuric chloride, silver nitrate, and the acetates of lead do
not produce even a cloudiness when added to solutions of pure hsemo-
globin, so long as this remains undecomposed. It has long been recog-
nised, too, that Lehmann's hypothesis, that the blood-colouring matter
was composed of colourless crystals tinted by a red pigment, was false ;
but as to the true nature of the albuminous residue, we have very little
knowledge, though the facts in our possession almost force us to the
conclusion that it is not identical in all animals, as shown by the
difference in the percentage of sulphur in the haemoglobin of the horse
and the dog.

The reagents which we employ to decompose the blood-colouring
matter yield us derivatives of the albuminous residue, not the body
itself ; we obtain acid albumin as a result of treatment with acids, alkaline
alljuminates as a result of treatment with alkalies. The most interest-
ing observations on the albuminous products of the decomposition of oxy-
hsemoglobin were published by Kiihne^ thirty years ago. He showed that
when CO2 is passed through solutions of pure oxyha^moglobin a flocculent
precipitate is thrown down, which does not possess, as had been errone-
ously asserted by A. Schmidt, fibrinoplastic properties, and which does
not behave as a glohulin. According to Kilhne, this precipitate possesses
so peculiar an appearance under the microscope that it cannot be mis-
taken for any other substance. It forms long colourless fibres which
are so like fibres of connective tissue that they might be taken for them.
This substance differs fundamentally from globuhn ; it is, for example,
insoluble in water containing oxygen in solution.

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 34 of 147)