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solution of blood prepared in this way is free from all turbidity, and
therefore more transparent than a pure aqueous solution, and undergoes
putrefactive alterations more slowly.

1 "On the Reduction and Oxidation of the Colouring Matter of the Blood," Proc. Boy.
Soc. London, 1864, vol. xiii. pp. 353-364 ; London, Minhurgh, and Dublin Phil. Mag.,
London, 1864, vol. xxviii. pp. 391-400.

" Stokes, op. cit., p. 357, par. 8.



2 30 • HAEMOGLOBIN.

1. By the action of reagents exerting a reducing action. —

It is an essential condition which all reagents to be emploj^ed in the
reduction of oxyhaemoglobin to hgemoglobin must fulfil, that they do not
act destructively on these substances, as is the case with acids and salts
possessed of an acid reaction.

Ordinary solutions of ferrous sulphate or stannous chloride cannot,
for instance, be employed, as they instantly lead to a decomposition
of the blood-colouring matter. The first and still the most generally
employed reducing agents, the use of which dates back to the researches
of Stokes on the blood-colouring matter, are ferrous and stannous salts
and the alkaline sulphides. Utilising the well-known property of citric
and tartaric acids to prevent the precipitation of the salts of iron and
tin by ammonia and the alkaline hydrates, Stokes indicated easy
methods of preparing active solutions of ferrous and stannous salts for
the study of the reduction of oxyhsemogldbin.

{a) Alkaline, solutions of ferrous salts {Stokes reagent). — To a solution
of a ferrous salt (usually ferrous sulphate or ferrous ammonium sulphate)
(re(]SI'H^)2(S04)2.6H20i), citric or tartaric acids or one of their alkaline
salts is added, and then ammonia, until the reaction is alkaline. A light
green solution is thus obtained, which rapidly darkens in the presence of
air by the absorption of atmospheric oxygen. Such a solution, which
must be freshly prepared, exerts a powerful and exceedingly rapid
reducing action on oxyhfemoglobin, even in the cold. Alkaline ferrous
solutions possess the disadvantage, in proportion as they absorb oxygen
and become oxidised, of becoming coloured, and absorbing the more
refrangible rays of the spectrum, interfering, therefore, with the accurate
study of the specific absorption due to the colouring matter.

(&) Alkaline solutions of stannous salts. — These are made as described
under a, by substituting a stannous (usually SnCl2) for a ferrous salt.
As they do not become coloured on salt being oxidised, these solutions
do not interfere with the accurate study of the absorption of the violet
rays. Like the analogous ferrous solutions, those containing tin rapidly
reduce haemoglobin even in the cold.

(c) Sohitions of the alkaline sulphides. — Solutions of these salts
(ammonium sulphide being almost invariably employed) effect the
reduction of oxyhsemoglobin, but much more slowly than is the case
with a and h, and their action is greatly accelerated by heat. Solutions
of ammonium sulphide for this purpose should be freshly prepared, and
be protected from the action of atmospheric oxygen and light, which
bring about chemical changes, and cause them to assume a yellow colour
and to absorb the violet end of the solar spectrum.

Sohitions of the crystalline sodium monosuliDhide (NagS) cannot be
employed with advantage as reducing agents for oxyhsemoglobin, as, according
to my experiments, they lead at once to the formation of sulphomethfemo-
globin, so that the pure spectrum of reduced hsemoglobin cannot be observed.

(/) Agitation with finely -divided iron} or with metallic iron reduced
by hydrogen — the so-called o^cmal ferrum redaetum.^

^ Rollett, "Versuclie ueber thatsachliche und vermeintliche Beziehungen d. Blutsauer-
stoffes," Sitzuncjsh. d. h. AJcad. d. Wissensch., Wieii, 1866, Bd. lii. Abth. 2, S. 246 et seq.

_ - Liidwig und Schmidt, "Das Verlialten der Gase welclie mit dem Blut durch den
reizbaren Silugethiermuskel stromen," Sitzuncjsb. d. Jc. Sachs. Oesellsch., Leipzig, 1868, Bd.
XX. S. 12-72.



HEMOGLOBIN {RED UCED HEMOGLOBIN). 231

{g) Solution of sodium liydrosulphite (NaHSOg).- — By the action of metallic
zinc on a solution of sodium sulphate, in the absence of oxygen, a solution of
intense reducing power is retained. Such a solution, which instantly decolor-
ises indigo and litmus, reduces oxyhaemoglobin.^

Methods of determining the percentage of haemoglobin have been based on
this reaction, 2 s though they have been abandoned as unreliable.'^

(h) Solution of hydrazin and its salts. — It was pointed out by Curtius ^ that
a solution of a salt of hydrazin reduces solutions of oxyheemoglobin with great
rapidity ; and Hlif ner ^ afterwards employed an aqueous solution of hydrazin
hydrate to effect the reduction of concentrated solutions of oxyhsemoglobin,
the advantages of this reducing agent being that the only products of its
decomposition are nitrogen and water, as shown in the following equation : —

H2N— NH2.H2O + 02 = ^2 + 3H2O.
(hydrazin hydrate)

2. By taking advantage of the reducing action exerted by pro-
ducts of putrefaction. — The solutions of oxyha?moglobin, or of diluted
blood, is set aside in sealed tubes, when, especially at temperatures ap-
proaching 40° C, reduction rapidly occurs. It is worthy of remark that,
whilst oxyhemoglobin or its solutions very rapidly undergo change at
temperatures above 0° C, such is not the case with reduced hsemoglobin,
which may be kept for many years in sealed tubes in the presence of
putrefactive bacteria and the products of their activity. On opening the
tubes and agitating with air, oxyhsemoglobin is at once formed, and under
favourable conditions may be crystallised.

3. By taking advantage of the conditions which favour the
"dissociation" of the compound of Og with haemoglobin. ^ — (a) By
boiling in a " Toricellian " or barometric vacuum ; (b) by subjecting
diluted blood or a solution of oxyhsemoglobin to the action of a
long-continued stream of a neutral gas, such as hydrogen, nitrogen, or
nitrous acid.

4. By temporarily arresting the circulation through a suffi-
ciently transparent part of the animal body, — It was first pointed
out by Vierordt,'' that the spectrum of oxyhsemoglobin can be satisfac-
torily demonstrated by bringing two fingers (preferably the fourth and
fifth) close together and passing a beam of sunlight through the com-
paratively thin layer of tissues at the boundaries of the adjacent fingers.
He further pointed out that, on placing caoutchouc rings at the base of
the first phalanges, after an interval varying between 40 and 300
seconds (?), the two bands of oxyhsemoglobin became replaced by the
single band indicative of reduced hfemoglobin.^

^ Scliiitzenberger and Risler, " Eecherches sur le pouvoir oxydant du sang," Compt. rend.
Acad. d. sc, Paris, 1873, tome Ixxvi. pp. 440-442, and pp. 1214-1216.

^ Rollett, loc. cit.

^ Ludwig and Schmidt, loc. cit.

■* Qninquaiid, "Sur un procdde de dosage dc I'h^moglobine dans le sang," Compt. rend.
Acad. d. sc, Paris, 1877, tome Ixxvi. p. 1489.

^ Jouo^n. f. prakt. Chem., Leipzig, 1889, Bd. xxxix. S. 27.

^ " Bestimmr.ng d. Sauerstoffscapacitat d. Blutfarbstoffs, " S. 156.

'' "Das Hamoglobiuspeetruni am lebenden Menschen," Ztsclir. f. Biol., Miinchen, 1876,
Bd. xi. S. 188; and " Die Sauerstoffzehrung der lebenden Gewebe," ibid., 1878, Bd. xiv.
S. 422.

^ Refer to the following papers by A. Henocque, " fitude spectroscopique du sang a la
surface sous-ungueale du pouce," Compt. rend. Soc. de hiol., Paris, Ser. 8, tome i. p. 671 ;
and also "Notes complementaires, " ibid., p. 700. According to this author, the average
time of reduction, when the circulation through the thumb is arrested, varies between fifty-
five and sixty-five seconds.



232 HEMOGLOBIN.

Preparation of crystallised hseraoglobin (reduced heemoglobin).—

It was shown almost simultaneously and independently by Kiihne^ and
by EoUett ^ that highly concentrated solutions of pure oxyhsemoglobin
may, after reduction, ho, made to crystallise, and that the crystals of
reduced haemoglobin, though differing in colour and spectroscopic char-
acters from the oxygen compound, are essentially identical with it in
crystalline form. Ktihne explained that the difficulty which is en-
countered, when attempting to crystallise reduced hsemoglobin, depends
upon its very great solubility.

Hoppe-Seyler was unable to crystallise reduced hsemoglobin ; ^ and Hiifner^
in 1880 published a note, in which he announced that he had succeeded in
obtaining crystals of reduced hsemoglobin, though he neither then nor after-
wards referred to the much more complete account published by Kilhne fifteen
years earlier.

In order to obtain crystals of reduced hsemoglobin for microscopic
examination, a pure and highly concentrated solution of oxyhemoglobin
in very dilute ammonia is placed in a gas chamber, and a stream of
chemically pm^e and thoroughly dried hydrogen is passed over it ; as the
solution evaporates crystals separate.^

Nencki and Sieber have obtained large quantities of crystals of
reduced hsemoglobin by reducing concentrated solutions of pure oxyhse-
moglobin of the horse through the agency of putrefactive bacteria, then
adding a sufficient quantity of 25 per cent, alcohol and exposing to cold.
The method which I employed more than twenty years ago, and which
appears to me to offer some advantages, is to place a magna of pure
oxyhsemoglobin crystals with a small quantity of the mother liquor
from which they have separated in a glass tube, so as nearly to fill the
latter, and then to seal it. The tube is heated for some days in an
incubator at about 35° C, and is then set aside in a cool place. After
some weeks of exposm'e to a winter temperature, the tube is found to
contain large quantities of crystallised and perfectly reduced hsemo-
globin.

No one has hitherto attempted to recrystallise reduced hsemoglobin,
though, with the conveniences at present at the disposal of the scientific
chemist, the process would present little difficulty.

Characters of the crystals of reduced haemoglobin. — In form
they are, as has been said, essentially identical with those of the
oxygen compound, and like these are doubly refracting. Hiifner
often obtained crystals 1 mm. long ; and Nencki and Sieber, working
with horses' blood, obtained crystals, mostly in the form of hexagonal
plates, 2 or 3 mm. in diameter. They are pleochromatic, appearing
of a dark red colour in some lights, and exhibiting a bluish or purple
tinge in others.

^ " Das A''orkonimen und die Ausscheidung des Harnoglobins aus deni Blute, " Vircliow's
Archiv, 1865, Bd. xxxiv. S. 423-436.

^ Log. cit.

^ Med. Chr/in. Uniersuch. , Berlin, S. 373.

■* "Ueber kiystallisclip, Hanioglobin," Zfschr. f. physiol. Chem., Strassburg, 1880,
S. 383. It is singular tliat Nencki and Sieber, in an interesting and really valuable
paper, sliould in 1887 have jiublished again, as a new discovery, the obtaining of crystals
of reduced hfcmoglobin, though they subsequently disclaimed all priority (see M. Nencki
and N. Sieber, "Venose Hiimoglobinkrystalle," Iler. d. dcutsch. chem. Gesellsch., Berlin,
1886, Bd. xix. S. 128 and 410).

^ Kiihne, op. cit.



COL O UR OF RED UCED HyEMO GL OB IN.



233



When the blood, crystals of horses' blood are prepared in closed
vessels, it happens very frequently that large quantities of hexagonal
tables of a dark red colour are found mixed with the well-known ordinary
prisms. If a drop of the liquid in which the crystals are suspended be
examined with the microscope, loithout a cover-glass, the hexagonal plates
are observed rapidly to liquefy, and simultaneously bundles of fine,
bright-red prismatic needles appear. Nencki long ago showed that the
dark red hexagonal tables are crystals of reduced hsemoglobin, whilst
the scarlet prisms are those of oxyhsemoglobin. Horses' blood appears
peculiarly apt to give crystals of the reduced blood-colouring matter.
In the preparation of the haemoglobin of the horses' blood by ordinary
methods, i.e. without special precautions in reference to the access of
air, both forms of crystals are usually obtained.^

The Absokption of Light by Solutions of Eeduced H/emoglobin.

Colour of solutions : dichroism. — In thick layers, or in thin layers
if concentrated, solutions of reduced hcemoglol^in present a dark cherry-
red colour, whilst very dilute solutions exhibit a green tint.



OXYH/EMOGLOBIN.



H/EMOGLOBIN.




34. — Graphic representation of the spectrum of — (]) oxylifenioglobin
and (2) hiieinoglobin. The numbers at the right-hand side of each
diagram indicate percentages.— After Rollett.



This dichroism is also characteristic of the blood of asphyxiated
animals, and was first observed by Brlicke. It is specially to he noted
that, lohilst solutions of reduced hamioglohiii are dichroic, solutions of the
Og- CO- and 1:^0 -compounds of hmvioglohin exhibit no trace of dichroism.

Cause of the differences observed in the colour of blood contrasted
with that of solutions of ho3moglobin. — The much brighter colour pre-
sented by blood, as contrasted with corresponding solutions of the blood-
colouring matter, depends upon the presence of the blood corpuscles.
Were we to conceive, as Eollett argues, the blood corpuscles suspended
in the liquor sanguinis or in serum, and retaining all their physical
properties save their colour, then, as a result of the repeated total reflec-
tions, due to the differences in the refractive indices of the corpuscles
and the fluid in which they float, blood would appear as white as milk.

^ Hlifner, oj). cit.. Arch. f. Physiol., Leipzig, 1894, S. 150.



234



HMMOGLOBIN.



But these total reflections do go on in the case of the actual coloured
corpuscles in a precisely similar manner to that which would occur in
the hypothetical case just discussed ; and the light reflected by them
is conditioned by, and corresponds to, the absorption of the spectral
colours exerted by the haemoglobin and the oxyhaemoglobin respectively.

The visible spectrum of reduced hseuioglobin. — It has already
been stated that, when a solution of oxyheemoglobin is reduced, the
two absorption-bands a and |8 disappear and are replaced by a single
one (7) situated between D and E, which is less deeply shaded and
possesses less sharply-defined edges (see Plate I., Spectrum 5). This
summary description must now be supplemented.

The right-hand diagram on p. 233 exhibits fairly accurately the
absorption of light by solutions of reduced haemoglobin of varying con-
centrations. The single absorption-band (7), though occupying in solutions
of from 0-2 to 04 per cent, and 1 cm. in thickness, the greater part of
the space between Frauenhofer's lines D and E, has its centre or darkest
region rather nearer D than E. According to my own measurements,
the darkest part of the band corresponds approximately to X 550.

It is to be noted that solutions of reduced haemoglobin have a much
greater absorptive power for the rays between A and B, and a smaller
absorptive power for those between F and G, than corresponding solu-
tions of oxyhaemoglobin.

The Specteophotometpjc Constants of Eeduged Hemoglobin.

In his most recent researches, Hiifner has determined the spectro-
photometric constants of haemoglobin for the same spectral regions as
were selected by him, in the same researches, for the determination of
the constants of oxyhaemoglobin.

In the case of reduced haemoglobin the respective extinction-co-
efficients are distinguished as g,. and g'^, and the corresponding absorp-
tion relations as A^ and A^.

The following are the results of Hiifner's determination -} —



Ar


^r


(A 554-A 556)


(A 531 •5-X 542-5)


0-001354


0-001778



The quotient — is a constant of special importance; it is 0'7617.



The value of the quotient - has also been determined by Hiifner ; ^ it

is 0-6541.

The determination of the amount of oxy- and reduced hsemo-
globin when both are present. — Vierordt pointed out tliat the absorption

^ G. Hiifner, "Neue Versuche zur Bestimmung der Sauerstoffcapacitat des Blutfarb-
stoffs," Arcli.f. Physiol., Leipzig, 1894, S. 140.



SPECTROPHOTOMETRIC DATA. 235

of light (as determined by the extinction-coefficient) in a definite spectral
region, exerted by a mixture of two or more colouring matters, is the sum of
the extinction-coefficients of each of its coloured constituents; and that in
the case of a solution containing tioo colouring matters, if we are acquainted
with the optical constants of each in iim and the same spectral regions, we are
able by the spectrophotometer to determine the relative and absolute amount
of each constituent. In a similar manner we should, according to theory,
be able to determine the amounts of three or of x colouring matters coexisting
in a solution, if we were acquainted with the value of A in three and the same,
or in X and the same spectral regions. The immense importance of a method
Avhich permits of the accurate determination of oxy- and reduced haemoglobin
in blood, and which furnishes us with essential data for calculating the
amount of oxygen present in combination with hsemoglobin, makes it
necessary that we should explain the nature of the very simple calculations
v/hich enable us, from the determination of the extinction-coefficients in two
spectral regions, to effect a determination which, so far as I know, cannot
be carried out with any pretence to scientific accuracy, or even with any claim
to be presumably correct, by any other process whatsoever.

We shall assume that, by following methods which we shall not attempt
to describe, but for which the reader is referred to Hlifner's original papers,
blood has been diluted with O'l per cent, of aqueous solution of NaOIi,
under conditions loliicJi ^preclude the possibility of contact ivith oxtjgen, and
that in the diluted blood solution the extinction-coefficients have been deter-
mined in the first and in the second regions selected by Hiifner. These
extinction- coefficients of a mixture of two colouring matters, we shall represent
by E and E.'

Let A,, be the absorption relation of (reduced) haemoglobin in the first

region (A 554 - A 556).
A\. that of the same body in the second spectral region (A 531*5 -

A 542-5).
Ag the absorption relation of oxyhsemoglobin in the first spectral

region.
A'g that of the same body in the second spectral region.

Then the percentage of (reduced) heemoglobin, which we may designate x, will
be found by the equation —

^_ A,.A\.{E'A\-EAg)

A'gA,.-AgA',,

and the percentage of oxyhsemoglobin by the following equation —

AgA'g{EA,-E'A',)



y-



A A,- — Ag A y



Having thus determined by spectrophotometry the amount of oxyhaemoglobin
by weight existing in a known volume, say 100 c.c. of blood, we can ascertain
the volume of the respiratory oxygen measured at 0° C. and 760 mm. pressure
(which could, but probably with less accuracy, be likewise determined with the
aid of the mercurial pump and subsequent analyses of the gases boiled out of the
blood) by multiplying each gramme of oxyhsemoglobin found by 1-338 (or 1-34).
In this manner Hiifner, having determined the relative and absolute amounts of
haemoglobin and oxyhsemoglobin in the blood, drawn simultaneously from the
main artery and vein of a limb, ascertained the amount of oxygen in each.
There is a strong presumption that determinations of oxygen made in this manner
are nearer the truth than those which the more complex and laborious methods
by means of the mercurial pump and gas analysis are capable of giving. In the
process of raising the blood to a temperature of at least 40° C. in the exhausted



236 HyEMOGLOBIN.

chamber connected with, the mercurial pump, some of the oxygen must be used
up in oxidising the readily oxidisable substances existing in the blood, and
especially in venous blood, and an error will be thereby introduced unequally
affecting different samples of blood, — an error which is influenced by the
duration and extent to which the heat is applied to the blood and the rapidity
with which the aqueous vapour and gases evolved by the blood are removed.

The photographic spectrum of reduced hsemoglobin. — When
the molecule of dissociahU oxygen is removed from oxyhaemoglobin,
either by the action of reducing agents, or by boihng in vacuo, the
absorption-band in the extreme violet is remarkably displaced towards
the less refrangible end of the spectrum, the centre of absorption
corresponding to ?. 426-0. The difference in the position of Soret's
band in the oxy- and in reduced hsemoglobin is shown in the photo-
type (Fig. 33). When we reflect that the addition of a molecule of
oxygen to the enormous molecule of hsemoglobin cannot affect in an
appreciable manner the mass of the molecule, we must conclude that
the displacement of the absorption-band towards the ultra-violet end
when haemoglobin combines with oxygen (all other conditions remaining
the same), indicates that this combination leads to a notable acceleration
of the motion of the intramolecular group of carbon atoms upon which
the extreme violet absorption-band depends.

The amount of oxygen "with -which haemoglobin combines to
form oxyhsemoglobin. — It is believed, on various grounds, that one
molecule of htemogiobin combines with one molecide of oxygen to form
the compound which we know as oxyhsemoglobin.

The most recent determinations made by Hlifner have shown that
1 grm. of reduced haemoglobin of the ox can link to itself 1-338 c.c. of
oxygen or carbonic oxide (measm^ed at 0° C. and 760 mm. pressure).
The molecular weight of the hsemoglobin of the ox (calculated from
Hlifner's most recent estimations of the iron which this body contains)
= 16669. The volume of oxygen absorbed by reduced hsemoglobin,
calculated from this molecular weight, should be 1-34 c.c, so that the
residt of experiment agrees in a surprising manner with theory.

Differences in chemical reactions bet-ween solutions of reduced
and oxyheemoglobin. — 1. Solutions of reduced hsemoglobin when boiled
in vacuo, or subjected to the action of CO, unhke solutions of oxyhsemo-
globin, yield no oxygen.

2. They are not decomposed even by long contact with trypsin, which
readily splits up oxyhsemoglobin into hsematin and the products of

• trypsin proteolysis.

3. They are unaffected by H2S, which, when acting for a sufficient
length of time upon oxyhsemoglobin, converts it into sulpho-methsemo-
globin.

4. Nitrites, potassium ferricyanide, and permanganate, and many
other oxidising and reducing agents, exert no action on reduced hsemo-
globin, whilst they convert oxyhsemoglobin into methsemoglobin.

5. When treated with alcoholic or watery solutions of acids or
alkalies, in the complete absence of free oxygen, hsemoglobin yields
purple-red solutions or precipitates. The lisemoglobin is, under these
circumstances, split up into an iron-containing coloured l)ody — hwmo-
chromogcn — and into an albuminous body or Ijodies. Oxyhsemoglobin,
under the same conditions, splits up into an iron-containing body —
hmmatin — and albuminous products.



COMPOUNDS OF HEMOGLOBIN WITH GASES. 237

Non-existence of the so-called " pseudo-hsemoglobin." — After
treating blood with reducing agents until the two bands of oxyhaemoglobin
were no longer visible, Siegfried ^ found that there yet remained oxygen
removable by boiling in a barometric vacuum. He therefore concluded that,
in addition to oxyhaemoglobin, there existed another oxygen compound of
haBmoglobin, and that this is characterised by the same absorption spectrum as
reduced haemoglobin. To this hypothetical body he gave the name of jjseudo-
luemoglohin. Its existence has been absolntehj dis'proved by Hiifner.^ The
mistake into which Siegfried fell illustrates the danger of drawing conclusions
from qualitative spectroscopic observations. Hiifner has shown that without
spectrophotometric determinations it is impossible to know whether a solution
of blood or of haemoglobin is completely reduced. The only reliable criterion
is to be obtained by determining the values of e,. and e',. so as to ascertain the

quotient — which should = 0'7617.

Blood which has been proved to be completely reduced in this manner,
yields no trace of oxygen when boiled in a mercurial pump.

The Compounds of Haemoglobin with Caebonic Oxide and Nitric
Oxide, and their relation to Oxyh/emoglobin.

Introductory remarks. — In a previous part of this article, I have
referred to oxyhemoglobin as an easily dissociated compound, formed



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 33 of 147)