SOME PRODUCTS OF PARTIAL HYDROLYSIS OF

SILK FIBROIN*
BY R. LORIMER GRANT AND HOWARD B. LEWIS
(From the Department of Physiological Chemistry, Medical School, Uni-
versity of Michigan, Ann Arbor)

(Received for publication, December 5, 1934)

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The chemistry of silk fibroin and of the various products of its
hydrolysis has been the subject of extensive investigation by Ab-
derhalden and his coworkers (1). Interest has been centered
around the “silk peptone” of Abderhalden and Steinbeck (2) and
the diketopiperazines, which Abderhalden believes to be primary
products of the hydrolysis of silk fibroin. Uchino (3) has studied
recently the products of partial degradation of fibroin by various
hydrolytic agents and has interpreted his data as evidence against
the existence of the diketopiperazines as primary products of hy-
drolysis.
The purpose of the present investigation was the study of the
composition and properties of peptones prepared from silk under
various conditions of hydrolysis. It was hoped that, from the
products of incomplete hydrolysis of silk, it might be possible to
obtain substances of widely different composition, which would
be of value in the study of the problems of structure.
EXPERIMENTAL

The material used for hydrolysis was silk waste of the grade
designated in commerce as No. 6 short nails,’ containing 4.73 per
* A preliminary account of this investigation was presented before the
Twenty-eighth meeting of the American Society of Biological Chemists
at New York, March 28-31, 1934 (Lewis, H. B., and Grant, R. L., J. Biol.
Chem., 106, Iii (1934)).
1 This product has been degummed to remove the sericin. We were
unable to extract any protein by prolonged treatment with boiling water
from the noils used in our experiments. We wish to express our indebted-
ness to the Cheney Brothers of South Manchester, Connecticut, for their
courtesy in supplying the silk used in these experiments.
667
668 Silk Peptones
cent moisture, 0.50 per cent ash, and 0.33 per cent of sulfur.
Other analyses of the silk are given in Table I.
Two methods were used for the determination of amino nitrogen,
the gasometric method of Van Slyke and the titration method of
Harris (4), a method by which it was possible to estimate carboxyl
groups also. Tyrosine was determined by the modified Folin-
Ciocalteau method suggested by Folin and Marenzi (5) and gly-
tine as the ester hydrochloride.

TABLE I

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Analyses of Silk Peptones Prepared by Method of Abderhalden and Steinbeck
(2) but with Varying Periods of Hydrolysis
For each hydrolysis, 10 gm. of silk were used. All values are calculated
on an ash-free moisture-free basis.
-
Amino N of total N
Yield Total N Tyrosine
Van Slyke Harris
._
Qm. per cent per cent per cm: per cent
Silkt 18.0 12.3
2-l 5.6 18.0 19.6 17.5 10.9
2-2 6.4 17.5 19.5 17.9 11.0
4-l 6.0 17.6 25.8 21.6 11.0
4-2 6.3 17.2 25.1 21.3 11.2
7-l 6.5 16.9 33.7 29.3 10.6
7-2 7.7 16.7 33.9 29.9 11.0
14-1 6.8 16.7 46.0 40.1 10.5
14-2 6.9 16.5 45.9 38.5 10.6
21-1 16.0 64.1 61.8 10.1
* The figures before the hyphen represent the duration in days of the
hydrolysis; thus Preparations 2-l and 2-2 are duplicate preparations which
were obtained after hydrolysis at room temperature for 2 days.
t Glycine, 31.6 per cent.

In five preparations of silk peptones, made by the method of

Abderhalden and Steinbeck (2), in which hydrolysis is effected by
70 per cent sulfuric acid at room temperature for 4 days, the nitro-
gen content ranged from 17.0 to 17.8 per cent; free amino nitrogen
(Van Slyke), from 18.9 to 25.3 per cent; tyrosine, from 9.1 to
10.3 per cent.
2 We have used the term peptone for convenience in referring to the
products obtained throughout with full realization that the products are
probably not homogeneous and certainly not uniform.
 R. L. Grant and H. B. Lewis 669
Peptones were also prepared similarly from the hydrolysates of
silk, the variable factor being the time of hydrolysis (2 to 14 days).
The analyses of the peptones prepared in this series are presented
in Table I. It is to be noted that as t,he period of hydrolysis at
room temperature increased, there was a progressive decrease in
the percentage of total nitrogen of the peptone and an increase in
the percentage of the total nitrogen present as free amino nitrogen
in the peptone. Table I also includes an analysis of a peptone
prepared from a 21 day hydrolysate. This product, analyzed by
the Van Slyke method, showed 64.1 per cent of the total nitrogen

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as free amino nitrogen. This high figure can best be explained by
the presence of free amino acids precipitated by the alcohol with
the peptone. However, all the free amino nitrogen values as
measured by this method may be too high since Abderhalden and
Van Slyke (6) have demonstrated that abnormally high results
for amino nitrogen may be obtained in the Van Slyke gasometric
procedure, if the free amino group is present in a glycine molecule
in peptide combination with glycine or other amino acids. Since
we do not know the exact composition of these peptones nor the
relative proportion of the free amino groups which may be present
in the glycyl radicals, it is impossible to estimate the magnitude of
any error involved in the Van Slyke determinations. That some
error is introduced into the Van Slyke method by the high content
of glycine of the silk peptones is indicated by the lower values for
amino nitrogen, as determined by the Harris method.
The tyrosine content of the peptones prepared after varying
periods of hydrolysis appeared to have no significant relation to
the duration of the hydrolysis. All the values, however, were
definitely lower than that of the original silk.
It appeared that the hydrolysis had progressed to the point
where fractionation of the peptones by simple methods was im-
possible. Accordingly, an attempt was made to find conditions
under which the products of hydrolysis might be separated into
fractions which would vary in composition. A product was ob-
tained after a much shorter period of hydrolysis than those previ-
ously employed, and separated into two fractions of widely differ-
ent tyrosine content. Since such a preparation and fractionation
has not been reported previously, the detailed procedure is given.
100 gm. of dry silk, from which all lumps had been removed by
pulling apart with the hands, were packed into a compact mass
670 Silk Peptones

in a 2 liter flask, 200 cc. of 70 per cent sulfuric acid were added,
and the flask was incubated at 30”. After 70 minutes, practically
all of the silk was in solution and the brown syrup was poured into
2 liters of ice water. A small amount of a gummy precipitate was
filtered off and discarded. The sulfuric acid was removed as the
insoluble barium salt and the clear light yellow filtrate and wash-
ings were concentrated at 40’ under diminished pressure to ap-
proximately 300 cc. After standing overnight in the refrigerator,
the syrup solidified.
The grayish white solid mass was ground with water in a mortar

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and transferred to a beaker with 1500 cc. of water. A consider-
able part of the material remained undissolved and the mass ap-
peared somewhat gelatinous. The mixture was centrifuged and
the insoluble fraction (Preparation 70-A) was washed several
times with water and twice with alcohol before it was removed
from the centrifuge cups and placed in 95 per cent alcohol. On
the following day, it was filtered off on a Buchner funnel, washed
with alcohol and ether, and dried in a vacuum oven at 60” before
analysis. The yield (corrected for ash and moisture) was 22.6 gm.
The supernatant fluid and all the washings from Preparation
70-A were combined and concentrated as before to 100 cc. The
syrup thus obtained was poured into 10 volumes of absolute alcohol
and a precipitate (Preparation 70-B) was obtained in a yield of
26.8 gm.
The less soluble fraction (Preparation 70-A) was a grayish white
powder, practically insoluble in cold water and dilute acids,
slightly soluble in hot water, and readily soluble in dilute alkali.
When dispersed in lithium bromide solution, it dialyzed readily
and completely through a collodion. membrane (7). This was in
contrast to the behavior of the original silk when similarly dis-
persed. Immunologically it was distinct from the product ob-
tained from the original silk (7). The more soluble fraction
(Preparation 70-B) was partially soluble in cold water. Both
products gave brilliant pink biuret tests. Analytical data are
presented in Table II.
In a second experiment, essentially the same procedure was
used except that the time of hydrolysis was 65 minutes. Two
similar fractions (Preparations 65-A and 65-B) were obtained. In
a third experiment by a slight variation of the procedure, three
 R. L. Grant and H. B. Lewis 671
fractions were obtained. The period of hydrolysis was 70 min-
utes at 30”. After removal of the acid and the excess barium,
the concentrated filtrate required 7 days in the refrigerator before
it solidified. The insoluble fraction (Preparation 70-A-2) was
separated, as already described. The filtrate after concentration
to 100 cc. was allowed to stand in the refrigerator for 24 hours. A
granular yellow precipitate was filtered off, washed with alcohol
and ether, and dried (Preparation 70-X). The filtrate and wash-

TABLE II

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Analysis of Silk Peptones Obtained after Short Periods of Hydrolysis and
Fractionated As Described in Text
The yields are expressed as gm. of ash-free moisture-free product ob-
tained from 100 gm. of silk. All chemical analyses are calculated on a
moisture-free ash-free basis.
-

Yield ‘I ‘otal N ryrosine Xycine

T Percentage equivalent of total N aa
-7

Amide 1hino N Car- Tyro-

N (Harris) boxy1 sine N
( HkWiS)
_ - . - --
am. 1,er cent per cent 7er cent
70-A 22.6 18.4 19.1 3.0* 3.2 8.0
70-B 26.8 18.9 5.7 10.7t 10.7 2.3
65-A 49.3 18.0 14.9 2.9 3.6 6.4
65-B 14.0 18.5 4.1 14.1 14.4 1.7
70-A-Q 27.4 18.4 17.9 30.5 1.9 1.7 2.7 7.5
70-x 11.8 18.8 8.4 33.0 2.4 1.8 3.0 3.4
70-B-2 10.6 18.3 2.5 31.0 4.1 17.1 19.1 1.0
-
* Amino nitrogen by the Van Slyke method, 3.0 per cent.
t Amino nitrogen by the Van Slyke method, 11.0 per cent.
1 Carbon, 49.0 per cent; hydrogen, 6.3 per cent; nitrogen (Dumas), 18.3
per cent. We are indebted to Mr. N. H. Fell for these analyses.

ings from this fraction were concentrated and poured into absolute
alcohol, from which the precipitate (Preparation 70-B-2) was fil-
tered off, washed, and dried as before.
From inspection of Table II it is evident that the more insoluble
fraction contained in each case more tyrosine than the original
silk (12.3 per cent), while in the more soluble fractions, the tyro-
sine content was low, comparable to that of the usual type of pro-
teins. Striking differences are also to be noted in the free amino
and carboxyl groups. Little free amino nitrogen was present in
672 Silk Peptones

the less soluble fractions, indicating that relatively few peptide

linkages have been broken by the acid, while in the more soluble
fractions, the higher content of free amino groups indicated a
considerable degree of hydrolysis.
Glycine determinations were made on the fractions of the third
experiment, Preparations 70-A-2, 70-X, and 70-B-2. The results
of the analyses, 30.5, 33.0, and 31.0 per cent respectively, failed
to demonstrate marked variations. in the glycine contents of these
fractions. All the glycine values were approximately the same as
that of the silk, 31.6 per cent.

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It is evident that the silk has been separated into fractions of
such complexity that typical biuret tests are obtainable, but of
widely different tyrosine content. In the case of Preparations
70-A and 70-B, 47 per cent of the tyrosine content of the original
silk was contained in the peptones isolated. Of this, approxi-
mately 74 per cent was present in the more insoluble fraction.
Similar results may be calculated in the case of the other fractions.
Such separations have never been obtained with silk proteins, so
far as is known to us. Abderhalden (1) has isolated from prod-
ucts of the hydrolysis of silk, a number of amino acid anhydrides,
the diketopiperazines. We have been unable to obtain evidence
of the presence of these in our products, since treatment with
ethyl acetate, a solvent for the common piperazines isolated from
silk, did not cause the extraction of any significant amount of
material from Preparations 70-A-2, 70-X, and 70-B-2.
Muldrew (8) has fractionated the products of peptic hydrolysis
of casein into a primary proteose containing neither tyrosine nor
tryptophane and a secondary proteose rich in both amino acids.
Jones and Gersdorff (9) have similarly obtained three fractions
from a peptic digestion of casein, in two of which cystine is absent.
While neither these produc& nor our own can be considered to
be definite chemical compounds rather than mixtures, by this
method it has been possible to concentrate one particular amino
acid in one fraction. In this connection, the comment of Hunter
(8) on the work of Muldrew is of interest. “It is an obvious ad-
vantage in dealing with a highly complex combination of amino
acids to be able thus to localize even two of them in one particu-
lar fragment of the whole.”
Further work on the chemical and biological properties of the
 R. L. Grant and H. B. Lewis 673
products of the partial hydrolysis of the proteins of silk is in
progress.
SUMMARY

Silk has been subjected to hydrolysis at 30” with 70 per cent

sulfuric acid for 65 to 70 minutes and the products of hydrolysis
studied. It has been possible to obtain two peptones of widely
different composition. The first had a low amino nitrogen con-
tent and a content of tyrosine higher than that of the 0rigina.l
silk. The second contained more amino nitrogen (10 to 17 per

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cent of the total nitrogen) and tyrosine in amounts similar to
many of the more common types of proteins (2.5 to 5.7 per cent).

BIBLIOGRAPHY

1. Cf. Abderhalden, E., 2. physiol. Chem., 120, 207 (1922) for recent, com-
plete analysis of silk fibroin; also Abderhalden, E., and Komm, E., 2.
physiol. Chem., 134, 121 (1924); 139, 147, 181 (1924).
2. Abderhalden, E., and Steinbeck, E., Z. physiol. Chem., 68, 312 (1910).
3. Uchino, T., J. Biochem., Japan, 20, 65 (1934).
4. Harris, L. J., Proc. Roy. Sot. London, Series B, 96, 500 (1923-24).
5. Folin, O., and Marenzi, A. D., J. Biol. Chem., 83, 89 (1929).
6. Abderhalden, E., and Van Slyke, D. D., 2. physiol. Chem., 74,505 (1911).
7. Fell, N. H., and Lewis, H. B., unpublished data.
8. Muldrew, A., unpublished data, cited by Hunter, A., Tr. Roy. Sot.
Canada, sect. 5, 19, 1 (1925).
9. Jones, D. B., and Gersdorff, C. E. F., J. Biol. Chem., 106, 707 (1934).
 SOME PRODUCTS OF PARTIAL
HYDROLYSIS OF SILK FIBROIN
R. Lorimer Grant and Howard B. Lewis
J. Biol. Chem. 1935, 108:667-673.

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