ENZYMATIC

(FTom the Department

HYDROLYSIS

OF GLYCOGEN.
of Toronto,

BY A. D. BARBOUR. of Biochemistry, University

Canada.)

Toronto,

(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 intermediate dextrins and maltose. Although the disappearance of glycogen under the action of pancreatic amylase has been studied 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 exploration of the possibility of reversion, and finally the endproducts 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 inactivated by 24 hours incubation at 37.
29

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30

Glycogen Hydrolysis
Stability of the Enzyme.

The enzyme was prepared by the method of Hunter and Dauphinee (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 phosA 10 cc. sample was taken phate buffer of pH 6.3 was prepared. 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+ hours. The glycogen was determined in these digests before and
TABLE I.

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St&L
No. Sample

of Glycogenase at 37 (zt : 6.1. pH

Period

of incubation en5yme. bra.

of

_-

Hydrolysis WC& 43 46 45 42 48

in 2$ ha

2 4 6 8 29 48

45 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 mixture 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 concentration 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 filtered through paper to remove the brown flocculent precipitate which separates. The filtrate was again heated to boiling and precipitated wit,h boiling alcohol. The supernatant liquid was decanted, 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 concentrations 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.

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The question of the identity of glycogen from different sources has been discussed at length by Norris (1) who describes experiments 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 trisaccharide, 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 somewhat 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.

Hydrolysis
pH . Source

at Varying
. .. . of glycogen.

Hydrogen Ion Concentrations Glycogen from Di$erent Sources.

. 6.0 1 6.2 1 6.4 1

with
6.6 formed.

Liver
/ 6.8

Extract
j 7.0

of

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Trisaccharide

Liver ...................... Muscle ..................... Scallop .....................

gm. 0.146 0.155 0.116

gm. 0.155 0.132

gm. 0.155 0.200

gm. 0.165 0.181 0.113

gm. 0.165 0.181 0.113

gm. 0.161 0.178 0.100

Estimation

of Glycogen.

The determination of glycogen was carried out by a modification of the method of Pfluger (10) and Imamura (11). A convenient 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~. It is then cooled, of concentrated hydrochloric acid, for 3 hours. 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 From the glucose below, further dilution being made if necessary. content of this solution the glycogen content of the original solution can be calculated. To avoid confusion, the results of glycogen determinations are expressed in terms of glucose, i.e., they are calculated as C6H120~.
TABLE III.

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Comparison

of
No.

Direct

Hydrolysis

and PJliiger o.f Glgcogen.

Methods

for

the Estimation

Solution

Glycogen by direct hydrolysis. per cent

Glyeoge~$ydPfliiger per cent

Difference. per cent

0.0197 0.0091 0.072 0.052 0.033 0.017

0.0199 0.090 0.073 0.052 0.030 0.017

+0.002 -0.001 +0.001 0.000 -0.003 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 Pfliigers 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 reagent, 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 potassium 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 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 constructed, 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 Hydrolysis. Ion Concentration for

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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 obtained 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
Correspondence between Increase in Eleducing Power and Disappearance of Glycogen.

35

During the study of the velocity of hydrolysis by muscle glycogenase,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 apTABLE IV.

Eflect of Hydrogen
Sample No.

Ion

Concentration
PH

on Enzymatic

Hydrolysis

of Glycogen.
Hydrolysis.

Glycogen after incubation. per

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cent

per

cent

2 3 4 5 6

between

5.53 6.04 6.24 6.54 7.27 Control.

TABLE V.

0.402 0.342 0.314 0.320 0.428 0.744

46 54 58 57 42

Correspondence

Increase

in Reducing

Power

and

Disappearance
100 x 30

of Glycogen.
Reducing POWX, glucose equivalents in 100 cc.
bra.

Increase in reducing power, glucose equivalents in 100 cc. gm.

Increase

trieacchsride equivalents in 100 cc. c7m.

vm.
2.06 1.80 1.57 1.08 0.44 0.16

gm.

gm.
0.26 0.49 0.98 1.62 1.90

0
3 6 12 24 36

0.014 0.091 0.233 0.291 0.510 0.660

0.077 0.142 0.277 0.496 0.646

0.25 0.47 0.92 1.64 2.13

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 purified, the osazone melts at 186 (corrected). This does not correspond to the melting point of the osazone of any sugar hitherto 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 melting 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 concentrated 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 correspond to the values for a trisaccharide osazone of the composition

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CZOH~,OI~N~.
On the assumption that only one product was present, determinations 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
Isolation of the Product of Hydrolysis.

37

The isolation of the sugar in the pure form presented considerable 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 481. The low molecular weight and reducing power suggested that the sugar had been converted into an anhydride of the constitution C,,HSoOlj with a theoretical molecular weight of 486, and this suggestion was supported by an analysis of the acetyl compound, 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 MunsonWalker (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 hydroxide 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 evaporated 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.

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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 trisaccharides 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 amylases were made. The results were quite different from those obtained with the muscle enzyme. In the case of salivary
Hydrolysis TABLE VI. of Glycogen by Salivary Amylase.

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Time.

Glyo~.s,",: in

Reducing pmw (asglucose) in 100 cc.

-min. 0 10 20 40 60 120 gm. 0.960 0.876 0.750 0.478 0.304 0.134 I. 0.000 0.084 0.210 0.482 0.656 0.826 am. 0.000 0.010 0.027 0.093 0.160 0.200 8.4 7.8 5.2 4.1 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. Investigation of the products of hydrolysis by the osazone method showed that the characteristic osazone of the trisaccharide described 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
IIydrolylsis by Salivary Amylnse.

39

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 melted sharply at 1.57 (corrected).

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Time.

Glyco~e destroyed in 100 cc.

min 0 10 20 40 60 135

om.
2.47 0 77 0.47 0.30 0.28 0 23

gm. 0.00

ym. 0.00

1.70 2.00 2.17 2.19 2.24

0 72 0.i8 0.85 0.88 1.00

IIydroly~sis

,with Pancreatic

Amylase.

50 cc. of 4 per cent glycogen solution were treated with 0.02 gm. of Mercks trypsin and incubated for 2 hours at 37, pH = 6.3. Samples were taken at intervals for glycogen and sugar determinations. The results arc shown in Table VII. The ratio of glycogen destroyed to reducing power with pancreatic 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 The characteristic crysosazone was examined microscopically. Some tals of isomaltose were identified as the principal product. crystals resembling those of glucosazone were also found. Repeating 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 characteristic crystals of the trisaccharide osazone be detected. EUfect of Increasing Glycogen Concentration on Hydrolysis.
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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 concentrated solutions of the product. A corollary of this expectation 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 concentration 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 In the that the inhibiting sufficient addition of Products on Hydrolysis.

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

A. D. Barbour
gether. This effect was observed both with the open chain trisaccharide 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 concentration 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 Added Subslance.

Glycogenase

in Presence

of

Added

substance.

Aversse glycogen content. 0 hr. 4 hrs. 24 hrs. --__

Hydrolysis. 4 hrs. 24 hrs. cent

per cent pet

centper centper centper

1.92 0.84 0.72 0.34 0.48 0.56 0 14 15 21 21 20

Trisaccharide.. . . Sucrose.. .... . . Glucose................................. Sodiumchloride .. . . .. . Urea .,...,.............................. Control . . .

. . .. . .

2.02 2.02 2.06 2.09 2.09 1.99

2.00 1.74 1.76 1.64 1.64 1.60

5 58 65 84 77 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 concentration 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 performed in which the trisaccharide was replaced by other substances. These were weighed out in sufficient amount to make a 0.2 M solution. 3 cc. of enzyme were added, and the volume It is made up to 10 cc. The results are shown in Table VIII. 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 inhibition. 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 presence and absence of the trisaccharide. This effect is illustrated in the following experiment. 5 cc. samples of 2 per cent glycogen solution were taken and the following substances dissolved:
TABLE IX.

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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.. 0.5 0.5 egg albumin.. Control.. .

+0.5gm.

albumin.

2.20 2.28 2.10 2.12

1.96 1.86 1.09 1.70

1.40 1.16 0.08 0.34

11 18 48 20

36 49 96 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 glycogenase, 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 rected), 186 (corrected), 187 (corrected); determinations average 186. gave: 186 (cor-

A. D. Barbour
Nitrogen Content of the Osazone.-Analyses (Coles modification) gave: 8.8 per cent average 8.7 per cent. Calculated for G,H~QO~~N., (trisaccharide) C~H320gN~ (disaccharide) Rotatory Power of Digest.-One digest cent (determined by acid hydrolysis and 2 dm. tube. (Y = +1.56; whence [a]ng = A second digest gave: Concentration a = i-1.79; whence [a]=, = +187. Anhydrotrisaccharide. Molecular Weight.--W = 10.0 gm., w = 0.0413 gm., A = 0.032, K (water) = 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. by the micro-Kjeldahl nitrogen, 8.6 per cent method nitrogen;

= 8.22 per cent. = 10.78 gave: Concentration = 0.438 per estimation of the glucose formed), +178. = 0.476 per cent, 2 dm. tube.

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Molecular Weight.-(In benzene solution.) Sample 1. W = 7.03 gm., Sample 2. IV= w = 0.0758 gm., A = 0.053, K (benzene) = 49.0, M = 996. 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 0 50.95. Calculated

gm. gave
for

0.1561

gm.

CO,,

0.0580 H 6.35,

gm. 0 50.79.

H20.

C 42.74,

H 6.31,

C18H32016. Barium

C 42.86,

Compound

of the Trisaccharide.

1. 0.1570 2. 0.1509 Calculated

Ba 21.0. gm. gave 0.0562 gm. B&O*. Ba 20.3. gm. gave 0.0521 gm. BaS04. Ba 20.8. for C18H3201E.Ba0.
CONCLUSIONS.

1. The hydrolysis of glycogen may be readily carried out by means of a glycerol extract of fresh muscle or liver tissue. Such an extract does not diminish in potency on long standing in a refrigerator, and its activity is only slightly diminished after incubation 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 ShafferHartmann 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 amyThe trisaccharide is not formed lases follows a different course. by these enzymes. Glucose and isomaltose have been identified among the products of these hydrolyses, but maltose apparently is not formed.

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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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