QUICK

METHODS
FOR
MOGLOBIN:
INHIBITORY
PHENOMENA,
FORM

OF

ETC.:
CHANGES
CRYSTALLIZATION.

BY EDWARD

I.

QUICK

METHODS

CRYSTALLIZING
OXYHJJZAND
ACCELERATOR

T.

IN

THE

REICHERT.

FOR CRYSTALLIZING

OXYHEMOGLOBIN.

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

quite so rapidly as in rats blood, yet within

a minute or two innumerable 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 heretofore 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 influenced 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 prevented 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 sometimes to a marked degree.
If crystals
of both kinds of oxyhaemoglobin 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=movlobin
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 rats

Quzkk

Methods

far

CrystaLking

Oxyhmogdobi~.

gg

oxyhzemoglobin
may appear in unaltered form, but most, if not all, of
those from the guinea-pigs
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
rats oxyhaemoglobin
will appear, and all of the crystals will be modified tetrahedra
and spzkdces, and transitional
forms between these.
The spindles resemble Charcots
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.