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upon them, and to recognise their true nature.

Kunde,' working at the same

time, made extensive observations from a comparative point of view, and was the discoverer of the exceptional form of the crystals in the guinea-pig and squirrel. Since then, many investigators have worked at the subject, notably Lehmann, Rollett, von Lang,' and Preyer, who has written an exhaustive treatise on the Subject.

The tetrahedral blood crystals of the guinea-pig were at one time supposed to belong to the regular system, but it was von Lang who showed that they are in reality rhombic.

A similar question might arise with regard to the hexagonal crystals of the squirrel and the hamster. May they not be rhombic crystals which have what

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FIG. 57. Suppose ABCD (a) to be the basal plane of a rhombic plate, and the angle A B C to be approximately 120°, the lines joining A C, B D being the axes. Then if the angles D) A B, DCB be replaced, as shown by the dotted lines, a hexagon will be produced differing but little from a regular hexagon.

mineralogists call a hexagonal habit® (see fig. 57 a)' or might they not be rhombic twins consisting of three parallelograms or six triangles (as shown in fig. 57 hand c)?

In order to settle this question it is necessary to examine the optical properties of the crystals.

Crystals may be divided, according to their optical properties, into three classes:

1. Isotropic. Those in which there is no distinction of different directions as regards optical properties. This includes crystals belonging to the regular system. They have but one refractive index, i.e. refract light, like amorphous bodies, singly.

2. Uniaral. Those in which the optical properties are the same for all directions equally inclined to one particular direction, called the optic axis, but vary according to this inclination. This class includes crystals belonging to the dimetric system (crystals with three rectangular axes, two of them being equal) and the hexagonal system. The optic axis corresponds with the principal crystallographic axis; that is, in the case of a hexagon the axis perpendicular to

Kunde, Zeitsch. f. rat. Med. N. F. Bd. ii. 1852, p. 276.

? Lehmann, Ber. d. k. sächs. Ges. d. Wissen. 1852, p. 22.
5 Rollett, Sitzungsber. d. Wien. Akad. Bd. xlvi. 1862, p. 65.
+ Lang, Ibid.

5 Preyer, Die Blutkrystalle, Jena, 1871.

6 Copper glance is an instance of this occurring in the mineral kingdom. In one form of mica also, crystals of the monoclinic system simulating hexagons are found.

the flat surface. In the direction of this axis a ray of light is refracted singly, and in other directions doubly.

3. Biaxal. This includes the remaining three systems of crystals, the trimetric or rhombic (three rectangular axes all unequal), the monoclinic, and the triclinic. In these there are always two directions along which a ray is singly refracted.

The best test as to whether a substance is doubly refractive or not is this: If between crossed nicols, which consequently appear dark, a substance be interposed that makes the darkness give place to illumination, however feeble, that substance is doubly refractive. This action is termed the depolarisation of the ray (see p. 38).

On submitting the squirrel's blood crystals to this test, they are found to remain dark in the dark field of the polarising microscope when they are examined with the apparent basal plane perpendicular to the axis of the instrument and rotated; nor when a quartz plate is inserted, do they produce any modification of the tint as the stage is turned.

Hence the presumption is, that they belong to the hexagonal system, as rhombic crystals of hexagonal habit or rhombic twins would produce some double refraction when examined in this way.

It is generally stated that blood crystals are doubly refracting and pleochromatic (i.e. exhibit tints as the upper nicol is rotated). We see it is necessary to make an exception to this rule in the case of hexagonal plates when lying flat.

It is found that the hexagonal crystals from squirrel's blood are too small and thin to allow of one applying the additional crucial test of the interference figures seen in convergent polarised light. These consist of a cross and circles, which are symmetrical in uniaxal crystals, asymmetrical in biaxal crystals.

We have, however, in the case of the hamster the occurrence of rhombohedral crystals; this confirms the view that the crystals are true hexagons, as the rhombohedron belongs to the hexagonal system.

It is found, however, that after recrystallising' squirrel's oxyhemoglobin several times, their hexagonal constitution is broken down, and the crystals obtained are either rhombic prisms or a mixture of these with rhombic tetrahedra. This leads us to believe that whatever the difference between the various forms of oxyhæmoglobin may be, it cannot be a very deep or essential one.

Have we then to deal with a case of polymorphism? The terms dimorphism and polymorphism cannot be applied to any substance which crystallises in two or more forms, unless the composition of that substance be exactly the same in all cases. Instances of dimorphism in the mineral world are carbon and sulphur among the elements, and sal ammoniac, potassium iodide, &c., among compounds. The conditions on which dimorphism depends are two: first, temperature; secondly, the solvent from which the substance crystallises. If, as in the case of many mineral salts, the compounds are united with different proportions of water of crystallisation, we have to deal with different hydrates, and the case is not one of true dimorphism; an instance of this is sulphate of soda.

1 Another peculiar result of recrystallising hæmoglobin has been pointed out by Kupffer and more recently by Krüger (Zeit. Biol. xxiv. 47), that is, that the absorption coefficient of oxyhemoglobin increases after recrystallisation. In determining the absolute amount of oxyhemoglobin by the spectrophotometer (see p. 50) it is best only to recrystallise once, as each recrystallisation increases the error of observation.

2 Halliburton, Quart. Journ. Mic. Science, xxviii. 181. Some remarkable forms of oxyhemoglobin 'crystals are also sometimes obtained by dissolving a mixture of the hæmoglobin of various animals and then crystallising.

The case seems to me to narrow itself down to this in the case of hæmoglobin; either we have here a case of polymorphism, or the crystalline forms are due to the combination with varying proportions of water of crystallisation. In the absence of a rational formula for hæmoglobin, it would be unsafe to affirm the former of these two alternatives. Moreover, the conditions that are known to produce dimorphism in minerals, namely, differences of temperature and of solvent, have in the case of hæmoglobin no influence.

If we then fall back on the latter alternative, the question which arises is whether there are any facts to support it. The explanation that the varying form of oxyhemoglobin is due to varying quantities of water of crystallisation may be otherwise expressed by saying that we have to deal with different hydrates of oxyhemoglobin. This would account for the varying solubilities of these substances in water and other reagents, and at the same time is not such an essential difference as to prevent the chief properties of oxyhæmoglobin from being universally the same.

Turning to Hoppe-Seyler's researches on this subject of water of crystallisation, it is seen that its amount varies considerably. The following is his table:-1

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In an earlier paper,2 the same author gives rather different percentages, viz. for guinea-pig's hæmoglobin 6, for goose's hæmoglobin 7, and for squirrel's hæmoglobin 9. C. Bohr3 has more recently made observations on the water of crystallisation of dog's hæmoglobin, and as the result of thirteen experiments he finds that its amount varies from 6.3 to 1.2 per cent. It is thus seen that great variations occur in the numbers obtained by these experiments. The reason for this variation seems to be the great difficulty of obtaining hæmoglobin in a pure state, and also possibly because the method adopted, which is the same as that carried out in similar investigations on inorganic salts, is not applicable to such a complex and much less stable organic compound as hæmoglobin; in other words, the temperature necessary to drive off the water of crystallisation is also sufficient to cause certain decomposition changes in the pigment.

My experiments have shown that squirrel's oxyhæmoglobin will under certain circumstances crystallise in forms other than the usual hexagonal form. A crucial experiment in order to see whether this is due to union with different amounts of water of crystallisation would have been first to ascertain the amount of this water in the hexagonal crystals, and then in the rhombic crystals obtained by recrystallisation. I have performed three such experiments, but the results obtained are conflicting, and exhibit variations as great as in Bohr's experiments, so that it is impossible to draw any conclusions from them, except the

1 Physiologische Chemie, p. 377.

2 Med. Chem. Untersuchungen, Heft iii. 1868, p. 370.

3 Experimentale Untersuchungen über die Sauerstoffaufnahme des Blutfarbstoffes, Kopenhagen (Olsen and Co.), 1885.

4 The same difficulty in obtaining concordant results in the estimation of water of crystallisation was found by J. G. Otto, Pflüger's Arch. xxxi. 240.

T

negative one that we cannot by our present methods of research make any definite statement with regard to the water of crystallisation of oxyhæmoglobin.

Even if it be found ultimately that the difference in crystalline form is dependent on varying amounts of water of crystallisation, the difficulty is only explained up to a certain point. What is left unexplained is the nature of the agency that causes the oxyhæmoglobin of some animals to unite with a certain amount of water of crystallisation, and that of other animals with a different It amount. That some such substance or agency does exist would seem to be the inevitable result of the recrystallisation experiments which have been related. may, however, be stated that this part is not played by any constituent of the The corpuscles of one animal may be obtained free from serum by centrifugalising and then mixing with the serum of some other animal whose blood crystals have another form. But it is found on subsequent crystallisation that the characteristic form of the blood crystals is not altered thereby. One can only suggest that it is some constituent of the stroma which exerts the influence in question.

serum.

Compounds of Hæmoglobin

Hæmoglobin forms at least four compounds with gases, viz.:

With oxygen: 1. Oxyhæmoglobin.

2. Methæmoglobin.

With carbonic oxide: 3. Carbonic oxide hæmoglobin.
With nitric oxide: 4. Nitric oxide hæmoglobin.

These compounds are isomorphous, they have similar crystalline forms; they each consist probably of a molecule of hæmoglobin combined with one of the gas in question. They part with the combined gas somewhat readily; but they are arranged in order of stability in the above list, the least stable first.

1. Oxyhæmoglobin.-This is the compound which exists in arterial blood. Many of its properties have been already mentioned. The oxygen linked to hæmoglobin, which is removed by the tissues through which the blood circulates, may be called the respiratory oxygen of hæmoglobin. The circumstances under which hæmoglobin combines with and parts from its respiratory oxygen in the body will be fully described under 'Respiration.' But the same processes may be imitated outside the body, using either blood or pure solutions of hæmoglobin. The respiratory oxygen can be removed, for example, in the Torricellian gram of hæmoglobin vacuum of an air pump. Preyer 1 estimated that 1 will combine with 1.27 c.c.2 of oxygen. Hüfner 3 gives almost the same number, viz. 1.28 c.c. of oxygen.

A. Schmidt at one time considered that the respiratory oxygen of

1 Die Blutkrystalle, p. 134.

2 Measured at 0° C. and 1 metre pressure; equivalent to 167 c.c. measured at 0° C. and 760 millimetres pressure.

3 Hüfner, Zeit. physiol. Chem. i. 317.

hæmoglobin was ozonised, and therefore more active than atmospheric oxygen. Pflüger has shown that this is not the case. When diluted blood is dropped on a filter paper which has been moistened with tincture of guaiacum and then dried, a blue ring sometimes forms at the edge of the drop. In fact it acts as ozone does, when liberated, for example, from hydrogen peroxide. But Pflüger has shown that when blood is poured on filter paper in the way just described, decomposition of the hæmoglobin almost instantly occurs, and it is the products of decomposition which occasion the reaction.

We have still the spectroscopic characters of oxyhæmoglobin to consider.

The various forms of spectroscope have been already described (p. 47). It will be sufficient here to repeat that the spectroscope is an instrument which enables us to tell the colour of a solution or transparent substance more accurately than we can with the unaided vision. White light passed through the coloured substance and then through a prism no longer gives a continuous spectrum, but certain parts of it are absorbed, hence the appearance of dark shadows or absorption bands in various parts of the spectrum. These bands remain constant in position for the same substance, and thus furnish us with a delicate test for that substance. We speak of the position of the absorption bands, either by their neighbourhood to certain of the black lines (Fraunhofer's lines) of the solar spectrum; or more accurately still by measuring their position in wave-lengths. The sign A denotes wave-length; in absorption spectra, the edges of the bands. are sometimes so ill defined, and vary in position with the concentration of the liquid, that more often the position of the centre of the band rather than that of its edges is given. A 500 means a wave-length equal to 500 millionths of a millimetre.

The two next figures illustrate a method of representing absorption spectra diagrammatically. The solution was examined in a layer one centimetre thick. The base line has on it at the proper distances the chief Fraunhofer lines, and along the right hand edges are percentages of the amount of oxyhæmoglobin present in I, of reduced hæmoglobin in II. The width of the shadings at each level represents the position and amount of absorption corresponding to the percentages. The characteristic spectrum of oxyhæmoglobin (first observed by Hoppe-Seyler) is seen as it actually appears through the spectroscope in the next figure (fig. 59, spectrum 2). There are two distinct absorption bands between the D and E lines; the one nearest to D (the a band) being narrower, darker, and with better defined edges than the other

1 Pflüger's Archiv, x. 252.

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