per

cent.

(the band). The centre of the a band corresponds to λ 579, of the B band to λ 553-8 (Gamgee'). As will be seen by looking at fig. 58, a solution of oxyhemoglobin of concentration greater than 0.65 and less than 0.85 per cent. gives one thick band overlapping both D and E, and a stronger solution still, only lets the red light through between the C and D lines.

A solution which gives the two characteristic bands must therefore be a dilute one. But, as before said, we are able with such solutions of

I

0-7

0-6

C-5

0-4

0.3

0.2

0-1

0

ABC

II

FIG. 58.-Graphic representations of the amount of absorption of light by solution of (I) oxyhæmoglobin, (II) of hæmoglobin, of different strengths. The shading indicates the amount of absorption of the spectrum; the figures on the right border express percentages (Rollett).

oxyhæmoglobin, or defibrinated blood will do equally well, to imitate the reduction of oxyhæmoglobin which occurs in the body. This was first pointed out by Professor Stokes,2 who employed for the purpose the reducing agent now known as Stokes's reagent.

The following are the means by which we can displace the respiratory oxygen in a solution of oxyhemoglobin :

(1) By boiling it in the Torricellian vacuum of a mercurial air pump. (2) By passing through the solution a neutral gas such as nitrogen, hydrogen, or carbonic acid.

(3) By the use of reducing agents.

(a) Stokes's reagent: a solution of ferrous sulphate, to which a

1 Gamgee, Physiological Chemistry. Where other wave-lengths are given subse quently, they are taken either from Gamgee's measurements, or from those made by MacMunn, and published in McKendrick's Physiology.

FIG. 59.-1, Solar spectrum. 2, Spectrum of oxyhæmoglobin (0.37 p.c. solution). First band, à 589564; second band, A 555-517. 3, Spectrum of hæmoglobin. Band, A 597-535. 4, Spectrum of COhemoglobin. First band, A 583-564; second band, à 547-521. 5, Spectrum of methæmoglobin (concentrated solution). 6, Spectrum of methæmoglobin (dilute solution). First band, X 647622; second band, à 587-571; third band, A 552-532; fourth band, à 514-490. 7, Spectrum of acid hrematin (ethereal solution). First band, A 656-615; second band, A 597-577; third band, A 557-529; fourth band, à 517-488. 8, Spectrum of alkaline hæmatin. Band from à 630-581. 9, Spectrum of hæmochromogen (reduced hæmatin). First band, à 569-542; second band, A 535-504, 10, Spectrum of acid hæmatoporphyrin. First band, A 607-593; second hand, A 595536. 11, Spectrum of alkaline hæmatoporphyrin. First band, à 633-612; second band, à 589-561third band, A 549-529; fourth band, à 518-488. The above measurements (after MacMunn) ne in millionths of a millimetre. The liquid was examined in a layer one centimetre thick. The edges of ill-defined bands vary a good deal with the concentration of the solutions.

little tartaric or citric acid has been added, and then ammonia till the reaction is alkaline. This reagent rapidly darkens in the air, and must be freshly made every time it is used.

(b) Instead of ferrous sulphate, stannous chloride may be used in the preparation of the foregoing. This has the advantage of not darkening, as it absorbs oxygen. It however must also be always freshly prepared before using.

(c) Ammonium sulphide. This on the whole is the most convenient reagent to use, though it is somewhat slower in its action than the two preceding; a little gentle warmth will however hasten its action.

Using any of these methods the colour of oxyhæmoglobin changes to the purplish tint of hæmoglobin, and by the spectroscope the two bands are now seen to be replaced by one, called the γ band; this band is not so well defined as either the a or the 3 band. Its position between the D and E lines is denoted in fig. 59 (spectrum 3); it is darkest about λ 550.

On dilution the band fades rapidly, so that in a solution of such concentration that both bands of oxyhemoglobin would be quite distinct, the single band of reduced hæmoglobin has disappeared from view. The oxyhæmoglobin bands can be distinguished in a solution which contains only one part of the pigment to 10,000 of water, and even in more dilute solutions which are apparently colourless, the a band is still visible.

On passing oxygen through a solution of hæmoglobin, or on shaking it up with the air, oxyhemoglobin showing its two bands, reappears. 2. Methemoglobin. This is a compound of hæmoglobin with oxygen which can be produced artificially; it also occurs in the body under certain circumstances, e.g. in certain diseased conditions it occurs in the urine (see Hæmoglobinuria), and after the administration of large doses of potassium or sodium chlorate it occurs in the blood, and death is the ultimate result.

It may be derived artificially from a solution of oxyhemoglobin in the following ways :-

(a) When a solution of oxyhemoglobin is exposed to the air in shallow layers for some time, it becomes acid in reaction, brown in colour, and exhibits the characteristic spectrum of methæmoglobin.

(b) On the addition of various oxidising agents the same occurs; potassium permanganate, potassium ferricyanide, nitrite of potassium, nitrite of amyl,' &c., act in this way. Hence the view originally

1 Hayem, Compt. rend. cii. 698, gives a long list of reagents that act in this way.

advanced by Sorby,' that methæmoglobin is more highly oxygenated than oxyhemoglobin; that it is in fact a per-oxyhæmoglobin.

(c) Methæmoglobin may however be prepared by removing part of the oxygen of oxyhæmoglobin by means of the mercurial air pump, or by means of palladium saturated with hydrogen. Hoppe-Seyler, who describes the above methods, therefore regards methæmoglobin as a sub-oxyhæmoglobin. Whichever view was held as to its constitution, it was admitted by all that the oxygen of methæmoglobin is more firmly combined than that of oxyhæmoglobin. Still it can be removed by reducing agents. The oxygen is however not removable by the air pump, nor by a stream of a neutral gas like hydrogen. On adding ammonium sulphide to a solution of methæmoglobin, the first change is to oxyhæmoglobin, and then to reduced hæmoglobin; these changes can be watched with the spectroscope.

More recently, however, Hüfner and Külz3 have advanced a third theory concerning the constitution of methæmoglobin, and that is that it contains the same amount of oxygen as oxyhæmoglobin, only in a closer state of combination. They are able to make this assertion from actual analyses; and these analyses were possible, inasmuch as they succeeded in obtaining pure methæmoglobin in a crystalline form. The method of obtaining these crystals is as follows : 5-Three or four cubic centimetres of a concentrated solution of ferricyanide of potassium are added to a litre of concentrated solution of hæmoglobin. A quarter of a litre of alcohol is added, and the mixture frozen. After one or two days' exposure to this low temperature, abundant crystals of a brown colour, which give the absorption spectrum of methæmoglobin, are deposited. They were obtained in this way from the hæmoglobin of the dog, pig, and horse, and their form is the same as that of the oxyhæmoglobin crystals of the same animals, i.e. rhombic prisms. Gamgee had prepared these crystals from dog's blood many years previously, but their true nature was not at that time recognised. His method was much the same as Hüfner's, the chief difference being that the nitrite of potassium or amyl was employed instead of ferricyanide of potassium. Jäderholm has also obtained these crystals from dog's blood by the ferricyanide method, and confirms Hüfner's statement that they are 1 Sorby, Quart. J. Mic. Science, 1870, p. 400.

2 Hoppe-Seyler, Zeit. physiol. Chem. ii. 150.

3 Zeit. physiol. Chemie, vii.

6

4 Hüfner and Kiilz employed the spectrophotometric method largely in their work.

5 G. Hüfner, Ueber krystallinisches Methämoglobin vom Hunde,' Zeit. physiol. Chem. viii. 366.

A. Gamgee, 'The action of Nitrites on Blood,' Philos. Trans. 1868, p. 589, et seq. 7 Zeitsch. für Biol. xx. 419. Jäderholm now agrees with Hüfner and Külz with regard to the composition of methæmoglobin.

rhombic prisms. He also figures some crystals of methæmoglobin obtained by Hammarsten from the horse by the same method, which were regular six-sided plates, and showed no double refraction if lying flat; they therefore presumably belonged to the hexagonal system; they were more insoluble in water than the crystals of dog's methæmoglobin.

When one wishes, however, to obtain a small quantity of crystals for microscopic examination, the following simple method may be employed. A few c.c. of the defibrinated blood of an animal (rat, guinea-pig, or squirrel) are taken, and an equal number of drops of nitrite of amyl added. The mixture is vigorously shaken for a minute or two. The colour changes to the dark chocolate tint of methæmoglobin, and spectroscopic observation shows the typical absorption bands of that compound. A drop of this liquid is then placed on a slide and covered; in a few minutes crystals form, which observation with the spectroscope shows to be composed of methæmoglobin. The edges of the cover-glass may then be sealed, and the crystals keep unchanged for several months.

The crystals obtained from guinea-pig's blood by this process are tetrahedra, which differ only in colour and spectroscopic appearances from those of oxyhæmoglobin from the same animal.

The crystals obtained from squirrel's blood are perfectly regular hexagonal plates, which remains dark between crossed nicols.

The crystals obtained from rat's blood are also perfectly regular hexagonal plates,3 which remain dark between crossed nicols, and which consequently are precisely similar to those of squirrel's methæmoglobin. This remarkable fact helps to show that the difference between the oxyhemoglobin of these two animals cannot be a very deep or essential one.

A

The spectrum of methæmoglobin shows three absorption bands, one in the red about half way between the C and D lines, and two others between the D and E lines which resemble in position those of oxyhæmoglobin, but on careful measurement are found to be different. fourth indistinct band in the blue has also been described (see fig. 59, spectra 5 and 6). On adding ammonia to a solution of methæmoglobin, the first two bands shift a little towards the violet end of the spectrum ;

1 Halliburton, Quart. Journ. Mic. Science, xxviii. 201.

2 This must be done immediately after the formation of the chocolate-coloured liquid, as in about a quarter of an hour the whole liquid sets into a gelatinous mass of the same colour, from which no crystals are obtainable.

3 A few triangles and forms intermediate between triangles and regular hexagons are also found.

4 Araki considers that these bands are due to admixture with oxyhæmoglobin (Zeit. physiol. Chem. xiv. 405).