HEMOGLOBIN OETSTALS OP BODENTS' BLOOD 181

On the Haemoglobin Crystals of Rodents' Blood.

By

W. I>. Halliburton, IMC.D., B .Sc ,Assistant Professor of Physiology, University College, London.

(From the Physiological Laboratory, University College; London.)

THE crystals of haemoglobin since their first discovery havebeen described by various observers as occurring in̂ no lessthan five out of the six crystallographic .systems. Subsequentinvestigators have reduced this number: to two, namely, therhombic system, in which the haemoglobin from.the blood ofmost animals crystallises; and the hexagonal system,:in whichthat from the blood of certain rodents is said to crystallise.

This research was undertaken at Professor Lankester's sug-gestion, in order, first, to ascertain whether these six-sidedcrystals really belonged to the hexagonal system ; and, secondly^.to find, if possible, an explanation of the difference of crystallineform that haemoglobin presents in different animals, while inits other chief properties haemoglobin is universally the same.

It will be convenient to take the Bubject under the following

1. Historical. , .2. Hexagonal blood-crystals.3. Influence of the other constituents of the blood on the

crystalline form of haemoglobin crystals.4. The crystalline forms of haemoglobin obtained by mixirig-

the blood from different animals.5. Can squirrel's haemoglobin be obtained in any form other

than hexagonal crystals ?6. Conclusions and remarks.

182 W. D. HALLIBURTON.

1. Historical.

Oxyhsemoglobin crystals were first described by Eeichert1 asoccurring in the uterus of a pregnant guinea-pig; by Leydig8

as occurring in the alimentary canal of the leech; and byKolliker,3 obtained from the blood of the dog, python, andother animals. Kolliker considered the crystals to be com-posed of a more or less modified haematin. Funke4 was, how-ever, the first to make complete observations upon them, andto recognise their true nature. Kunde,5 working at the sametime, made extensive observations from a comparative point ofview, and was the discoverer of the exceptional form of thecrystals in the guinea-pig and squirrel. Since then manyinvestigators have worked at the subject, notably Lehmann,8

Rollett,7 von Lang,8 and Preyer,9 in whose exhaustive treatisea complete bibliography of the subject up to 187] is given.

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

a. In the great majority of animals10 in which haemoglobin

1 Reichert, ' Miiller's Arohiv,' 1849, p. 197-s Leydig, • Zeitsch. f. wiss. Zool.,' Bd. i, 1849, p. 116.3 Kolliker, ' Zeitsch. f. wiss. Zool.,' Bd. i, 1849, p. 266.4 Funcke, ' Zeitsch. f. nat. Med.,' N. P., Bd. i, 1851, p. 184; Bd. ii,

1852, p. 204 and p. 288. "De sanguine venaj lievates," 'Diss. Lipsise,"1851.

6 Kunde, ' Zeitsch. f. nat. Med.,' N. F., Bd. ii, 1852, p. 276.6 Lehmann, ' Ber. d. k. Sach's Ges. d. Wissen.,' 1852, p. 22.7 Rollett, ' Sitzungsber. d. Wien. Akad.,' Bd. xlvi, 1862, p. 65.8 Lang, ibid.9 Preyer, ' Die Blutkrystalle,' Jena, 1871.10 To the animals falling under this rule I can add several, the crystalline

form of the hemoglobin of which have not been hitherto recorded. I ammuch indebted for specimens of the blood of these animals to my friendMr. F. E. Beddard, of the Zoological Gardens.

Opossum (Didelphys cancrivora).—Very large and dark red crystals,can be readily obtained. They belong to the rhombic system.

Kangaroo (Macropus giganteus).—Crystals are more soluble, andso less readily obtained. They are rhombic prisms, slenderer than in theopossum.

HEMOGLOBIN CRYSTALS OF RODENTS* BLOOD. 183

occurs, vertebrate and invertebrate, crystals of it can beobtained in the form of prisms and plates belonging to therhombic system.

b. The exceptions to this rule hitherto noted are thefollowing:

i. Guinea-pig. Haemoglobin crystals from the blood of thisanimal are tetrahedra, once supposed to belong to the regularsystem, but now shown by von Lang to be in reality rhombic.

ii. Lehmann mentions that similar tetrahedra may be ob-tained from the blood of the mouse and rat. This has notsince been confirmed.

iii. In several birds the crystals obtained are also tetrahedra.iv. In three animals—the squirrel, the hamster, and the

mouse—six-sided plates have been described.v. In one of these, the hamster, rhombohedra are described

as occurring also.

2. Hexagonal Blood-crystals.We will take the three animals in which the haemoglobin is

said to crystallise in the hexagonal form one by one.a. Squirrel.—The discovery of the fact that haemoglobin

crystals from this animal are six-sided plates was made byKunde (1852). Writing in the same year, Lehmann assertsthat though these crystals are six-sided they do not belong tothe hexagonal system. He gives, however, no reasons for thisassertion. Lang and Preyer arrived at the opposite conclusioni. e. that they do belong to the hexagonal system, from the studyof their optical properties.

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

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

Bear (Ursus syriaous).—Bunches of rhombic needles, easily obtained.They are slenderer than those obtained from dog's blood as a rule, some beingalmost 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.

184 W. D. HALLIBURTON.

My own observations are as follows:—The crystals can beobtained with the greatest ease by simply adding a drop ofwater to a drop of defibrinated blood on a slide, and coveringit; in less than a minute crystals appear. I have also preparedthem by other methods;l but in all cases the crystalline formis the same. When first formed the crystals are six-sidedplates, many equilateral, but many not. After recrystallisation,however, the crystals are then all but perfectly regular. Thequetions then arises, Do they belong to the hexagonal system ornot ? To this question one of the three following answers mustbe the correct one.• 1. They do belong to the hexagonal system.

2. They do not belong to the hexagonal system, but arerhombic crystals, having a so-called "hexagonal habit." Inmineralogy instances are known of such occurrences. This isthe case with copper-glance, some of whose crystals so closelyresemble hexagonal ones that several mineralogists believedthat there were two kinds, one being hexagonal. Again, micais an instance of a monoclinic crystal with "hexagonalhabit."

FIG. 1. \ / FIG. 2. Fio. 3.

Suppose A B c D (fig. 1) to be the basal plane of a rhombicplate, and the angle A B C to be approximately 120°, the lines

1 The method that I have found best for the preparation of blood-crystalsiri most animals is to add to defibrinated blood a sixteenth of its volume ofether, and then to shake for two or three minutes until the liquid becomes ofa clear lake colour; in the course of time, varying from five minutes to threedays, crystals form in abundance (' Gamgee's Physiological Chemistry,' p. 87)

HAEMOGLOBIN OEYSTALS OF EODENTS* BLOOD. 185

joining A C, B D being the axes. Then if the angles D A B ,D c B be replaced, as shown by the dotted lines, a hexagonwill be produced differing but little from a regular hexagon.

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

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

Crystals may be divided, according to their optical proper-ties, into three classes:

1. Isotropic.—Those in which there is no distinction ofdifferent directions as regards optical properties. This includescrystals belonging to the regular system. They have butone refractive index, i. e. refract light like amorphous bodiesdo, singly.

%. Uniaxal.—Those in which the optical properties arethe same for all directions equally inclined to one particulardirection, called the optic axis, but vary according to this in-clination. This class includes crystals belonging to thedimetric system (crystals with three rectangular axes, two ofthem being equal) and the hexagonal system. The opticaxis corresponds with the principal crystallographic axis.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 systemsof crystals, the t r imetr ic or rhombic (three rectangular axesall unequal), the monoclinic, and the t r ichinic . In thesethere are always two directions along which a ray is singlyrefracted.

The best test, as to whether a substance is doubly refractiveor not, is this : If between crossed nicols, which consequentlyappear dark, a substance be interposed that make's the dark-ness give place to illumination, however feeble, that substanceis doubly refractive. This action is termed the depolarisationof the ray.

The crystals of squirrel's haemoglobin I submitted to this

186 W. D. HALLIBURTON.

test, with the result that no depolarisation of the light can bedetected, when they are examined with the apparent basalplane perpendicular to the axis of the instrument and rotated;nor when a quartz plate is inserted do they produce any modi-fication of the tint, as the stage is turned. The instrumentused was a Zeiss polarising microscope.

Hence the presumption is that they belong to the hexagonalsystem, as rhombic crystals with hexagonal habit or rhombictwins would produce some double refraction examined in thisway.

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

" The observation under the microscope between crossednicols, so far as it goes, is rather in favour of the ' crystalsbeing hexagonal, that is, presupposing that the field remainsdark when the crystal is rotated in the field of view. However,this is not quite conclusive, and in such cases greater certaintywould be obtained if the crystals were placed under a Ber-trand's polarising microscope, to see the shape of the inter-ference rings and cross."

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

Acting on Professor Lewis's advice, I submitted the crystalsto Professor Judd, who with Mr. Fletcher's co-operationexamined them, aud gave me the following report, for which Iam much indebted to him :—"I have every reason to believethe crystals belong to the hexagonal system from their form,and their extinction between crossed nicols. I regret, however,

1 The cardinal directions are, however, believed not to be the same for theether as for the material of the crystal.

HEMOGLOBIN CRYSTALS OP EODBNTS* BLOOD. 187

to find that their minute size, and especially their extremetenuity, prevents our applying the crucial test of the inter-ference figures seen in convergent polarised light.

"Bertrand devised a form of microscope which enables theseinterference figures to be studied in the minute crystals seenin their rock sections, and von Lasaulx has improved thisapparatus. We have what I believe to be the best form of theBertrand-Lasaulx apparatus constructed by Nachet; but evenemploying 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 thistest. I fear that no instrument exists which will accomplishwhat you desire; and Mr. Fletcher, on theoretical grounds,doubts whether it would be possible under any conditions toapply the test to such minute crystals."

The largest crystals of squirrel's haemoglobin that I haveobtained were those formed by the addition of water to thedefibrinated blood; they varied in size from 4001 to "005 m. inbreadth.

Since receiving Professor Judd's report, I have tried toobtain larger crystals by Gscheidlen's1 method. He sealsdefibrinated blood in narrow glass tubes, which are then keptat a temperature of 37° C. for several days. On opening thesetubes and emptying their contents into a watch glass, crystalsof great size are formed from dog's blood after evaporation hasoccurred.

With squirrels' blood, however, I have not obtained largercrystals by this method than by the first. The reason for thisseems to be the extreme readiness with which squirrels' haemo-globin crystallises. It is a well-known fact that bodies thatcrystalise rapidly crystallise in small and numerous crystals.If some method could be devised for retarding, but not pre-venting, the crystallisation of squirrel's haemoglobin, we mightthen be able to obtain crystals of it large enough to which toapply this crucial test.

1 ' Physiologische Methodik,' p. 361,

188 W; D. HALLIBURTON. '

The matter must therefore be left incomplete up to thispoint for the present. The probability, however, is greatly infavour of the crystals being-true hexagons.

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

I shall also presently show that it is possible by the inter-mixture of the blood of different animals to obtain crystalsclosely resembling hexagons, but which are not so, as is shownby their optical properties.

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

Bqjanowski1 was the next to make observations on thesecrystals. He describes and figures them as six-sided platesresembling in form those from squirrel's blood, of a fleshcolour, and very soluble in water. He prepared them by theaddition of a mixture of equal parts of alcohol and ether tothe blood. No description of their optical properties is given.He remarks, " I have not been able to observe the fine needlesdescribed by Kunde."

Preyer repeated these experiments, and confirmed the obser-vations of Kunde, not those of Bojanowski. He obtainedsmall prismatic crystals.

I have myself experimented with the blood of eighteenmice, and the result has been again to confirm Kunde's obser-vations. The crystals are exceedingly difficult to obtain, andin some cases 1 have had to repeat the process of freezing andthawing many times after the addition of alcohol, before suc-ceeding in obtaining them. They are very soluble in water.The crystals are exceedingly small rhombic prisms. They arenearly colourless, and it is only when they are heaped togetherthat any red tinge at all can be perceived in them. In onecase in which by the addition of ether to the blood I obtainedcrystals of fair size after allowing the mixture to stand for five

1 Bojanowski, 'Zeitscli. f. wiss. Zool.,' Bd. xii, 1863, p. 333.

HEMOGLOBIN CRYSTALS OF EODENTS BLOOD. 189

days, the crystals still showed this same peculiarity, namely, inbeing nearly colourless. I have successfully employed Boja-nowski's method for the preparation of the crystals, namely,the addition of a mixture of alcohol and ether to the blood; butin no case did hexagonal crystals form. Mouse's haemoglobinalso differs from squirrel's in being very soluble in water ; thisis admitted by Bojanowski; one would therefore expect ap r io r i that its crystalline form would be different.

c. H a m s t e r (Cricetus vulgaris).—My remarks underthis heading will be only historical. I have not myself beensuccessful in obtaining one of these animals. The crystallineform of the haemoglobin was first described by Lehmann, whofound rhombohedra and six-sided plates. His experimentswere repeated by Preyer,! whose observations on the subject arevery complete. He fornd both crystalline forms, viz. six-sided plates, and rhombohedra. This is interesting since therhombohedron belongs to the hexagonal system. By examina-tion between crossed nicols he found that the six-sided plateshad no action in " depolarising " the ray, and he therefore con-cludes that they, like squirrel's haemoglobin crystals, are truehexagons.

d. Conclusions.—The presumption in favour of thehaemoglobin crystals of the squirrel and hamster being truehexagons is exceedingly great. In the case of the mouse, itseems to be almost equally certain that the crystals are not asa rule hexagonal. I should not like, however, to deny thathaemoglobin may sometimes in the case of the mouse crystallisein this way, because of some observations I have made on thehaemoglobin crystals of the rat.

Crystals are obtained from the blood of this animal withgreat ease; mere addition of water to the blood causes almostimmediately an abundant crop of crystals. On this accountthe blood of this animal is used by the students in the practicalclasses at University College for the preparation of haemoglobincrystals. Professor Schafer told me that on looking over thestudents' preparations he had occasionally seen hexagons to-

1 ' Die Blutkrystalle,' p. 262.

190 W. D. HALLIBURTOlT.

gether with the ordinary rhombic prisms and plates. Inorder to verify this, I have made numerous specimens of thecrystals from the blood of about fifteen rats. As a rule, nohexagons were present; but on three occasions I have detectedhexagonal plates—very few in number, perhaps not more thanone or two on the slide—among the rhombic crystals. Thereappeared to be nothing special either about the animal usedor the method employed in these cases. The diameter ofthese crystals averaged about the same as in squirrel's blood(003—"003 m.). Between crossed nicols they also behavedthe same as squirrels' haemoglobin crystals, viz.-remained darkin all positions.

In addition to this, if crystallisation be watched under themicroscope, a single corpuscle will often be observed to setinto a minute hexagon. This is what Preyer calls intraglobularcrystallisation. He describes it as occurring in the blood ofthe hamster. It can also be observed in the blood of therat. The crystals apparently so formed last but a few seconds,the corpuscles then becoming shrunken, or irregular, and veryoften under the subsequent action of water, globular. It istherefore possibly a stage in the crenation of the corpuscle.But, apart from this, it is undoubtedly the fact that hexagonalcrystals are occasionally found in the blood of the rat.1 It

1 Since writing the above, I have received the following in a letter from Mr.Sheridan Lea, of Cambridge. He says :—" When I was showing a class howto put up permanent specimens of haemoglobin crystals from rat's blood, weobtained uniformly hexagons, instead of prisms. This I have neither evernoticed or heard of before, and I thought it might be of interest to you.The method employed was that of Stein (' Centralb. f. d. med. Wiss.,' 1884,No. 23, and ' Virchow's Archiv,' 97, 483)." I had myself occasionally usedStein's method of preparing crystals from rat's blood, but had always obtainedthe usual rhombic prisms. On receiving Mr. Lea's letter I made a largenumber of preparations of haemoglobin crystals by this method. The methodconsists in simply mounting a drop of deiibrinated blood in a drop of Canadabalsam. In the case of some animals, among which were man and the mouse,I was not able to get any crystals at all. In the commoner mammals, dogand cat, the crystals obtained were very fine specimens of rhombic prisms.In the guinea-pig and squirrel they presented the usual tetrahedral andhexagonal shapes respectively. With rat's blood, however, the results were

HEMOGLOBIN CRYSTALS OP EODUNTS* BLOOD. 191

would therefore be possible that such crystals occasionally mayoccur in the blood of other animals, such as the mouse, theusual form of whose blood-crystals is, however, rhombic.

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

3. Influence of the other Constituents of the Blood on theCrystalline form of Haemoglobin Crystals.

These experiments, as well as those in the next section ofthis paper, were undertaken at the suggestion of ProfessorSchafer.

The blood-crystals of an animal have the same form whetherthey be obtained from the fresh blood, or from the blood fromwhich the fibrin has been removed. Fibrin, or its precursorin the blood-plasma fibrinogen, has then no influence on theform of the blood-crystals.

The following experiments were undertaken to ascertainwhether the other constituents of the blood-plasma, which areall contained in the serum, have any effect in influencing theform of the crystals.

The method of experimentation was as follows:—Defibrinatedblood is taken in a tube and centrifugalised for about half an

very strange. In the majority of cases the usual rhombic needles were formed ;but in a few cases I confirmed Mr. Lea's observations, and obtained perfectlyregular hexagons; in some oases the hexagons would occupy one part of theslide only, while the remainder was filled with the ordinary prisms. Hexagonsseemed to form where the proportion of blood to balsam was small, and theywere formed especially at the edges of a preparation where the drop of bloodhad probably had time to dry somewhat before being covered with Canadabalsam. These hexagons remained dark in the dark field of the polarisingmicroscope. After a day or two they cracked in a peculiar way, and seemedthen to be made up of minute needles radiating from a centre. This may ormay not indicate the way in which they are formed. The fact that theyoccurred most in parts of the field where there was leaBt water seems, how-ever, to confirm the theory advanced later in the paper, viz. that the differenceof crystalline forms in haemoglobin is due to different amounts of water ofcrystallisation.

192 W. D. HALLIBURTON.

hour; the corpuscles settle at the bottom of the tube, andthe supernatant serum is pipetted off. To the corpuscles theblood-serum of some other animal is added, the mixture shaken,and the mixture again centrifugalised; the serum is againpipetted off, and more added. After repeating this processseveral times, the corpuscles of one animal are obtained in theserum of another animal without any of the serum of the firstanimal being in the mixture. Haemoglobin crystals are thenprepared from this mixture. In some cases the foreign serumdissolves the haemoglobin and disintegrates the corpuscles.This was first pointed out by Landois.1

Mere addition of the blood-serum of one animal does notas a rule cause the formation of blood-crystals. It does so,however, sometimes.2 This is explicable on the assumptionthat the blood-serum used is very watery, and the haemoglobinof the other animal crystallises very readily. I have myselfcome across no case in which it occurred.

My results may be best given in the form of the followingtable. I have given not only the effect of the foreign serum on thecrystalline form of haemoglobin, but also the effect on the cor-puscles themselves, as to whether they are disintegrated or not.

Corpuscles of

RatSquirrelSquirrelRatGuinea-pigGuinea-pigMouse

In Serum of

SquirrelRutDogGuinea-pigCatDogCat

Effect on the Corpuscles.

Much dissolvedVery little dissolvedVery little dissolvedLittle if any dissolvedNearly entirely dissolvedMuch dissolvedLittle dissolved

Effect on thD Crystalline Formof the Hemoglobin.

Nil.Nil.Nil.Nil.Nil.Nil.Nil.

The result of these experiments is to show that the serum ofone animal has no influence in causing a change of the haemo-globin crystals of another animal.

I next examined in a qualitative manner the serum of certain1 ' Die Transfusion des Blutes,' Leipzig, 1874.2 An instance of such action is recorded by Professor Sctaafer (" Blood

Transfusion," ' Trans. Obst. Soc. London,' 1879, p. 317).

HEMOGLOBIN CRYSTALS OF KODENTS' BLOOD. 193

rodents with regard to the proteids or albuminous substancescontained in it. I obtained similar results in all animals,results which show, too, that the serum proteids of rodentsagree with those in other mammalian animals which I hadpreviously investigated.1 The proteids, the most importantbodies in the blood-plasma, being similar, the serum wouldnot on a pr ior i grounds be suspected of influencing thecrystalline form of haemoglobin. The results I have obtainedwith regard to the heat-coagulation temperatures of thesebodies is shown in the following table.

Tem pera tures of Coagulat ion of the Pro te ids in theBlood of cer ta in Rodents .

"Same of Proteiil.

Globulins—Fibrinogeu. .Serum globulins .

C. i C. C.56° : 56° ' 56°7i>0'75° 75°

Guinea-pig. |

C.

Squirrel.

c.

75°

C.

75"

Albumins—73° , 70° '• 70°-l°|72° !72°-3'76° '• 77° • 78° i77° :77°(smallinamount)77°'

y 84° i 84° , 84° 87° (trace)84° (very abundant) " "

The stromata of the red blood-corpuscles might, however,possibly be supposed to have some influence on the crystallineform of the haemoglobin. We have seen that crystallising thehaemoglobin of one animal from the serum of another yieldednegative results; squirrel's haemoglobin remained hexagonal,rat's and guinea-pig's rhombic pi-isms and tetrahedra respec-tively, whatever the serum in which they had been dissolved.A similar result followed crystallisation from a fluid consistingof serum plus the dissolved stromata of the corpuscles of someother animal. This was obtained by adding to the blood onesixteenth of its volume of ether, and letting it stand; thecrystals of haemoglobin which formed were filtered off, and theether evaporated from the filtrate which consisted of the serumwith the stromata of the corpuscles dissolved in it.

1 Halliburton, "Periods of Serum," 'Journal of Physiology,' vol. v, p. 152.VOL. XXVIII, PART 1. NEW SER. N

191 W. D. HALLIBURTON.

So far then these experiments seem to show that the differ-ence of crystalline form is due to some inherent quality of thehaemoglobin itself, and not due to any agency in the bloodexternal to the hsemoglobin.

4. The Crystalline forms of Haemoglobin obtained bymixing the Blood from different Animals.

By mixing the defibrinated blood from two animals, whosehaemoglobin crystallises differently, and then preparing crystals,I thought I might obtain some new forms resulting from themixture. Here my experiments have yielded mostly negativeresults, but the one positive result I have obtained from suchexperiments warrants me in recording the whole. The bloodof two animals were mixed in about equal proportions, shakenthoroughly, and then haemoglobin crystals prepared by theether method.

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

Form of Hremoglobiii Crystals prepared from the Mixture.

RatRat

Squirrel

Dog

Dos

SquirrelGuiuea-pig

Guinea-pig

Squirrel

Guinea-pig

Both rbombic prisms and hexagous present.No rhombic prisms of the shape usually seen in rats'

blood present. No tetrahedra. Crystals are allrhombic prisms with hexagonal habit.

Hexagonal phtes and tetrahedra both present. Manytetrahedra imperfect. The tetrahedra were all re-duced to about half the size of those prepared fromthe unmixed blood of the same guinea-pigs.

Fine rhombic needles and hexagonal plates both pre-sent in abundance.

The greater number of the crystals formed are verysmall tetrahedra, about a quarter the size of thoseprepared from the blood of the same guiuea-pigs.The optical properties are, however, the same.Rhombic prisms very slender, like those of dog'sblood, also seen.

The second case, that of mixing blood from the rat andguinea-pig, is interesting, and demands further description.It shows that it is possible to obtain a new form of haemoglobin

HEMOGLOBIN CRYSTALS OF EODRNTS* BLOOD. 195

by mixing that from two animals in which the crystalline formis different. I t also shows that rhombic haemoglobin crystalsmay assume a hexagonal type (fig. 4). These crystals are not,however, perfect or equilateral hexagons, two of the sidesbeing longer than the other four.

E

The side A B = E D = -0019 m. (average).The sides B C = C D = E F = F A = '00125 m. (average).

This irregularity is possibly to be accounted for by the factthat, in rats' hEemoglobin crystals, the angles corresponding toB C D , A F E, are 51°. In order to obtain perfect hexagonsof a rhombic type it is necessary, as before stated, that thisangle be 60°.

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

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

5. Can Squirrel's Haemoglobin be obtained in any form otherthan Hexagonal Crystals ?

Another set of experiments was performed with the objectof breaking down the hexagonal constitution of the haemo-globin of squirrels' blood. The first method tried was that ofdriving off the water of crystallisation, and of then addingwater to the dehydrated hsemaglobin.

The hsemaglobin was obtained in a state of purity and driedover sulphuric acid until it lost no more weight. Then it wasexamined, and found to have its normal spectroscopic proper-

196 W. B. HALLIBURTON.

ties. It was heated to 100° C. in a water oven, and againexamined. It had lost but a slight amount of weight. It wasrather more insoluble in warm water than previously, but thespectroscopic properties, and the form of the crystals obtainedfrom the solution, remained as before. This confirms the obser-vation previously made by Hoppe-Seyler that dry haemoglobinis not decomposed by a temperature of 100° C. It was againheated in the water oven at 100° C. until there was no furtherloss of weight. I t was then heated to ]20° C. in an air-bath,and again examined. It was found to have lost considerablyin weight, to have lost its crystalline lustre, to he brown incolour (hjematin) and to be insoluble in water. That is, itparts with its water of crystallisation at a temperature whichdecomposes it, with the formation of heematin, the proteidmatter becoming at the same time coagulated and insoluble.

Experiments were then tried with the object of ascertainingwhether a lower temperature will remove the water of crystal-lisation in a Torricellian vacuum. This I did by means of aPfliiger's mercurial air-pump. The action of the vacuum aloneconverted the dried haemoglobin, at any rate partially, into theconditions of methaemoglobin. The water of crystallisationseemed to be completely lost at a temperature of 50°—60° C,as subsequent heating to 120° C. produced no further loss ofweight. But this temperature was also sufficiently high todecompose the haemoglobin in such a way as to render itinsoluble, or almost so, in water, and therefore no crystals couldbe subsequently obtained from it.

The next method adopted was to convert the haemoglobin byvarious reagents into methsemoglobin ; then by reducing agentsto form once more hsemoglobin, and then obtain crystals ofthis. But the reducing agents used were found to hinder theformation of crystals.

The third and simplest method was to repeatedly recrystallisethe haemoglobin, when it was found after three or four re-crystallisations that no six-sided crystals were obtained, but amixture of rhombic needles and tetrahedra, and in some casesthe latter were absent. This is interesting in connection with

HEMOGLOBIN CRYSTALS OF BODENTS* BLOOD. 197

the reverse experiment already related, in which crystals simu-lating hexagons were obtained by mixing together the blood ofthe rat and guinea-pig, and in which the same result wasobtained from a mixture of the solutions of the pure hsemo-globin of the same animals.

6. Conclusions and Remarks.

What the difference between the various forms of, haemo-globin may be, it cannot be a very deep or essential one. Thedifference in crystalline form is associated with a difference ofsolubility in water and other reagents; but the spectroscopiccharacters, the decomposition products, the compounds it forms,of which hsemin is a readily obtained example, are universallythe same. Not only so, but Hoppe-Seyler has shown1 that iuvarious animals dried haemoglobin has the same or nearly thesame elementary composition.

Have we then to deal •with a case of polymorphism? Theterms dimorphism and polymorphism cannot be applied to anysubstance which crystallises in two or more forms, unless thecomposition of that substance be exactly the same in all cases.Instances of dimorphism in the mineral world are carbon andsulphur among the elements, and sal ammoniac, potassiumiodide, cuprous oxide, &c, among compounds. The conditionson -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 areunited with different proportions of water of crystallization, wehave to deal with different hydrates, and the case is not one oftrue dimorphism ; an instance of this is sulphate of soda.

The case seems to me to narrow itself down to this in thecase of haemoglobin; either we have here a case of poly-morphism, or the crystalline forms are due to the combina-tion with varying proportions of water of crystallisation. Inthe absence of a rational formula for haemoglobin it wouldbe unsafe to affirm the former of these two alternatives. More-over, the conditions that are known to produce dimorphism in

1 'Pliysiologisclie Chemie,' p. 377.

198 W. D. HALLIBURTON.

minerals, namely, differences of temperature and of solvent, havein the case of haemoglobin no influence.

If we then fall back on the latter alternative, the questionwhich arises is whether there are any facts to support it. Theexplanation that the varying form of oxyhsemoglobin is due tovarying quantities of water of crystallisation may be other-wise expressed by saying that we have to deal with differenthydrates of oxyhsemoglobin. This would account for thevarying solubilities of these substances in water and otherreagents, and at the same time is not such an essential differ-ence as to prevent the chief properties of haemoglobin frombeing universally the same.

Turning to Hoppe-Seyler's researches on this subject of waterof crystallisation, it is seen that its amount varies consider-ably. The following is his table i1

Dog's hsemoglobin .Guinea-pig's ,,Squirrel's „Goose's

Per<ventage of Water of Crystnllisot3 to 479-49'4

In an earlier paper,2 the same author gives rather differentpercentages, viz. for guinea-pig's haemoglobin 6, for goose'shsemoglobin 7, and for squirrel's haemoglobin 9. Dr. ChristianBohr3 has more recently made observations on the water ofcrystallisation of dog's hsemoglobin, and as the result ofthirteen experiments he finds that its amount varies from 6*3to 1"2 per cent. It is thus seen that great variations occur inthe numbers obtained by these experiments. The reason forthis variation seems to me to be the great difficulty of obtain-ing htemoglobin in a pure state, and also possibly because themethod adopted, which is the same as that carried out insimilar investigations on inorganic salts, is not applicable tosuch a complex and much less stable organic compound as

1 ' Pliysiologische Chemie,' p. 377.3 'Med. Chem. TJntersuckungen/ Heft iii, 1S68, p. 370.' ' Experimental TJntersuchungen iiber die Sauerstoffaufnahme des Blut-

farbstoffes,' Kopenhagen (Olsen and Co.), 1885.

HAEMOGLOBIN CRYSTALS OF RODENTS* BLOOD. 199

haemoglobin; in other words, the temperature necessary todrive off the water of crystallisation is also sufficient to causecertain decomposition changes in the pigment.

My experiments have shown that squirrel's haemoglobin willunder certain circumstances crystallise in forms other than theusual hexagonal form. A crucial experiment in order to seewhether this is due to uniou with different amounts of water ofcrystallisation would have been first to ascertain the amountof this water in the hexagonal crystals, and then in therhombic crystals obtained by recrystallisation. I have per-formed three such experiments, but the results obtained are soconflicting, and exhibit variations as great as in Bohr's experi-ments, that it is impossible to draw any conclusions fromthem, except the negative one that we cannot by our presentmethods of research make any definite statement with regardto the water of crystallisation of haemoglobin.

Even if it be found ultimately that the difference in crystal-line form is dependent on varying amounts of water of crystal-lisation, the difficulty is only explained up to a certain point.What is left unexplained is the nature of the agency thatcauses the haemoglobin of some animals to unite with a certainamount of water of crystallisation, and that of other animalswith a different amount. That some such substance or agencydoes exist would seem to be the inevitable result of the recrys-tallisation experiments which have been related.