ARTICLE:

A SIMPLIFIED PROCEDURE FOR THE

ISOLATION OF LYSINE FROM
PROTEIN HYDROLYSATES

Access the most updated version of this article at

http://www.jbc.org/content/131/1/1.citation
Find articles, minireviews, Reflections and
Classics on similar topics on the JBC Affinity
Sites .
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/131/1/1.citation.full.html
#ref-list-1

Downloaded from http://www.jbc.org/ by guest on November 11, 2014

Eldon E. Rice
J. Biol. Chem. 1939, 131:1-4.

A SIMPLIFIED
LYSINE

PROCEDURE
FOR THE ISOLATION
FROM PROTEIN
HYDROLYSATES
BY

(From

the Ilivision

ELDON

E.

OF

RICE

of Biochemistry,
Noyes Laboratory
versity
of Illinois,
Urbana)

of Chemistry,

Uni-

(Received for publication, July 13, 1939)

EXPERIMENTAL

Three 4 liter (4.4 kilos) lots of blood corpuscle paste were refluxed for 24 hours, each with 4 liters of 50 per cent (by volume)
sulfuric acid. The sulfate ions in two of these hydrolysates
were
1

Downloaded from http://www.jbc.org/ by guest on November 11, 2014

A method has been developed for the preparation of lysine from

protein hydrolysates
by a direct precipitation
of the amino acid as
the picrate.
The procedure greatly reduces the time required
for the isolation of lysine and eliminates the electrolysis which is
an essential part of the method of Cox, King, and Berg (1929).
After one or two recrystallizations
the lysine picrate obtained by
direct precipitation
decomposes at 263-266, which is the same as
t,he decomposition
point of the product isolated by the more
elaborate procedure.
Moreover,
the yield and quality of the
lysine monohydrochloride
prepared by the proposed method are
Histidine
can be
as high as those obtained after electrolysis.
separated as a by-product
in both procedures.
The method is
adaptable to hydrolysates
that have been neutralized with calcium
hydroxide as well as those that have been treated with baryta.
The greater ease of removal of t,he calcium sulfate by filtration, as
well as the low cost of calcium hydroxide, makes this the more
practical way of preparing a neutral hydrolysate
if large quanbities
of lysine are needed.
Triplicate lots of blood corpuscle paste have been used for the
preparation of lysine monohydrochloride
by the method of Cox,
King, and Berg (1929), and by two modifications
of the direct
procedure.
The results are outlined below.

Isolation

of Lysine

CsH,4N202~HC1.

Calculated,

N 15.33;

found,

N 15.30

Lot B was treated directly with 150 gm. of solid commercial

picric acid, and heated with frequent shaking on a steam cone

Downloaded from http://www.jbc.org/ by guest on November 11, 2014

removed with exactly the requisite quantity of barium hydroxide,

as in the procedure of Berg and Rose (1929), and the filtrate was
concentrated in vacua to 3 liters. The insoluble amino acids which
separated during the concentration
were removed by filtration.
The syrupy liquid remaining, though free of barium ions, was definitely alkaline to litmus.
It was divided into two equal parts,
hereafter referred to as Lot A and Lot B.
The third hydrolysate
was diluted to 17 liters, and neutralized
by the addition of 2700 gm. of calcium hydroxide suspended in 8
liters of water.
The filtrate and washings from the calcium sulfate were concentrated in vacua to a volume of 8 liters.
Calcium
hydroxide was then carefully added until the solution was alkaline
to litmus.
After the solution was filtered again, the hydrolysate
was concentrated
in vacua to a volume of 1500 cc., and allowed
to stand overnight in an ice box. The insoluble amino acids which
separated were removed by filtration.
The syrup which remained
will be referred to hereafter as Lot C.
From Lot A lysine dihydrochloride
was isolated and recrystallized exactly as described by Cox, King, and Berg (1929).
This
product was dissolved in the minimum volume of boiling alcohol
and converted into lysine monohydrochloride
by treatment with
pyridine in absolute alcohol (45 gm. of pyridine for each 100
gm. of lysine dihydrochloride).
After standing overnight in an
ice box, the crude monohydrochloride
was collected on a Buchner
funnel, and washed with a little absolute alcohol, and then with
ether.
Purification was accomplished by slowly adding 3 volumes
of alcohol to a solution of the material in an equal weight of
water.
The alcohol must be added very carefully in order to
promote crystallization.
On the other hand, unless the addition
is complete within about 10 minutes, some lysine monohydrochloride dihydrate
precipitates.
After refrigeration
for 12 hours the final product was collected
on a filter, washed with absolute alcohol and ether, and then
dried for 4 hours at 50 in a vacuum oven. The yield of lysine
monohydrochloride
was 54.0 gm.

E. E. Rice

C~HUN~O~.HCI.

Calculated,

N 15.33;

found,

N 15.42

Lot C, which had been neutralized with calcium hydroxide, was

treated in the same manner as Lot B. It yielded 55.2 gm. of
lysine monohydrochloride.
CsHr,NzOz.HCl.

Calculated,

N 15.33;

found,

N 15.26

Two similar series of isolations, comparing results by the three

methods, were carried out. These runs agreed closely in all respects with those reported above. The average yield of pure
lysine monohydrochloride
obtained by each of the different
procedures was by the method of c/ox, King, and Berg 58.4 gm.,
by the direct method (neutralization
with baryta) 57.9 gm., by
the direct method (neutralization
with calcium hydroxide) 55.6 gm.
Each sample of the lysine monohydrochloride
was analyzed in
duplicate by the Van Slyke procedure, and in no case did the
amount of nitrogen found differ from the theoretical by more than
0.10 per cent. Each of the products gave negative Sakaguchi
1 2 kilos of sodium
chloride
(Gilson,
1938) were added
to this solution
and the histidine-mercury
complex
was then precipitated
by the method
of Foster
and Shemin
(1938).
This material
was converted
into histidine
dihydrochloride
by the procedure
outlined
by Hanke
and Koessler
(1920).
The yield was 81.9 gm. of analytically
.pure product.

Downloaded from http://www.jbc.org/ by guest on November 11, 2014

at 50 until all of the solid had dissolved.

The lysine picrate
which precipitated as the solution cooled (12 hours in a refrigerator) was collected on a 20 cm. Buchner funnel and washed twice
with 200 cc. portions of cold water.
The mother liquor was set
aside for the preparation of histidine.
A solution of the precipitate in 2500 cc. of water was boiled for 5 minutes with 70 gm. of
norit and then filtered through a hot Buchner funnel.
As the
filtrate cooled, a solid mass of bright yellow lysine picrate formed.
Since the decomposition point of this material was 261, the product was used in the next step without
further purification.
In
other runs the picrate obtained at this point decomposed at 255260, but an additional recrystallization
invariably yielded a picrate with the correct
decomposition
point (265-266).
The
lysine picrate obtained from this lot was converted into the monohydrochloride
in the manner previously
described.
The yield
was 57.4 gm.

Isolation

of Lysine

SUMMARY

A procedure for the

chloride from protein
been described.
This
equipment, and greatly
tion of the amino acid.
as high as those of lysine
Histidine dihydrochloride
hydrolysate.

preparation
of pure lysine monohydrohydrolysates
by direct precipitation
has
method eliminates the need for special
reduces the labor involved in the isolaThe yield and quality of the product are
obtained by more complicated procedures.
may be readily prepared from the same

BIBLIOGRAPHY

Berg, C. P., and Rose, IV. C., J. Biol. Chem., 82, 479 (1929).
Cox, G. J., King,
H., and Berg,
C. P., J. Biol. Chem., 81, 755 (1929).
syntheses,
Foster,
G. L., and Shemin,
D., in Fuson,
R. C., et al., Organic
New York,
18, 43 (1938).
Gilson,
L. E., J. Biol. Chem., 124, 281 (1938).
Hanke,
M. T., and Koessler,
K. K., J. BioZ. Chem., 43, 521 (1920).
Hunter,
G., Biochem. J., 16, 637 (1922).
Jorpes,
E., Biochem. J., 26, 1504 (1932).

Downloaded from http://www.jbc.org/ by guest on November 11, 2014

tests for arginine (Jorpes, 1932). The samples of lysine monohydrochloride

prepared from twice recrystallized
lysine picrate
gave negative tests for histidine (Hunter,
1922). On the other
hand, lysine monohydrochloride
prepared from picrate that had
not been recrystallized
twice gave positive tests by the Hunter
method, regardless of the method of preparation.
In view of
this fact lysine picrate should always be recrystallized
at least
twice regardless of its decomposition
point.
Lysine can also be prepared from dried blood fibrin by the direct
method of precipitation.
In demonstrating
this fact, three 2.5
kilo lots of ground fibrin were hydrolyzed
by refluxing for 16
hours, each with 8 liters of 25 per cent (by volume) sulfuric acid.
The resulting solutions were neutralized
with baryta, concentrated, and treated with 130 gm. of picric acid per lot. The
picrates were converted int,o the final product as previously
described, and yielded an average of 46.5 gm. of analytically
pure
lysine monohydrochloride.
Two additional hydrolysates
which
had been neutralized with calcium hydroxide yielded an average
of 44.5 gm. of the pure product.
33.2 gm. of lysine monohydrochloride were obtained from 2.5 kilos of commercial casein. An
attempt to prepare lysine from gelatin was unsuccessful.