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

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rays of the solar spectrum, oxidised haemochromogen is, in solutions of much
greater concentration, remarkably transparent for the ultra-violet.

Hoppe-Seyler made the observation that perfectly pure solutions of haematin
are quite unaffected by reducing agents, but that the addition of certain foreign
matters {e.g. albumin) renders reduction possible. I can, from my own re-
peated observations, emjDhatically confirm this fact.

It has been stated above that diluted blood and solutions of oxyhaemoglobin
treated with acids exhibit a band in the red between C and D (of which the
centre is approximately situated at A 640), though it varies somewhat with the
nature of the acid which has effected the decomposition. If, however, blood
be treated with glacial acetic acid, and the mixture at once shaken with ether,
the latter subsequently separates, holding so much of an acid compound of
hcematin in solution as to possess a deep red colour. This ethereal solution,
in addition to the characteristic band of acid hsematin, exhibits three other
bands whose positions and relative intensities are indicated in Plate II.,
Spectrum 6.

Hsematin hydrochloride {syn. haeniin). — When a minute drop of
blood on a glass slide is mixed v^ith a drop or two of glacial acetic
acid, and tiie mixture is boiled over a tiny flame, and then allowed
to evaporate, the residue is found on microscopic examination to
contain innumerable reddish-brown prismatic crystals, which were
formerly constantly referred to as Teichmann's^ crystals (after their
discoverer). Such crystals may be obtained from any old blood stain
on cloth, linen, wood, metal, etc. The stained tissue or the scrapings
of the stain are heated, as above, with glacial acetic acid. It is neces-
sary, however, in the case of stains which may have been subjected
to the action of water, to add a minute crystal of sodium chloride
to the glacial acetic acid before boiling. Hoppe-Seyler ^ subsequently
discovered methods of obtaining Teichmann's crystals in quantities,
which enabled him to examine their physical properties with some
degree of completeness and to analyse them, and he w^as able to show
that litemin is a compound of hsematin and hydrochloric acid, to which,
as a result of his more recent researches, he ascribed the empirical
formula Cg^HgjN^FeOgHCl. Nencki and Sieber,^ on the other hand,
assigned to hajmin the formula C32H3(,N4re03HCl, corresponding to the
formula C32H3oN4Ee03, which they assign to haematin.

Metliod of 'preparing iuemin in hulk. — A method for preparing hsemin in
bulk was, as has been said, first devised by Hoppe-Seyler, and other methods
were described by I^encki and Sieber. These methods demand the ex-
penditure of much time, labour, and patience ; and none of them, as I know
from my own abundant personal experience, yield a product which can
compare in the absolute uniformity of its crystallisation and the complete
absence of all amorphous matter with the one described by Schalfijew, which
is as follows : —

One volume of defibrinated and strained blood is added to four volumes of
glacial acetic acid, previously heated to 80° C. As soon as the temperature has
fallen to 55°-60°, the liquid is again heated to 80° C. On cooling, crystals at
once separate, and can be seen floating in the liquid, presenting a charac-

1 Ztschr.f. rat. Med., 1853, Bd. iii. >S. 375, and Bd. viii. S. 141.

^ VircJiow's Archiv, 1864, Bd. xxix. S. 597-600; "Das Hamin," Med.-chem. Unter-
such., Berlin, S. 379-385.

^ Jrch.f. expcr. Path. u. PharmakoL, Leipzig, 1884, Bd. xviii. S. 401 ; 1886, Bd. xxi.
S. 325 ; 1888, Bd. xxiv. S. 430.


teristic silky lustre and a dark blue colour. The crystals are allowed to settle
for at least twelve hours, and the clear dark brown mother-liquid is syphoned
off. The blue sediment, if care was taken to avoid the presence of any
blood clots in the defibrinated blood used in the preparation, is found on
microscopic examination to be entirely composed of crystals of hsemin. It
is repeatedly washed by decantation with water ; then thrown on a filter,
and, after renewed washing with distilled water, it is subjected to long-con-
tinued washing with spirit and ultimately with absolute alcohol. This washing
with alcohol must be continued so long as the alcohol assumes a brown colour,
and is a very long process. The blue mass remaining on the filter is ultimately
washed with ether and alcohol, and in the first instance is allowed to dry by
exposure to the air, and afterwards by heating to 115° C.^

It was stated by Schalhjew that by his process 5 grms. of pure hsemin can
be obtained from 1 litre of defibrinated blood. This yield, which would be
approximately equivalent to the theoretical yield, on the assumption that the
blood contains in the mean 1 2 per cent, of haemoglobin, is from my own experi-
ence never realised, 1 litre of blood yielding on the average 3"5 grms. of
pure hsemin.

Physical and chemical properties. — Whilst presenting in mass a blue
colonr, and exhibiting, when floating in a liquid, a silky lustre, on micro-
scopic examination heemin crystals appear dark brown elongated rhombic
plates and prisms belonging to the tricliuic system. They are arranged
singly or in groups. They are strongly doubly-refracting. They are
quite insoluble in water, alcohol, ether, or chloroform.

When pure uniformly crystallised hsemin is boiled in pure glacial
acetic acid, the latter dissolves an appreciable quantity, assuming a dark
brownish-red colour. From this solution the htemin is in great part de-
posited, on cooling, in perfect crystals, without any admixture with
amorphous substances. I find, however, that if the process of re-crystal-
lisation be repeated, the substance deposited on cooling consists of
hsemin crystals mixed w4th some amorphous colouring matter.

Ha?min is very easily soluble in highly dilute solutions of the caustic
alkalies and their carbonates ; from these solutions heematin is pre-
cipitated on the addition of an acid. If nitric acid be used as the
precipitant, the chlorine which had originally been combined with the
hsematin, and which is now present in the filtrate as an alkaline
chloride, can be precipitated by silver nitrate.

When hasmin crystals are heated, they remain unchanged up to
about 200° C. ; more strongly heated, they glow and leave an ash com-
posed of pure iron oxide. When pure hsemin is intimately mixed, as by
pounding, with pure concentrated sulphuric acid, hydrochloric acid is

Nencki and Sieber, who employed amyl alcohol in the preparation of
hsemin, found that when prepared in this way the crystals contained amyl
alcohol, and that their composition corresponded to the formula (C3.,IIooN4

The existence of a definite compound of hsemin and amyl alcohol is,
however, doubted by Hoppe-Seyler."

1 M. Schalfijew. I have not seen the original paper in the Journ. russk. fiz.-chhn.
Obsh., St. Petersburg, 1885, S. 30-37. See abstracts in Bei: d. deutsch. chem. GeseUsch.,
Berlin, 1885, Bd. xviii. (Referat Bd.), S. 232-233 ; also in Jahresb. ii. d. Fortschr. d. TMer-
Ohem., Wiesbaden, 1885, Bd. xv. S. 138.

^"Ueber Bhitfarbstotfe und ihre Zersetzungsproducte," Ztschr. f. i^liysiol. Chem.,
Strassburg, 1882, Bd. x. S. 331.


The compounds of hgematin with acids, e.g. hsematin-hydrochloride,
present, even in solutions of great dilution (1 : 25,000-1 : 50,000), an
intense absorption - band, which encroaches more and more on the
ultra-violet, as the strength of the solution increases. In a solution con-
taining one part of crystallised htematin hydrochloride in 20,000 parts
of glacial acetic acid, the band extends between h and M, the most
intense absorption between A and L. The less refrangible border of this
band is sharply defined, whilst the more refrangible border is less
definite. As the solution is diluted the band becomes narrower,
through less and less of the ultra-violet being absorbed. In highly dilute
solutions the band which is still intense absorbs both H and K.^

The acid compounds of hsematin exhibit, therefore, an absorption-
band, which is exactly on the boundary of the ultra-violet proper, and
which extends further and further into the ultra-violet as the con-
centration of the solution increases.


Fig. 37. — The photogi-aphic spectrum of hfemiii.

It has abeady been explained that Hoppe-Seyler employed the
name haemochromogen to denote the very remarkable Ijody which he
was the first to study with care, and which results from the decomposi-
tion of reduced haemoglobin, in the absence of all oxygen, by acids, and
especially by alkalies, and of which the solutions present absorption-
bands in the visible spectrum, which are identical with those of the
reduced hsematin of Stokes.

The latter name had been applied by Stokes to the chemical
substance assumed to be the cause of the characteristic absorption-
spectra which are exhibited by solutions of the blood-colouring matter,
and likewise by impure solutions of htematin when subjected to the
action of reducing agents. It now remains to describe the methods of
preparing solutions of haemochromogen, the body itself and its properties
(so far as these are known to us), its combinations, and especially to
refer to tlie \aews which Hoppe-Seyler advanced and held, in reference

1 Gamgee, Proc. Roy. Soc. London, 1896, vol. lix. p. 276.



to the relations of hajmochromogen to hsemoglobin, and the part which
it plays in relation to the optical properties of, and the chemical affinities
for gases manifested by, the complex molecule of hsemoglohin.

Methods of preparing solutions containing hcemochromogen hy the direct
decomposition of hcemoglohin. — Without referring to a more complicated
and in some respects more satisfactory method of decomposing haemo-
globin in the absence of oxygen,^ the following very simple method,
which, like the first, we owe to Hoppe-Seyler,^ will be described.

A solution of oxyhemoglobin is placed in a glass tube, and then a
smaller glass tube containing a solution of sodium or potassium hydrate,
or, if desired, of tartaric or phosphoric acid, is introduced into the larger
tube, the open end of which is then drawn out and sealed in the blow-
pipe flame. The apparatus thus prepared is then subjected to gentle
heat, taking care not to incline the tubes so as to cause their contents
to mix.

The oxyhsemoglobin contained in the larger, outer tube first becomes
reduced, and thereafter the oxygen contained in the air of the tube is
absorbed by the hasmoglobin. When many days have elapsed, and the
whole of the haemoglobin is again reduced, the tubes are inverted and
their contents mixed, when the formation of haemochromogen may be
followed by the changes in colour and in the spectrum, which the
colouring matter undergoes.

Physical and chemical properties. — When acted upon by dilute solu-
tions of the caustic alkalies, haemochromogen gives rise to a beautiful
cherry-red solution, which, when sufficiently diluted, exhibits two
absorption-bands apparently identical with those of Stokes' reduced
haematin, which have already been referred to.

The visible spectrum of solutions of haemochromogen in alkahne
solutions is distinguished from all others by the extraordinary intensity
and sharpness of the absorption-band nearest to D. The second ab-
sorption-band, which is very much less intense, has less sharply-defined
borders. The solution, even when concentrated, absorbs very little of
the red.

The following are measurements of the position of the absorption-
bands in the visible spectrum by Hoppe-Seyler and myself : —

Gamgee's measurements ^ (1878) X 567-547 X 532-518

Hoppe-Seyler's „ * (i889) A 565-547 A 527-514

My study of the photographic spectrum of haemochromogen has led
to the following results : ^ — Solutions, even of very great dilution, exhibit
an absorption-band between h and g. This band has the same position
as the band of CO-haemoglobin, but is much more intense. With one
part of hffimochromogen in 25,000 parts of water, a stratum 10 mm. thick
being examined, an intense absorption-band occupies the region between
X.410-0 and X430'0. From the examination of solutions of various strengths
it results that the mean ray absorbed corresponds to about \ 420-0.

By heating to 110° C. a solution of haemochromogen mixed with
a sufficiently concentrated solution of sodium hydrate, haemochromogen

1 Hoppe-Seyler, Med.-eliem. Untersuch., Berlin, S. 540 and 541 ; and Gani<^ee's
"Physiological Chemistry," voL i. jjp. 118 and 119.

2 "PhysioL Chem.," 1878, S. 390.

'^ "Physiological Chemistry," 1880, vol. i. p. 111.

•* Ztschr.f. physiol. Cliem., Strassburg, 1889, Bd. xiii. S. 496.

■"' Gamgee, Proe. Roy. Soc. London, 1896, vol. lix. p. 276.



separates as a violet-grey powdery precipitate, which dissolves again in
the hqnid from which it had separated, as soon as this cools. It is quite
erroneous to state, as is asserted in all text-books,^ that Hoppe-Seyler
succeeded in separating htemochromogen in a crystalline condition. He
only succeeded {at most) in obtaining crystals of the CO-compound,
and concluded that hsemochromogen itself must be a crystalline body, but
he never even asserted that he had actually obtained the crystals, and a
promise made in 1889 ^ to describe the assumed crystalhne hsemo-
chromogen, though implying that he had already obtained the body in
this condition, was never fulfilled. Moreover, in the last systematic
account of hsemochromogen which he published in 1893, Hoppe-Seyler ^
does not refer to its being crystalhne, but, on the contrary, speaks of it
(as he had done in 1889) as separating in the form of a violet-grey
powdery precipitate.




38. — The photographic spectrum of oxygenized htemochromogen and of

Acids, even when very dilute, lead in the first instance to the forma-
tion of hsemochromogen from reduced haemoglobin, in the absence of
oxygen ; they, however, decompose a part of the hsemochromogen with
great rapidity, removing its iron and gi^T-ug rise to ha?matoporphyrin.
This explains, according to Jaderholm,^ the complex (four-banded) nature
of the spectrum of hsemochromogen, as at first described by Hoppe-
Seyler,^ when prepared hj the action of acids on hemoglobin.

^ Hammarsten, " Lehrbuch d. phys. Chem.," Dritte Aufiage, 1895, S. 122 ; Neumeister,
"Lehrbuch der physiol. Chem., etc.," 1895, Bd. ii. S. 154 ; Halliburton, "A Text-Book
of Phys. Chemistry," 1891, p. 290; Sheridan Lea, "The Chemical Basis of the Animal
Body," Appendix to Foster's "Physiology," 1892, p. 2-32.

- Hoppe-Seyler, Ztschr. f. physiol. Chem., Strassburg, 1889, Bd. xiii. S. 495.

^Hoppe-Seyler und Thierfelder, " Handbuch d. phys. u. path. Chem. Analyse,"
Berlin, 189-3, S. 214, 215 (" Hamochromogen ").

* See Abstract by Hammarsten in Jahrcsh. ii. d. Fortschr. cl. Thier-Ohem., Wiesbaden,
1874, Bd. iv. S. 102.

^ Med. -chem. Untcrsuch., Berlin, S. 542. In his later descrijjtions of the spectrum of
acid solutions of htemochromogen no mention is made of four bands.


When subjected to the action of such reducing agents as tin and
hydrochloric acid, hsemochromogen gives rise to coloured products, which
are obviously nearly related to, though not identical with, such bodies as
the so-called urobilins.

It was stated that when blood saturated with CO, or a concentrated
solution of CO-hffimoglobin, is treated with a concentrated solution of
sodium hydrate, a bright red precipitate separates. Jaderholm stated
that this precipitate consisted of a compound of CO with hsematin, and
could be prepared directly by the action of the gas on a solution of
reduced hsematin ; he further asserted that the visible absorption-
spectrum of the CO-hsematin closely resembled that of CO-hsemoglobin,
the bands occupying the same position; though he described them as
being less intense in the hsematin compound, and as differing from the
CO-hsemoglobin compound in the fact that the two bands a and /S
exhibit equal intensities.

By causing an alkaline hydrate to act upon CO-hsemoglobin in the
absence of oxygen (method with double tubes previously described), and
heating to 100° C, Hoppe-Seyler separated the body which Jader-
holm had described as CO-hsematin, but which appears really to be
CO-hsemochromogen. Like hEemochromogen itself, its CO-compound,
which has been deposited at 100° C, dissolves again when
the liquid from which it separates cools. The CO-compound of
hsemochromogen is described by Hoppe-Seyler as a crystalline body,
though none of its physical characters have been subjected to even a
superficial examination. The visible spectrum of its solution is, accord-
ing to Hoppe-Seyler, absolutely undistinguishable from that of CO-

The most interesting and weighty observation made by Hoppe-
Seyler on this subject was, however, that concerning the volume of CO
which combines with hsemochromogen to form its CO-combination. He
found that the same volume of CO combines with hsemochromogen as
would be required to convert an equivalent weight of reduced hsemo-
globin into the CO-compound. This unquestionably interesting ob-
servation, taken in connection with the fact that crystals form under
certain circumstances in solutions which contain CO-hsemochromogen
(there is no absolute proof that the crystals represent this substance),
led Hoppe-Seyler to form certain hypotheses of extraordinary boldness,
for which the experimental bases are as yet altogether wanting, but
which have been accepted with misplaced confidence ; these hypotheses
he looked upon as legitimate conclusions from his own experiments, and
formulated as follows : —

" We are justified in concluding that in crystallised CO-hsemoglobin,
as well as in the colouring matter of the blood corpuscles, there is
present a particular group of atoms which combines with and retains
carbonic oxide, which is characterised by the special manner in which it
absorbs light, and which, after separation from the albuminous residues,
passes unchanged into CO-hsemochromogen.

" Without possibility of doubt, this group of atoms is identical with
the one which, in the arterial blood-colouring matter,^ and in crystallised
oxyhsemoglobin, holds two atoms of oxygen in combination, in the place
of a molecule of CO.

" The oxyhsemoglobins, the hsemoglobins, and the CO-hsemoglobins, as

1 Reference is here made to the hypothetical "arterin."
VOL. I. — 17


well as the colouring matters of the red-l)lood corpuscles, all contain
haemochromogen, and this body can be obtained from them all by a
process of simple decomposition, even in the crystalline condition, and
almost in theoretical proportions." ^

In other words, Hoppe-Seyler announced that, from his experiments
it might be concluded that htemochromogen represented an iron-con-
taining coloured radical, which, by linking itself to an albuminous residue
or albuminous residues, forms hiemoglobin, and that haemochromogen in
the latter body combining with a molecule of oxygen forms oxyhemo-
globin ; with a molecule of carbonic oxide, carbonic-oxide haemoglobin,
etc. — these substances containing oxyhaemochromogen and CO-heemo-
chromogen respectively.

Not only are the facts wanting which would be needed in order to
prove this hypothesis, but there are many others which appear to me
to indicate that whilst, tolun once formed, haemochromogen, as indeed
heematin, includes the specific atomic group upon which the character-
istic optical and physico-physiological properties of the blood-colouring
matter depend, probably haemochromogen does not exist preformed in
haemoglobin and its compounds. I trust shortly to throw more light
on this question.

Linossier ^ described comiDOunds of hcematiii and reduced haematin with
nitric oxide as well as Avith carbonic oxide. On re ideating his experiments,
I convinced myself that (as had been shown by Jaderholm and by Hoppe-
Seyler in the case of CO) jSTO exerts no action on hsematin, but appears to form
a compound with hasmochromogen, which is possessed, as Linossier describes,
of a fine red colour, and exhibits two absorption-bands between D and E,
similar to those of oxyhee-moglobin. This ^0-hsemochromogen awaits a careful

Hoppe-Sejder has speculated in reference to the condition in which the
iron exists in hcemochromogen and hsematin respectively, and has emitted the
opinion that the iron in haemochromogen is present in a ferrous and in hcematin
in a ferric condition, but the grounds for an opinion do not actually exist.^


Methods, of ineiKiration. — When either haematin or hsemin is
thoroughly mixed with concentrated sulphuric acid, it dissolves, and
by filtering through asbestos a clear and beautiful purple-red solution
is obtained. When this solution is poured into a large quantity of
water, the greater part of the dissolved colouring matter is precipitated
in the form of a brown flocculent precipitate, the quantity of which
increases if alkalies be added so as to neutralise the acid. This colour-
ing matter is impure haimatoporphyrin. In this operation the acid
separates the whole of the iron from the hcematin, and it is found in
solution in the state of a ferrous salt. In the process of decomposition
of hffimatin by sulphuric acid there is no evolution of hydrogen gas.

Erom hsematin and hsemin hsematoporphyrin can also be obtained —
(1) by the action of strong HCl in sealed tubes heated to 130° C.

1 Ho]5pe-Seyler, Ztschr.f. physiol. C'hem., Strassburg, Bd. xiii. S. 492 and 493.

^ " Sur une combinaison de I'hematine avec le bioxyde d'azote," Com2)t. rend. Acad. d.
sc, Paris, tome civ. p. 1296.

^ For the discussion of the question, see Hoppe-Seyler, Med.-chcm. Untersuch., Berlin,
S. 546-559.



(Hoppe-Seyler) ; (2) by the action of acetic acid saturated with HBr,
aided by heat (Neiicki and Sieljer).

Htemochromogen is, in the absence of oxygen, converted even by the
weakest acids into h&ematoporphyrin, the iron being found in the
solution as the ferrous salt of the acid employed. Although occurring
more slowly, the decomposition of CO-hsemochromogen by acids also
yields htematoporphyrin.

According to Hoppe-Seyler, the composition of htematoporphyrin is
represented by the formula C34H3,;N40|3.

According to Nencki and Sieber, who have made the most complete
investigation of tliis body, it has the composition C^i^H^gN^Og, and they
explain its origin from hsematin by the following equation, in which
they adopt their own as distinguished from Hoppe-Seyler's formula for


According to Nencki and Sieber, hamatoporphyrin is isomeric with


Fig. 39. — The jiliotographic spectrum of liEeniatoporpliyrin.

Physical and chemical properties. — Heematoporphyrin forms beauti-
ful crystalline compounds with Na and with HCl.

It is insoluble in pure distilled water, slightly soluble in dilute acids,
more soluble in strong acids, and readily soluble in alkaline solutions,
weak and strong. It is also readily soluble in acid and alkaline alcohol.

Solutions of hpematoporphyrin in acidulated alcohol have a beautiful
purple colour, and assume a bluish violet tint when the solution is made
very strongly acid. Alkaline solutions are of a fine red, but in the
presence of a great excess of alkali exhibit a violet tint. Solutions of
haematoporphyrin, even if extraordinarily dilute, exhibit a magnificent
red fluorescence, which strangely enough is not referred to in text-books,
though it seems to me to be their most remarkable characteristic.

An alcoholic solution of hciematoporpliyrin, acidvilated with hydro-


chloric or sulphuric acids, exhibits in the %dsible spectrum two absorption-
bands, of which one, which is the narrower and the weaker, is situated
between C and D and immediately adjoins D. The second, which is
much more intense, more sharply defined and broader, hes nearly mid-
way between D and E ; but nearer the former than the latter.

Alkahne solutions exhibit in the visible spectrum fom' absorption-
bands, to wit, a weak band midway between C and D, an equally
weak Ijand between D and E, but nearer to the former, a more strongly
marked band nearer to E, and lastly a fourth liand, darkest of all, which

Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 36 of 147)