Of the chemical constituents found in the blood corpuscles, the coloring matter is by far the most important. To it alone the blood owes one of its most important functions - the respiratory.

Oxyhemoglobin is a chemical compound of great complexity, of which the percentage composition is given as -

Carbon,...............53,85

Hydrogen,..............7.32

Nitrogen,..............16.17

Oxygen,...............21.84

Sulphur, ................................................................39

Iron................. .43

Its rational formula is unknown, but the following has been proposed as approximate, C6ooH960N154FeS3O179. It may be regarded as a form of globulin, associated with a colored material containing iron, called haematin. Its chief peculiarities are (i) that, although it contains a colloid substance, it crystallizes more or less readily in all vertebrates when removed from the stroma of the corpuscles; (2) the considerable amount of iron it contains (0.4 per cent.); (3) the remarkable manner in which it is combined with oxygen to form an unstable compound; and (4) the ease with which it yields its oxygen to the tissues and takes it from the air.

The readiness with which the oxyhaemoglobin crystals are formed varies much in different animals and under different circumstances, as may be seen from the following list: -

Most readily - rat, guinea pig, mouse. Readily - cat, dog, horse, man, ape, rabbit. With difficulty - sheep, cow, pig. Not at all - frog.

The presence of oxygen causes the crystals to form more rapidly, so that a stream of oxygen passed through a strong solution of haemoglobin causes small crystals of oxyhaemoglobin to form.

The crystals always belong to the rhombic system, being most frequently plates (man, etc.) and prisms (cat), and rarely tetrahedra (guinea pig) and hexagonal plates (squirrel).

The color of the crystals and their solution varies according to the light by which they are looked at. By reflected light they are bluish red or greenish in color, and by direct light, scarlet.

The preparation of oxyhemoglobin crystals is accomplished by first separating the coloring matter from the corpuscles by freezing, or the addition of water or ether, and then rendering it less soluble by evaporation, cold, and the addition of alcohol.

For microscopic observation it generally suffices to kill a rat with ether, and expose a drop of the blood diluted with distilled water on a slide until half dried, and then cover. Crystals appear in the fluid as it becomes concentrated.

The combinations which haemoglobin enters into are numerous and throw much light upon the function of the corpuscles.

As already stated, the coloring matter, when exposed to the air, combines with oxygen to form a loose chemical compound called oxyhaemoglobin. This is the condition in which the coloring matter of the blood is generally found. Although so prone to combine with oxygen, the oxyhaemoglobin very readily parts with some of it. In the circulation it is always united with oxygen, normally leaving the lungs in a state of. saturation. On its way through the capillaries of the tissues, some of it parts with a little of its oxygen, becoming partially reduced (haemoglobin), but even the most venous blood always contains some oxyhaemoglobin.

The oxygen can be removed by reducing the pressure under an air pump, or by exposing the solution to a mixture of nitrogen and hydrogen. Various reducing agents rob the oxyhaemoglobin of its oxygen; and if blood or a solution of oxyhaemoglobin be sealed in a glass tube so as to exclude the air, the loose oxygen is taken up by some of the other constituents of the blood, and the oxyhemoglobin becomes gradually reduced to haemoglobin, after which it undergoes no further change or decomposition. The reduction in the sealed tube depends on the putrefactive changes in the proteids, and may be prevented by careful aseptic precautions. If the reduced haemoglobin be shaken for a few moments with air, the bright color characteristic of oxyhemoglobin soon reappears, and if the reducing agent be not injurious to the blood, the reduction and reoxidation may be repeated several times, the haemoglobin going through the changes which take place in it during normal respiration.

Crystals of Haemoglobin from different animals, showing the variety inform of crystals.

Fig. 106. Crystals of Haemoglobin from different animals, showing the variety inform of crystals.

The Spectra of Oxyhemoglobin, Reduced Haemoglobin and CO haemoglobin (Gamgee) 1,2', 3 and 4.

Fig. 107. The Spectra of Oxyhemoglobin, Reduced Haemoglobin and CO-haemoglobin (Gamgee) 1,2', 3 and 4. Oxyhemoglobin increasing in strength or thickness of solution. 5. Reduced Haemoglobin. 6. CO haemoglobin.

The union of oxygen with haemoglobin solutions is not mere absorption of the oxygen by the liquid, but a definite chemical combination. This is proved by the following facts: (1) When the pressure is removed, the oxygen does not come away from the solution in accordance with the law which governs the escape of absorbed gas, but all comes off suddenly when the pressure is lowered to about 1/12 of an atmosphere {vide p. 244). (2) The two substances give a different absorption band when examined with the spectroscope. The reduced haemoglobin gives one wide diffuse band, which lies between the D and E lines of the solar spectrum, and much of the violet end is cut off. The single band, which is characteristic of reduced haemoglobin, is replaced by two when the haemoglobin combines with oxygen - one broad band in the green near E, and a narrow one, more clearly defined, in the yellow close to D line; both bands lie between D and E. With strong solutions the spectrum is darkened at either extremity, and the two bands become wider and tend to fuse into one. (3) Further, the oxygen may be replaced by other substances which unite with the haemoglobin. One of the most important of these is carbonic oxide, which forms a much more stable compound with haemoglobin than oxygen. It is of a bright cherry-red color and has two absorption bands in the spectrum very like those of oxyhemoglobin; that in the yellow is, however, removed a greater distance from the D line toward the violet end.

It is this compound which is formed in poisoning with carbonic oxide. The CO occupying the place of the oxygen, destroys the function of the blood corpuscles. CO-haemoglobin may be distinguished from O-haemoglobin by not being reduced by reagents greedy of oxygen, and by the bright red color which persists when 10 per cent, solution of caustic soda is added, and the mixture heated. O-haemoglobin gives a muddy-brown color under the same treatment.