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of the iron-containing part of the molecule in the hemoglobin from the
most different animals, we are, it appears to me, driven to the con-
clusion that the difference in solubility mu&t he due to differences in
the albuminous residue in the hemoglobin molecule.

Sohibility in liquids other than water. — Oxyhemoglobin is soluble in
highly diluted solutions of ammonia, and the other caustic alkalies, and
their carbonates. These solutions resist decomposition much longer
than aqueous solutions of haemoglobin.^ Kiilme states that a highly
dilute ammoniacal solution of oxyhemoglobin will remain in great part
unchanged for several weeks at ordinary temperatures. Stronger
solutions of the caustic alkalies or their carbonates induce decom-

1 W. Preyer, "Die Blutkrystalle," S. 55.

2 Based upon these facts is the method, introduced by Hlifner, of diluting blood or
solutions of oxyhaemoglobin witli solutions containing 0"1 per cent, of NaOH. Such
solutions are much more transparent than purely aqueous solutions, and are therefore
most valuable for the purposes of spectroscopic researches.



A CTION OF RE A GENTS ON OX YH.EMO GL OB IN. 2 o 7

position of oxyliifinogiobin with a rapidity which depends upon their
concentration.

Oxyhemoglobin is soluble in highly diluted alcohol, the solutions
resisting putrefaction much longer than aqueous solutions. By contact
with even highly dilute alcohol, crystals of oxyheemoglobin become much
more sparingly soluble in water. Oxyhfemoglobin is insoluble in absolute
alcohol. When crystallised oxyhemoglobin is treated with a large excess
of absolute alcohol, it is under favourable circumstances converted into an
insoluble crystalline modification, to which Nencki and Sieber have
given the name of 'paralimmoglobin?- This body cannot be looked upon
as a chemical individual. Oxyhaimoglobin is insoluble in chloroform,
benzol, and carbon disulphide.

4. Diffusibility. — Oxyhtemogiobin offers a remarkable example of a
soluble crystalline body, which, judged by its power to pass through a
septum of parchment paper, must be declared to be absolutely non-
diffusible. This character depends upon the enormous size of its
molecule.

Comparison of the action of certain reagents on solutions of
oxyhsemoglobin and on solutions of albuminous bodies. — It has
already been incidentally stated that in hemoglobin an iron-containing
body is hnked to an albuminous body or bodies, and reference has been
made to the fact that, under the action of various agents, oxyhemoglobin
breaks up into the iron-containing hematin, and into albuminous bodies.
Although the decomposition of hemoglobin and its products will be con-
sidered in some detail in a future section, it is convenient in this place
to refer to this point, and to state that when oxyhemoglobin is decom-
posed so as to yield hematin and albuminous substances, the former
amounts approximately to 4 per cent, and the latter to 96 per cent, of
the original hemoglobin.

Such being the case, it is of particular interest to contrast the
action of certain reagents on solutions of albuminous bodies, and on
solutions of oxyhemoglobin.

Solutions of oxyhemoglobin differ remarkably from solutions of
albuminous bodies in their behaviour towards a large number of
reagents.

As Ktihne pointed out long ago,- all those tests for albumin which
do not immediately bring about a decomposition of oxyhemoglobin,
furnish a negative result when applied to aqueous solutions of this
body. Cupric and ferrous sulphates, mercuric chloride, silver nitrate,
neutral and basic acetates of le^d, all of which precipitate albuminous
solutions, occasion (so long as the body remains undecomposed) no pre-
cipitate — not even cloudiness — when added to solution of oxyhemo-
globin. So soon, however, as the red colour of oxyhemoglobin has
disappeared under the action of any one of the above salts, and the
brown colour due to hematin has appeared (a result which they all
sooner or later bring about), the characteristic albuminous precipitates
appear.

1 M. Nencki und N. Sieber, " Untersuch. iieber die Blutfarbstoff, " Ber. d. deutsch.
chem. Geselhch., Berlin, 1885, Bd. xviii. S. 392 ; M. ISTencki iind B. Lachowitz, "Ueber das
Parahamoglobin," ihid., Bd. xviii. S. 2126. The reader is referred for a criticism of Nencki
and Sieber's researches on paralifemoglobin, to a paper by Hoppe-Seyler, entitled "Ueber
Blutfarbstoffe und ihre Zersetzungsproducte," ^tecAr. /. »7z,«szo^. Chem., Strassburg, 1886,
Bd. X. S. 531.

^ "Lehrbuch der physiolog. Chemie," Leipzig, 1866, S. 207.



2o8 HyEMOGLOBIN.

Other reagents which bring about an instant decomposition of
oxjhaemoglobin, and, consequently, instantly set free the albuminous
matter, exhibit also, as might have been anticipated, the characteristic
albumin reactions, i.e. behave towards a solution of haemoglobin as if
it were a solution of a native albumin. This remark apphes to acetic
acid and potassium ferrocyanide, to mercuric nitrate, to the concentrated
mineral acids — reagents, all of which precipitate a solution of oxyliEemo-
globin as they do solutions containing albuminous bodies.

When subjected to the action of heat, solutions of oxyhaemoglobin
coagulate like solutions of the native albumins; but, doubtless, before
the temperature of coagulation (64° to 68°'o C.) is reached, the complex
hyemogiobin molecule has already been decomposed — a supposition
wliich is suggested by the following observation : ^ — If to an aqueous
solution of crystalhsed oxyhfemoglobin of the dog a small quantity
of sodium carbonate be added, on applying heat no coagulation occurs,
even though the temperature be raised to 100° C. When, however, the
temperature reaches 54° C, the colour of the solution instantly changes to
deep brown, and spectroscopic examination indicates that the spectrum
of oxyhcemogloljin has been replaced by that of alkaline haematin.



The Absokption of Light by Oxyhemoglobin.

(a) The visible spectrtiin.— Historical notes. — The researches of
Brewster and Herschel had shown that absorption-bands occur in the
spectrum of light Avhich has been passed through certain coloured gases,
vapours, and coloured sohitions, and the so-called absorption spectra of indigo
and chlorophyll had been described before the time when Hoppe - made the
discovery of the beautiful absorption spectrum of blood, distinguished by
two very characteristic absorption-bands, situated in the region which inter-
venes between the Imes of Frauenhofer, D and E.

This discovery at once enabled Hoppe to affirm that haematin, which had
up to that time been generally looked upon as the true blood-colouring matter,
does not exist as such in the blood corpuscles, but that it is a product of the
decomposition of the colouring matter ; that the latter, to which he afterwards
gave the name of hsemoglobin, and which he recognised as forming the so-called
blood crystals described by Kunde, Lehmann, and Funke, is the cause of the
absorption-bands which he had discovered in the spectrum of diluted blood,
and that this colouring matter, under the influence of heat, acids, and various
other chemical agents, splits up into hcematin and an albuminous substance or
substances.

There can be no question that, although Hoppe, in a certain measure,
appreciated the immense value of the knowledge which he had gained by his
study of the optical properties of the blood, the full light which it was
destined to shed on the function of the blood-colouring matter was only
recognised Avhen Professor Stokes, two years later, pubhshed his paper " On
the Reduction and Oxidation of the Colouring Matter of the Blood." ^ The
new facts acquired by the combination of chemical and optical methods in
this research, and which at once shed a flood of light on phenomena which
liad until then been shrouded in darkness, enlisted as workers in this field

1 Preyer, "Die Blutkrystalle," S. 61.

"^ Hoppe only assumed tlie name of Hoppe-Seyler in ISG-l-. The paper containing
his first observations on the spectrmii of the blood bore the ibllowing title : — Professor
Hoppe in Tubingen, "Ueber das Verhalten des Blutfarbstott'es im Spectrum des
Sonnenlichtes," Virchow" a Archiv, 1862, Bd. xxiii. S. 446-449.

^ Proc. Roy. Soc. London, 1864, vol. xiii. p. 357.



VISIBLE SPECTRUM OE OXYH.EMOGLOBIN. 209

many persons of distinction in all countries, amongst the first and most
successful of whom were W. Preyer ^ in Germany, and Sorby and Ray
Lankester in England. Amongst all, however, Avho by their work have
contributed to the spectroscopic investigation of the blood, two appear to
me to stand out pre-eminently — these are Vierordt and Hiifner. By the dis-
covery of the first practical method of determining the extinction-coefjicient
of coloured liquids, and his elaboration of a general method for the
quantitative analysis of colouring matters, a method capable of surprising
refinement and accuracy, and which is based upon the relation which exists
between the extinction-coefficient and concentration, Vierordt has placed
both the sciences of physics and physiology under a lasting obligation.-
To Hiifner belongs the merit of having developed and perfected the
methods of spectrophotometry, but especially of employing it so as to obtain
results of paramount importance to physiology, and which would have been
unattainable without its aid. Not only has he, by his own long-continued
researches, and those of his pupils, determined the spectrophotometric con-
stants of hsemogiobin and its compounds with oxygen and carbonic oxide,
but he has by spectrophotometry succeeded in determining the absolute
and relative amounts of reduced and oxyhsemoglobin existing side by side
in the blood. He has further shown that, as we now know the volume of
oxygen which can combine with 1 grm. of haemoglobin, by determining the
amount of haemoglobin and of oxyhaemoglobin coexisting in any specimen of
blood, we possess data enabling us to calculate the volume of the dissociable
or respiratory oxygen of the blood, without having recourse to direct deter-
minations by means of the mercurial pump and gas analysis.

Further, by the method of spectrophotometry, combined with the results
of chemical investigation, Hiifner has furnished us with the proof that, in
spite of the differences in many physical characters, and even in centesimal
composition presented by the blood-colouring matter of different animals, the
coloured iron-containing group existing in hsemoglobin, upon which its essen-
tial physiological functions depend, is identical in all.^

General description of the visible spectrum of oxyhsemoglobin.
— Instruments required. — For the study of the visible, as distinguished from
the photographic spectrum of the blood, or of oxyhsemoglobin, the spectro-
scopes which are in common use in physical and chemical laboratories may be
employed, providing the dispersion of their prisms be not too great. A
spectroscope of the ordinary Bunsen type, provided with a single good flint-
glass prism, is infinitely to be preferred for the study of absorption spectra to
an instrument with two prisms, for, with the greater dispersion, absorption-
bands appear much less clearly defined than with the smaller. Direct vision
spectroscopes of the Browning or Hofmann patterns, or microspectroscopes,
i.e. direct vision spectroscopes adapted to the eyepiece of the compound
microscope, may be employed ; and the second class of these instruments
renders great services in the investigation of minute quantities of colouring
matters — as, for instance, in the examination of the optical characters of
the colouring matters of the tissues.

It is advisable, indeed for the purposes of original research indispensable,
that the spectroscope employed should furnish means of determming accur-

1 Preyer's monograph, entitled "Die Blutkrystalle," Avhich appeared in Jena in 1871,
still continues indispensable to tlie physiological chemist. It is replete with original
observations of great value, and establishes that Preyer had no unimportant share in the
development of our knowledge of the blood-colouring matter.

^ Karl Vierordt, "Die Anwendung des Spektral-apparates zur Photometric der Absorp-
tionsspektren und zur quantitativen chemischen Analj^se," Tubingen, 1873; "Die
quantitative Spektralanalyse in ihrer Anwendung auf Physiologie, Physik, Chemie, und
Technologic, " Tubingen, 1876.

^ As the chief of Htifner's papers have been already quoted, or will be referred to sub-
sequently in detail, their dates and titles are not given in this place.
VOL. I. — 14



2 1 o H^MO GL OB IN.

ately the position of any line or the boundaries of any absorption-band
observed in the spectrum, it being usual to express the position in terms of
the wave length of the light corresponding to it. With this object the
spectrum of sunlight is observed, and the position of the principal lines of
Frauenhofer is determined in reference to the divisions of the photographic
scale, or, in the case of the finer spectroscopes and spectrometers, in reference
to the divisions of the graduated circle of the instrument. From the results
of these observations a curve is readily plotted, enabling the experimenter at
any time to convert the readings of the arbitrary scale of his instrument into
Avave lengths. 1

For all exact spectroscopic work the eyepiece of the spectroscope should
be provided with cross-threads ; and, when employed in the investigation of
absorption spectra, if possible with the arrangement employed in spectro-
photometry, which enables the observer to limit, by a variable slit in the
eyepiece, any particular spectral region and to shut out of the field of view
the remainder of the spectrum.

As a source of light, for some investigations the light of the sun reflected
from the mirror of a heliostat driven by clock-work is desirable ; for general
purposes the light of the sun, reflected from a white surface, may be employed.
Artificial sources of illumination possess the great advantage of being available
at all times, and susceptible of considerable constancy. A gas lamp, furnished
Avith the Auer incandescent burner, is the best of all lamps for the examina-
tion of absorption spectra.

In examining the absorption-spectra of liquids, it is convenient to employ
cells or troughs with perfectly parallel glass or quartz sides, which are a
definite Avidth apart. Such vessels are made according to the model of
Hoppe-Seyler, and sold under the name of hcematinometers (Fig. 23), the
internal surface of the parallel glass plates being exactly 1 cm. apart, and the
little trough being so arranged as to be readily taken to pieces for cleaning.
The small troughs employed in spectrophotometry, and which are usually
constructed with great care, are well adapted to the general purposes of the
spectroscopist.

Instead of a vessel of Avhich the sides are at a constant and known dis-
tance apart, it is convenient for many purposes to employ the so-called
hcematoscope, or hcevioscope, of Hermann,^ as shown in the accompanying
woodcut (see Fig. 24). F is a glass plate, forming the anterior wall of the
tube D, which is supported on the stand A. C is a metallic tube, sliding in
and out of the tube D, and closed anteriorly by a glass plate parallel to F. E
is a funnel communicating Avith the interior of D F B, Ey sliding the piston
C in and out of the tube D, the capacity of the vessel D F B and the
depth of a stratum of liquid contained betAveen the tAvo glass plates, may be
modified at Avill within Avide limits.

The depth of the stratum is read off by the aid of a millimetre scale,
engraved on the sliding tube C.

As the absorption of light passing through a coloured liquid depends
upon the number of absorbing molecules in its path, by doubling the thick-
ness of the stratum of a coloured liquid examined, Ave obtain the same result
as by examining a solution of double concentration. With such a contrivance
as the hgematoscope, Ave are, Avithin certain limits, able therefore to obtain the
same result with a solution of constant concentration as Avith a large number
of solutions of Avhich the concentration varies in known proportions.

1 In a work intended for the advanced student of physiology, it appears superfluous to
enter into such details concerning the construction of the s|)ectroscope, or the method of
working with it, as can be learned in all courses of practical physics, or may be found in
any elementary treatise devoted to this branch of science.

- " Notizen flir Vorlesungs uud andere Versuche," Arch. f. d. qes. Physiol., Bonn,
Bd. iv. S. 209.



VISIBLE SPE CTR UM OF OX YHyEMO GL OB IN. 2 1 1

The spectrum as seen with solutions of varying concentration.—

When well-arterialised defiljrinated blood (containing on an average
from 12 to 14 per cent, of oxyheemoglobin) is dilnted with nine times
its vohnne of distilled water, and a stratum 1 cm. thick is brought before
the slit of the spectroscope, it will be found that the whole of the
spectrum is absorbed, with the exception of the red end, or rather of
those rays having a wave length greater than about 600 millionths of
a millimetre (/. 600).

If, now, the blood solution be gradually diluted, a point is reached
at which the spectrum is (proceeding from the red end) clear up to 1)
(a 598), and a strip of green is visible between h and F (x 518-3-X 486-1).
Between D and & the absorption is intense (see Plate I., Spectrum 4),
and beyond F no trace of light appears. On diluting still further, that





Fig. 23. — The hpeinatiuometer.



Fig. 24. — Tlie hamatoscope.



which appeared as a single wide absorption-band between D and ?), and
afterwards as the solution was progressively diluted between D and E,
is seen to resolve itself into two distinct absorption-bands, separated by
a green interspace ; the violet end of the spectrum is still powerfully
absorbed (Plate I., Spectrum 3).

Of the two absorption-bands just referred to, the one next to D is
narrower than its fellow ; it has more sharply defined borders, and to the
eye ai^inars more intense ; its centre corresponds to X 579, and we may
conveniently distinguish it as the absorption-band a in the spectrum of
oxyhemoglobin.

The second of these absorption-bands, i.e. the one next to E, which
we shall designate the band /3, is broader, has less sharply-defined edges,
and its centre corresponds approximately to X 553'8. Between the two
bands is a green interspace.

On diluting the solution more and more largely, and continuing to
examine a stratum 1 cm. thick, the absorption of the violet end becomes



2 1 2 H.^MO GL OB IN.

less and less, and the whole spectrum as far as G- appears beautifully
clear, except where the two absorption-bands are situated (Plate I.,
Spectrum 2). If dilution be pushed still further, these disappear ; before
they vanish they appear as faint shadows across the limited region which
they occupy. The band a is said to disappear last. I find, however, that
whenever I can detect a I am able to detect a faint shadow in the
position of X 540->. 550. When the bands are just perceptible, there is
no obvious absorption of either the red or the violet end of the spectrum.
The two absorption-bands of oxyhcemogiobin are seen in greatest
perfection when a stratum 1 cm. thick of a solution containmg 1 part
per 1000 of oxy haemoglobin is examined; this corresponds to a solution
made by dilutmg from 1-2-1-4 parts of blood to 100. They are still
perceptible when the solution contains 1 part oxyhasmogiobm in 100,000
parts of water (1 grm. m 10 htres).



OXYILEMOGLOBIN.



H^MOGLOBIK.




Fig. 25. — Graphic representation of the spectrum of oxyhfemoglobin and
hemoglobin. The numbers on the right are percentages. — After
Kollett.



The above figure illustrates a method of representmg graphically
the variations in the spectrum of the blood-colouring matter, correspond-
ing to all concentrations (a stratum of 1 c.c. being examined).^

In these diagrams the position of the principal Frauenhofer lines
is shown ; the numbers on the right indicate percentages of the blood-
colouring matter. The shaded part of the diagram indicates absorp-
tion of light. By drawing lines parallel to the abscisste we at once
observe the character of the absorption spectrum which corresponds
to the concentration indicated at the right-hand side of each diagram.
Thus, by inspection of the left-hand diagram, we learn that solutions of
oxyhpemoglobin, containing more than 0"65 per cent., exhibit a single
broad absorption-band in the visible spectrum, owing to the fact that
the two absorption-bands a and /3 have run together, and that the
green interspace between }> and F is shown only by solutions of less
concentration than from 0-8 to 0-9 per cent. When the absorption of
this part of the spectrum is complete, only orange and red remain
unabsorljed.

By placing the solution of oxyhcemogiobin in a wedge-shaped cell,

^ A. Rollett, "Physiologiedes Blutes," Hermann's "Handbiich," Leipzig, 1880, Bd. iv.
Th. 1, S. 48.



THE OR V 6- ME THODS OF SPE CTR OP HO TOME TRY. 213

the slit being perpendicular to the edge of the wedge, the accuracy of
the diagram can be realised objectively, each section of the slit forming
a spectrum corresponding with a given thickness of stratum, which
increases in a continuous manner from the edge towards the base of the
wedge. This method of examination was first employed by J. H. Glad-
stone.^

The theory and methods of spectrophotometry. — The spectro-
photometric constants of oxyhsemoglobin — (a) The theory. — Inte-
resting and attractive though it undoubtedly is, the exammation of an
absorption-spectrum, or the comparison of allied absorption-spectra, by
the unaided sense of sight, may be singularly deceptive.

The impression which the unaided eye enables us to form of the
boundaries, the breadth, the intensity of an absorption-band, or of the
extent and depth of a less defined general absorption, is often very
fallacious. When, for instance, the absorption of a definite region of the
spectrum commences and ceases abruptly, the band appears to the eye
more intense than when the absorption commences and ceases more
gradually.^ The most striking illustration of the truth of these remarks
is indeed furnished by the two oxyhsemoglobin bands. The first, less
refrangible band (a), has always been described as much more intense
than the second, which is broader and less sharply defined, and un-
questionably this is the impression which we form by ordinary methods
of examination. Vierordt ^ has, however, shown that, in opposition to
the visual impression, a greater percentage of light is absorbed in the
spectral region which corresponds to the second band than in that
corresponding to the first band. Measuring, spectrophotometrically, the
percentage of light remaining unabsorbed, after traversing a stratum 1 cm.
broad of a solution containing 1 per cent, of defibrinated mammalian
blood, he found that in the region of the first, apparently more intense
band, 87 per cent, of the light was absorbed and 13 per cent, trans-
mitted ; whilst in the region of the second, apparently less intense band,
90 per cent, of the light was absorbed, and only 10 per cent, transmitted.

This result at once suggests the necessity of a method of determin-
ing quantitatively the amount of light absorbed by any medium whose
absorption-spectrum forms the subject of investigation, instead of trusting
to our unaided sense of sight. When, however, we are made acquainted
with the remarkable and far-reaching conclusions which can be legiti-
mately drawn from an accurate determination of the percentage of light
of a definite wave length, absorbed by colouring matters existing in
solution, the beauty and the importance of the method of spectrophoto-
metry become apparent. Until Vierordt's discovery, those coloured
bodies whose visible spectrum presented no definite absorption-bands,
were held to be beyond the scope of spectroscopic research. Now, how-
ever, we know that a photometric study of the spectrum affords us not
only the means of identifying them, but supplies us with a method
for the quantitative analysis of colouring matters, surpassing all others
in accuracy, and permitting, in certain cases, of the accurate determination
of data not to be ascertained in any other way.

^ J. H. Gladstone, "On the Use of the Prism in Qiialitative Analysis," Journ. Chem.
Soc, London, 1858, vol. x. p. 79.

- A. Rollett, "Pliysiologie des Bhites," Hermann's "Handbuch," Leipzig, 1880, Bd. iv.



Online LibraryE. A. (Edward Albert) Sharpey-SchäferText-book of physiology; (Volume v.1) → online text (page 30 of 147)