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QUICK METHODS FOR CRYSTALLIZING OXYHJJZ-

MOGLOBIN: INHIBITORY AND ACCELERATOR

PHENOMENA ETC.: CHANGES IN THE

FORM OF CRYSTALLIZATION.

BY EDWARD T REICHERT

I. QUICK METHODS FOR CRYSTALLIZING OXYHEMOGLOBIN.

T

HE most expeditious known method for preparing crystals of

the oxyhemoglobin of the blood of the dog is to lake the blood

with ether and subject it to cold. Crystals appear within a variable

period rangin g from a few minutes to many hours. I have found

that if to the blood there be added before or after laking from I to

5 per cent of ammonium oxalate crystallization invariably begins

immediately and that any quantity of crystals can be obtained within

a few hours at ordinary room temperature. If a drop of this blood

be placed under the microscope crystals will be seen to form at once

near the margin of the drop and to be deposited so rapidly that a

solid mass is formed in a few minutes.

The blood of the horse does not yield so readily to this treatment.

If a drop of blood so prepared be examined under the microscope it

will be found that crystallization will not begin until after from fifteen

to forty minutes or more and that it will proceed slowly. Better

results can be obtained if the blood be oxalated and set aside for the

corpuscles to subside The supernatant liquid is then poured off

and the remainder laked with ether.

Defibrinated blood of the rat laked with water on a slide and

covered with a cover-glass after the margin of the drop has become

dried usually crystallizes very readily as is well-known. Better

results can be obtained if the blood be oxalated before or after laking ;

and even more rapid crystallization occurs if the blood be laked with

ether instead of water. Crystals form so rapidly in the oxalate-ether

blood that a magma is formed in the test-tube within a few minutes.

The oxalate-ether process applied to the blood of the guinea-pig

gives most satisfactory results. Crystallization does not proceed

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quite so rapidly as in rat’s blood yet within a minute or two innu-

merable tetrahedra appear and any quantity can be obtained within

a couple of hours.

The blood of the necturus crystallizes readily when so treated

which is a matter of especial interest because of the difficulty hereto-

fore experienced in obtaining crystals of oxyhemoglobin from the

blood of cold-blooded animals. The crystals resemble those of the

triple phosphates.

The rapidity with which crystallization begins and proceeds is in-

fluenced decidedly both by the method of laking and the percentage

of oxalate. Ethyl ether is a much better laking agent than water

and acetic ether is stronger than ethyl ether. The presence of any

quantity of oxalate up to saturation increases crystallizability but

I have thus far found from I to 5 per cent to be the best; the larger

the quantity the more is crystallization hastened. When more than

5 per cent is used the oxalate also tends to crystallize upon the slide.

If the blood be prevented from drying as in the test-tube the oxalate

remains in solution.

Asphyxial blood yields crystals more readily than normal blood.

II

. INHIBITORY AND ACCELERATOR PHENOMENA ETC.

If to the blood of one species the blood plasma or serum of another

species be added the laking of the blood may be retarded accelerated

or unaffected according to the character of the mixture. The period

required for laking may be prolonged for five minutes or more.

The crystallization of the oxyhaemoglobin may be hindered or pre-

vented in such mixtures. Thus by varying the proportions of a

mixture of the bloods of the dog and guinea-pig crystals from one or

both may appear but the process is invariably retarded and some-

times to a marked degree. If crystals of both kinds of oxyhaemo-

globin are deposited those of one usually begin forming some time

before those of the other and the crystallization of one may be

complete before crystals of the other are seen.

III. CHANGES IN THE FORM OF CRYSTALLIZATION.

The typical forms of the crystals of certain kinds of oxyh=mo-

vlobin may be modified or completely changed when the bloods of two

b

species are mixed. Thus if to the blood of the rat there be added a

definite percentage of the blood of the guinea-pig crystals of the rat’s

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Quzkk Methods far CrystaLking Oxyhmogdobi~. gg

oxyhzemoglobin may appear in unaltered form but most if not all of

those from the guinea-pig’s blood will be changed ; in fact if any

perfect tetrahedra are found they will have been formed at the very

end of the crystallization. If the proportions of the mixture be

properlv modified not a single crystal of what can be identified as

rat’s oxyhaemoglobin will appear and al l of the crystals will be modi-

fied tetrahedra and spzkdces and transitional forms between these.

The spindles resemble Charcot’s crystals in form but not in color ;

they vary in size some of them being very large and some may have

small spindles attached to them ; and they can be obtained having

sharp angles if crystallization has not been too rapid.

This complete change in the form of the crystals of oxyhaemoglobin

when the bloods of two species are mixed and the spindle-shaped

form of the crystals are I believe unique facts in the crystallography

of this most important substance.