ENZYMATIC HYDROLYSIS OF GLYCOGEN.

BY A. D. BARBOUR.
(FTom the Department of Biochemistry, University of Toronto, Toronto,
Canada.)

(Received for publication, August 26, 1929.)

The products of the enzymatic hydrolysis of glycogen have been

assumedin the past to be maltose and glucose arising from inter-
mediate dextrins and maltose. Although the disappearance of
glycogen under the action of pancreatic amylase has been studied

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carefully by Norris (1) and the effect of salivary amylase has been
investigated by Rona and Van Eweyk (2), there has apparently
been no published account of an attempt to isolate or identify the
product. It has been usually assumedto be maltose. Meyerhof
and his collaborators (3) studied the production of lactic acid in
digests of glycogen with ground frog muscle or muscle extracts,
and Lohmann (4) has described the formation of a sugar in these
digests which he believes to be similar to, or identical with, the
“amylotriose” which Pringsheim (5) obtained as a product of the
breakdown of glycogen and amylopectin with concentrated
hydrochloric acid.
The importance of glycogenolysis merits a more detailed study
of the process. An attempt was therefore made to obtain further
information concerning the phenomenon from a study of the
enzymatic hydrolysis in vitro. The first step was the preparation
of glycogenase under standard conditions. Some of the factors
controlling the hydrolysis were then studied, including an ex-
ploration of the possibility of reversion, and finally the end-
products with different enzymes were identified.
An extract of rabbit muscle with glycerol was employed for the
hydrolysis, since this extract contains an enzyme which is a
probable factor in the normal breakdown of glycogen in the
animal organism. It was found that this glycerol extract did not
deteriorate during 6 months in the refrigerator, and was not in-
activated by 24 hours incubation at 37’.
29
30 Glycogen Hydrolysis
Stability of the Enzyme.
The enzyme was prepared by the method of Hunter and Dauph-
inee (6), the muscle being ground in a meat chopper as finely as
possible before extraction. To determine whether the enzyme
undergoes autodestruction, the following experiment was made.
A mixture of equal volumes of muscle extract and’ 0.25 M phos-
phate buffer of pH 6.3 was prepared. A 10 cc. sample was taken
immediately after mixing with buffer and incubated with 10 cc.
of 2 per cent glycogen for 23 hours. The enzyme mixture was
incubated for 48 hours at 37”. At intervals 10 cc. samples were
taken and incubated with 10 cc. of 2 per cent glycogen for 2+

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hours. The glycogen was determined in these digests before and
TABLE I.
St&L of Glycogenase at 37’ (zt :pH 6.1.
-
SampleNo. Period of incubation of Hydrolysis in 2$ ha
en5yme.
_-
bra. WC&
0 43
2 46
4 45
6 42
8 48
29 45
48 34
-

after incubation, with the results shown in Table I. There is no

autodestruction of the enzyme on incubation at 37’ for periods
up to 24 hours. A slight loss in potency is observed in 48 hours.

Preparation of Glycogen.
The glycogen used was prepared and purified by a modification
of the method of Pfliiger (7). The livers of healthy rabbits were
found to be the best source of material. The animals were well
fed with carrots, and about 4 hours later were killed by a blow on
the head. The livers were removed as quickly as possible, and
immediately covered with an equal weight of caustic potash
solution of sp. gr. 1.44 (42 per cent by weight KOH). The mix-
ture was heated for 3 hours on a boiling water bath, cooled, diluted
with an equal volume of water, and the glycogen precipitated by
 A. D. Barbour 31
the addition of sufficient 95 per cent alcohol to make the final con-
centration of alcohol 66 per cent. After standing for a short time,
the supernatant fluid was decanted from the sticky precipitate, and
the latter was dissolved in a quantity of water equal to the volume
of the original solution. This solution was heated to boiling and
filtered through a cotton or asbestos plug. The glycogen was
again precipitated with alcohol. This second precipitate, after
the supernatant fluid was decanted, was redissolved in the same
volume of water, and carefully adjusted with glacial acetic acid
to the turning point of phenolphthalein. 2 drops of glacial acetic
acid were added in excess, and the solution heated to boiling and

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filtered through paper to remove the brown flocculent precipitate
which separates. The filtrate was again heated to boiling and pre-
cipitated wit,h boiling alcohol. The supernatant liquid was de-
canted, the precipitate dissolved in cold water in which it is readily
and completely soluble, and precipitated with cold alcohol. This
last precipitate was washed by decantation with increasing con-
centrations of alcohol, collected on a Buchner funnel, washed
with absolute alcohol, and dried in an open dish over boiling water.
The product obtained in this way is ash-free, and consists of
99 to 100 per cent glycogen monohydrate (C&H,,OS.HzO),,, which
according to Slater (8) is the stable form of glycogen at ordinary
temperatures.

Identity of Glycogen from Di$erent Sources.

The question of the identity of glycogen from different sources
has been discussed at length by Norris (1) who describes experi-
ments showing differences in the rate of hydrolysis of glycogen
obtained from dog liver, rabbit liver, scallops, and yeast. In
order to ascertain whether glycogen from these different sources is
hydrolyzed at different rates under optimum conditions, the
following experiments were carried out with equal concentrations
of glycogen from scallops, rabbit liver, and rabbit muscle.
Solutions were prepared cont,aining 6 cc. of 1 per cent glycogen,
3 cc. of phosphate buffer, and 3 cc. of glycerol extract of liver.
After incubation for 1 hour, samples were taken and the protein
precipitated by the method of Folin and Wu (9). The sugar in
the filtrates was determined. The results, expressed as trisac-
charide, are shown in Table II.
32 Glycogen Hydrolysis
These results show that the rate of hydrolysis is approximately
the same for rabbit liver and rabbit muscle glycogen, and some-
what lower for scallop glycogen. The results also show that there
is no significant variation in the rate of hydrolysis caused by
changing the Cn+ during the 1st hour of incubation. Judged by
the criterion of rate of hydrolysis liver and muscle glycogen from
the same animal are identical, and scallop glycogen differs from
mammalian glycogen. The product of hydrolysis of scallop
glycogen with muscle glycogenase is, however, identical with that
of liver glycogen.
TABLE II.

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Hydrolysis at Varying Hydrogen Ion Concentrations with Liver Extract of
Glycogen from Di$erent Sources.

pH . . .. . . 6.0 1 6.2 1 6.4 1 6.6 / 6.8 j 7.0

Source of glycogen. Trisaccharide formed.

gm. gm. gm. gm. gm. gm.

Liver ...................... 0.146 0.155 0.155 0.165 0.165 0.161
Muscle ..................... 0.155 0.200 0.181 0.181 0.178
Scallop ..................... 0.116 0.132 0.113 0.113 0.100

Estimation of Glycogen.
The determination of glycogen was carried out by a modifica-
tion of the method of Pfluger (10) and Imamura (11). A con-
venient quantity for the determination is 5 cc. of solution. The
sample is treated with an equal weight of a solution of KOH of
sp. gr. 1.44, and heated on a boiling water bath for 3 hours under
an air condenser. The resulting mixture is treated with an equal
volume of water, and a sufficient volume of 95 per cent alcohol is
added to make the final concentration of alcohol 66 per cent.
The sample is then allowed to stand overnight to complete the
precipitation of the glycogen.
The supernatant liquid is decanted through a filter paper,
care being taken that as little as possible of the precipitate gets
on the paper. The precipitate is washed by decantation, once
with 20 cc. and then twice with 10 cc. of a saturated solution of
sodium chloride in 66 per cent alcohol. The precipitate is then
dissolved in hot water, in the same flask. The hot water is poured
in through the filter, in order to dissolve the last trace of glycogen.
 A. D. Barbour 33

The solution, which should be approximately 50 cc. is heated on

the water bath, under an air condenser, with the addition of 3~.
of concentrated hydrochloric acid, for 3 hours. It is then cooled,
a drop of phenolphthalein added, neutralized with concentrated
KOH, made slightly acid with HCl, and made up to 100 cc. in a
volumetric flask. The glucose was determined in the filtrate by
the modification of the Shaffer-Hartmann (12) method described
below, further dilution being made if necessary. From the glucose
content of this solution the glycogen content of the original solu-
tion can be calculated. To avoid confusion, the results of glyco-
gen determinations are expressed in terms of glucose, i.e., they

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are calculated as C6H120~.
TABLE III.
Comparison of Direct Hydrolysis and PJliiger Methods for the Estimation
o.f Glgcogen.

Solution No. Glycogen by direct Glyeoge~$ydPfliiger Difference.

hydrolysis.
-
per cent per cent per cent
0.0197 0.0199 +0.002
0.0091 0.090 -0.001
0.072 0.073 +0.001
0.052 0.052 0.000
0.033 0.030 -0.003
0.017 0.017 0.000

The accuracy and sensitivity of the method were determined as

follows: Solutions of glycogen of varying concentrations were
prepared. Samples were taken in triplicate and hydrolyzed at
once by hydrochloric acid. At the same time the glycogen was
determined in triplicate samples by Pfliiger’s method. The
average results are given in Table III.

ModiJication of the Xha$er-Hartmann Method.

The original method of Shaffer and Hartmann was found to be
unsatisfactory for very accurate work with small amounts of
material. Solutions containing less than 0.002 per cent of glucose
give no reduction with the reagent and only approximate results
are obtained with 0.004 per cent. By the use of a modified re-
agent, employed by Professor Shaffer and suggested by him, in
34 Glycogen Hydrolysis

which the potassium iodide is omitted, it is possible to estimate

accurately as little as 0.0005 per cent of glucose. The procedure
is identical with that of the original Shaffer-Hartmann method
except that after the samples are cooled, 5 cc. of 1 per cent potas-
sium iodide solution are added immediately before the addition of
the sulfuric acid.
The method of heating in open test-tubes was discarded owing
to the danger of reoxidation and also to difficulties in titration.
The solutions were heated in 100 cc. flasks, closed with a stopper
containing a Bunsen valve. Two types of valves were used;
the first, a glass tube with a very small opening covered by a ring

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of cigarette drainage tube; the second, a thick walled rubber tube
closed at the top, and with a 3 inch slit in one side. Both types
were equally satisfactory. A sugar-thiosulfate table was con-
structed, based on experiments in which known concentrations of
pure glucose prepared by the method of Hudson and Dale (13)
were used. The glucose content of solutions of glucose of unknown
concentration, determined by this method, always gave results
which were identical with those obtained by determinations of the
optical rotation.

Determination of Optimum Hydrogen Ion Concentration for

Hydrolysis.
Five mixtures were made, each containing 10 cc. of 5 per cent
glycogen solution, 5 cc. of enzyme, and 5 cc. of phosphate buffer.
Two controls were included containing 10 cc. of distilled water in
place of the buffer and enzyme. Toluene was added to prevent
bacterial decomposition. The mixtures were incubated at 37”
for 5 hours, after which the glycogen content was estimated and
the pH determined electrometrically. The results are shown in
Table IV.
The optimum hydrogen ion concentration for hydrolysis is at a
pH of approximately 6.3. This agrees closely with values ob-
tained for other amylases by Rona and Van Eweyk (2), by
Paechtner (14), and by Norris (1).
The velocity of the hydrolysis is fairly slow. With moderate
concentrations of glycogen, and at the optimum CH+, complete
hydrolysis is effected in about 15 hours.
 A. D. Barbour 35

Correspondence between Increase in Eleducing Power and

Disappearance of Glycogen.
During the study of the velocity of hydrolysis by muscle glyco-
genase,an exact correspondence was observed between the rate of
disappearance of glycogen and the rate of appearance of reducing
power of the digest. It was found that at any time during the
course of the hydrolysis, the reducing substance which had ap-
TABLE IV.
Eflect of Hydrogen Ion Concentration on Enzymatic Hydrolysis of Glycogen.
Sample No. Glycogen after Hydrolysis.
PH

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incubation.
per cent per cent
1 5.53 0.402 46
2 6.04 0.342 54
3 6.24 0.314 58
4 6.54 0.320 57
5 7.27 0.428 42
6 Control. 0.744
-
TABLE V.
Correspondence between Increase in Reducing Power and Disappearance
of Glycogen.
Reducing Increase in 100
Increase x 30
POWX, reducing power,
glucose glucose ’ trieacchsride
equivalents equivalents equivalents
in 100 cc. in 100 cc. in 100 cc.

bra. vm. gm. gm. c7m. gm.

0 2.06 0.014
3 1.80 0.091 0.077 0.25 0.26
6 1.57 0.233 0.142 0.47 0.49
12 1.08 0.291 0.277 0.92 0.98
24 0.44 0.510 0.496 1.64 1.62
36 0.16 0.660 0.646 2.13 1.90

peared, calculated as glucose, corresponded to almost exactly 30

per cent of the quantity of glycogen which had been destroyed
during the same period. If the initial products of hydrolysis are
dextrins, such a correspondence is improbable; rather, one would
expect a rapid conversion of the glycogen into dextrin, followed by
hydrolysis of the dextrin to reducing sugar. As will be shown
36 Glycogen Hydrolysis
later, such a sequence occurs during the hydrolysis of glycogen by
salivary and pancreatic amylases.
Results obtained in the hydrolysis of a 2 per cent glycogen
solution are shown in Table V.

Identification of the Product of Hydrolysis.

Preparation of the phenylosazone of the product of hydrolysis
of glycogen by the muscle enzyme gave a product consisting
entirely of small star-shaped aggregates of needles. When puri-
fied, the osazone melts at 186’ (corrected). This does not corre-
spond to the melting point of the osazone of any sugar hitherto

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described as a product of the breakdown of glycogen. The same
product was obtained whether the glycogen used was prepared
from rabbit liver or from scallops. In view of opinions in the
literature to the effect that, since the melting points of the osazones
extend over a range of several degrees and are not far apart for the
different sugars, the determination of the melting points is of
doubtful value, it may be stated that no difficulty was experienced
in this respect. This compound, as well as other osazones which
were prepared for comparison with it, on being rapidly heated
to within about 10” below the melting point, the temperature then
being raised slowly, began to darken somewhat at 2-3’ below the
melt’ing point, and then melted sharply within a range of less
than lo.
Considerable difficulty was experienced in obtaining a sufficient
amount of the osazone for quantitative work. The material is not
much more soluble in hot water than in cold, and is not readily
crystallized from non-aqueous solvents. By making a very con-
centrated solution of the material in boiling pyridine or alcohol,
and adding boiling water until precipitation commenced, it was
found possible, however, to separate the osaxone in pure form.
The molecular weight and nitrogen content of the osazone corre-
spond to the values for a trisaccharide osazone of the composition
CZOH~,OI~N~.
On the assumption that only one product was present, deter-
minations on glycogen digests showed that the product must have
an optical rotatory power represented approximately by [(~]n~ =
182” corresponding to [a], = 154’, with a reducing power 30 per
cent of that of glucose.
 A. D. Barbour 37

Isolation of the Product of Hydrolysis.

The isolation of the sugar in the pure form presented consider-
able difficulty since the digests contained a complex mixture of
protein, glycerol, and sugar. The first attempts consisted in the
coagulation of the proteins by heat, or their precipitation by
alcohol, concentration of the filtrate to the least possible volume,
and precipitation of the sugar by absolute alcohol. This material
was purified by acetylation and regeneration from the acetyl
compound and was finally precipitated with absolute alcohol.
The purified product showed [ol]ng = 187“, reducing power 8.5
per cent of that of glucose, and a molecular weight of approximately

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481.
The low molecular weight and reducing power suggested that
the sugar had been converted into an anhydride of the constitu-
tion C,,HSoOlj with a theoretical molecular weight of 486, and
this suggestion was supported by an analysis of the acetyl com-
pound, showing 47.5 per cent (CO. CH,) (theoretical = 47.5
per cent).
It is interesting to note that the anhydrotrisaccharide closely
resembles the “grenzdextrin” obtained by Pringsheim and Beiser
(15) from the digestion of amylopectin with malt amylase. The
latter compound had [aID = 160”, the molecular weight of an
anhydrotrisaccharide, and a reducing power by the Munson-
Walker (16) method corresponding to 4 per cent of that of glucose.
The conversion of the sugar into its anhydride apparently tAkes
place during the concentration of the glycerol-containing digest
filtrate. This is shown by the fact that during this process the
reducing power rises more slowly, in proportion, than the volume
diminishes; and hence a conversion of the sugar into a compound
of lower reducing power must be taking place.
The sugar itself was more effectively isolated by precipitation
as a barium compound on the addition of excess barium hy-
droxide from a solution in 70 per cent alcohol. This compound
was dissolved in water, decomposed by a current of COS, the last
trace of barium removed with sulfuric acid, and the filtrate evapo-
rated to dryness. The sugar was purified by a repetition of this
procedure, and showed a molecular weight of 505, an elementary
composition corresponding to the formula C18H32016, [ol]ng = 181”,
and reducing power 31 per cent of that of glucose.
38 Glycogen Hydrolysis
Thus, the sole product of the hydrolysis of glycogen by a glycerol
extract of muscle is a trisaccharide, with the physical constants
given above, forming a characteristic osazone, melting at 186”.
The characteristics of this compound differentiate it from the tri-
saccharides described by Pringsheim (5) and by Ling and Nanji
(17).
Digestion with Other Amylases.
To compare the effect of muscle glycogenase with those of other
amylases, digests of glycogen with salivary and pancreatic amy-
lases were made. The results were quite different from those

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obtained with the muscle enzyme. In the case of salivary

TABLE VI.
Hydrolysis of Glycogen by Salivary Amylase.

Glyo~.s,",: in Reducing pmw

Time. (asglucose) in
100 cc.

--
min. gm. I”. am.
0 0.960 0.000 0.000
10 0.876 0.084 0.010 8.4
20 0.750 0.210 0.027 7.8
40 0.478 0.482 0.093 5.2
60 0.304 0.656 0.160 4.1
120 0.134 0.826 0.200 4.1

amylase the increase in reducing power did not coincide with the
disappearance of glycogen, being proportionately less at first and
increasing steadily. The products also were different. Investi-
gation of the products of hydrolysis by the osazone method
showed that the characteristic osazone of the trisaccharide de-
scribed above was absent. The chief product was isomaltose.
Maltose could not be detected in any case, and glucose was only
occasionally present. Since the reducing power of the digests
was not at any time nearly enough to account for all the glycogen
destroyed, it would appear that dextrins are formed also.
 A. D. Barhour 39
IIydrolylsis by Salivary Amylnse.
100 cc. of 1 per cent, glycogen were treated with 0.5 cc. of
filtered saliva at, pH = 6.3 for 2 hours at 37’. Samples were
taken at intervals for the determination of glycogen and sugar.
The results are shown in Table VI. It is evident that the ratio
of sugar formed is not constant, but increases continually during
the course of the hydrolysis.
Treatment of the hydrolyzed residue with phcnglhydrazine
resulted in the formation of an osazonc having the characteristic
crystalline form of the osazone of isomaltosc. No other osazone
could be detected. The osazonc when rcprccipitatcd and dried

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melted sharply at 1.57’ (corrected).

Glyco~e”
Time. destroyed in
100 cc.

min om. gm. ym.

0 2.47 0.00 0.00
10 0 77 1.70 0 72
20 0.47 2.00 0.i8
40 0.30 2.17 0.85
60 0.28 2.19 0.88
135 0 23 2.24 1.00

IIydroly~sis ,with Pancreatic Amylase.

50 cc. of 4 per cent glycogen solution were treated with 0.02 gm.
of Merck’s trypsin and incubated for 2 hours at 37”, pH = 6.3.
Samples were taken at intervals for glycogen and sugar determi-
nations. The results arc shown in Table VII.
The ratio of glycogen destroyed to reducing power with pan-
creatic amylase is constant and it therefore appears that there is
no serial degradat,ion of the glycogen, just as in the caseof muscle
glycogenase. The ratio, however, is different, averaging 2.4 in
the case of pancreatic amylase and 3.3 in the case of muscle
glycogen.
40 Glycogen Hydrolysis

The residue from the hydrolysis with pancreatic amylase was

heated with phenylhydrazine and acetic acid and the resulting
osazone was examined microscopically. The characteristic crys-
tals of isomaltose were identified as the principal product. Some
crystals resembling those of glucosazone were also found. Re-
peating the procedure, with larger amounts of material, enabled
a fraction to be separated which showed a melting point of 206’.
The bulk of the material, when purified, melted at 157”.
In neither of the experiments quoted above could the charac-
teristic crystals of the trisaccharide osazone be detected.

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EUfect of Increasing Glycogen Concentration on Hydrolysis.
Since the hydrolysis of glycogen belongs to the type which
yields a number of products from the destruction of 1 molecule,
it might be expected, as pointed out by Moore (18) that reversion
of the reaction, i.e. synthesis, could be easily attained in con-
centrated solutions of the product. A corollary of this expecta-
tion is that in concentrated solutions of glycogen, hydrolysis
would proceed at a slower rate than in dilute solutions. This has
already been shown to be true for the hydrolysis of proteins (19).
For the hydrolysis of glycogen by muscle glycogenase the velocity
of hydrolysis was found to fall off rather rapidly with increasing
concentrations of glycogen, varying from 80 per cent hydrolysis
in 24 hours in a 1.20 per cent solution of glycogen to 70 per cent
in a 2.32 per cent solution. In these experiments the concentra-
tion of the enzyme was kept proportional to the concentration of
the glycogen, and was such a concentration that under the given
conditions further addition of enzyme caused no increase in the
rate of hydrolysis. Similar considerations would lead to the
expectation that hydrolysis is readily retarded by the addition of
products.

E$ect of Addition of Products on Hydrolysis.

In the hydrolysis of glycogen by muscle enzyme it was found
that the addition of the products of hydrolysis exerted a decided
inhibiting effect. The addition of 5 per cent of the products was
sufficient to reduce the rate of hydrolysis over 50 per cent, and the
addition of 10 per cent practically inhibited the hydrolysis alto-
 A. D. Barbour

gether. This effect was observed both with the open chain tri-
saccharide and with the anhydride.
Since the products of hydrolysis inhibit the process, it was
thought that in solutions containing a sufficient concentration of
the trisaccharide and enzyme, it might be possible to attain the
enzymatic synthesis of glycogen, as has been done in the case of
protein. Nevertheless, under these conditions synthesis did not
occur. Although attempts were made with solutions which
varied in concentration from 20 per cent to the highest concen-
tration obtainable, and at hydrogen ion concentrations from pH 3 to
11, no increase was noted in the glycogen content of any of these

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TABLE VIII.
Rate of Hydrolysis of Glycogen by Muscle Glycogenase in Presence of
Added Subslance.

Aversse glycogen
content. Hydrolysis.
Added substance.
0 hr. 4 hrs. 24 hrs. 4 hrs. 24 hrs.
--__
per cent pet centper centper centper cent

Trisaccharide.. . . . 2.02 2.00 1.92 0 5

Sucrose.. .... . . . 2.02 1.74 0.84 14 58
Glucose................................. 2.06 1.76 0.72 15 65
Sodiumchloride .. . . .. . .. . . 2.09 1.64 0.34 21 84
Urea .,...,.............................. 2.09 1.64 0.48 21 77
Control . . . 1.99 1.60 0.56 20 72

solutions. Even when solutions containing the sugar and enzyme

were evaporated to a syrup in vacua over PZ05, and then held for
a considerable time at 37”, no glycogen was found. The specific
inhibition which is observed may not therefore be a simple con-
centration effect, but may be due, for instance, to a combination
of the enzyme with the added products.
As controls for the experiments, where retardation of hydrolysis
was observed on adding trisaccharide, experiments were per-
formed in which the trisaccharide was replaced by other sub-
stances. These were weighed out in sufficient amount to make a
0.2 M solution. 3 cc. of enzyme were added, and the volume
made up to 10 cc. The results are shown in Table VIII. It is
evident from these results that the effect of trisaccharide is specific
42 Glycogen Hydrolysis
and not merely due to osmotic effects, for solutions of equal molar
concentrations of other substances caused relatively no inhibi-
tion. Sucrose and glucose showed a very slight inhibition, while
a slight acceleration of hydrolysis was observed with sodium
chloride and urea.
During the course of these experiments a curious effect was
observed. It was found that the addition of moderately high
concentrations of egg albumin to glycogen digests had the effect
of markedly accelerating the rate of hydrolysis, both in the pres-
ence and absence of the trisaccharide. This effect is illustrated
in the following experiment. 5 cc. samples of 2 per cent glycogen

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solution were taken and the following substances dissolved:
TABLE IX.
Effect of Addition of Protein on Rate of Hydrolysis of Glycogen

Substance added.
0 hr. 4 hrs. 24 hrs. 4 hrs. 24 hrs.
-----
per cent per cent per cent per cent per cent
0.5 gm. trisaccharide.. 2.20 1.96 1.40 11 36
0.5 “ “ +0.5gm. albumin. 2.28 1.86 1.16 18 49
0.5 “ egg albumin.. 2.10 1.09 0.08 48 96
Control.. . 2.12 1.70 0.34 20 84

(1) 0.5 gm. of trisaccharide, (2) 0.5 gm. of trisaccharide + 0.5

gm. of egg albumin, (3) 0.5 gm. of egg albumin, (4) control. 3
cc. of enzyme were added to each sample and sufficient water to
make the volume 10 cc. They were incubated at 37” for 24 hours
with the results shown in Table IX.

Chemical and Physical Properties of the Trisaccharide.

In view of the importance of the conclusion that a trisaccharide
is the sole product of the hydrolysis of glycogen by muscle glyco-
genase, the chemical and physical properties of the trisaccharide
were investigated as completely as possible and the following
data were obtained.
Melting Point of Ihe Osazone.-Three determinations gave: 186” (cor-
rected), 186” (corrected), 187” (corrected); average 186”.
 A. D. Barbour
Nitrogen Content of the Osazone.-Analyses by the micro-Kjeldahl method
(Cole’s modification) gave: 8.8 per cent nitrogen, 8.6 per cent nitrogen;
average 8.7 per cent.
Calculated for G,H~QO~~N., (trisaccharide) = 8.22 per cent.
“ “ C~H320gN~ (disaccharide) = 10.78 “ “
Rotatory Power of Digest.-One digest gave: Concentration = 0.438 per
cent (determined by acid hydrolysis and estimation of the glucose formed),
2 dm. tube. (Y = +1.56”; whence [a]ng = +178”.
A second digest gave: Concentration = 0.476 per cent, 2 dm. tube.
a = i-1.79’; whence [a]=, = +187”.

Anhydrotrisaccharide.

Molecular Weight.--W = 10.0 gm., w = 0.0413 gm., A = 0.032”, K (water)

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= 18.6; whence M = 481. Calculated for C18H30011 = 486.
Rotatory Power.-Concentration = 0.4127 per cent, 2 dm. tube. (x =
3.09”; whence [oI]+ = 187”.
Reducing Power.-Concentration = 0.4127 per cent (diluted 1 : 40 for
determination); reduction (as glucose) = 0.0017; reduction by the same
concentration of glucose = 0.0206. The reducing power is 8.5 per cent
of that of glucose.

Acetyl Derivative of the Anhydrotrisaccharide.

Molecular Weight.-(In benzene solution.) Sample 1. W = 7.03 gm.,

w = 0.0758 gm., A = 0.053”, K (benzene) = 49.0, M = 996. Sample 2. IV=
8.98 gm., w=O.O696 gm., A = 0.041”, K = 49.0, M = 926. Calculated for
C~~HMOU(CO.CH&~ = 906.
Acetyl Content.
1. 0.0255 gm. required 2.80 cc. 0.1 N NaOH. (CO.CH,) = 47.5 per
cent.
2. 0.0566 gm. required 6.25 cc. 0.1 N NaOH. (C0.CH3) = 47.6 per
cent.
Calculated for CisH2,0r6(C0.CH,),r,. (CO.CH,) = 47.5 per cent.

Trisaccharide (Open Chain).

Molecular Weight.-(In water.) W = 11.53 gm., w = 0.1848 gm., A =

0.059”, K (water) =. 18.6, M = 505. Calculated for C18Ha2016 = 504.
Rotatory Power.-Concentration = 0.052 per cent, 2 dm. tube. 01 =
1.82”, [aJag = 181”.
Reducing Power.-Concentration = 0.502 per cent (diluted 1 : 40 for
determination); reducing power (as glucose) = 0.0075; reduction by same
concentration glucose = 0.0251. The reducing power is 31 per cent of that
of glucose.
Ultimate Analysis.
1. 0.1215 gm. gave 0.1911 gm. CO,, 0.0697 gm. HzO. C 42.96, H 6.37,
0 50.67.
 Glycogen Hydrolysis
2. 0.1020 gm. gave 0.1561 gm. CO,, 0.0580 gm. H20. C 42.74, H 6.31,
0 50.95.
Calculated for C18H32016. C 42.86, H 6.35, 0 50.79.

Barium Compound of the Trisaccharide.

1. 0.1570 gm. gave 0.0562 gm. B&O*. Ba 21.0.

2. 0.1509 gm. gave 0.0521 gm. BaS04. Ba 20.3.
Calculated for C18H3201E.Ba0. Ba 20.8.

CONCLUSIONS.

1. The hydrolysis of glycogen may be readily carried out by

means of a glycerol extract of fresh muscle or liver tissue.

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Such
an extract does not diminish in potency on long standing in a
refrigerator, and its activity is only slightly diminished after in-
cubation for 48 hours at 37”.
2. The optimum hydrogen ion concentration for glycogen
hydrolysis with the tissue enzyme is about pH 6.3.
3. The sole product of the hydrolysis of glycogen by muscle
extract appears to be a trisaccharide. This compound possesses
30 per cent of the reducing power of glucose, by the Shaffer-
Hartmann method, and is dextrorotatory, [o~]n~ = j-181”. The
sugar is rather readily converted into an anhydride having 8.5
per cent of the reducing power of glucose, and [a] rig = 187”.
4. The hydrolysis of glycogen by the muscle enzyme is greatly
inhibited by the addition of the trisaccharide or its anhydride to
the digest. Sucrose and glucose have a slight inhibiting effect,
and sodium chloride, sodium or potassium phosphate, and urea
have a slight accelerating effect. The addition of 5 per cent
albumin produces a marked acceleration, both in the presence and
absence of the trisaccharide.
5. Attempts at the enzymatic synthesis of glycogen from the
trisaccharide and its anhydride were unsuccessful.
6. The digestion of glycogen by salivary and pancreatic amy-
lases follows a different course. The trisaccharide is not formed
by these enzymes. Glucose and isomaltose have been identified
among the products of these hydrolyses, but maltose apparently
is not formed.
 A. D. Barbour

The author wishes to express his indebtedness to Professor H.

Wasteneys for suggesting this problem and directing the progress
of the work, and to Dr. H. Borsook for many helpful suggestions.

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209 (1924).
7. Pfliiger, E., Arch. ges. Physiol., 96,1 (1903).

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8. Slater, W. K., J. Physiol., 67, pp. xxxviii, lxxvii (1923).
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13. Hudson, C. S., and Dale, J. K., J. Am. Chem. Sot., 39,320 (1917).
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 ENZYMATIC HYDROLYSIS OF
GLYCOGEN
A. D. Barbour
J. Biol. Chem. 1929, 85:29-45.

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