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ADSORPTION AND HYDROLYSIS OF GLYCOGEN

BY GEORGE BANCROFT AND EDITH G. FRY


(From the Cancer Research Laboratories, University of Pennsylvania
Graduate School of Medicine, Philadelphia)

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(Received for publication, November 17, 1932)

In an earlier paper (1) the effect of adsorption on the course of


enzyme reactions was discussed. Since the study of enzyme
hydrolysis in homogeneous solution tacitly disregards this effect
which may be of considerable importance in such a heterogeneous
system as the body, some of the characteristics of glycogen in
adsorption and hydrolysis were investigated by means of the
system charcoal-glycogen-water and acid or enzyme. It is clear
that the properties of glycogen when adsorbed must depend largely
on the type and extent of adsorption. If the adsorption is re-
versible, the rate of hydrolysis would be checked only if there was
very complete adsorption, for then the amount in solution would
be nearly negligible at all times.
Adsorption of Glycogen by Charcoal-The general method of
procedure for determining the adsorption and hydrolysis of gly-
cogen was as follows: 1.00 gm. of purified charcoal’ was suspended
in 15 cc. of glycogen solution2 of various concentrations in a flask.
This was evacuated to remove adsorbed air and to insure thorough
wetting of the particles. After standing overnight, the charcoal
was filtered off through a Gooch crucible, two 3 cc. portions of
water being used to transfer the solid to the crucible, and the
glycogen was determined in the filtrate by hydrolysis. The
* Since impurities often cause conflicting results with charcoal (Miller
(2)) and since purified charcoal is a better adsorbent, Pfanstiehl’s norit
was sifted through a 250 mesh sieve and purified by the HzFg treatment as
described by Miller (3). The purified charcoal contained less than 0.2
per cent ash, which was deemed sufficiently ash-free. The very slight re-
sidual reducing value was allowed for in a blank.
2 The glycogen used in these experiments was prepared from rabbit livers
by the trichloroacetic acid extraction method of Sahyun and Alsberg (4).
255
256 Adsorption and Hydrolysis of Glycogen

adsorption could then be calculated by difference. It is recog-


nized that this method of determining the adsorption, although
perhaps not showing the true adsorption of glycogen, gave con-
sistent and accurate results and permitted the study of the hy-
drolysis of glycogen with hydrochloric acid on the same sample.
Glycogen was determined as glucose either by the Hagedorn-
Jensen method (5) or the Hanes (6) modification for use with
larger quantities after the glycogen had been hydrolyzed l+ to
2 hours with N hydrochloric acid. This method of hydrolysis

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requires much less time than the customary method and further-
more has been recommended by Sahyun and Alsberg (7). Ex-
cept when so stated, glycogen values are given as the glucose
value multiplied by the customary factor 0.927.

TABLE I
Adsorption of Glycogen on 1 Gm. of Purified Charcoal (Norit)
Glycogen
Glycogen used Glycogen in solution Glycogen adsorbed in solution after
dilution

m7. WT. WI. ml.


100.0 49.10 50.90 2.65
50.0 1.00 49.00 0.87
30.0 0.13 29.87 0.56
20.0 0.10 19.90 0.41
10.0 0.08 9.92 0.22

The data in Table I show quite clearly that glycogen is very


strongly adsorbed by charcoal. The type of adsorption is some-
what unusual in that charcoal apparently takes up glycogen
almost quantitatively to a saturation point and then can take up
no more. The tenacity of the adsorption is indicated from the
figures in the fourth column which were obtained from an experi-
ment described later. In this case charcoal containing adsorbed
glycogen was suspended in 35 cc. of dilute acid and heated 2 hours
on a water bath. The figures show the amount of glycogen
present in the solution after that treatment. The exchange of 35
cc. of water for 20 cc. of equilibrium mixture in itself amounts to
considerable dilution yet only 2.65 mg. of glycogen appeared in
solution when more than 50 mg. were adsorbed on the charcoal and
considerably less in the other concentrations.
G. Bancroft and E. G. Fry 257

Hydrolysis of Glycogen by Hydrochloric Acid in Presence of


Charcoal-The completeness of the adsorption of glycogen indi-
cated that the effect on hydrochloric acid hydrolysis might be
large. In order to determine whether glycogen adsorbed on
charcoal was stable to hydrochloric acid, 1 gm. samples of char-
coal containing different amounts of adsorbed glycogen were sus-
pended in 35 cc. of water containing 10 cc. of 1.008 N HCl. These
mixtures were heated 2 hours on a water bath, decanted through
Gooch crucibles, cooled, and aliquots analyzed for acidity, glucose,

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and glycogen. Since hydrochloric acid is also adsorbed by char-
coal, the acidity was determined in order to know what was the

TABLE II
Hydrolysis of Diferent Quantities of Adsorbed Glycogen by 0.688 N
Hydrochloric Acid
Glycogen in Glucose formed by
Glycogen adsorbed Acidity of solution solution expressed hydrolysis
as glucose

ml. N WJ. ml.


50.90 0.274 2.86 1.93
49.00 0.277 0.94 0.82
29.87 0.277 0.60 0.52
19.90 0.278 0.44 0.23
9.92 0.280 0.24 0.17

effective concentration of acid in solution. As the concentration


of acid is not sufficient for complete hydrolysis, glycogen in solu-
tion can be determined by further hydrolysis of an aliquot. The
results are given in Table II.
The fact that even with 50.9 mg. of glycogen only 1.93 mg. of
glucose were formed shows how completely adsorption alters the
course of hydrolysis.3 The amount of glucose formed is about
what would be expected from the equilibrium concentration of
2.86 mg. of glycogen Cexpressed as glucose) alone with that strength
of acid.
In order to see whether the hydrolysis was dependent upon the
concentration of both unadsorbed glycogen and unadsorbed acid
3 Although norit adsorbs some glucose from solution, the adsorption in
the presence of glycogen is so much smaller as not to be a significant factor
in explaining the reduced hydrolysis in the presence of charcoal.
258 Adsorption and Hydrolysis of Glycogen
in solution, the amount of glycogen was kept constant and the
quantity of acid varied. As only dilute acid was used, which
would not hydrolyze glycogen completely, it was not necessary to
determine the adsorption separately. Glycogen was adsorbed on
the charcoal in the usual way, but after standing overnight acid
and water were added to give the desired concentration in a vol-
ume of 20 cc. without filtering off the charcoal. The mixture was
then heated on a water bath exactly 2 hours, decanted through a
Gooch crucible, and cooled rapidly in ice, and 5 cc. aliquots were

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5-

‘0 -

5-

o-

5-

L
CONCN. OF HCL N
FIG. 1. Hydrolysis of glycogen in the presence of charcoal. o indicates
glucose and hydrochloric acid in solution after heating with charcoal; A,
glucose and hydrochloric acid with 26.5 mg. of glycogen.

taken for the determination of glucose, acidity, and total sugar or


glycogen. The amount of adsorption could then be calculated
from the amount of glycogen in solution by difference from the
original amount of glycogen added.
If the hydrolysis depends upon the amount of free glycogen and
free acid in solution, the same values should be obtained by hy-
drolyzing similar amounts of glycogen with similar quantities of
acid without the presenceof charcoal. Rather than determine the
G. Bancroft and E. G. Fry 259

hydrolysis for each separate case, and since the variations in the
amount adsorbed indicated that an average value would be more
accurate, the average amount of glycogen present in the solution
after adsorption was hydrolyzed with different concentrations of
acid, and from these values a curve was drawn from which the
amounts of glucose for the exact amounts of acid used could be
determined. This curve was constructed from the values ob-

TABLE III

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Hydrolysis of Approximately 96.5 Mg. of Glycogen in Presence of Charcoal by
Various Concentrations of Acid
GlUCOS2
HCl concentra- Adsorbed Glycogen in Glucose produced from
tion, free dwxen, by solution concentration in 26.5 mg.
in solution difference solution glycogen’
-
N m9. m9. m9.
0.239 68.0 31.8 25.3 25.3
0.194 68.1 28.4 20.8 20.7
0.136 73.7 22.8 14.5 14.5
0.083 72.4 24.1 8.8 8.8
0.070 70.2 26.3 7.0 7.4
0.044 72.1 26.1 4.6 4.7
* The glucose values for the exact concentrations of hydrochloric acid
were obtained from Fig. 1.

tained by hydrolyzing 26.5 mg. of glycogen with various concen-


trations of acid in a volume of 20 cc.
Concentration of acid, N.. 0.25 0.20 0.15 0.09
Glucose, mg.. . 26.5 21.1 16.0 9.47

The close relationship between the hydrolysis of glycogen in the


presence of charcoal and the hydrolysis of 26.5 mg. of glycogen,
the average amount free in solution, by acid from a corresponding
solution, leaves no doubt that the hydrolysis depends upon the
concentration of unadsorbed glycogen and unadsorbed acid in
solution and is independent of the rather large amounts of glycogen
and acid adsorbed on the charcoal. Miller and Bandemer (8)
have obtained, moreover, similar results with the hydrolysis of
sucrose by acids in the presence of charcoal. The comparison is
given in the fourth and fifth columns of Table III and is shown
diagrammatically in Fig. 1.
260 Adsorption and Hydrolysis of Glycogen

Hydrolysis of Glycogen by Amylase in Presence of Charcoal-


It has been shown that adsorbed glycogen is not appreciably hy-
drolyzed by hydrochloric acid, and, that with an excess of glycogen
and hydrochloric acid above that which is adsorbed, the amount of
hydrolysis depends upon the quantity of free glycogen and free
acid in solution. Adsorption has induced stabilization of glycogen
rather than activation. In the present section, the hydrolysis of
glycogen with amylase as the catalyst has been carried out.
Since the activity of amylase changes considerably with the pH

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of the solution, care must be taken in such experiments to insure
a constant pH.* The hydrolyses were carried out at 38” in a total
volume of 75 cc5 The course of hydrolysis was determined by
means of samples removed at intervals, heated in boiling water to
inactivate the enzyme, filtered, and 3 cc. aliquots taken for analy-
sis by the maltose method of Hulme and Narain (12). The
values obtained were multiplied by 25 to give the total reducing
value of the solutions. The zero determination acted as the blank
and was assumed to be constant throughout the period of hy-
drolysis. The amylase6 usually was added last and marked the
start of the experiment except in the experiments with completely
adsorbed enzyme when the addition of glycogen marked the start.
The data obtained by the hydrolysis of glycogen in the presence
and absence of charcoal, with and without the addition of butyl
alcohol or chloroform, substances which increase glycolysis in
muscle, are given in Fig. 2.
4 It is well known (Miller (9), Miller and Bandemer (lo), Kolthoff (11))
that ash-free charcoal by adsorption of the acid constituents of a buffer
and hydrolytic adsorption of neutral salt solutions will change the pH of the
solution. Since a Serensen buffer of pH 6.4 is changed in the presence of
charcoal and NaCl to pH 6.6, all experiments with charcoal were made with
a buffer having an initial pH of 6.4 and those without charcoal having a pH
of 6.6. Enough NaCl solution was present to activate amylase despite any
adsorption of chloride by charcoal.
6 The following is a typical mixture: 1.00 gm. of charcoal, 10 cc. of gly-
cogen solution (approximately 1 per cent), 15 cc. of phosphate buffer (h1/7.5)
pH 6.4, 7.5 cc. of 0.5 N NaCl solution, 32.5 cc. of distilled water, and 10 cc.
of enzyme suspension containing 100 mg. of dry enzyme.
6 The amylase was a sample of “raw Degomma dl,” a pancreatic amylase
obtained from Rohm and Haas Company. It contained no maltase as far
as could be determined. When dissolved it always contained some sus-
pended protein which could be filtered off without apparently changing the
activity of the enzyme.
G. Bancroft and E. G. Fry 261

In all the enzyme experiments a large excess of enzyme above


that necessary for hydrolysis was used first in order to avoid the
possibility of the products formed poisoning the catalyst enough to
check the reaction before equilibrium was reached, and second,

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FIQ. 2. Maltose produced from 97.4 mg. of glycogen by 100 mg. of amylase
in the presence and absence of 1.0 gm. of purified norit with and without the
addition of 0.75 cc. of butyl alcohol or chloroform. No formation of mal-
tose was ever detected when either the glycogen or the amylase was com-
pletely adsorbed. When completely adsorbed glycogen or enzyme was
used, the substance was adsorbed on the charcoal, the suspension filtered,
and the charcoal containing adsorbed material then suspended in the usual
mixture. The amount of glycogen adsorbed (26.7 mg.) was determined in
the usual way. The quantity of enzyme adsorbed was not accurately
determined although qualitative tests indicated that practically all of the
20 mg. was adsorbed.

in order that sufficient enzyme would be present in the solution


even though considerable amounts were adsorbed by the charcoal.
Fig. 2 shows the very marked effect of charcoal on the course of
hydrolysis of glycogen by amylase. The data show the following
facts quite clearly: (1) Butyl alcohol and chloroform have no
effect on the normal hydrolysis of glycogen by amylase. (2)
262 Adsorption and Hydrolysis of Glycogen

In the presence of charcoal considerable amounts of glycogen are


still adsorbed and stable even with butyl alcohol or chloroform.
(3) No detectable reaction takes place when either glycogen or
amylase is completely adsorbed on the charcoal. (4) The form of
the curve in the presence of charcoal indicates the presence of a
competing reaction with the hydrolysis which is, no doubt, the
adsorption of maltose. Since the rate of adsorption increases with
the amount of maltose in solution, the curve shows roughly four
parts: first, where the hydrolysis predominates; second, where

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both reactions proceed about equally; third, where the adsorption
predominates; and fourth, where both reactions have reached
equilibrium.
TABLE IV
Adsorption of Glycogen from 75 Cc. of Solution Bu$ered to pH 6.6 on 1.0 Gm.
of Charcoal at 38”

mg. ml. mg. WI.


Plain......................... 97.4 56.6 52.6 44.8
Butyl alcohol. 97.4 54.2 50.2 47.2

The larger amount of hydrolysis with butyl alcohol and charcoal


than with charcoal alone might be due solely to adsorption of
maltose or it might be due to reduced adsorption of glycogen under
these conditions. These possibilities were investigated. As it is
evident from Table IV that glycogen adsorption on charcoal is not
materially reduced by butyl alcohol, this explanation of the differ-
ences observed is definitely eliminated. However, maltose ad-
sorption on charcoal under these conditions was found to vary
markedly with small changes in the amount of glycogen present,
for, with an initial concentration of 30.2 mg. of maltose the ad-
sorption dropped from 27.7 to 19.7 mg. in the presence of glycogen.
Moreover, no maltose was adsorbed in the presence of butyl
alcohol. As the exact amount of glycogen present was unknown,
an accurate estimation of the extent of the adsorption and hence
the effect on the hydrolysis could not be obtained in this way,
but, by running a normal hydrolysis in the presence of charcoal
until a constant value had been obtained, the amount of maltose
G. Bancroft and E. G. Fry 263

present and adsorbed on the charcoal could be determined by


adding butyl alcohol. As shown in Table V, the solution after
20 minutes was divided into two parts; one was inactivated by
heating before butyl alcohol was added and to the other butyl
alcohol was added directly.
In the former case the amount of maltose which had already
been formed was determined, but in the latter case the hydrolysis
continued until the values obtained when butyl alcohol was added
at the start of the experiment were approached. Adsorption of

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maltose alone is not the complete answer for the difference in
hydrolysis in the presence and absence of charcoal.

TABLE V
E$ect of Addition of Butyl Alcohol on Glycogen Hydrolysis by Amylase in
Presence of Charcoal at 38”

Time Maltose in solution


I

min. ml.
0 0.0
20* 7.8
Not inactivated Inactivated

30 43.5 31.6
40 45.7 34.0
60 46.9 33.0

* After 20 minutes, 0.01 cc. of butyl alcohol per cc. of solution was added
to each part.

DISCUSSION

Examination of the figures in a semiquantitative manner shows


that the 82.3 mg. of maltose formed by hydrolysis is 84.5 per cent
of the total value of 97.4 mg. of glycogen. From Table IV on
adsorption of glycogen, 52.6 mg. remained free in solution in the
presence of butyl alcohol. 84.5 per cent of this value would be
44.5, which is very close to the value 46.0 obtained in the hydrolysis
experiments with butyl alcohol. This indicates that in the pres-
ence of butyl alcohol the hydrolysis is proportional to the amount
of free glycogen in solution. Since only 33 mg. of maltose were
formed in the presence of charcoal alone, this relationship of the
hydrolysis to the amount of free glycogen does not hold. It would
264 Adsorption and Hydrolysis of Glycogen

require further investigation to determine the cause of this


difference but it seems probable that the adsorbed enzyme is so
oriented on the surface of the charcoal that it can combine with
some of the unadsorbed glycogen but cannot split it to maltose.
The action of butyl alcohol, therefore, would be either to reduce
the adsorption of this complex or to facilitate its splitting.
Przylecki (13) in studying the increased hydrolysis of glycogen
with butyl alcohol in the presence of charcoal ascribed the results
to decreased adsorption of glycogen. Lesser and Zipf (14) on

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the other hand believed the increase to be caused by reduced
adsorption of enzyme. However, Unna (15) was unable to find
any amylase in solution after addition of butyl alcohol. More-
over, both explanations depend upon the tacit assumption that ad-
sorbed enzyme or glycogen will react with unadsorbed glycogen or
enzyme respectively. However, since it has been shown that no
detectable reaction takes place with completely adsorbed enzyme
or glycogen in the presence of an excess of the other, these ex-
planations are inadequate.
No attempt was made to purify the enzyme and as Miller (16)
has shown with invert,ase that adsorption and subsequent activity
varies with a number of factors including age, concentration, and
purity of enzyme, it is possible that under certain conditions ad-
sorbed amylase might be active.
The type of adsorption exhibited by glycogen on charcoal has
been pointed out as being somewhat unusual and quite different
from such molecularly dispersed substances as sucrose, and the
inorganic acids. This may be relat,ed to the colloidal nature of
the glycogen, just as Miller found a different type of adsorption for
invertase which is also colloidal in nature.

SUMMARY

1. Glycogen is shown to be very strongly adsorbed by charcoal;


the type of adsorption where the charcoal takes up glycogen
nearly quantitatively to a saturation point and then can take up
no more may be due to the colloidal nature of the polysaccharide.
2. The amount of hydrolysis with hydrochloric acid depends
upon the concentration of free glycogen and free hydrochloric acid
in solution and is independent of the rather large amounts of
glycogen which are present but adsorbed on the charcoal.
G. Bancroft and E. G. Fry

3. No detectable reaction takes place when either glycogen or


enzyme is completely adsorbed on charcoal even in the presence
of an excess of the other.
4. The increase in hydrolysis with butyl alcohol or chloroform is
not due to reduced adsorption of glycogen or enzyme as suggested
by Przylecki and Lesser respectively, but only in their presence
does the amount of hydrolysis approach that expected from the
amount of glycogen which is unadsorbed.
5. The low amount of glycogen hydrolyzed in the presence of

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charcoal may be due to the combination between unadsorbed
glycogen and adsorbed enzyme.

BIBLIOGRAPHY

1. Bancroft, W. D., and Bancroft, G., J. Physic. Chem., 36, 194 (1931).
2. Miller, E. J., J. Physic Chem., 31, 1197 (1927).
3. Miller, E. J., J. Physic. Chem., 30, 1031 (1926).
4. Sahyun, M., and Alsberg, C. L., J. Biol. Chem., 89, 33 (1930).
5. Hagedorn, H. C., and Jensen, B. N., Biochem. Z., 136, 46 (1923).
6. Hanes, C. S., Biochem. J., 23,99 (1929).
7. Sahyun, M., and Alsberg, C. L., J. Biol. Chem., 93, 235 (1931).
8. Miller, E. J., and Bandemer, S. L., J. Am. Chem. Sot., 49, 1686 (1927).
9. Miller, E. J., in Weiser, H. N., Colloid symposium monograph, New
York, 6, 49 (1928).
10. Miller, E. J., and Bandemer, S. L., J. Physic. Chem., 32, 829 (1928).
11. Kolthoff, I. M., Rec. trav. chim. Pays-Bas, 46, 549 (1927); 2. Elektro-
them., 33, 497 (1927).
12. Hulme, A. C., and Narain, R., Biochem. J., 26, 1051 (1931).
13. Przylecki, St. J., and Niedzwiedzka, H., Biochem. J., 21, 1025 (1927).
14. Lesser, E. J., and Zipf, K., Biochem. Z., 140, 435 (1923).
15. Unna, Z., Biochem. Z., 1’72, 393 (1926).
16. Miller, E. J., J. Physic. Chem., 34,2666 (1930).