ON THE REDUCTION AND OXIDATION OF THE COLOURING

MATTER OF THE BLOOD.

[From the Proceedings of the Royal Society, June 16, 1864.]

1. SOME time ago my attention was called to a paper by Professor Hoppe*, in which he has pointed out the remarkable spectrum produced by the absorption of light by a very dilute solution of blood, and applied the observation to elucidate the chemical nature of the colouring matter. I had no sooner looked at the spectrum, than the extreme sharpness and beauty of the absorption-bands of blood excited a lively interest in my mind, and I proceeded to try the effect of various reagents. The observation is perfectly simple, since nothing more is required than to place the solution to be tried, which may be contained in a testtube, behind a slit, and view it through a prism applied to the eye. In this way it is easy to verify Hoppe's statement, that the colouring matter (as may be presumed at least from the retention of its peculiar spectrum) is unaffected by alkaline carbonates and caustic ammonia, but is almost immediately decomposed by acids, and also, but more slowly, by caustic fixed alkalies, the coloured product of decomposition being the hæmatine of Lecanu, which is easily identified by its peculiar spectra. But it seemed to me to be a point of special interest to inquire whether we could imitate the change of colour of arterial into that of venous blood, on the supposition that it arises from reduction.

2. In my experiments I generally employed the blood of sheep or oxen obtained from a butcher; but Hoppe has shown that the blood of animals in general exhibits just the same bands. To obtain the colouring matter in true solution, and at the same time to get rid of a part of the associated matters, I generally allowed the blood to coagulate, cut the clot small, rinsed it well, and extracted it with water. This, however, is not essential, and

* Virchow's Archiv, Vol. xxIII, p. 446 (1862). [Cf. ante, p. 240.]

blood merely diluted with a large quantity of water may be used; but in what follows it is to be understood that the watery extract is used unless the contrary be stated.

3. Since the colouring matter is changed by acids, we must employ reducing agents which are compatible with an alkaline solution. If to a solution of protosulphate of iron enough tartaric acid be added to prevent precipitation by alkalies, and a small quantity of the solution, previously rendered alkaline by either ammonia or carbonate of soda, be added to a solution of blood, the colour is almost instantly changed to a much more purple red as seen in small thicknesses, and a much darker red than before as seen in greater thickness. The change of colour, which recalls. the difference between arterial and venous blood, is striking enough, but the change in the absorption spectrum is far more decisive. The two highly characteristic dark bands seen before are now replaced by a single band, somewhat broader and less sharply defined at its edges than either of the former, and occupying nearly the position of the bright band separating the dark bands of the original solution. The fluid is more transparent for the blue, and less so for the green than it was before. If the thickness be increased till the whole of the spectrum more refrangible than the red be on the point of disappearing, the last part to remain is green, a little beyond the fixed line b, in the case of the original solution, and blue, some way beyond F, in the

case of the modified fluid. Figs. 1 and 2 in the accompanying woodcut represent the bands seen in these two solutions respectively.

4. If the purple solution be exposed to the air in a shallow vessel, it quickly returns to its original condition, showing the two characteristic bands the same as before; and this change takes place immediately, provided a small quantity only of the reducing agent were employed, when the solution is shaken up with air. If an additional quantity of the reagent be now added, the same effect is produced as at first, and the solution may thus be made to go through its changes any number of times.

5. The change produced by the action of the air (that is, of course, by the absorption of oxygen) may be seen in an instructive form on partly filling a test-tube with a solution of blood suitably diluted, mixing with a little of the reducing agent, and leaving. the tube at rest for some time in a vertical position. The upper or oxidized portion of the solution is readily distinguished by its colour; and if the tube be now placed behind a slit and viewed through a prism, a dark band is seen, having the general form of a tuning-fork, like Figs. 1 and 2, regarded now as a single figure, the line of separation being supposed removed.

6. Of course it is necessary to assure oneself that the single band in the green is not due to absorption produced merely by the reagent, as is readily done by direct observation of its spectrum, not to mention that in the region of the previous dark bands, or at least the outer portions of it, the solution is actually more transparent than before, which could not be occasioned by an additional absorption. Indeed the absorption due to the reagent itself in its different stages of oxidation, unless it be employed in most unnecessary excess, may almost be regarded as evanescent in comparison with the absorption due to the colouring matter; though if the solution be repeatedly put through its changes, the accumulation of the persalt of iron will presently tell on the colour, making it sensibly yellower than at first for small thicknesses of the solution.

7. That the change which the iron salt produces in the spectrum is due to a simple reduction of the colouring matter, and not to the formation of some compound of the colouring matter with the reagent, is shown by the fact that a variety of reducing agents

of very different nature produce just the same effect. If protochloride of tin be substituted for protosulphate of iron in the experiment above described, the same changes take place as with the iron salt. The tin solution has the advantage of being colourless, and leaving the visible spectrum quite unaffected, both before and after oxidation, and accordingly of not interfering in the slightest degree with the optical examination of the solutions, but permitting them to be seen with exactly their true tints. The action of this reagent, however, takes some little time at ordinary temperatures, though it is very rapid if previously the solution be gently warmed. Hydrosulphate of ammonia again produces the same change, though a small fraction of the colouring matter is liable to undergo some different modification, as is shown by the occurrence of a slender band in the red, variable in its amount of development, which did not previously exist. In this case, as with the tin salt, the action is somewhat slow, requiring a few minutes unless it be assisted by gentle heat. Other reagents might be mentioned, but these will suffice.

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8. We may infer from the facts above mentioned that the colouring matter of blood, like indigo, is capable of existing in two states of oxidation, distinguishable by a difference of colour and a fundamental difference in the action on the spectrum. It may made to pass from the more to the less oxidized state by the action of suitable reducing agents, and recovers its oxygen by absorption from the air.

As the term hæmatine has been appropriated to a product of decomposition, some other name must be given to the original colouring matter. As it has not been named by Hoppe*, I propose to call it cruorine, as suggested to me by Dr Sharpey; and in its two states of oxidation it may conveniently be named scarlet cruorine and purple cruorine respectively, though the former is slightly purplish at a certain small thickness, and the latter is of a very red purple colour, becoming red at a moderate increase of thickness.

9. When the watery extract from blood-clots is left aside in a corked bottle, or even in a tall narrow vessel open at the top, it

[* It was in the year 1864, the date of the present paper, that Hoppe proposed the name haemoglobin, now universally employed, Virchow's Archiv, xx1x, p. 223.]

presently changes in colour from a bright to a dark red, decidedly purple in small thicknesses. This change is perceived even before the solution has begun to stink in the least perceptible degree. The tint agrees with that of the purple cruorine obtained immediately by reducing agents; and if a little of the solution be sucked up from the bottom into a quill-tube drawn to a capillary point, and the tube be then placed behind a slit, so as to admit of analyzing the transmitted light without exposing the fluid to the air, the spectrum will be found to agree with that of purple cruorine. On shaking the solution with air it immediately becomes bright red, and now presents the optical characters of scarlet cruorine. It thus appears that scarlet cruorine is capable of being reduced by certain substances, derived from the blood, present in the solution, which must themselves be oxidized at its expense.

10. When the alkaline tartaric solution of protoxide of tin is added in moderate quantity to a solution of scarlet cruorine, the latter is presently reduced. If the solution is now shaken with air, the cruorine is almost instantly oxidized, as is shown by the colour of the solution and its spectrum by transmitted light. On standing for a little time, a couple of minutes or so, the cruorine is again reduced, and the solution may be made to go through these changes a great number of times, though not of course indefinitely, as the tin must at last become completely oxidized. It thus appears that purple cruorine absorbs free oxygen with much greater avidity than the tin solution, notwithstanding that the oxidized cruorine is itself reduced by the tin salt. I shall return to this experiment presently.

11. When a little acid, suppose acetic or tartaric acid, which does not produce a precipitate, is added to a solution of blood, the colour is quickly changed from red to brownish red, and in place of the original bands (Fig. 1) we have a different system, nearly that of Fig. 3. This system is highly characteristic; but in order to bring it out a larger quantity of substance is requisite than in the case of scarlet cruorine. The figure does not exactly correspond to any one thickness, for the bands in the blue are best seen while the band in the red is still rather narrow and ill-defined at its edges, while the narrow inconspicuous band in the yellow hardly comes out till the whole of the blue and violet, and a good