A NOTE ON THE CRYSTALLIZATION OF FREE LYSINE.

*
BY HUBERT BRADFORD VICKERY AND CHARLES S.
LEAVENWORTH.
(Fhwn the Laboratory of the Conn&icut Agricultural EzprrimmJ Station,
New Haven.)
(Received for publication, December 6, 1927.)

Downloaded from http://www.jbc.org/ by guest on February 19, 2018

With the exception of lysine, all of the amino acids derived from
proteins by hydrolysis have been obtained in the crystalline form.
In view of the ease with which it is possible to crystallize free
arginine,’ there seemed to be no reason to suppose that lysine would
not crystallize from its solutions provided that material of sufhcient
purity could be secured. The chief impurity to be feared is the
carbonate, since lysine is so strong a base that it readily absorbs
carbon dioxide from the air.* Other amino acids may also con-
taminate a lysine fraction prepared in the usual way’ and it is
eaaential, in order to purify this substance, to carry out the crys-
tallization of a At. The picrate is by far the most suitable.
For the following work a number of preparations of lysine
picrate obtained from various proteins were combined. Each of
these had been crystallized at least once and all decomposed with
a slight explosion at temperatures above 260”. The material
(60 gm.) was dissolved in hot water, boiled with a liberal quantity
. of norit, filtered, and evaporated until crystallization commenced
at the surface of the solution. On standing overnight a large
crop of fine needles wss obtained which decomposed at 265-266’:
l The expenees of thie investigstion were sh8re.d by the Connecticut
Agricultural Experiment Station and the Carnegie Institution of Waahing-
ton, D. C.
1 Vickery, II. B., 8nd Leavenworth, C. S., J. Biol. Chem., 1927, lxxii, 403.
* A dilute solution of free lysine ie intensely alkeline to phenolphthdein.
s Leavenworth, C. S., J. Biol. Chem., 1924, lxi, 316.
’ Lysine picrate begin8 to drrrken at 240” and becomes very derk at 250”.
No obvious melting occurs but the slight explosion et 265-266’ ia etrikingly
chsr8cterietic. The decomposition tempereture is little &ected by the
rata of heeting.
437
43s Crystallization of Free Lysine
on evaporation of the mother liquor a small, but equally pure,
second crop separated. The second mother liquor yielded, on
further concentration, a third crop of less pure material and,
finally, dark and manifestly impure crystals.
The f&t two crops were united and recrystallized from hot
water whereby well developed needles, many of them at least 1
cm. long, were secured. These decomposed at 265-266” as before.
A second crop from the alcoholic washings and mother liquor of
the first crop was equally well crystallized and decomposed at the
same temperature. The combined weight of these two crops was

Downloaded from http://www.jbc.org/ by guest on February 19, 2018

54 gm., representing a 90 per cent recovery.
The picrate was dissolved in 150 cc. of hot 10 per cent sulfuric
acid, the picric acid, which crystallized on cooling, was filtered off,
and the solution was extracted with ether until no further picric
acid could be removed. In spite of the careful purification of the
picrate this solution was faintly yellow. It was therefore diluted
and boiled with norit, which removed every trace of color, and
then treated with a hot solution of three times recrystallized
barium hydroxide until the reaction was no longer acid to Congo
red but was still distinctly acid to litmus. The barium sulfate
was removed and washed and the solution concentrated to about
200 cc. in vacua.
The 3 liter distillation flask containing the lysine solution
was fitted with a stopper carrying an air inlet tube, a dropping
funnel, and a short length of open glass tubing through which a
sampling pipette could be introduced. A strong stream of air,
freed from carbon dioxide, was passed through the apparatus
during all the subsequent operations. Cold saturated barium
hydroxide solution was slowly added until, on warming and allow-
ing the barium sulfate to set&, no further precipitation could be
observed. Small samples were then withdrawn, centrifuged, and
tested for excess of either sulfuric acid or barium hydroxide. The
reagent was added until a point was found at which no precipitate
appeared on adding dilute sulfuric acid or barium hydroxide to
separate samples. The test samples were not returned to the
flask.
The precipitated barium sulfate was allowed to settle and was
washed twice by decantation with liberal amounts of hot boiled
water, the fluid being siphoned off each time into B flask contain-
 H. B. Vickery and C. S. Leavenworth 439
ing purified air. After concentration in vacua to about 500 cc.
the solution was drawn through a thick pad of paper pulp on a.
sintered glass filter in a system protected from the outside air by
soda-lime. The perfectly clear filtrate was concentrated to a
thick, slightly greenish yellow sirup. Crystallization began
during the last stages of the concentration and, on standing over-
night, a considerable part of the lysine separated as a solid mass
of crystals. After turning the flask on its side the remaining sirup
slowly ,drained away from the crystals and the appearance is
shown in Fig. 1. A small sample of the magma was drawn, with

Downloaded from http://www.jbc.org/ by guest on February 19, 2018

every precaution against the access of carbon dioxide, into a
covered cell constructed of microscope slides cemented together

FIG. 1. Mass of crystalline free lysine at bottom of flask. One-half

natural size.

with paraffin and provided with inlet and exit tubes. A photo-
micrograph of the crystals is shown in Fig. 2. The crystals are
fine transparent needles which aggregate into irregular, striated
plate-like forms and also into radiating groups. Owing to the
necessity of photographing through a thick layer of sirup, many of
the crystals in the figure are out of focus.
A portion of the sirup was treated with 20 to 30 volumes of
absolute alcohol. A white precipitate separated, which, on warm-
ing, dissolved to a slightly turbid solution. On cooling, lysine
separated at once in tiny needles which are shown in Fig. 3. It
was found necessary to employ absolute alcohol that had been
refluxed over potassium hydroxide since ordinary absolute alcohol
 Crystallization of Free Lysine
promptly assumed a yellow color when heated with lysine and the
crystals which separated on cooling were impure. Even with this
precaution it was found difficult to secure a white preparation
with a theoretical nitrogen content. The best preparation con-
tained 19.06 per cent of nitrogen, theory 19.17 per cent, and was
free from carbonate.
On slow heating, free lysine darkens above 210’ and decomposes
at 224-225” (Anschiitz thermometer). By more rapid heating
the apparent decomposition point may be raised lo or 2”.
Some of the sirup was allowed to stand exposed to the air until

Downloaded from http://www.jbc.org/ by guest on February 19, 2018

a superficial layer of crystals which contained carbonate had

FIG. 2. Free lysine crystallized from water. X 80

separated. These were pressed out dry and were found to be-
have, on heating, in the sameway as the crystals of the free base.
Another portion of the sirupy suspension of crystals was re-
moved and diluted with boiled water. A nitrogen determination
indicated that the solution contained 6.49 per cent of lysine. The
specific rotation was [ar]: = +14.6”. Since this solution, on
being tested with barium hydroxide, gave a barely visible pre-
cipitate of barium carbonate, the amount of carbon dioxide which
had been absorbed by the lysine throughout the operations was
negligible.
 H. B. Vickery and C. S. Leavenworth 441

Another preparation of crystallized lysine was made from a

sample of lysine chloride (20 gm.) obtained, via the picrate, from
casein, to ascertain whether the precautions that were taken to
purify the material and eliminate carbon dioxide were really
necessary. The chloride was treated with exeess of silver oxide
in a solution maintained faintly acid with sulfuric acid, the silver
chloride was removed, and the excess of silver precipitated by
hydrogen sulfide. The solution was then concentrated slightly,
decolorized with norit, and the sulfuric acid exactly removed by

Downloaded from http://www.jbc.org/ by guest on February 19, 2018

FIG. 3. Free lysine crystallized rapidly from somewhat dilute alcohol.

X 80.

purified barium hydroxide, the work being done in an open beaker

in the usual way. The barium sulfate was centrifuged off, the
solution concentrated to about 500 cc. in VWUO, and then filtered
perfectly clear through paper pulp on an open filter. It contained
a trace of carbonate. On concentration in vucuo to a sirup, crys-
tallization readily took place. The crystals thus secured were
typical and as finely developed as the previous sample. A few
thin and irregular plates were present; the edges of some of these
formed angles indicating that, if fully developed, the plates would
be hexagonal. On adding several volumes of purified absolute
alcohol to the magma and warming, a somewhat turbid solution
442 Crystallization of Free Lysine

resulted, probably due to the presence of a trace of carbonate.

Consequently if pure free lysine is desired the precautions de-
scribed above are necessary, but they may be omitted if a demon-
stration sample only of crystallized lysine is required.
The turbid alcoholic solution, on standing several days deposited
lysine in hexagonal plates (Fig. 4) from which the still turbid
mother liquor could readily be decanted. This was filtered per-
fectly clear and enough absolute alcohol added to throw out a
small white flocculent precipitate which dissolved to a clear solu-

Downloaded from http://www.jbc.org/ by guest on February 19, 2018

tion on warming. When cooled, needles were promptly deposited

FIG. 4. Free lysine crystallized slowly from dilute alcohol. X 80.

on the glass at the surface of the fluid while the flocculent precipi-
tate slowly crystallized into tiny hexagonal plates at the bottom
of the flask. Lysine therefore deposits crystals of two apparent
types from alcoholic mother liquors. In the presence of much
alcohol, which facilitates rapid crystallization, tiny needles form.
From a somewhat lessstrongly alcoholic mother liquor, crystalliza-
tion is slower and well formed hexagonal plates, often occurring
in small piles of diminishing size, separate. Examination of these
plates under the microscope showed that some of them are formed
of aggregates of needles.
 H. B. Vickery and C. S. Leavenworth 443

SUMMARY.

Free lysine, when adequately purified and protected from car-

bon dioxide, crystallizes from a concentrated aqueous solution in
needles which aggregate into striated irregular plate-like forms or
into radiating bunches. The crystals decompose at approxi-
mately 224’ and possess a specific rotation of [C-K]: = +14.6”.
Lysine may also be crystallized in tiny needles or in hexagonal
plates from slightly diluted, purified, warm alcohol. It is ex-
tremely soluble in water but practically insoluble in cold absolute

Downloaded from http://www.jbc.org/ by guest on February 19, 2018

alcohol.
Pure lysine picrate decomposes with a characteristic explosion
at approximately 266’.
A NOTE ON THE CRYSTALLIZATION
OF FREE LYSINE
Hubert Bradford Vickery and Charles S.
Leavenworth
J. Biol. Chem. 1928, 76:437-443.

Access the most updated version of this article at

Downloaded from http://www.jbc.org/ by guest on February 19, 2018

http://www.jbc.org/content/76/2/437.citation

Alerts:
• When this article is cited
• When a correction for this article is posted

Click here to choose from all of JBC's e-mail

alerts

This article cites 0 references, 0 of which can be

accessed free at
http://www.jbc.org/content/76/2/437.citation.full.ht
ml#ref-list-1