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

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Hoppe-Seyler was the first ^ to observe that solutions of oxyhsemo-
globin exposed to the air, or filter papers saturated with such solutions,
often assume a brown colour. Under these cu^cumstances, the solution

1 "Lehrbuch," 1866, S. 206, 207.

2 Centralhl. f. d. med. TFissensch. , Berlin, 1864, No. 5-3. See also Med.-chcm. Uiiter-
stick., Berlin, S. -378, imd " Die Ziisammensetzung des Methanioglolnu, iind seine Umwand-
lung zu Oxyhamoglobin," Zischr. /. 2}hysiol. Chem., Strassburg, 1878, Bd. ii. S. 150, 155.


is found to have become acid and to exhibit a spectrum in which, in
addition to the. two bands of oxyhfemoglobin, one is seen in the red,
occupying much the position of the band of acid hematin.

Hoppe-Seyler applied the name of methamoglobin to the very
indefinite and problematical body whose solutions possessed the above
characters, and held it to be a product of the partial reduction of
oxyhciemoglobin, derived from it by the removal of a portion of the
dissociable oxygen of that compound.

I myself, soon after, investigated the changes l^rought about in the
properties of oxyhsemoglobin under the influence of nitrites, and in a
memoir,^ of which the experimental facts have, so far as they have yet
been controlled, been confirmed in every particular, pointed out the
remarkable phenomena which attended the conversion of oxyhcemoglobin
into methsemoglobin, though I committed the error of believing that the
changes described by me were due to the combination of nitrites with
oxyhsemoglobin, and not to an action which was afterwards shown to be
possessed by a large number of both oxidising and reducing substances.
I showed that Ijlood which had been acted upon by nitrites, in addition
to marked and definite changes in colour and spectrum, had almost
entirely lost its power of absorbing oxygen from the atmosphere ; that,
under the influence of nitrites, the oxygen of oxy haemoglobin is not
removed, but passes into a condition in which it is no longer removable
by boiling in vacuo or by the action of carbonic oxide. The action
of reducing agents reveals, however, as I showed, that the molecule
of loose oxygen of oxyhsemoglobin is still present in blood which
has been acted upon by nitrites, for, in the absence of all traces
of oxygen, reducing agents first of all and instantaneously hberate
oxyheemoglobin, which is only afterwards reduced. I pointed out that
the chocolate-coloured nitrite blood can be crystallised, the colouring
matter being isomorphous with haemoglobin and its compounds, and
that the crystals contain the nitrite which has brought about the
change, though I showed that the composition of these molecular com-
pounds of oxyhaemoglobin is not a constant one. After innumerable
contradictions, it has been proved, though without a word of acknow-
ledgment, mainly by the researches of Hlifner and his pupils, that my
account of the changes which characterise the formation of methsemo-
globin was, in every particular, exact, whilst the comparatively recent
statement, by Robert, of the existence of combinations of hydrocyanic
acid and cyanides with methtemoglobin is an illustration of the class
of compounds of oxyhsemoglobin which I was the first to discover and
describe, and of which doubtless a large number will be obtained.

Mode of preparatio7i.- — A large number of inorganic and organic bodies,
acting u23on sohitions of oxyhsemoglobin, convert it into methse.moglobin.
The chief of these are potassixmi ferricyanide — which, on account of the
rajDidity of its action, is to be preferred to all others — nitrites, chlorates,
potassium permanganate, nitrobenzol, pyrogallol, pyrocatechin, acetanilid, etc.

In order to study the spectroscopic characters of methsemoglobin, a
solution of diluted blood is treated with a few drops of a strong solution
of potassium ferricyanide, when the change in colour and spectrum is seen to
occur almost instantly. To prepare the crystalline colouring matter, 2 or
3 c.c. of a saturated solution of potassium ferricyanide or of a nitrite is

1 A. Gamgee, "On the Action of Nitrites on Blood," Phil. Trans., London, 1868, vol.
clviii. pp. 589-626.


added to a litre of saturated aqueous solution of crystals of oxyhaemoglobin,
and after the conversion into metlisemoglobin has occurred, about 25 per cent,
of alcohol added. The mixture is then exposed to a temperature below 0° C.
I succeeded in recrystallising metlisemoglobin prepared by the action of
potassium nitrite and of ethyl and amyl nitrites on oxyhaemoglobin.

Chemical and physical characters. — Crystals of methfemoglobin are
more sparingly soluble than those of oxyhaemoglobin, and the colorific
intensity of theu' sohitions is less.

It is to be noted that, whilst solutions of reduced and oxyhaemoglobin
are not precipitated by either neutral or basic lead acetates, these
reagents added cautiously, with careful avoidance of an excess, precipi-
tate methcemoglobin, hmmatin, and hoimatoporphyrin, and may be em-
ployed for the separation and detection of traces of oxyhaemoglobin
when mixed with and concealed by any of the above-mentioned bodies.

Solutions of methsemoglobin, when of a neutral or a slightly acid
reaction, possess a chocolate-brown colour. When the solution is
rendered alkaline, its colour changes to red without a tinge of the

The acid solution is found to present a spectrum in which the oxy-
haemoglobin bands a and /3 are very weak or even not visible, whilst an
absorption-band is seen in the red between C and D, and nearer the
former. This band occupies nearly, though by no means exactly, the
position of a similar band in the spectrum of acid hsematin (see Plate
II., Spectrum 5).

On now rendering the solution alkaline by means of ammonia, the
band in the red disappears, and is replaced by a faint absorption-band
immediately on the red side of D. By changing the reaction of the
solution, the alterations in its colour and spectrum may be repeated
indefinitely (Gamgee).

If a solution of methasmoglobin be placed in a deep test tube, in
front of a spectroscope, and arrangements be made for allowing a stream
of solution of ammonium sulphide to flow to the bottom of the
liquid, it can be readily shown that at the very moment of the
contact of the reducing and the methaemoglobin solution, the spec-
trum of oxyhemoglobin appears ; to be subsequently and much more
slowly replaced by that of reduced haemoglobin, which in its turn,
when shaken with air, yields oxyhemoglobin.

A study of the photographic spectrum of methsemoglobin has led
me to results of great interest. The conversion of oxyhaemoglobm into
methaemoglobin is attended by a shifting of the band of Soret from the
extreme violet to the ultra-violet properly so called (Fig. 36). The most
persistent part of the band in very dilute solutions, coincides, indeed,
with the H and K bands, but the band extends more and more into the
ultra-violet, as the concentration of the solution increases. The position
and characters of this band in the case of methaemoglobin absolutely
corresponds with those of the acid compounds of haematin, and not
with those presented by hceinoglobin and its compounds, or by
haemochromogen (see Fig. 38).

This syjectroscopic character certainly seems to lend weight to the
evidence of other kinds, which indicates that methsemoglobin is a first
product of the decoinposition of the oxyhaemoglobin molecule, and that
this is a decomposition which leads to the separation of a compound of
htematin, and not of haemochromogen. Hoppe-Seyler, indeed, expressed


the opinion that tlie colouring matter in nietlisemoglobin is in the same
state as in hsematin, the iron being, as he thought, in the condition of a
ferric compound, whilst in oxyhasmoglobin and in hgemochromogen he
believed it to exist in a ferrous state, though the grounds for these very
definite statements are certainly wanting.

The researches of Hiifner on the oxygen of metheemoglobin.

— I had shown, that the action of methsemoglolDin, as produced by the action
of nitrites, could not be attended by a profound alteration in the constitution
of oxyhsemoglobin, seeing that the addition of certain reagents at once caused
all the effects of the action to disappear, and revealed the continued existence
of oxidised haemoglobin. Nitrites (for these we should now read all agents
cajDable of transforming oxyhaemoglobin into methaemoglobin) had, by my
experiments, been shown to resemble in no way those agents which thrust
oxygen out of the blood ; on the other hand, I had shown that the action of


Fig. 36. — The photographic spectrum of oxyhsemoglobiii and methfemoglobin.

nitrites resulted in ihe locking up of the oxygen of the blood, so as to render
it irremovable by carbonic oxide, or by a vacuum. But although I had dis-
covered that metheemoglobin, when treated with reducing agents, at once
liberates oxyhsemoglobin, I had not been able to show that when the latter
substance is converted into the former the whole of its oxygen is locked up
without loss, and may be subsequently liberated. This was reserved for

When nitric oxide acts upon a solution of methsemoglobin, the brown
colour is changed to bright red, the spectrum of the red solution being
identical with that of I^Odiaemoglobin. Reflecting on this experiment, Hiifner
thought that perhaps NO possesses the power of becoming oxidised to ^02,
at the expense of the oxygen locked up in methsemoglobin (i.e. oxygen of
the original oxyhaemoglobin which had passed into a more stable combination).

As such might be the case, it occurred to Hiifner to determine the volume
of NO2 produced (for this would bear a definite relation to the abstracted
from methsemoglobin), by causing the nitrous acid (HjS[0.2), which would
be produced by the action of the water of the blood on NOg, to decompose
urea, the N liberated being a measure of the oxygen derived from methsemo-


globin. The ingenious conception of Hlifner will be rendered evident by the
three following equations : —

(1) 6X0 + 2(Hb-0,) = i'^0, + 2(Hb-X0).

(2) 4(N02) + 2(H,6) = 2(N6.H) + 2(N0.H).

(3) 2(N02H) + Q:^^,P = 3(H20) + 200*2 + 2(^2).

From these equations it results that each molecule of nitrogen liberated
will correspond to a molecule of oxygen which had become fixed in methaemo-

Whether the more firmly combined oxygen of methaenioglobin Avere capable
of oxidising nitric oxide or not, the oxygen of oxyheemoglobin would certainly
be able to do so, and Hiifner proceeded to compare the amount of N liberated
in the above reaction by solutions of exactly corresponding concentration of
oxyhsemoglobin and of alkaline methsemoglobin. The results left no room for
doubt, and led to the conclusion that when oxyheemoglobin is converted into
methsemoglobin, tlie icliole of its oxygen passes into a state of more intimate
combination, so that it can no longer be removed either by CO nor by a
vacuum, but is yet available to oxidise such bodies as ^O.y.

The compounds of raethseraoglobin "with nitrites. — I showed, as
has already been stated, that Avhen a solution of pure oxyhsemoglobin is
treated with a solution of a nitrite, so as to produce the change in colour
and spectrum which Ave noAV knoAV to be characteristic of methsemoglobin,
the blood-colouring matter crystallised out of the solution is found to con-
tain the nitrite, though the proportion in which the latter combines with
the haemoglobin is not constant. The discordance in results did not appear
to me surprising, and that " as in the case of other combinations of a molecular
kind, such as the \mion of salts with their water of crystallisation, of bases
Avith sugar, of albumin with metallic oxides, of iodine with the compound
ammonias, the amount of the simpler body added to the more complex should
vary within wide limits." I further speculated on the probability of a large
number of similar combinations to that of oxyhsemoglobin Avith nitrites

The compounds of methsemoglobin "with HON and cyanides. —
It has long been noticed that hypostatic marks on the bodies of men and
animals poisoned by prussic acid or metallic cyanides, as well as the mucous
membrane of the stomach, present a striking bright red colour. Kobert ^
surmised that this coloration might be due to combination of methsemo-
globin Avith HCIS" or metallic cyanides, a hypothesis of which he thinks
he has obtained confirmation from his experiments. Kobert found that on
adding solutions of HCJST of extreme dilution to a 1 or 2 per cent, solution
of methsemoglobin, these assume a beautiful bright red colour, Avhilst the
absorption band or bands of methasmoglobin have disappeared, and are
replaced by a single broad absorption-band betAveen D and E, occupying about
the position of the band of reduced hsemoglobin. This band cannot, however,
be made to disappear by the action of oxygen.

According to Kobert, this band is not affected by the addition of ammonium
sulphide. He believes the body which is produced by the action of HON on
methsemoglobin to be a compound of the tAvo bodies, and he ascribes to it the
name " cyanogenmethsemoglobin," and represents it for brevity by the symbol
CNH'MetHb. He further lays claim to have discovered for the first time
similar compounds Avith nitrites (! !). But Kobert's vieAV of the nature of the
action of HCX on metiisemoglobin has not been universally accepted. The
absorption-spectrum Avhich he has described as characteristic of his new
compound is identical Avith that described by Preyer as resulting from the

^ " Ueber Cyaiiniethamoglobin iiud den Nachweis der Blausaure," Stuttgart, 1891.


action of HCN on oxyhsemoglobin, and by Nawrocki and Lankester as pro-
duced when KCN acts upon blood, especially with tlie aid of gentle heat, and
which has generally been held to be a compound of cyanogen and hsematin

Szigeti^ maintains that Kobert's cyanogenmethaemoglobin is in reality
cyanhcematin, the first step in the action of HCN being to split up the
mothaemoglobin molecule into heematin and an albuminous substance. I do
not, however, take this view, and, in spite of the evidence which Robert has
adduced being in many respects incomplete, I am inclined to think that the
view which he has advanced is correct. In the first place, the certain existence
of compounds of the nitrites with metheemoglobin afibrds presumptive
evidence of the strongest kind that similar compounds with such bodies
as cyanogen, hydrocyanic acid, and cyanides exist ; in the second, the almost
instantaneous action of solutions of hydrocyanic acid of plienomenal dilu-
tion renders it highly improbable that the action of hydrocyanic acid on
methasmoglobin is one in which decomposition into hsematin is a preliminary

There can be no question that HCN acting in the cold, and, for a short
time upon, blood or on solutions of oxyhaemoglobin, produces no change in the
spectrum, and it is against all experience and analogy from the action of other
dilute acids on either oxyhsemoglobin or methsemoglobin to conclude that
solutions of HCjST of extraordinary dilution should be able — and almost instan-
taneously — to split up the oxyorthomethsemoglobin molecule. Kobert has
found that a solution containing 0-000003 grm. of HCN is able to produce the
characteristic change in 1 c.c. of a 1 per cent, solution of metheemogiobin.
He has further shown that his assumed cyanogenmethsemoglobin contains
HCN which can be recovered from it without loss by distilling with sulphuric

CO-methsemoglobin. — According to Weyl and v. Anrep,'-^ this com-
pound is produced when aqueous solutions of iodine and potassium iodide,
or solutions of potassium permanganate, continue to act upon a solution of
CO-hsemoglobin for several days. This body is said to retain the red colour
of CO-hsemoglobin, and to present the same absorption-bands in its spectrum.
I fail to understand the grounds for believing in its existence.

Sulpho - metheemog'lobin. — This hypothetical body was believed by
Hoppe-Seyler ^ to be the cause of the green coloration observed on the
surface of putrefying organs.

Sulphuretted hydrogen has no action on reduced hsemoglobin. When
acting in small quantities on neutral solutions of pure oxyhsemoglobin, it
reduces these. If, simultaneously, a stream of sulj^huretted hydrogen and
oxygen be passed through blood or neutral solutions of pure oxyheemoglobin,
the solution assumes a green colour in thin, and a red colour in thick layers,
and becomes turbid. These solutions are characterised by the presence of two
absorption-bands in the red, one on the red side of, but quite close to, C ; the
other is about midway between C and D, the two bands being united together
by a shadow.

It appears to me that there is not the slightest ground for believing
that the phenomena above described are due to a definite body, — "sulpho-

1 " Ueber Cyanhamatin," Vrtljschr. f. gerichlt. u. off. Med., Berlin, Supp. Bd. vi.
S. 9-35. I only know this paper from the abstract by Andreasch in Jahresh. il. d. Fortschr.
d. Thicr-Chcm., Wiesbaden, 1883, Bd. xxiii. S. 620.

^ "Ueber Kohlenoxyd-Hamoglobin," 1. Oxydation von CO-Hb zu Meth-Hb, Arch./.
Physiol., Leipzig, 1880, S. 227-240.

^ C'entralbl. f. d. med. JVissensch. , Berlin, 1868, No. 28; "Ueber die Einwirkung
des SchwefelwasserstofFs auf d. BlntfarbstofT," Mcd.-cliem. Untersuch., Berlin, S. 651 ;
Araki, "Schwefelmethsemoglobin," Ztec/ir. /. ^j/it/sz'oZ. Ghem., Strassburg, 1890, Bd. xi. S.


methsemoglobin " ; they are almost certainly caused, by a mixture of de-
composition products of oxyhsemoglobin, brought about by the action of H2S
upon it.

H^MATIN (Cs^HssN^FeO^, Hoppe-Seyler) ; (CgaHgoN^FeOg, Nencki

and Sieber).

As has been already stated, liEematin is the coloui'ing matter
which results from the decomposition of oxyhsemoglobin by acids and
alkalies. In acid and alkaline solutions the body is characterised by
certain spectroscopic appearances, and especially by yielding, under
suitable conditions, when treated with reducing agents, a body possessing
the optical characters, when examined with the spectroscope, which
were originally described by Stokes as those of " reduced hcematin," now
known as " hcemochromogen " (Hoppe-Seyler).

Mode of "preparation. — As we are now in possession of an easy and in
all respects admirable method of preparing, in a state of great purity, the
crystalline hydrochlorate of hamatin or "hcemin" (see p. 252), the latter
body should invariably be employed in the preparation of pure htematin.

Pure crystallised hsemin (prepared by Schalfijew's process) is dissolved
in a 'highly dilute solution of potassium hydrate, and the alkaline solution
is precix^itated by means of dilute hydrochloric acid. The flocculent-
brown precipitate is washed with hot distilled water until the washings
give no turbidity with silver nitrate. The hsematin thus precipitated is
first dried at the temperature of 100°, and then at 115°, or even higher.

Physical and chemical properties. — Hsematin has not hitherto been
crystallised. In the condition of utmost purity it possesses a bluish-black
colom', and a very pronounced metallic lustre. When finely powdered it
appears as a dark brown powder, which is distinctly pleochromatic.

It is insoluble in water, alcohol, ether, and chloroform, but slightly
soluble in glacial acetic acid ; also in acidulated alcohol, but absolutely
insoluble in aqueous solutions of acids. It is very readily soluble in all,
even highly dilute, alkaline solutions.

Hsematin forms a crystalline compound with hydrochloric acid
(haematin hydrochloride, or haemin), which, because of its importance,
will be separately described, and also others with hydrochloric and
hydrobromic acids.^

It combines with potassium and sodiimi, as well as with calcium,
barium, and other metals. The calcium and barium compounds are
obtained by precipitating ammoniacal solutions of hsematin by means
of solutions of calcium or barium chloride, but they have not been yet
obtained in a state of purity, and have not been analysed.

Hsematin may be strongly heated to 180° C. without undergoing
decomposition. When heated further it is carbonised without previously
melting or taking fire, and liberates hydrocyanic acid, leaving a
residue of pure oxide of iron, which amounts to 12"6 per cent, of
the hsematin incinerated.

When boiled with concentrated potassium hydrate, hsematin under-
goes no perceptible change ; when fused with caustic potash, it is very
slowly decomposed, and evolves ammonia. It is only attacked by

^ M. C. Husson, Comfl. rend. Acad. d. sc, Paris, tome Ixxxi. p. 477 ; V. D. Harris,
Journ. Physiol., Cambridge and London, 1885, vol. v. p. 209 ; D. Axenfield, Centralhl.
f. d. vied. Wissensch., Berlin, 1885, No. 47.

H^AfATIN. 251

concentrated hydrochloric acid, at a temperature above 150° C. Con-
centrated sulphuric acid dissolves it, without any gas being evolved,
giving rise to a dark red solution, from which water precipitates the
substance known as " hasmatoporphyrin " (see p. 258), which, as it
contains no iron, has been sometimes spoken of as iron-free haematin.
This body is soluble in alkaline solutions, and both its acids and
alkaline solutions exhibit very characteristic absorption-spectra.

Alkaline solutions of hsematin in thick layers, when examined by
transmitted light, appear red, whilst thin layers appear of an olive-
green colour. Acid solutions, whatever the thickness of the stratum
examined, always appear of a brown colour.

When the spectrum of light transmitted through alkaline and acid
solutions of htematin is examined by the photographic as well as by the
direct method, it is seen that the last rays of the spectrum to be
absorbed are the red rays up to B ; that the solutions are characterised
by a defined absorption-band between C and D, which is shifted towards
D in the case of the alkaline, towards C in the case of the acid solutions ;
that alkaline solutions, even when extremely diluted, effect a general
absorption of the whole ultra-violet, violet, etc., rays ; that acid solutions,
even when very highly diluted, whilst not exerting a general absorption
of the ultra-violet, exhibit an absorption-band at the junction of the
extreme violet and the ultra-violet, properly so called.

The absorption-bands in the visible spectrum of both alkaline and
acid solutions of haematin are shown in Plate II., Spectra 2, 4, and 6.
The alkaline solutions exhibit one absorption-band between C and D, of
which the more refrangible border adjoins D, whilst acid solutions exhibit
an absorption-band also between C and D, of which the less refrangible
border adjoins C, though the position of the band is somewhat in-
fluenced by the particular acid which has been employed. Attention
is directed to the fact that the band between C and I) in the spectrum
of methajmoglobin differs in position from the band in the spectrum
of acid as well as from that of alkaline haematin. Whilst the absorption-
band of the former is close to C and that of the latter close to D, the
band of methEemogiobin, in acid solutions, is separated by a marked
interval both from C and D, though it is closer to the former than to
the latter.

Alkaline solutions of ha3matin in the presence of certain foreign
matters, when treated with reducing agents, exhibit a spectrum which
is apparently identical with that which will be described under " Hsemo-
chromogen," and which was first described by Stokes as the spectrum of
reduced hmmatin. The band in the red disappears, and two characteristic
bands appear in the green (Plate II., Spectrum 3). On now shaking
the reduced liquid with air, the two l^ands first referred to disappear,
and are replaced by the original hsematin band.

This experiment would appear to show that haematin is but oxidised hsemo-
chromogen, a conchision which is false, and which is an iUustration of the
mistakes into which observers may be led who conclude as to the identity of
two colouring matters from the identity of prominent absorption-bands in their

A strong proof that oxidised hsemochromogen is not identical with hsematin
is derived from my own observations on the absorption of the extreme violet and
ultra-violet. Whilst haematin possesses even in solutions of great dilution the
power of absorbing the whole of the ultra-violet, the violet and even the blue

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 35 of 147)