THE HYDROLYSIS OF GLYCOGEN BY GLYCEROL

EXTRACT OF MUSCLE
BY ALBERT CARRUTHERS AND WE1 YUNG LEE
(From the Department of Biochemistry, Peiping Union Medical College,
Peiping, China)

(Received for publication, July 24, 1934)

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Barbour (1) has claimed that muscle amylase acting on glycogen
gives a trisaccharide as the sale product of hydrolysis. This find-
ing is strikingly different from those which have been obtained
with pancreatic, salivary, and malt amylases which, it is generally
agreed, produce maltose and, when conversion to disaccharide is
incomplete, also dextrin (2). Most investigations, it is true, have
been carried out with starch as substrate but the studies of Pring-
sheim (3) and Kuhn (4) lead one to suppose that there is no signifi-
cant difference between amylopectin, a constituent of starch, and
glycogen. Sodium fluoride extracts of muscle acting on glycogen
were found by Osborne and Zobel (5) to produce dextrins and
maltose and probably some glucose. Among the products of
glycogen hydrolysis by potassium chloride extracts of frog muscle
Lohmann (6) found glucose and a sugar identified as an amylo-
triose. If glycerol extracts of muscle split glycogen entirely and
solely to a trisaccharide, these extracts must have peculiar proper-
ties distinguishing them from other amylase systems. Case (7),
without giving any experimental data, stated that he was able to
confirm Barbour’s results. Experimental work reported here is
not in agreement with Barbour’s conclusions.

EXPERIMENTAL

Esperiment I-30 cc. of glycerol extract of rabbit muscle, pre-

pared by the method Barbour employed (1)) were mixed with 10 cc.
of phosphate buffer, pH 6.4, and 8.0 gm. of glycogen dissolved in
160 cc. of water. Toluene was added and the mixture was incu-
bated 46 hours. Protein was then removed by heat coagulation.
525
526 Glycogen Hydrolysis

The addition of 2 or 3 drops of dilute acetic acid facilitated coagu-

lation and filtration. To the clear filtrate 2 volumes of alcohol
were added to remove unchanged glycogen. After standing 24
hours in the ice box, the mixture was filtered and the filtrate con-
centrated under reduced pressure and at temperatures below 50”.
To the syrupy concentrate 95 per cent alcohol was added, which
gave a precipitate. After this precipitate was separated by centri-

TABLE I
Properties of Sugar Fractions Isolated from
-
Glycogen-Muscle
-
Extract Digests

I I

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GlllCOS9 Specific Acetyl product
equivalent rotation
t
Nature of fraction and
I
method of separation Acetyl
,
Mol. wt. con-
I tent
4
-- ~-
am. am. Pm Per
cent cent
Powder by alcohol 0.203 0.613 33
mtn. “ “ 0.3590.990 34 173
I Powder by alcohol- 0.632 0.695 90 126 70
ether pptn.
Total concentrate 4.4003.320 53 140
Powder by alcohol 1.295 2.905 45 162 60 1235 (A)
ppt*- 663 (B) 51.75
Residual syrup 50 161 62 602 49.40
Total concentrate 2.5926.176 42 171 65
Powder by alcohol 0.574 1.914 30 198 59 1414 (A)
mtn. 735 (B) 51.74
Powder by alcohol- 0.502 1.396 36 168 59
ether pptn.
I Residual syrup 1.205 1.890 64 133 60 625 52.55
-

fuging, the liquor was reconcentrated and absolute alcohol was

added to the concentrate. A second precipitate was separated
and the liquor was again concentrated. This time a mixture of
equal parts of alcohol and ether was added and a third precipitate
was obtained. These precipitates are referred to as Fractions I,
II, III in Table I, Experiment 1. All the fractions were very
sticky, owing to contamination with glycerol, and therefore the
analytical results, presented in Table I, were probably not reliable.
 A. Carruthers and W. Y. Lee 527

Repeated washing of the precipitate with absolute alcohol and

ether removed some of the stickiness. Attempts to purify frac-
tions by precipitation with barium hydroxide were unsuccessful.
On account of the difficulty experienced in isolating sugar frac-
tions from the digestion mixtures it was decided to remove glycerol
from the extract by dialysis. Extracts were dialyzed in collodion
sacks for 1 hour against running tap water, followed by 2 hours
against distilled water. The latter was changed at intervals of 15
minutes. The dialyzed extracts were found to be quite active and
therefore no sodium chloride was added to the digestion mixtures.
Buffer also was omitted in the hope that this would facilitate the

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isolation of pure fractions.
Experiment S-60 cc. of glycerol extract, treated with toluene
and the toluene layer removed, were dialyzed as described and the
dialyzed preparation was mixed with 10 gm. of glycogen dissolved
in water. The volume of the mixture was 200 cc. A few drops of
toluene were added and the mixture was incubated for 46 hours at
37”. Protein and unchanged glycogen were removed as in Experi-
ment 1. The aqueous sugar solution was concentrated to a small
volume and absolute alcohol was added, giving a precipitate,
Fraction I. This precipitate was obtained as a white powder and
in contrast to the fractions of Experiment 1 the preparation was
not sticky. As Fraction I had a much higher glucose equivalent
than could possibly be given by a trisaccharide, no further frac-
tionation was attempted. The liquor from Fraction I was con-
centrated to a syrup and analyzed. Results of this experiment
are summarized in Table I.
Experiment S-50 cc. of glycerol extract, prepared as before,
were dialyzed and mixed with 8.0 gm. of glycogen in water and the
volume made to 200 cc. Toluene was added and the mixture was
incubated at 37” for 36 hours. Protein and unchanged glycogen
were removed and an aqueous concentrate prepared as in Experi-
ment 2. The results of this experiment are summarized in Table
I and full analytical details follow.
Analysis of Aqueous Concentrate-The total volume was 40.5
cc. 2 cc. of the concentrate were diluted to 20 cc. and the optical
activity was determined. To 10 cc. of the 20 cc. solution 0.66 cc.
of concentrated HCl was added and the mixture heated for 3 hours
in a boiling water bath, neutralized, and diluted to 20 cc. The
528 Glycogen Hydrolysis
optical activity was again determined. Reducing power of the
solution before and after acid hydrolysis was also determined.
Reducing power throughout this work was measured by the
Shaffer-Hartmann method as modified by Barbour (1).
(a) The glucose equivalent (by reducing power) in 40.5 cc. of
concentrate before hydrolysis = 2.592 gm.; (b) after hydrolysis =
6.176 gm.; (a)/(b) = 42 per cent.
Rotation before Hyldrolysis-a! = 5.23” 2 dm tube* C1 =
6.176 X 2/40.5 X 5 = 1.525; [c& = 17;‘. ’ ’
Rotation after Hydrolysis--a = l.OO”, 2 dm. tube; C = 1.52512 =
0.7625; [a]~~ = 65”.

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Separation of Fractions-To the aqueous concentrate (38 cc.)
200 cc. of absolute alcohol were added, which gave a white precipi-
tate (Fraction I). To the alcoholic liquor, obtained after centri-
fuging off Fraction I, 100 cc. of ether were added, when a further
precipitate was obtained and, by centrifuging, this gave Fraction
II. The ether-alcohol mixture was not clear on separation, even
after prolonged cooling and it was therefore concentrated in vacua
below 50” to a syrup (Fraction III).
Analysis of Fractions-Fraction I was washed by stirring twice
with absolute alcohol and once with ether and then dried in a desic-
cator over sulfuric acid. Yield, 2.21 gm. 50 mg. dissolved in
20 cc. of water gave (a) a glucose equivalent before acid hydroly-
sis = 13.0 mg.; (b) after acid hydrolysis = 43.3 mg.; (a)/(b) = 30
per cent.
Rotation-a = 0.86”, 2 dm. tube; C = 43.3 X 5 = 0.2165;
[cf]Hg = 198”.
15 cc. of the above solution were hydrolyzed, neutralized, and
diluted to 20 cc. a! = 0.19”, 2 dm. tube; C = 15/20 X 5 X 43.3 =
0.162; [c& = 59”.
50 mg. of powder heated with 60 per cent KOH gave no reducing
substance after addition of alcohol and hydrolysis with acid,
showing the absence of glycogen.
About 1.0 gm. of substance was acetylated after the method of
Pringsheim (3). The acetylated product, after standing 2 days in
water, was thoroughly broken up, stirred with water, and the mix-
1 Concentration throughout this work was calculated on the basis of
reducing power after acid hydrolysis. This is not strictly correct, but is
satisfactory as an approximation.
 A. Carruthers and W. Y. Lee 529

t’ure centrifuged. The brownish colored residue was dissolved in

about 20 cc. of chloroform and the chloroform solution was shaken
three times with water. To the washed chloroform extract cal-
cium chloride was added and the clear extract filtered after stand-
ing several hours. The filtrate was evaporated in vacua to dryness
and the residue was taken up in hot absolute alcohol. The alcohol
solution was decdorized by repeated boiling with charcoal, the
solution being filtered through charcoal each time. The alcohol
solution, about 200 cc., was allowed to stand for several days at
room temperature. With Fraction I a few mg. of a grayish white
powder separated, sufficient only for molecular weight determina-

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tions. Molecular weight determinations by Rast’s method (8)
on this and all other fractions are given in Table II.

TABLE II
Molecular Weight Determinations on Acetyl Derivatives of Sugar Fractions

Fraction Weight of M. p.
substance depression Mol. wt.

%?. mg. “C.

Precipitate A, Frac- 5.710 1.053 4.95 1416
tion I 4.317 0.758 4.32 1412
Residue B, Fraction I 4.298 0.508 6.30 711
4.867 0.740 7.60 760
Fraction III 3.478 0.514 9.3 603
3.790 0.582 9.0 648

After removal of this small amount of precipitate no more sub-

stance separated and therefore the alcohol solution was evaporated
to dryness. Residue B was thoroughly dried in a desiccator over
sulfuric acid. The determination of acetyl content of this frac-
tion was performed by the method of Pringsheim (3). The results
are given in Table III.
Fraction II-After washing with alcohol and ether and drying
over sulfuric acid, the weight of powder obtained was 1.50 gm.
150 mg. dissolved in 15 cc. of water gave (a) a glucose equivalent
before acid hydrolysis = 50.2 mg.; (b) after acid hydrolysis =
139.0 mg.; (a)/(b) = 36 per cent.
Rotation before Hydrolysis--cu = 3.11”, 2 dm. tube; C = 0.139 X
100/15 = 0.927; [culHB = 168”.
530 Glycogen Hydrolysis
Rotation after Hydrolysis300 mg. of powder, dissolved and
hydrolyzed and the volume after hydrolysis made up to 20 cc.,
gave (Y = 1.64, 2 dm. tube; C = 1.39; [cy]ng = 59”.
Fraction III-The syrup dissolved in 20 cc. of water gave (a)
a glucose equivalent before acid hydrolysis = 1.205 gm.; (b)
after acid hydrolysis = 1.890 gm.; (a) /(b) = 64 per cent.
Rotation before Hydrolysis-2 cc. of solution diluted to 25 cc.
gave a! = 2.02”, 2 dm. tube; C = 0.756; [~y]n~= 133”.
Rotation after Hydrolysis-10 cc. of the diluted solution hydro-
lyzed and the volume made to 20 cc. gave Q! = 0.45”, 2 dm. tube;
c = 0.378; [a]& = 60”.

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TABLE III
Determination of Acetyl Content of Acetyl Derivatives of Sugar Fractions

Fraction‘ Weight of substance 0.1 N NaOH A&y: content

I

gm. cc. per cent

Residue B, Fraction I 0.0934 11.21 51.62
0.0825 9.95 51.86
Fraction III 0.0772 9.50 52.90
0.1006 12.20 52.20

Part of the aqueous syrup was dried as thoroughly as possible,

finally over phosphorus pentoxide, and an acetyl product was
prepared and analyzed (seeTables II and III).
DISCUSSION

The results of Experiment 1 were considered unreliable because

the sugar fractions isolated were very impure. The results of
Experiment 2 seemedto indicate that the digestion had produced
a mixture of sugars containing mainly disaccharide with, possibly,
somemonosaccharide. The fraction isolated by alcohol precipita-
tion gave a glucose equivalent much higher than that possible
for a trisaccharide. The results of Experiment 3, however, illus-
trated the mixed nature of the products and showed the presence
of polysaccharide more complex than disaccharide, of disaccharide,
and of monosaccharide. The significance of the analytical values
is made clearer by comparing them with the values given, either
theoretically or experimentally, by glucose, maltose, and a possible
trisaccharide. The latter are summarized in Table IV.
 A. Carruthers and W. Y. Lee 531
Considering Experiment 3, it is seen that after removal of un-
changed glycogen the digestion mixture contained 6.176 gm. of
sugar expressed as glucose. The specific rotation and reducing
power were nearly those of maltose. Crude fractionation with
alcohol and ether, however, indicated the presence of more than
one substance. The first precipitate by alcohol, Fraction I, gave
a lower reducing power and a higher specific rotation than the
original mixture, suggesting the presence in this fraction of either
a trisaccharide or a mixture of polysaccharides, some more complex
than disaccharide, with possibly some monosaccharide. From

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this fraction a few mg. of acetyl derivative (Fraction I, A) were
obtained which, while probably not a pure substance, gave a

TABLE IV
Properties of Glucose, Maltose, and a Trisaccharide
I Specific rotation A&y1 product
GlUC~e
equivdenl t-
by “;+i ;‘c”l”d’
reducing Acetyl
powder h ydrolysis hydrolysi. s Mol. wt. content
Ia’lHg [OIH,
- --
degrees degrees per cent
Glucose (calculated). ... ... 100 62 390 55.10
Maltose “ . .. ... ... 163 62 678 50.70
(‘ (found). . . . . . . . . . . 43 161 66 664 51.17
Trisaccharide (calculated). . 62 966 48.96
- -

molecular weight indicating a high polysaccharide. The bulk of

the acetyl derivative (Fraction I, B), separated after concentration
of the alcohol solution, gave a molecular weight and an acetyl
number closely approximating those for a disaccharide. Fraction
I therefore seemed to be predominantly disaccharide mixed with
possibly some polysaccharide such as dextrin. The sugar sepa-
rated by alcohol-ether, Fraction II, showed a slightly higher reduc-
ing power and a lower specific rotation consistent with there being
lesspolysaccharide than in the first fraction. The residual syrup,
after removal of Fractions I and II, showed a relatively high
reducing power and a low rotation, suggesting that here propor-
tionately more monosaccharide was present. In conformity with
this suggestion an acetyl product from this syrup gave a molecu-
532 Glycogen Hydrolysis
lar weight slightly lower and an acetyl number slightly higher than
those for a disaccharide. The original sugar mixture contained
6.176 gm. and the fractions totaled 5.194 gm. As all of the
products isolated were amorphous, the analytical data did not
establish the absolute identity of any particular compound.
However, failure to obtain crystalline products was to be expected,
since a mixture of substances, probably containing dextrin, was
being dealt with. As a control crystalline octaacetyl maltose
was prepared from pure maltose with comparative ease; the analy-
sis of this compound is presented in Table IV. Of the evidence
outlined the molecular weight determinations most convindingly

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support the view that a disaccharide and not a trisaccharide was
the main constituent of the digestion mixture. It is certain, how-
ever, that a mixture of sugars was present.
Barbour observed that the glucose equivalent of a glycogen-
muscle extract determined at any time over 36 hours corresponded
almost exactly to 30 per cent of the glycogen hydrolyzed. (In
Table V of Barbour’s paper the increase in reducing power from
0 to 6 hours should be 0.219 and not 0.142. The corrected value
gives a glucose equivalent of 45 per cent.) Pringsheim et al.
(9) have repeatedly found amylases to yield 70 to 80 per cent only
of the theoretical amount of maltose. The remaining 20 to 30 per
cent of the polysaccharide, glycogen or starch, is converted to a
“limit dextrin.” The reducing power of maltose, as measured by
the modified Shaffer-Hartmann procedure adopted by Barbour,
gives a glucose equivalent of 43 per cent. If muscle amylase
behaved as other amylases in yielding say 70 per cent of the
theoretical amount of maltose, the glucose equivalent of the mix-
ture would be 0.7 X 43 = 30.1 per cent. Such a value therefore
would not necessarily indicate the formation of a trisaccharide.
Barbour claimed, however, that the isolated product (open chain
compound) also gave a glucose equivalent of 30 per cent and this
would suggest that the muscle amylase did not behave as do
other amylases, but unfortunately he did not present evidence to
establish the absolute purity of the isolated sugar.
Barbour stated that from aqueous glycerol concentrates an
anhydrous trisaccharide was isolated, the formation of the anhy-
dride being attributed to the action of glycerol during concentra-
tion. This we were not able to confirm. It should be pointed out,
 A. Carruthers and W. Y. Lee

however, that from the data given by Barbour the molecular

weight and the specific rotation are incorrectly calculated and
should be 240 and 374 respectively and not 481 and 187 as stated.

SUMMARY

While the arguments and the evidence presented here do not

disprove the possibility that a trisaccharide may be one of the
products of glycogen hydrolysis by glycerol extracts of muscle,
they prove that trisaccharide is not the sole product and they
suggest that maltose (a disaccharide) is the main product.

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BIBLIOGRAPHY

1. Barbour, A. D., J. Biol. Chem., 86,29 (1929-30).

2. Pringsheim, H., The chemistry of the monosaccharides and of the poly-
saccharides, New York, 247 (1932).
3. Pringsheim, H., Ber. them. Ges., 67, 1581 (1924).
4. Kuhn, R., Ann. Chem., 443, 1 (1925).
5. Osborne, W. A., and Zobel, S., J. Physiol., 29, 1 (1903).
6. Lohmann, K., Biochem. Z., 178,444 (1926).
7. Case, E. M., Biochem. J., 26, 561 (1931).
8. Rast, K., Ber. &em. Ges., 66, 1051 (1922).
9. Pringsheim, H., and Fuchs, W., Bet-. them. Ges., 66, 1762 (1923). See also
Pringsheim, H., and Schmalz, K., Biochem. Z., 142, 108 (1923).