Unpublished
The Separation of the Substances in Liver which are Reticulocytogenic
in the Guinea Pig.
II. The Isolation of a Peptide Fraction*
Y. Subbarow and Bernard M. Jacobson
(From the Biochemical Laboratory, Harvard Medical School, and the. Medical
Clinic, Macsachusetts General Hospital, Boston)
* This investigation has been supported. by grants from the DeLamar Mobile
Research Fund and the Proctor Fund, of Harvard Medical School; by
Therapeutic Research Grants Nos. 206 and 244 of the Council on Pharmacy and
Chemistry of the American Medical Association; and by a grant from the Lederle
Laboratoeies, Inc.
Kept on the alkaline side does not exceed 10 minutes.) This elute contains 4.3 mg.
of total nitrogen, and approximately 25,000 g.p.u. of biological activity, per 100
gm. of liver. To the acidified elute is added 0.3 gm. of norit charcoal, for every 100
gm. of liver, the mixture stirred 60 minutes, filtered, and after the adsorbate is
washed several times until the washings are free of chloride the charcoal is
eluted 3 times with hot 60 per cent alcohol, each time with 10 c.c. for each
100 gm. of liver. The combined elutes are concentrated in vacuo to a volume of 0.25
c.c. per 100 gm. of liver. At this point the solution is light brown in color, contains
only 2.2 mg. of total nitrogen per100 gm. of liver, and exhibits the same biological
activity shown prior to the charcoal adsorption. Three procedures may now be
followed.
(I) To the solution is added an equal volume of saturated picric acid.
An oily
precipitate is immediately brought down, which on standing in the refrigerator overnight
settles out as syrup.
The supernatant fluid is then removed, and the syrup is
thoroughly stirred with anhydrous ether, in order to remove the excess picric acid. The
first two ether washings are discarded. The remaining material is light yellow in color.
After drying the precipitate is obtained in a yield of 8 mg. from 100 gm. Of fresh liver,
with a biological activity of approximately 19,000 g.p.u. hen examined microscopically
the material is seen to consist of a mixture of several variously shaped types of flaky
particles.X -ray patterns demonstrate it to consist principally of amorphous.
(II) To the solution are added 20 volumes of a mixture of equal volumes of 95 per
cent ethyl alcohol and ether. After remaining in the refrigerator for 2 days the
precipitate is filtered off. This precipitate contains 1.4 mg. o f total nitrogen
and exhibits an activity of approximately-20,000 g.p.u. The precipitate is
dissolved in water at a concentration of 0.25 c.c. Per100 gm. of liver, and from
this point procedure A (I) is followed.
The yield of picrate is the same as th at
obtained by procedure A (I).
(III) The solution of the alcohol-ether precipitate (procedure A (II))'is it
acidified to CHO3 and diluted to a concentration of 5 c.c. Per100 gm. of liver. To
each 5 c.c. are added 2 c.c. of a saturated solution of Reinecke salt; a heavy
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granular precipitate comes down immediately.
When the suspension is gently
warmed to 45-50 C the precipitate goes into solution, with the exception of a small
amount of an oily brown residue. The supernatant fluid is poured off. 2 c.c. of
water, for every 100 gm. of liver, are added to the residue, the mixture
warmed to 450 C and the solution is combined with the above supernatant fluid.
The combined solutions are kept at room temperature for 30 minutes, and then in
the refrigerator overnight. The resultant precipitate is made up of well-formed
needles. The yield is mg. of Reineckate from 100 gm. of liver.
The ethyl alcohol solution is brought to a concentration of 2 c.c. Per100
gm. of liver. To it are added 20 volumes of 95 per cent ethyl alcohol and 20
volumes of ether. After 48 hours in the refrigirator the precipitate is filtered
off, and is a dissolved in a volume of 10 c.c. of water for every 100 gm. of
liver. At this stage the solution is dark brown in color, contains about 5.5 mg.
of total nitrogen, and exhibits an activity of approximately 35,0.0 g.p.u.
per 100 gm. of liver. To the solution is added 0.5 gm. Of worit charcoal for every 100
gm. of liver, and after 1 hour of occasional stirring the charcoal is filtered off,
washed with water., and is eluted 3 times, each time with 10 c.c. of 60 per
cent ethyl alcohol, per 100 gm. of liver, at 80 for C for 5 minutes. The combined
elutes are concentrated in Vacuo at 40 C to a concentration of 0.25 c.c. per 100 gm. of
liver.
At this point the solution is light brown in color contains 2.9 mg. of total
nitrogen per 100 gm. of liver. ‘Any one of the throe procedures of method A may now be
appled to the solution. The yields of picrate and of Reineckate are the same as those of
obtained by means of method A.
Liethod C.
The ethyl alcohol solution is concentrated to 2 c.c. per 166 gm. Of liver.200 c.c. of
the concentrate (derived from 10 kgm. Of liver) are acidified to pH 3, warmed to 40 C,
and to the warm solution are added 10 gm. Of solid Reinecke salt; the salt is Egitated
ntil it completely dissolves. 1 c.c of In HCL is then added to the solution. After
remaining the refrigirator for 48 hours the precipitate is filtered off, and is washed with
ice-cold water until the washings are nearly colorless. The precipitate is then treated
with 1 liter of 40 per cent alcohol, and the insoluble residue is filtered off and
discarded. To the alcoholic filtrate are added 15 c.c of dimethylaniline (3), followed by
mechanical stirring for 15 minutes. Subsequently 800 c.c. of water are added, and the
mixture is strires again for 10 minutes, and is-then kept in the refrigirator overnight.
The precipitate is lts red off by suction and is discarded. The filtrate is concentrated in
vacuo at 40C.
In the separution from liver of the factor or factors which are therapeutically
effective in pernicious anemia we have used us a biological tost method the
reticulocyte response of guinea pigs (l). The separation of the materials in liver,
which are reticulocytogenic in the guinea pig, is thus. Far only partially complete. It has
already been reported (2) that of the 528,000 guinea pig units (g.p.u.), per 100 gnu of
fresh liver, contained in the crude liver extract used as a starting point in the
separation, 48,000 g.p.u. have been isolated in pure form as tyrosine, and
140,000 g.p.u. Have been separated in pure form as a complex purine.
The
present communication describes in detail the isolation of approximately 19,000 g.p.u.
in the form of a peptide. A complete study of the properties of the peptide will be
presented in a subsequent communication. The starting point in the isolation of the
peptide is the filtrate of the ethyl alcohol elute remaining after the removal of the
73
fraction C (2). This solution contains l3.3 mg. of total nitrogen and a biological
activity of 55,000 g.p.u.,. per 100 gnu of fresh liver from which the extract was
derived.
Three alternative methods of separating the peptide fraction from this
solution are given below.
A.
The solution is diluted so that 4 c.c.represents the material from obtained
from 100 gm. of fresh liver, and is brought to pH 2 with HC1. For each 100 gm.
of liver 0.5 gnu of English Puller's earth is added, the mixture stirred for 35 to
45 minutes, and filtered.
The adsorfeate is washed until the washings are
colorless, and is then suspended in water, 5 c.c. For each 100 gm. of liver. To each 5
c.c. of the suspension is added 1 c.c. of 0.45 N Na OH, the mixture stirred for
5 minutes, filtered rapidly, and the liltrate is promptly brought to pH 6 with
HCL. (The total length of time during which the solution is to a volume of 400 c.c. Tho
concentrate- is transferred to a sepratory funnal and is shaken with 500 c.c. of amyl
alcohol.
The extraction is repeated until the amyl alcohol layer is colorless.
Tho
solution is concentrated to a volume of 50 c.c, and to it are added.10 volumes of 95
per cent ethyl alcohol and 10 volumes of ether. After standing in the refrigirator
for 48 ours the precipitate is filtered off. The yield of precipitate is 1.2 gm. from 10
kgm. of fresh liver ( 12 mg. From100 gm. of liver); it exhibits an activity of
35,000 g.p.u. per 100 gm. of liver. The precipitate is then converted to the picrate,
as described in method A. The yield of picrate is the same as that obtained by
method A.
Properties of the picrate.
The picrate, when examined microscopically, is seen to consist of several variously
shaped types of flaky particles. X-ray patterns demonstrate principally amorphous
material.
Composition: C 44.4, H 5.4, N 17.1, S 4,2. The picrate is slightly soluble
in cold water, readily soluble in hot water, but often in water forms a gummy
substance. It is partially soluble in hot ethyl and methyl alcohols. After regeneration of
the picrate in acid solution with ether and subsequent removal of the acid by
concentration the material gives c biuret and diazo reactions, but does not give
glyoxylic or Millon's reactions. The peptide is not precipitated by sulphosalicylic acid,
but is brought down by by trichloracetic acid in excess, by saturated ansmonium
sulphate, and by tannic, phosphotungstic, picrolonic, and flavianic acids.
Discussion
There has been described above the isolation in relatively pure form of a poptide,
from a liver extract that is therapeutically effective in pernicious anemia. The
description of the biological action of this peptide has been limited to its
reticulccytogenic efrect in the guinea pig. The possible role of this material in the
therapeutic effect of liver in pernicious anemia will be reserved for another
communication. Peotinent, however, Jr. this connection is the observation of
Cohn, McMeekin, and Minot (4) that picric acid precipitates of liver extract have
been therapeutically efrective in pernicicus anemia, in one case following the
parenteral administration of 140 mg. of the regenerated picrate. A similar
observation was later made by Dakin and West (3); in the latter instance a dose of
150 mg. proved therapeutically notent. On the other hand, the properties of the picrate
desribed an this paper differ in many respects from those reported by the above
authors.
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Summary
From a liver extract that is therapeutically effective in pernicious anemia there has
been separated in pure form a peptide, as a picrate in a yield of 0 mg., and as a
Roineckate in a yield of 10 mg., respectively, from 100 gm. of fresh liver. The picrato
biologiceal aetoon reticulocytogenic action in the guinea pig of approximately 19,000
g.p.u.
The poptido contains 4? per cont of sulphur, gives biurot and diaao
roactions, but does not give glyoxylic or Millon's reactions.
Bibliography
1.
2.
3.
4.
Jacobson, B.M., J. Clin. Investigation,14, 665, (1955).
Subbarow;, Y., Jacobson, B.M., and Fiske, C.H., New Eng. J. Med.,21
Cohn, E.J., MoMeekin, T.L., and Minot, G.R., Trans. Ass. of An. Phys., 45, 543
(1930)
Dakin, H.D., and West, R., J. Biol. Chem.,109, 489, (1955).
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