An enzymic method for determination of the average chain lengths of glycogens and amylopectins

ANALYTICAL

An

BIOCHEMISTRY

Enzymic Chain

39,

373436

Method Lengths

(1971)

for of

Determination

Glycogens

J. H. CARTER Department

AND

University

of Biochemistry, Miami,

Florida

and

of the

Average

Amylopectins

E. Y. C. LEE of Miami 33162

School

of

Medicine,

Received July, 1, 1970

Glycogen and amylopectin are related polysaccharides in that they are homopolymers of a-glucose containing 1 + 4- and 1 + 6-linkages. They are both branched structures consisting of linear chains of glucose units linked 1+ 4 which are joined by l+ 6-linkages at the branch points (Fig. 1). A characteristic of these structures is their average chain length, which is in the range of 10 to 16 for glycogen and 20 to 24 for amylopectins (1,2). The average chain length may be defined as the average number of glucose units in an uninterrupted sequence of 1 + 4bonds. Methods for the determination of average chain length revolve around the determination of the number of chain-ends, i.e., of end-group analysis. Reference to Fig. 1 shows that there are two types of end-group, the nonreducing end group and the glucosyl end involved in the 1 + 6linkage which becomes the reducing end on hydrolysis of that bond. Of the end-group methods so far developed for glycogen and amylopectin, chemical methods, e.g., methylation (3) and periodate oxidation (4), determine the nonreducing end group. Enzymic methods determine the potential reducing end. The two enzymic methods most widely used both rely on the use of two enzymes, the first an exoenzyme specific for the hydrolysis of the 1 -+ 4-linkages, the second a debranching enzyme specific for the 1 + 6-linkage. The reducing end glucose unit is released as free glucose, distinct from the other segments of the chain. The first method, that of Gori and Larner (5), utilizes rabbit muscle phosphorylaee and the rabbit muscle debranching enzyme system. The latter, amylo-l,&glucosidase/oligo-1,4+ 1,4-glucantransferase,l is composed of two enzymic activities which are both essential for the debranching process and which are associated with a single protein (6,7). Cori and Larner demonstrated that phosphorylase a.nd their preparation of debranching enzyme caused the conversion of glycogen and amylol Abbreviated

to glucosidase-transferase

in the rest of the text. 373

374

FIG. 1. Schematic representation of glycogen structure. Key: (-) = a-l +4 bond; (+) = or-1 + 6 bond; (80) = glucose unit; (a) = nonreducing glucose endgroup; (0) = glucose unit at glucosyl end of a-1 --) 6 bond, the potential reducing end-group on hydrolysis of the a-l +6 bond.

pectin into glucose l-phosphate and glucose in the proportions (n - 1) : 1, where 72 is the average number of glucose units per chain. The second method, that of Lee and Whelan (8), utilizes sweet-potato /3-amylase and the bacterial debranching enzyme, pullulanase. This combination of enzymes caused the conversion of the branched polysaccharides into maltose and glucose in the proportions (n - 1) : 1, where 12is the average number of glucose units per chain. We have developed a third enzymic based on the same principles, and which is a combination of the earlier methods in that /3-amylase is used in combination with glucosidase-transferase. Our method was tested by determining the average chain lengths of a number of glycogens and amylopectins already standardized by the periodate oxidation method. The chajn lengths of these samples were also determined by the Cori and Larner method. The advantage of the modified method is that it utilizes more convenient enzymes. These are the glucosidase-transferase from yeast (9,10), which is relatively easier to isolate than the rabbit muscle system, and sweet-potato fl-amylase, which is more stable than phosphorylase. Also the time taken for the determination is much less than in the Lee and Whelan method (8). M~ZTERISLS

AND

METHODS

Maltose was determined by the reducing value assay of Nelson (11). The preparation of the reagents and the method of assay were performed as described by Robyt and Whelan (12)) with the difference that the

ENZPMIC

DETERMINATION

OF

CHAIK

LEKGTHS

375

assay volumes were decreased to one-quarter. The reagent was standardized against maltose and reducing sugar values in the chain length assay were expressed as pmoles of maltose. Glucose was determined with glucose oxidase using a modification of the method of Fleming and Pegler (13,14). Each preparation of the reagent was calibrated against glucose. Determination of glycogen and amylopectin was performed by hydrolysis with amyloglucosidase (8). The amyloglucosidaae used was a freeze-dried preparation of the Aspergillus niger enzyme whose specific activity, when assayed with glycogen as the substrate, was 10 I.U./mg. Sweet-potato b-amylase was obtained from Worthington as a crystalline suspension in 0.6 saturated ammonium sulfate. This was diluted to approximately 0.5 mg/ml in a dilution buffer consisting of 50 m&f Ea acetate pH 5.0, 0.5 miM reduced glutathione, and 0.5 mg human serum albumin/ml. This was used as a stock solution and usually contained approximately 500 I.U. p-amylase/ml. A further dilution to 20 I.U./ml in 10 n&l Na acetate, pH 5.0, 0.5 mg human serum albumin/ml was made for inclusion in the chain length incubation mixtures. The enzyme was assayed in a 1 ml incubation mixture at 30” containing 0.570 soluble starch in 50 mM Na acetate, pH 5.0; production of maltose was assayed by reducing value and the units of ,8-amylase activity are expressed as pmoles maltose released per minute. It should be noted that the definition of this unit is not, the same as that specified originally by Hobson et al. (15). In terms of activity 1 I.U. is equivalent to approximately 10 of their units. Glycogen phosphorylase b was prepared from rabbit muscle by the method of Fischer et al. (16), and converted into phosphorylase a with phosphorylase b kinase (17). The phosphorylase a was purified by recrystallization nine times and was free of .a-amylase or glucosidase activity. The enzyme was stored as a crystalline suspension in 50 ml11 Tris HCl-40 mM mercaptoethanol, pH 6.8. A stock solution for use in the chain length method was prepared by centrifugation of the crystals and resuspension in 50 m:M glycerophosphate, pH 6.8, 0.5 rnnl mercaptoethanol, 1 mM AMP to give a solution containing at least 100 I.U./ml. The nine-times recrystallized enzyme was found to be very insoluble and the AMP was added since it was found to enhance the solubility of phosphorylase a. Thus, a stock suspension of about 2 mg phosphorylase a/ml in 50 mM glycerophosphate, pH 6.8, 0.5 mM mercaptoethanol remained as a suspension even after warming to 30” for several minutes. Addition of AMP led to the immediate formation of a clear solution. The mercaptoethanol concentration was such that. in the chain length incubation mixtures it was low enough not to interfere with

376

CARTER

AND

LEE

the glucose oxidase assay (18). Phosphorylase activities were assayed by the method of Hedrick and Fischer (19) with the difference that 40 mM glycerophosphate buffer was used. Rabbit muscle glucosidase-transferase was prepared by the method of Brown and Brown (6), with the modification that the enzyme was repeatedly chromatographed on DEAE cellulose to remove phosphorylase b, and finally chromatographed on Sephadex G-200. This preparation was free of #a-amylase, branching enzyme, and phosphorylase. A material of specific activity 7.6 I.U./mg was used in these experiments. Glucosidasetransferase activity was assayed as by Lee et al. (10). Ye,ast glucosidase-transferase was prepared from Fleischmann’s baker’s yeast (Standard Brands) by a modification of the method of Lee et al. (10) in that the yeast was lysed with ethyl acetate rather than homogenized in a colloid mill. The following describes the extraction procedure. Four pounds of yeast (previously at O-5”) was crumbled into a 4 liter beaker. Ethyl acetate (180 ml) was added and the beaker placed into a water bath at 45”. The mixture was stirred for 15 min during which time the yeast liquefied and the temperature rose from 5” to about 30”. Water (1800 ml, room temperature), phenylmethyl sulfonyl fluoride (Sigma Chemical Co., to a final concentration of 0.1 mM based on a volume of 3600 ml), toluene (5 ml), and thymol (1 gm) were added and the suspension was stirred at room temperature overnight. The suspension was then centrifuged to remove cell debris (65OOg, 2”). All further operations were carried out in the cold (2-5”‘). This operation was carried out with 5 batches of yeast and the combined supernatants were adjusted to pH 4.7 with 1 N acetic acid. After 30 min the precipitate which formed was collected by centrifugation (65009) and resuspended in 200 ml of water. The pH was adjusted to 7.0 with 1 N sodium hydroxide and the solution stored overnight. The solution was adjusted to pH 5.6 with 1 N acetic acid and the precipitate which formed removed by centrifugation. The preparations were then assayed for activity and for protein; at this stage the preparations were The results from three such prepreferred to as the “4.7 precipitate.” arations are shown in Table 1. This material was found to be much more convenient as a starting material for further purification than the 4.7 precipitate obtained after disruption of the yeast cells in a colloid mill, since the specific activities were about 10 times higher than those observed by Lee et al. (10). The batchwise TEAE cellulose extraction of Lee et aE. (10) was omitted and the 4.7 precipitate chromatographed on DEAE cellulose (Whatman DE 52). A column (2.5 X 45 cm) was equilibrated with 50 mM Tris-HCI, pH 7.6. The enzyme was eluted with a linear gradient

ENZYMIC

Specific Activities

DETERMINATION

of Yeast

OF

CHAIN

TABLE 1 Glucosidase-Transferase Preparations Stage (20 lb Yeast) Total act.ivity (I.E.1

Preparation 1 2 3 Lee et al. (10)

492 640 344 -

377

LENGTHS

at the 4.7 Precipitate

Total protein (gm)

Sp.act. (I.U./mg prot.ein)

1.64 4.07 2.74 -

0.31 0.16 0.13 0.017

consisting of 1 liter of the equilibrating buffer and 1 liter of 0.5 M NaCl in the same buffer. The fractions containing the glucosidase-transferase were eluted at approximately 0.2 144chloride concentration. The fractions containing the activity were combined and concentrated to a small volume (lo-20 ml) by ultrafiltration using a Diaflo PM-10 membrane (Amicon Corportion). Yields were of the order of 75% of the activity applied to the column, and specific activities of the order of l-2 I.U./mg. The final purification step was chromatography on Sephadex G-200 in 50 mM Na citrate-phosphate buffer, 1 mM EDTA, pH 7.2. Columns (2.5 X 100 cm) were used at a flow rate of 10 ml/hr maintained by a peristaltic pump. The material obtained after the DEAE cellulose chromatography was split into batches of no more than 50 mg in 6-10 ml for chromatography on Sephadex. The fractions containing the activity were pooled and concentrated to a small volume (5-10 ml) by ultrafiltration and stored in the refrigerator, where they were stable for several months. Recoveries of activity after Sephadex chromatography were in the order of 80% and the specific activities in the range 6.0-6.3 I.I-./mg. Determination of average chain lengths of glycogen and amylopectin by use of P-amylase and glucosidase-transferase. The incubation mixtures contained approximately 1 mg polyssccharide, 0.125 mg human serum albumin, 2.0 I.U. /3-amylase, 0.2 I.U. glucosidase-transferase in a final volume of 1 ml 50 mM Na citrate-phosphate buffer, pH 6.5. These were incubated at 30” for 3 hr. Samples were removed and assayed in duplicate for maltose by reducing value (0.1 ml samples) and glucose (0.2 ml samples) with glucose oxidase. Values were expressed as pmoles and the average chain length calculated from the expression: average

chain

length

2 X fimoles maltose -1 pmoles glucor e

= -

The explanation for the subtraction factor is that the free glucose would be estimated in the reducing value assay as though it were maltose

378

CARTER

AWD

LEE

(20) ; the total polysaccharide (assuming complete cnzymic degradation) in terms of the amount of glucose is then given by the expression (2 x pmoles maltose - pmoles free glucose). The degree of enzymic degradation achieved was determined by comparison with the value for the total polysaccharide present as determined by hydrolysis of a sample of the incubation mixture with amyloglucosidase (see above). The control incubation mixtures were: (a) one in which the glucosidase-transferasc was omitted and (5) one in which the polysaccharide was omitted. The first control confirmed that there was no detectable maltase activity in the P-amylase preparation under these conditions (21). Determination of average chain lengths of glycogen and mnylopectin by use of phosphorylase a and glucosidase-transferase. The incubation mixtures contained about 1 mg polysaccharide, 0.125 mg human serum albumin, 2.0 I.U. phosphorylase a, and 0.2 I.U. glucosidase-transferase in a final volume of 1 ml 50 mM Na phosphate buffer, pH 6.8. These were incubated at 30” for 3 hr. Samples (0.2 ml) were assayed for free glucose with glucose oxiclase. The glucose l-phosphate plus the free glucose were determined together after brief acid hydrolysis of the phosphate ester as by Lee et al. (10) using the following conditions: Samples (0.1 ml) were diluted to 1 ml with water. Samples of the d&&on (0.2 ml) were mixed with 0.2 ml 0.2 AT sulfuric acid and heated in a boiling water bath for 10 min and immediately cooled in ice. The solution was neutralized, made up to 1 ml with water, and assayed for glucose with glucose oxidase. The average chain lengths were calculated as: total free glucose after acid hydrolysis free glucose of the degree of enzymic hydrolysis were based of the total polysaccharide by amyloglucosidase:

average chain length = Determinations determination

on

the

total free glucose after acid hydrolysis total glucose by amyloglucosidase (The acid hydrolysis conditions led to the produc.tion of glucose from the hydrolysis of glucose l-phosphate only, and under these conditions no free glucose was liberated from a control containing glycogen alone.) percentage

hydrolysis

=

RESULTS

AND

DISCUSSION

The practical application of enzymic methods for the determination of average chain length is strongly dependent on the availability of the requisite enzymes in high purity. To this end the glucosidase-transferase system from yeast was chosen for use in the method, together with P-amylase, which is commercially available. In our experience the yeast

EKZSMIC

DETERMIXATION

OF

CHAIh-

379

LEKGTHP

glucosidase-transferase is relatively easier to purify than the rabbit muscle enzyme, and we describe a simplified procedure based on that of Lee et nl. (10) for the isolation of the enzyme system (see “Experimental”). The use of glucosidase-transferase and /3-amylase also offered another potential advantage in that preparations of both enzymes are free of cy-amylase. We have found that stringent procedures are necessary to ensure that phosphorylase and pullulanase preparations arc free of this activity, which can interfere severely with known enzymic methods for the determination of average chain length. It had to be proved first that a combination of ,8-amylase and glucosidase-transferase would in fact bring about the total degradation of glycogen and amylopectin. In theory it would be expected that the 1 + 4 linked glucose unit,s would be converted into maltose, and that free glucose would arise only from the potential reducing ends of the chains (Fig. 1) , so that the relative amounts of maltose and glucose formed would be in the ratio (n - 1)/2: 1, where n is the average chain length. From this relationship the value of n may be derived as:

In an experiment in which a 60-fold excess of ,8-amylase over the yeast glucosidase-transferase in terms of activities was used, a complete degradation of a sample of shellfish glycogen into maltose and glucose was achieved (Table 2). Paper chromat,ography of the incubation mixture confirmed that gIucose and maltose were the sole products. Maltose was determined by reducing value, and glucose with glucose oxidase. It should be noted that the reducing value assay would determine the glucose present as though it were maltose. In order to determine a true value for maltose, the experimental value for maltose (M,) must be Degradation of Shellfish G’ucosidase-Transferase

TABLE 2 Glycogen by Combined and Sweet-Potato

Time (hr)

Maltose (pmoles)

Free glucose (rmoles)

1 2 3

13.7 14.3 14.0

2.28 2.56 2 56

Actions oc Yeast B-Amvlase”

Degradat)ionb ry;, 9s 101 100

Average chain length 11.0 10.2 9.9

D Incubation conditions were the ame as those indicated in the text for the determination of average chain length except that this was a 5 ml incubation mixture containing 2.0 I.U./ml of P-amylase and 0.034 1.U /ml of yeast glucosidase-transferase. * Based on the determination of total glycogen by hydrolysis wi’h amyloglrlcosidase.

380

CARTER

AND

LEE

corrected by subtraction of the value for glucose (G), where M, and G are the values in pmoles. Substitution of the experimental values in the equation for the average chain length given above yields:

Reference to Table 2 shows that the chain length calculated on this basis is in good agreement with the value of 9.7 previously determined for this glycogen by L’ee et al. (10) using the Cori and Larner (5) method. In another experiment the ability of the rabbit muscle glucosidase-transferase to act in combination with ,@-amylase was tested, and it is seen (Fig. 2) that it is equally effective.

0

40

80

120

160

TIME (minutes) FIG. 2. Combined actions of yeast or rabbit muscle glucosidase-transferase and P-amylase on shellfish glycogen. The incubation mixtures were the same as those used for the determination of chain length except that the amounts of enzyme used were 0.1 I.U. of glucosidase-transferase/ml and 2.0 I.U. of p-amylase/ml. Values for rabbit muscle enzyme are indicated by triangles, and values for yeast enzyme by circles.

The ability of a combination of glucoaidase-transferase and pamylase to effect the total conversion of glycogen into maltose and glucose merits some discussion. In the presence of p-amylase it might be expected that the structure on which debranching enzyme must act is that of a ,+limit dextrin (Fig. 3). The side chains of the J-limit

ENZYMIC

DETERMINATION Maltose

OF

CHAIN

381

LENGTHS

removed

--/%A by

transfer (6) Maltotriose removed

by

transfer

I

$qi

Maltose removed by B -ally I ase

A+ CC)

Fro. Fig.

3. Action of glucosidase-transferase 1. A and D represent outer chain

on p-amylase limit dextrin. Key as structures

of a /3-amylase

limit

in

dextrin.

dextrin (22) are an equal mixture of maltosyl and maltotrisoyl residues (A and D). In order for the maltotriosyl side chains to be prepared for debranching they must be shortened to glucosyl residues (D + B) by maltosyl transfer. Shortening of the maltotriosyl residues by maltosyl transfer (D+ B) is compatible with the reported capabilities of the rabbit muscle and yeast debranching enzyme systems, but neither system is reported to effect the glucosyl transfer (10,23) required for the debranching of the maltosyl side chains (A+ B). However, the maltosyl side chains could be extended by maltosyl transfer from structure D, to give structure C, and this could then give the desired structure B by maltotriosyl transfer. It might be expected that a reaction C +A would ensue in the presence of excess P-amylase,that the conversion A + C + B would thereby be thwarted,and debranching would be incomplete. In practice this does not happen. Complete conversion of glycogen was noted even when the ratio of @amylase:debranching enzyme was increased to 2OO:l. Our interpretation is that the formation of the maltosyl stubs by ,&amylase is so slow that transferase action on longer side chains occurs so as to obviate maltosyl formation. It might be noted that full attenuation by &amylase of the outer chains of glycogen, if not amylopectin, is a very slow process. With ,Q-amylase alone, in an amount 50 times in excess of that used here, it required 47 hr to achieve limiting degradation of glycogen (24). Since we achieve the complete degradation of glycogen with /3-amylase and glucosidase-transferase within 3 hr, it seems likely that the maltosyl side chains are not formed as intermediates or end-products.

382

CARTER

AND

LEE

In the light of the demonstrated ability of a combination of glucosidase-transferase and ,!3-amylase to effect the total degradation of shellfish glycogen to maltose and glucose in the expected proportions, it appeared that this now provided the basis for a new method for the determination of the average chain lengths of glycogens, if not amylopectins as well. The method was tested by determination of the average chain lengths of a number of glycogen samples isolated from different sources. Most of these were generously supplied to us by Professor D. J. Manners, who had determined their chain lengths by the periodate oxidation method (4). In our method we use a final concentration of 0.2 I.U. of glucosidase-transferase and 2.0 I.U. of &amylase/ml. For comparative purposes the average chain lengths of these same samples were also determined by the use of yeast glucosidase-transferase and phosphorylase (see “Experimental”) in a variation of the Cori and Larner method. The results are shown in Table 3. It is seen that the chain lengths determined by all three methods are in good agreement, the widest deviation being of sample 9, where the chain length determined by our method was 14.2 compared with 16.0 and 17 by the Cori and Larner and periodate methods, respectively. The same result was obtained when the assay was repeated. A second point of note is that the degrees of degradation of the polysaccharides by our method appear to be close to completion in most cases, while those obtained with the phosphorylase plus glucosidase-transferase method were consistently lower. It is not known whether this reflects some undetected systemat’ic error on our part or is due to an inherent property of the method. Cori and Larner reported that total degradation was achieved but that the ratio glucose: glucose l-phosphate was unchanged even when conditions were deliberately chosen so that degradation was incomplete (5). Three samples of amylopectin also provided by Professor D. J. Manners were assayed in the same way (Table 3). Again a good agreement for the values of chain length was obtained, despite the fact that the degrees of degradation by both methods were strikingly low. It would appear from our results that the ability of the combinations of enzymes to degrade the polysaccharides totally is not. an important factor in obtaining an accurate chain length; a similar observation was made by Cori and Larner in regard to their method (5). It might be expected that an accurate value for average chain length could be obtained only if the total degradation of the polysaccharides is achieved, since only then would all the end group be expressed as the desired product. In both enzymic methods, however, calculation of average chain length is dependent only on the ratio of the amounts of the products of degradation and not on the absolute amounts. It seems clear that the degraded

ENZTMIC

Determination

DETERMINATION

of Average

Chain Enzymic

OF

CHAIN

TABLE 3 Lengths of Glycogens method

Ie

Enaymic

Degradation Sample

CL

(76)

383

LENGTHS

and Amylopectins met)hod Degradation

IF CL by periodate

CL

(5%)

13.8 11.7 14.4 12.7 5.5

89 93 96 95 89

13 11 13 11 6 13 15 14 17

Gl ycogens

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Cat liver I Human muscle II Rabbit muscle III Ascaris lumbricoides I Human liver, type III glycogenosis Skate liver I Trichomonus foelus Rabbit liver VII Horse diaphragm (prerigor) Rabbit, liver @limit dextrin Human liver, type IV glycogenosis

15.0

101

11.1

99

13.4 12.5 4.9

99 93 78

11.2 14.2 13.4 14.2

96 99 101 99

11.9 15 5 14.7 16.0

93 92

8.2

108

8.5

98

21.7

98

24.9

80

-

19.9 9.7

Y! 67

21.0 10.2

88 58

20 n-10

20.4

89

22.6

87

98 94

dmylopectim

1. Waxy 2. Potato p-limit 3. Waxy

maize starch I amylopectin dextrin sorghum starch

II

n p-Amylase + yeast glucosidase-transferase. b Phosphorylase a + yeast glucosidase-transferase.

portions of the polysaccharide are representative of the total structure of the enzyme. As to why this is so, possible explanations are, first, that only some of the polysaccharide molecules are degraded and, second, that the polysaccharide might contain elements of structure resistant to enzymic action. A small amount of this element would lead to a disproportionately large part of the structure being protected from further degradation, since we are dealing with what are essentially exoenzymes. It is relevant that in recent studies on the use of amyloglucosidase for the degradation of glycogen and amylopectin it has been shown that purified amyloglucosides is not capable of total degradation unless n-amylase is added (25,26), so that. the inability of exoenzymes to degrade these polysaccharides completely. may be more general than was previously thought to be the case. Consideration of the possible effects of #cY-amylase on our method

384

CARTER

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LEE

shows that they ought to be minimal and might even be beneficial in the case of the amylopectins in allowing the enzymes to bypass the blockages. Accordingly, this was tested in an experiment in which 0.006 I.U. and 0.06 I.U./ml of salivary a-amylase were added to the incubation mixtures in the chain length determination of amylopectin. The results (Table 4) show that the extent of degradation was increased from 92% to 1057% without greatly affecting the chain length value. Since glucosidase-transferase is capable of action on low molecular weight substrates it is not surprising that the dissection of t’he polysaccharlde structure by the endo-action of ,a-amylase did not affect the method. In contrast, addition of 0.006 I.U./ml of a-amylase to the incubation mixture in the determination of the average chain length of amylopectin with phosphorylase and glucosidase-transferase reduced the yield of #a-glucose l-phosphate by 35% without changing the yield of glucose. The effect of a-amylase on the method of Lee and Whelan (8) would also bc to increase the apparent amount of end-group.

Effect

of or-Amylase

TABLE 4 on Determination of Average Chain p-Amylase and Glucosidase-Transferase”

cu-Amylase (I.U./ml) 0 0.006 0.06

added

Degradation (5%) 92 105 105

Length

of Amylopectin

with

Average chain length 30.5 31.7 23.6

a Experimental conditions were those described in the text for the determination of average chain length except that the incubation mixtures contained in addition 10 mil4 NaCl and the stipulated amount of human salivary or-amylase.

The reproducibility of our method was tested by determining the average chain length of shellfish glycogen. A mean value of 9.8 with a standard deviation of 0.06 for six assays was obtained. The sensitivity of the method is such that we used 1 mg of glycogen for our determination; smaller samples could be used by a reduction of the assay volumes. A potential source of error in the method is the reported presence of small amounts of maltase activity in sweet-potato J3-amylase preparations (21). In the method of Lee and Whelan (8) erythritol is used to inhibit this activity. We have found this to be unnecessary in our method since the levels of p-amylase activity used are much lower and the incubation time is relatively short. It is nevertheless recommended that the ,&amylase preparations be tested for the presence of maltase; the use of a control incubation mixture in which glycogen and fl-amylase

ENZYMIC

DETERMINATION

OF

CHAIN

LENGTHS

385

alone are present is sufficient (see “Experimental”). A second potential source of error is the effect of reduced glutathione on the glucose oxidase assay (18). Reduced glutathione is reported to be necessary for the stability of &amylase (24). We have omitted reduced glutathione from the dilute /?-amylase solutions and have found the activity to be stable over the short incubation period which we use. SUMMARY

A new method for the determination of the average chain lengths of glycogens and amylopectins is described. The method utilizes yeast glucosidase-transferase and sweet-potato p-amylase, and was tested successfully against a number of samples of polysaccharides of known chain lengths. The met,hod has several advantages over previous enzymic methods in that (1) it is relatively insensitive to the presence of ar-amylase, (2) the enzymes required are readily available-sweet-potato /3-amylase is commercially available and we describe a relatively simple method for the isolation of t’he yeast glucosidase-transferase, (3) the time required for the assay is short, relative to the method of Lee and Whelan (8). ACKNOWLEDGMENTS This

work was supported by a grant from the National Institutes of Health (AM 12532). We are indebted to Professor D. J. Manners for his generous gift of glycogen and amylopectin samples, and Professor W. J. Whelan for his helpful discussion of this work. E. Y. C. Lee is an Investigator of the Howard Hughes Medical Institute. REFERENCES 1. WHELAN, W. J., in “Encyclopaedia of Plant Physiology” (W. Ruhland. ed.), Vol. 6, p. 154. Springer-Verlag, Berlin, 1958. 2. MANNERS, D. J., Advan. Carbohydrute Chem. 12, 261 (1957). 3. BOURNE, E. J., FANTES, K. H., AND PEAT, S., J. Chem. Sot. 1949, 1169. 4. MANNERS, D. J., AND WRIGHT, A., J. Chem. Sot. 1961, 2861. 5. CORI, G. T., AND LARNER, J., J. Biol. Chem. 188, 264 (1951). 6. BROWN, D. H., AND BROWN, B. I., Methods Enzymol. 8, 515 (1966). 7. NELSON, T. E., KOLB, E., AND LARNER, J., Biochemistry 8, 1419 (1969). 8. LEE, E. Y. C., AND WHELAN, W. J., Arch. Biochem. Biophys. 116, 162 (1966). 9. LEE. E. Y. C., NIELSEN, L. D., AND FISCHER, E. H., A&. Biochem. Biophys. 121, 245 (1967). 10. LEE, E. Y. C., CARTTAR, J. H., NIELSEN, L. D., AND FISCHER, E. H., Biochemistry 9, 2347 (1970). 11. NELSON, N., J. Biol. Chem. 153, 375 (1944). 12. I~OBYT, J. F., AND WHELAN, W. J., in “Starch and Its Derivatives” (J. A. Radley, ed.), p. 430. Chapman & Hall, London, 1968. 13. FLEMING, I. D., AND PEGLER, H. F., Analyst 88, 967 (1963).

386 14.

CARTER

20.

21. 22. 23. 24. 25. 26.

LEE

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