On the Hæmoglobin Crystals of Rodents’ Blood

This research was undertaken at Professor Lankester’s suggestion, in order, first, to ascertain whether these six-sided crystals really belonged to the hexagonal system; and, secondly^. to find, if possible, an explanation of the difference of crystalline form that hæmoglobin presents in different animals, while in its other chief properties hæmoglobin is universally the same.

It will be convenient to take the subject under the following heads:

1. Historical.

2. Hexagonal blood-crystals.

3. Influence of the other constituents of the blood on the crystalline form of hæmoglobin crystals

4. The crystalline forms of hæmoglobin obtained by mixing the blood from different animals.

5. Can squirrel’s hæmoglobin be obtained in any form other than hexagonal crystals ?

6. Conclusions and remarks.

Oxyhæmoglobin crystals were first described by Reichert¹ as occurring in the uterus of a pregnant guinea-pig; by Leydig² as occurring in the alimentary canal of the leech; and by Kölliker,³ obtained from the blood of the dog, python, and other animals. Kölliker considered the crystals to be composed of a more or less modified hæmatin. Funke⁴ was, however, the first to make complete observations upon them, and to recognise their true nature. Kunde, ⁵ 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,⁶ Rollett,⁷ von Lang,⁸ and Preyer,⁹ in whose exhaustive treatise a complete bibliography of the subject up to 1871 is given.

Our present knowledge of the crystalline form that haemoglobin assumes may now be summarised as follows:

• In the great majority of animals¹⁰ in which haemoglobin occurs, vertebrate and invertebrate, crystals of it can be obtained in the form of prisms and plates belonging to the rhombic system.

• The exceptions to this rule hitherto noted are the following:

  • Guinea-pig. Haemoglobin crystals from the blood of this animal are tetrahedra, once supposed to belong to the regular system, but now shown by von Lang to be in reality rhombic.

  • Lehmann mentions that similar tetrahedra may be obtained from the blood of the mouse and rat. This has not since been confirmed.

  • In several birds the crystals obtained are also tetrahedra.

  • In three animals—the squirrel, the hamster, and the mouse—six-sided plates have been described.

  • In one of these, the hamster, rhombohedra are described as occurring also.

We will take the three animals in which the hæmoglobin is said to crystallise in the hexagonal form one by one.

—The discovery of the fact that hæmoglobin crystals from this animal are six-sided plates was made by Kunde (1852). Writing in the same year, Lehmann asserts that though these crystals are six-sided they do not belong to the hexagonal system. He gives, however, no reasons for this assertion. Lang and Preyer arrived at the opposite conclusion i. e. that they do belong to the hexagonal system, from the study of their optical properties.

Belideus breviceps (a marsupial).—Crystals similar to those of the opossum.

Seal (Phoea vitulina).—Rhombic prisms, many of them very short and simulating hexagons. Easily obtained.

Bear (Ursus syriacus).—Bunches of rhombic needles, easily obtained. They are slenderer than those obtained from dog’s blood as a rule, some being almost silken in appearance.

Hydromys leucogaster (white-bellied beaver rat).—Rhombic prisms.

Sus leucomystax (white-whiskered swine).—Rhombic prisms.

Water-vole (Arvicola aquatica).—Crystals are obtained easily by adding water to the blood. They are of the usual rhombic shape.

My own observations are as follows:—The crystals can be obtained with the greatest ease by simply adding a drop of water to a drop of defibrinated blood on a slide, and covering it; in less than a minute crystals appear. I have also prepared them by other methods;¹ but in all cases the crystalline form is the same. When first formed the crystals are six-sided plates, many equilateral, but many not. After recrystallisation, however, the crystals are then all but perfectly regular. The quetions then arises, Do they belong to the hexagonal system or not ? To this question one of the three following answers must be the correct one.

1. They do belong to the hexagonal system.

2. They do not belong to the hexagonal system, but are rhombic crystals, having a so-called “hexagonal habit.” In mineralogy instances are known of such occurrences. This is the case with copper-glance, some of whose crystals so closely resemble hexagonal ones that several mineralogists believed that there were two kinds, one being hexagonal. Again, mica is an instance of a monoclinic crystal with “hexagonal habit.”

Suppose A B c D (fig. 1) 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 DAB, D c B be replaced, as shown by the dotted lines, a hexagon will be produced differing but little from a regular hexagon.

3. The third alternative is that they may belong to the rhombic system by being twins, consisting, of three parallelograms or six triangles, as is shown in figs. 2 and 3. Twins are, however, rare in the rhombic system.

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:

—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 do, singly.

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

—This includes the remaining three systems of crystals, the trimetric or rhombic (three rectangular axes all unequal), the monoclinic, and the trichinic. 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.

The crystals of squirrel’s hæmoglobin I submitted to this test, with the result that no depolarisation of the light can be detected, 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. The instrument used was a Zeiss polarising microscope.

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

I submitted the question as to whether this was conclusive to Professor Lewis, of Cambridge, and he kindly wrote to me in answer as follows:

“The observation under the microscope between crossed nicols, so far as it goes, is rather in favour of the ‘crystals being hexagonal, that is, presupposing that the field remains dark when the crystal is rotated in the field of view. However, this is not quite conclusive, and in such cases greater certainty would be obtained if the crystals were placed under a Bertrand’s polarising microscope, to see the shape of the interference rings and cross.”

It should be here stated that uniaxal crystals in the direction of their optic axis exhibit a symmetrical cross and circular rings; in biaxal crystals the rings are oval, or at any rate not circular, and the cross is not symmetrical. This is the case, because the resistance to displacement in the three cardinal directions called the axes of elasticity are all unequal in biaxal crystals. This is true, not only for the crystalline substance itself, but also for the luminiferous ether that pervades it.¹

Acting on Professor Lewis’s advice, I submitted the crystals to Professor Judd, who with Mr. Fletcher’s co-operation examined them, and gave me the following report, for which I am much indebted to him:—”I have every reason to believe the crystals belong to the hexagonal system from their form, and their extinction between crossed nicols. I regret, however, to find that their minute size, and especially their extreme tenuity, prevents our applying the crucial test of the interference figures seen in convergent polarised light.

“Bertrand devised a form of microscope which enables these interference figures to be studied in the minute crystals seen in their rock sections, and von Lasaulx has improved this apparatus. We have what I believe to be the best form of the Bertrand-Lasaulx apparatus constructed by Nachet; but even employing an immersion objective magnifying 650 diameters, the crystals are still so small as to give neither rings, nor cross, nor brushes.

“I greatly regret that we have not been able to apply this test. I fear that no instrument exists which will accomplish what you desire; and Mr. Fletcher, on theoretical grounds, doubts whether it would be possible under any conditions to apply the test to such minute crystals.”

The largest crystals of squirrel’s hæmoglobin that I have obtained were those formed by the addition of water to the defibrinated blood; they varied in size from ·001 to ·005 m. in breadth.

Since receiving Professor Judd’s report, I have tried to obtain larger crystals by Gscheidlen’s¹ method. He seals defibrinated blood in narrow glass tubes, which are then kept at a temperature of 37° C. for several days. On opening these tubes and emptying their contents into a watch glass, crystals of great size are formed from dog’s blood after evaporation has occurred.

With squirrels’ blood, however, I have not obtained larger crystals by this method than by the first. The reason for this seems to be the extreme readiness with which squirrels’ hæmoglobin crystallises. It is a well-known fact that bodies that crystalise rapidly crystallise in small and numerous crystals. If some method could be devised for retarding, but not preventing, the crystallisation of squirrel’s hæmoglobin, we might then be able to obtain crystals of it large enough to which to apply this crucial test.

The matter must therefore be left incomplete up to this point for the present. The probability, however, is greatly in favour of the crystals being true hexagons.

We have seen that in order to have a rhombic plate with hexagonal habit, it is necessary that one of its angles be approximately 120°; I measured the angles in the rhombic plates found in the rat, and found that they averaged 129°.

I shall also presently show that it is possible by the intermixture of the blood of different animals to obtain crystals closely resembling hexagons, but which are not so, as is shown by their optical properties.

b. Mouse.—Kunde was the first to describe the hæmoglobin crystals of this animal. He made eighteen observations, and the crystals he found were fine needles and prisms.

Bojanowski¹ was the next to make observations on these crystals. He describes and figures them as six-sided plates resembling in form those from squirrel’s blood, of a flesh Colour, and very soluble in water. He prepared them by the addition of a mixture of equal parts of alcohol and ether to the blood. No description of their optical properties is given. He remarks, “I have not been able to observe the fine needles described by Kunde.”

Preyer repeated these experiments, and confirmed the observations of Kunde, not those of Bojanowski. He obtained small prismatic crystals.

I have myself experimented with the blood of eighteen mice, and the result has been again to confirm Kunde’s observations. The crystals are exceedingly difficult to obtain, and in some cases 1 have had to repeat the process of freezing and thawing many times after the addition of alcohol, before succeeding in obtaining them. They are very soluble in water. The crystals are exceedingly small rhombic prisms. They are nearly colourless, and it is only when they are heaped together that any red tinge at all can be perceived in them. In one case in which by the addition of ether to the blood I obtained crystals of fair size after allowing the mixture to stand for five days, the crystals still showed this same peculiarity, namely, in being nearly colourless. I have successfully employed Bojanowski’s method for the preparation of the crystals, namely, the addition of a mixture of alcohol and ether to the blood; but in no case did hexagonal crystals form. Mouse’s haemoglobin also differs from squirrel’s in being very soluble in water; this is admitted by Bojanowski; one would therefore expect a priori that its crystalline form would be different.

—My remarks under this heading will be only historical. I have not myself been successful in obtaining one of these animals. The crystalline form of the haemoglobin was first described by Lehmann, who found rhombohedra and six-sided plates. His experiments were repeated by Preyer,¹ whose observations on the subject are very complete. He fornd both crystalline forms, viz. six-sided plates, and rhombohedra. This is interesting since the rhombohedron belongs to the hexagonal system. By examination between crossed nicols he found that the six-sided plates had no action in “depolarising “the ray, and he therefore concludes that they, like squirrel’s haemoglobin crystals, are true hexagons.

The presumption in favour of the haemoglobin crystals of the squirrel and hamster being true hexagons is exceedingly great. In the case of the mouse, it seems to be almost equally certain that the crystals are not as a rule hexagonal. I should not like, however, to deny that haemoglobin may sometimes in the case of the mouse crystallise in this way, because of some observations I have made on the haemoglobin crystals of the rat

Crystals are obtained from the blood of this animal with great ease; mere addition of water to the blood causes almost immediately an abundant crop of crystals. On this account the blood of this animal is used by the students in the practical classes at University College for the preparation of haemoglobin crystals. Professor Schafer told me that on looking over the students’ preparations he had occasionally seen hexagons together with the ordinary rhombic prisms and plates. In order to verify this, I have made numerous specimens of the crystals from the blood of about fifteen rats. As a rule, no hexagons were present; but on three occasions I have detected hexagonal plates—very few in number, perhaps not more than one or two on the slide—among the rhombic crystals. There appeared to be nothing special either about the animal used or the method employed in these cases. The diameter of these crystals averaged about the same as in squirrel’s blood (·002–·003 m.). Between crossed nicols they also behaved the same as squirrels’ hæmoglobin crystals, viz. remained dark in all positions.

In addition to this, if crystallisation be watched under the microscope, a single corpuscle will often be observed to set into a minute hexagon. This is what Preyer calls intraglobular crystallisation. He describes it as occurring in the blood of the hamster. It can also be observed in the blood of the rat. The crystals apparently so formed last but a few seconds, the corpuscles then becoming shrunken, or irregular, and very often under the subsequent action of water, globular. It is therefore possibly a stage in the crenation of the corpuscle. But, apart from this, it is undoubtedly the fact that hexagonal crystals are occasionally found in the blood of the rat.¹ It would therefore be possible that such crystals occasionally may occur in the blood of other animals, such as the mouse, the usual form of whose blood-crystals is, however, rhombic.

The rats employed in the above experiments were the common house rat, and also tame rats.

These experiments, as well as those in the next section of this paper, were undertaken at the suggestion of Professor Schafer.

The blood-crystals of an animal have the same form whether they be obtained from the fresh blood, or from the blood from which the fibrin has been removed. Fibrin, or its precursor in the blood-plasma fibrinogen, has then no influence on the form of the blood-crystals.

The following experiments were undertaken to ascertain whether the other constituents of the blood-plasma, which are all contained in the serum, have any effect in influencing the form of the crystals.

The method of experimentation was as follows:—Defibrinated blood is taken in a tube and centrifugalised for about half an hour; the corpuscles settle at the bottom of the tube, and the supernatant serum is pipetted off. To the corpuscles the blood-serum of some other animal is added, the mixture shaken, and the mixture again centrifugalised; the serum is again pipetted off, and more added. After repeating this process several times, the corpuscles of one animal are obtained in the serum of another animal without any of the serum of the first animal being in the mixture. Haemoglobin crystals are then prepared from this mixture. In some cases the foreign serum dissolves the haemoglobin and disintegrates the corpuscles. This was first pointed out by Landois.¹

Mere addition of the blood-serum of one animal does not as a rule cause the formation of blood-crystals. It does so, however, sometimes.² This is explicable on the assumption that the blood-serum used is very watery, and the haemoglobin of the other animal crystallises very readily. I have myself come across no case in which it occurred.

My results may be best given in the form of the following table. I have given not only the effect of the foreign serum on the crystalline form of haemoglobin, but also the effect on the corpuscles themselves, as to whether they are disintegrated or not.

The result of these experiments is to show that the serum of one animal has no influence in causing a change of the haemoglobin crystals of another animal.

I next examined in a qualitative manner the serum of certain rodents with regard to the proteids or albuminous substances contained in it. I obtained similar results in all animals, results which show, too, that the serum proteids of rodents agree with those in other mammalian animals which I had previously investigated.¹ The proteids, the most important bodies in the blood-plasma, being similar, the serum would not on a priori grounds be suspected of influencing the crystalline form of hæmoglobin. The results I have obtained with regard to the heat-coagulation temperatures of these bodies is shown in the following table.

The stromata of the red blood-corpuscles might, however, possibly be supposed to have some influence on the crystalline form of the hæmoglobin. We have seen that crystallising the hæmoglobin of one animal from the serum of another yielded negative results; squirrel’s hæmoglobin remained hexagonal, rat’s and guinea-pig’s rhombic prisms and tetrahedra respectively, whatever the serum in which they had been dissolved. A similar result followed crystallisation from a fluid consisting of serum plus the dissolved stromata of the corpuscles of some other animal. This was obtained by adding to the blood one sixteenth of its volume of ether, and letting it stand; the crystals of hæmoglobin which formed were filtered off, and the ether evaporated from the filtrate which consisted of the serum with the stromata of the corpuscles dissolved in it.

So far then these experiments seem to show that the difference of crystalline form is due to some inherent quality of the hæmoglobin itself, and not due to any agency in the blood external to the hæmoglobin.

By mixing the defibrinated blood from two animals, whose hæmoglobin crystallises differently, and then preparing crystals, I thought I might obtain some new forms resulting from the mixture. Here my experiments have yielded mostly negative results, but the one positive result I have obtained from such experiments warrants me in recording the whole. The blood of two animals were mixed in about equal proportions, shaken thoroughly, and then hæmoglobin crystals prepared by the ether method.

It will be convenient here again to give my results a tabular arrangement.

The second case, that of mixing blood from the rat and guinea-pig, is interesting, and demands further description. It shows that it is possible to obtain a new form of hæmoglobin by mixing that from two animals in which the crystalline form is different. It also shows that rhombic haemoglobin crystals may assume a hexagonal type (fig. 4). These crystals are not, however, perfect or equilateral hexagons, two of the sides being longer than the other four.

This irregularity is possibly to be accounted for by the fact that, in rats’ haemoglobin crystals, the angles corresponding to BCD, A F E, are 51°. In order to obtain perfect hexagons of a rhombic type it is necessary, as before stated, that this angle be 60°.

Under crossed nicols these crystals appear perfectly bright, so contrasting with the true hexagons obtained from the blood of the squirrel and hamster.

This result was not, however, always obtained; in one or two cases I obtained as a result of mixing the blood of these two animals a mixture of crystals; that is prisms and tetrahedra.

Another set of experiments was performed with the object of breaking down the hexagonal constitution of the haemoglobin of squirrels’ blood. The first method tried was that of driving off the water of crystallisation, and of then adding water to the dehydrated hæmaglobin.

The hæmaglobin was obtained in a state of purity and dried over sulphuric acid until it lost no more weight. Then it was examined, and found to have its normal spectroscopic properties. It was heated to 100° C. in a water oven, and again examined. It had lost but a slight amount of weight. It was rather more insoluble in warm water than previously, but the spectroscopic properties, and the form of the crystals obtained from the solution, remained as before. This confirms the observation previously made by Hoppe-Seyler that dry haemoglobin is not decomposed by a temperature of 100° C. It was again heated in the water oven at 100° C. until there was no further loss of weight. It was then heated to 120° C. in an air-bath, and again examined. It was found to have lost considerably in weight, to have lost its crystalline lustre, to be brown in colour (liæmatin) and to be insoluble in water. That is, it parts with its water of crystallisation at a temperature which decomposes it, with the formation of hæmatin, the proteid matter becoming at the same time coagulated and insoluble.

Experiments were then tried with the object of ascertaining whether a lower temperature will remove the water of crystallisation in a Torricellian vacuum. This I did by means of a Pflüger’s mercurial air-pump. The action of the vacuum alone converted the dried haemoglobin, at any rate partially, into the conditions of methaemoglobin. The water of crystallisation seemed to be completely lost at a temperature of 50°—60° C., as subsequent heating to 120° C. produced no further loss of weight. But this temperature was also sufficiently high to decompose the haemoglobin in such a way as to render it insoluble, or almost so, in water, and therefore no crystals could be subsequently obtained from it.

The next method adopted was to convert the haemoglobin by various reagents into methaemoglobin; then by reducing agents to form once more haemoglobin, and then obtain crystals of this. But the reducing agents used were found to hinder the formation of crystals.

The third and simplest method was to repeatedly recrystallise the haemoglobin, when it was found after three or four recrystallisations that no six-sided crystals were obtained, but a mixture of rhombic needles and tetrahedra, and in some cases the latter were absent. This is interesting in connection with the reverse experiment already related, in which crystals simulating hexagons were obtained by mixing together the blood of the rat and guinea-pig, and in which the same result was obtained from a mixture of the solutions of the pure hæmoglobin of the same animals.

What the difference between the various forms of. hæmoglobin may be, it cannot be a very deep or essential one. The difference in crystalline form is associated with a difference of solubility in water and other reagents; but the spectroscopic characters, the decomposition products, the compounds it forms, of which hæmin is a readily obtained example, are universally the same. Not only so, but Hoppe-Seyler has shown¹ that in various animals dried hæmoglobin has the same or nearly the same elementary composition.

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, cuprous oxide, &c., among compounds. The conditions on which dimorphism depend 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 crystallization, we have to deal with different hydrates, and the case is not one of true dimorphism; an instance of this is sulphate of soda.

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 oxyhæmoglobin is due to varying quantities of water of crystallisation may be otherwise expressed by saying that we have to deal with different hydrates of oxyhæmoglobin. 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 hæmoglobin 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

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. Dr. Christian Bohr ³ 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 me 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 hæmoglobin 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 so conflicting, and exhibit variations as great as in Bohr’s experiments, that it is impossible to draw any conclusions from them, except the negative one that we cannot by our present methods of research make any definite statement with regard to the water of crystallisation of hæmoglobin.

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 hæmoglobin of some animals to unite with a certain amount of water of crystallisation, and that of other animals with a different amount. That some such substance or agency does exist would seem to be the inevitable result of the recrystallisation experiments which have been related.