the 6-o-methylglucose-containing lipopolysaccharide of ... · analyzed by paper chromatography....

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 243, No. 16, Issue of August 25, pp. 4319-4331, 1968

Printed in U.S.A.

The 6-O-Methylglucose-containing Lipopolysaccharide of

Mycobacterium phlei

STRUCTURE OF THE REDUCING END OF THE POLYSACCHARIDE*

(Received for publication, March 29, 1968)

MILTON H. SAIER, JR. AND CLINTON E. BALLOU

From the Department of Biochemistry, University of California, Berkeley, California 9.&%20

SUMMARY

The 6-0-methylglucose-containing polysaccharide (MGP), from mycobacterial species, is known to have a branched structure, with 3-0-methylglucose and glucose at the nonreducing termini. A glucose unit at the reducing terminus is linked glycosidically to the Z-hydroxyl of D-glyCerk acid. This paper describes further studies which are aimed at defining the structure of the reducing end of the polysaccharide.

The aglycon of MGP has been converted to the methyl ester which was reduced with sodium borotritide. a-Gluco- pyranosyl-(1 --) 2)-glycerol-3H and ar-ghlcopyranosyl- (1 -+ 6)-cr-glucopyranosyl-(1 4 2)-glycerol-3H were isolated from a partial acid hydrolysate of the radioactive product, thus establishing the configuration of the glucosidic linkage to glyceric acid.

The aglycon of MGP was removed by a Lossen rearrangement, and the glucose residue at the reducing terminus was reduced with sodium borotritide to yield r-MGP*. A radioactive disaccharide, which accumulated during partial acid hydrolysis of r-MGP*, was isolated in 25% yield. It was characterized as isomaltitol. Acid hydrolysis of methylated r-MGP* gave 1,2,3,4,5-penta-0-methylglucitol as the only radioactive product. Three other radioactive oligosaccharides, a tri-, tetra-, and pentasaccharide, derived from the reducing end of the polysaccharide, were isolated from a partial acid hydrolysate of r-MGP*. The trisaccharide was characterized as /3-Glc&(I + 3)-cr-Glcp- (1 + 6)-glucitol-3H; the tetrasaccharide was B-O-methyl- cu-Glcp-(1 +4)-P-Glcj-(1 4 3)-a-Glcp-(1 + 6)-glucitol-3H; and the pentasaccharide was a-Glc@-(1 + 3)-6-O-methyl- ar-Glcp-(1 +4)-p-Glcp-(1 + 3)-a-Glcp-(1 + 6)-glucitol-3H. Coupled with the results outlined in the previous paragraph, the structures of these oligosaccharides detine the configurations and linkages for the first 5 residues at the reducing end of the molecule.

* This work was supported by Grants AM884, AM8845, AM- 10109. and TI-GM31 from the United States Public Health Service and Grant GB-5566 from the National Science Foundation. Taken in part from the doctoral thesis of Milton H. Saier, Jr.

A procedure employing well known reactions was adapted for the selective stepwise degradation of oligosaccharides from the reducing end. This involved reduction of the reducing terminal sugar residue and selective oxidation of the resulting polyol with dilute periodate. The oxidized fragment was reduced and the small reduced aglycon was then cleaved from the remainder of the molecule by mild acetolysis. A single application of the method to isomaltotriose and the radioactive trisaccharide from r-MGP* gave isomaltose and laminaribiose, respectively.

A re-examination of the methanolized products of methylated MGP by gas liquid chromatography provided direct evidence for a single (1 -+ 6) linkage in the polysaccharide. The sugars, in an acid hydrolysate of methylated cr-amylase- digested MGP, were reduced with sodium borotritide, oxidized by sodium periodate, and reduced a second time. The products contained radioactive 2,4,6-tri-o-methylglucitol, 2,3-di-0-methylthreitol, and 2,3,4-tri-o-methylxylitol plus 2,3,4,6-tetra-O-methylglucitol in the approximate molar ratios 1:8:3. Therefore, a single (1 + 3) linkage is also present in the main chain of MGP. With the exception of this linkage, the (1 -+ 6) linkage mentioned above, and the branch point linkage, all remaining linkages in the polysaccharide appear to be of the (1 + 4) type.

The substrate specificities of two enzymes used for structural analyses, an cr-glucoamylase from Aspergillus niger and an a-glucosidase from the yeast, Debaryomyces vanryi, were investigated. At low substrate concentrations the former is highly specific for a-(1 + 4)-linked glucosyl residues, while the latter hydrolyzes a much broader spec- trum of a-glucosides.

Previous publications from thislaboratory (l-3) have described the isolation and partial characterization of a mycobacterial lipopolysaccharide which contains glucose, 60-methylglucose,

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4320 Lipopolysaccharide of Mycobacterium phlei Vol. 243, No. 16

and 3-0-methylglucose. The “methylglucose polysaccharide” obtained by deacylation of the methylglucose lipopolysaccharide contains about 18 sugar units linked glycosidically to each other with one branch in the chain, and the polysaccharide is linked to the 2-hydroxyl of n-glyceric acid at what would otherwise be the reducing end of the polymer.

In this paper we describe the isolation and characterization of oligosaccharides derived from the reducing end of the molecule. Three glucose units initiate the chain. The first is attached to glyceric acid by an a!-(1 + 2) linkage, and the second and third are linked ar-(1 --f 6) and ,&(l + 3), respectively. The fourth sugar unit, the first 6-O-methylglucose in the chain, is con- nected to the preceding trisaccharide moiety by an ol-(1 --t 4) linkage. The branch, a glucose residue, is linked a-(1 --) 3) to this 6-0-methylglucose unit. All other linkages appear to be &(l + 4).

EXPERIMENTAL PROCEDURE

Enzymes-Porcine pancrease cu-amylase, /3-glucosidase (emul- sin), and glucose oxidase (Glucostat Special) were purchased from Worthington. 6-0-Methylglucose is not a substrate for the glucose oxidase. Glucoamylase I from Aspergillus niger (4) was generously supplied by Dr. John Pazur. One unit of glucoamylase activity is defined as that amount of enzyme which hydrolyzes 1 I.tmole of maltose per hour with the maltose concentration at 1 InM and other conditions as described in Table VI.

Yeast a-glucosidase was isolated as follows. Debaryomyces van@ (obtained from Dr. H. J. Phaff) was grown at 30” in 2 liters of the medium described by Stewart and Ballou (5) except that glucose was omitted, and 2% glycerol (w/v) and 1% maltose were included. Twenty-four hours after inoculation with 50 ml of stationary phase cells, the cells, in late log phase, were har- vested. Little a-glucosidase activity was present in the cultural filtrate. Cells were suspended in 100 ml of 0.05 M phosphate buffer, pH 7. Thirty g of Superbrite glass beads (Minnesota Mining and Manufacturing Company) were added, and the solution was chilled to 0”. The yeast cells were broken open by homogenizing for 15 min at maximum speed in a Sorvall Omni- Mixer, the temperature being maintained below 4”. Cellular debris was removed by centrifugation, and to the supernatant were added 20 ml of 1% protamine sulfate, pH 7. After 15 min in the cold, the suspension was centrifuged. Ammonium sulfate, 50 g, was added slowly to the clear yellow supernatant, and after 15 min at 4” the precipitated protein was collected by centrifugation. This was dissolved in 10 ml of 0.05 M phosphate buffer, pH 7, and was dialyzed overnight against neutral phosphate buffer containing 10 InM mercaptoethanol. Various glycosides were incubated with 0.25 ml of this enzyme preparation for 5 hours at 30”, after which 1 ml of methanol was added and protein was denatured by heating at 100” for 1 min. Ions were removed from the centrifugal supernatant withthe carbonate form of a mixed bed resin (Dowex 501), and the products were analyzed by paper chromatography. Under these conditions the enzyme preparation was found to have a broad substrate specificity for ol-glucopyranosides but did not hydrolyze /j’- glucopyranosides (see “Discussion”). With maltose as substrate, the enzyme was optimally active at neutral pH values. It was stable for at least 1 week at 4” and was not inactivated by repeated freezing and thawing.

Standard Compounds-Cellobiose and gentiobiose were from Pfanstiehl Chemical Company, Waukegan, Illinois. Nigerose was prepared by partial acid hydrolysis of A. niger nigeran (a

gift from Dr. Pazur) (6). Hydrolysis was carried out in 1 N

sulfuric acid for 3 hours at 85”. Sulfuric acid was neutralized with barium carbonate, the precipitate was removed by filtration, and remaining salts were removed with a mixed bed resin (Dowex 501). Nigerose was purified by preparative paper chromatography (Table I, Solvent B). Laminaribiose was obtained from Dr. E. Percival. Isomaltose and isomaltotriose were from Dr. W. J. Whelan. Maltotriose was isolated by gel filtration (Sepha- dex G-25) from a deacetylated partial acetolysate of amylose. The preparation of the two isomers of 6-0-methylmaltose has been described (3). The reduced forms of the above oligosaccharides were prepared by sodium borohydride reduction. After reduction was complete, cations were removed with Dowex 50 (H+) and boric acid was removed by repeated evaporation from methanol. Remaining salts were removed with a mixed bed resin.

2,3,4-Tri-0-methylglucose, 2,3,6-tri-0-methylglucose, 2,4,6- tri-O-methylglucose, and 2,3,4,6-tetra-0-methylglucose were available in crystalline form. The corresponding methyl glycosides were prepared by heating the sugars in 5% methanolic HCl at 100” for 8 hours. The radioactive reduced compounds were obtained by reduction of the parent sugars with sodium borotritide. The precursor for 2,3,4-tri-0-methylxylitol was prepared by methylation of xylose and hydrolysis of the methyl glycoside. Radioactive 2,3-di-0-methylthreitol resulted from periodate oxidation and sodium borohydride reduction of 2,3,6- tri-0-methylglucitol-1-3H.

Radioactive cu-n-glucopyranosyl-(1 + l)-ethyleneglycol and cr-isomaltosyl-(1 + 1) -ethyleneglycol were synthesized by selective periodate oxidation and sodium borotritide reduction of isomaltitol and isomaltotriitol, respectively (7). Oxidation of the glucitol moiety was carried out for 6 hours in 0.1 mM

sodium periodate, conditions which did not oxidize the sugar residues. The products were characterized by acid hydrolysis, which released glucose and radioactive ethyleneglycol. Radio- active ol-D-glucopyranosyl-(1 4 2) -glycerol, P-n-glucopyranosyl- (1 --t 2)-glycerol, cr-n-glucopyranosyl-(1 -+ 2)erythritol, and P-n-glucopyranosyl-(1 + 2)erythritol were prepared from maltitol and cellobiitol, respectively, as follows. The reduced disaccharides (3 mg) were oxidized for 6 hours in 600 ml of 0.1 mM sodium periodate. Excess periodate was destroyed with ethyleneglycol, and the solutions were concentrated to 10 ml before exposing the products to sodium borotritide. The major radioactive product, RGlc = 0.79 (Solvent B), in each of the reaction mixtures was isolated by paper chromatography. Acid hydrolysis released glucose and radioactive erythritol. The minor radioactive products were characterized as glucopyranosyl- (1 + 2)-glycerol (10%) and glucitol (10%). The major product presumably resulted because of cyclization (7). Glucitol must have arisen from overoxidation of glucosyl-(1 --f 2).tartronalde- hyde. The glucopyranosyl-(1 + 2)-erythritol anomers were again subjected to periodate oxidation and borotritide reduction as above. The only radioactive product in each case had RGL~ = 1.00 (Solvent B) and RGlc = 0.82 (Solvent D). Acid hydrolysis released glucose and radioactive glycerol. With the conditions described in the legend to Fig. 10, /3-glucosidase hydrolyzed completely the two products derived from cellobiitol to yield glucose and the radioactive polyol, glycerol or erythritol. The

glucosides from maltitol were not hydrolyzed by this enzyme, but they were hydrolyzed by the yeast a-glucosidase. Standard 6-O-methyl-c-n-glucopyranosyl-(1 + 2).erythritol, used as a


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chromatographic standard for thecharacterizationof theperiodate oxidation products of r-Penta*,* and cr-n-glucopyranosyl-(I -+ l)-glycerol were obtained from cr-amylase-digested MGP by periodate oxidation, reduction, and mild acid hydrolysis. The isolation and characterization of these compounds will be described in a subsequent paper.

Preparation of r-MGP*-ar-Amylase-digested MGP, which had been subjected to a Lossen rearrangement for the selective removal of the glyceric acid (3),2 was reduced partially with sodium borotritide and then heated at 60” for 1 hour in the presence of a large excess of sodium borohydride to complete the reduction. The reduced radioactive product, designated r- MGP*, was passed through a Sephadex G-50 column. All of the radioactivity eluted with the polysaccharide, and the amount of radioactivity in each tube was proportional to the amount of carbohydrate.

Reagents-Sodium borotritide (specific activity, 2.6 mC per mg) was purchased from New England Nuclear. It was used without dilution unless otherwise stated. Bis(2-methoxyethyl) ether (diethyleneglycol dimethyl ether) was purchased from Matheson Coleman and Bell. Other materials were as described previously (3).

Analytical Procedures-Total carbohydrate was determined by the phenol-sulfuric acid method with glucose as the standard (8). Methylation of carbohydrates was carried out as before (3), and products were purified by passage through a Sephadex LH-20 column with methanol as the eluting solvent. Column effluents were analyzed for carbohydrate by the phenol-sulfuric acid assay and for radioactivity by counting aliquots of the fractions in 10 ml of Bray’s solution (9) in a Packard Tri-Carb liquid scintillation counter.

Reduction with Sodium Borotritide-The sample to be reduced was exposed to excess sodium borotritide for 10 min at 60”. Dowex 50 (H+) resin was added, and the solution was shaken for 5 min with the resin which was then removed by filtration and washed 3 times with aqueous methanol. The combined filtrate was evaporated to dryness under vacuum, and boric acid was removed as methyl borate by evaporating it three times with methanol. I f salts remained, or if the level of background radioactivity was high, the solution was treated with a mixed bed resin, and resin was removed by filtration and washed three times with aqueous methanol. The solution was again concentrated to dryness and evaporated with methanol.

Chromatography and Electrophoresis-Descending paper chromatography was done on Whatman No. 1 filter paper. Whatman No. 3MM filter paper, previously washed for 24 hours with 2% aqueous ammonia, was used for preparative separations. The following solvents (compositions in volume ratios) were used: A, 1-butanol-pyridine-water, 10 : 3 : 3; B, ethylacetate-acetic acid- formic acid-water, 18 : 3 : 1: 4; C, ethylacetate-pyridine-water, 5:3:2; D, 1-butanol saturated with water; E, benzene-ethanol-

1 The abbreviations used are: MGLP, methylglucose lipopolysaccharide: MGP, methvlglucose nolvsaccharide obtained bv deacylating MGLP; r-MGP;, MGP &om which glyceric acid has been removed by a Lossen rearrangement, and inwhich the glucose residue at the reducing terminus has been reduced to glucitol-l- 3H; r-glycerol-MGP*, MGP in which glyceric acid has been reduced to glycerol-1JH; r-Di*, r-Tri*, r-Tetra*, and r-Penta*, reduced oligosaccharides obtained by partial acid hydrolysis of r-MGP*. The structures are shown in Fig. 12.

2 The modified Lossen rearrangement in Reference 3 is based on HOARE, D. G., OLSON, A., AND KOSHLAND, D. E., JR., J. Amer. Chem. Sot., 90, 1638 (1968).

water, 170 : 47 : 15 (organic phase) ; F, ethylacetate-pyridine- water, 10:4:3; G, 5% methanol in benzene.

Sugars and polyols were detected on paper chromatograms with the aniline hydrogen phthalate reagent (specific for sugars) (lo), alkaline AgN03 (the chromatogram was held over steam to increase sensitivity for polyols and glycosides) (II), and the periodate-benzidine reagent (12). Radioactive compounds were detected on paper chromatograms with a Nuclear-Chicago scanner, model 1006. Chromatograms were also surveyed for radioactivity by cutting them into horizontal strips which were placed in vials with 10 ml of Bray’s solution (9) and counted in a liquid scintillation counter. This procedure was about 100 times more sensitive than the former. Paper electrophoresis was carried out for 2 hours at 1250 volts in 0.1 M sodium borate at pH 10 or in 0.1 M sodium molybdate at pH 5 (13).

Thin layer chromatography was done with 0.5-mm layers of Silica Gel H or G (Merck). Compounds were generally detected by spraying the plates with 50% aqueous sulfuric acid and heating them at 125” for 1 hour. Radioactive compounds on the thin layer plates were detected by scraping horizontal bands of the gel from the plates into vials containing 10 ml of scintillation fluid and counting the vials in a scintillation counter.

Gas liquid chromatography was performed with a Varian Aerograph model 1200 gas chromatograph, attached to a hydrogen flame ionization detector; and a stainless steel column (5 feet x 0.125 inches) packed with 10% carbowax on Aeropak 30 was used.

RESULTS

Identification of an cr(1 + 6) Linkage in MGP

Radioactive Isomaltitol from a Partial Acid Hydrolysate of r-

MGP*-The kinetics of the release of radioactive oligosaccharides from r-MGP* during acid hydrolysis was studied, Solvent B being used for chromatographic analysis. Total acid hydrolysis gave glucitol as the only radioactive product (3), while all of the activity remained at the origin before hydrolysis. The distribution of radioactivity on the chromatogram at inter- mediate times of hydrolysis is illustrated in Fig. 1. No radioactive compound migrated with 6-0-methylglucosyl-(1 -+ 4)- glucitol. A compound with the approximate RF of maltitol accumulated, which, after 50 hours in 1 N HCl at 63”, accounted for about 250/, of the total radioactivity.

ar-Amylase-digested r-MGP* (10 pmoles, specific activity, 600,000 cpm per pmole by scintillation counting) was subjected to hydrolysis in 1 N HCl at 63” for 50 hours. The compound with the approximate RF of maltitol was isolated by preparative paper chromatography (Solvent B) and purified by gel filtration. A single sharp peak of radioactivity emerged from a calibrated Sephadex G-25 column with the elution volume of a disaccharide. The substance, designated r-Di*, was obtained in a 23% yield based on the radioactivity of the starting material. About 450 pg of carbohydrate (phenol-sulfuric acid assay) was present.

r-Di* (50 pg) was hydrolyzed for 4 hours at 100” in 1 N HCI, and the hydrolysate was chromatographed in Solvent B. Glucose and glucitol were present in about equal amounts. 6-0- Methylglucose was not detected. r-Di* (10 pg) and nonradioactive maltitol (100 pg) were mixed and treated with glucoamylase. The enzyme slowly released glucose and glucitol from the reduced disaccharide mixture, but no radioactive glucitol was produced. This suggested that r-Di* and maltitol were not identical. This was confirmed by chromatography in Solvent B


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4322 Lipopolysaccharde of Mycobacterium phlei Vol. 243, No. 16

I I I A

I B

I C D - - --

5 hours

50 hours

IV

I I I 0 10 20 30 40 50 60

DISTANCE ALONG CHROMATOGRAM (cm)

FIG. 1. Kinetics of the partial acid hydrolysis of r-MGP*. Peaks I, II, III, and IV correspond to products carrying a labeled glucitol residue. Bars correspond to areas on the chromatogram occupied by references. A, origin; B, maltitol; C, 6-O-methyl-ol- n-glucosyl-(1 --f 4)-n-glucitol; D, glucitol. Hydrolysis was carried out in 1 N HCl at 63” for the periods of time indicated in the figure.

for 5 days, during which maltitol migrated 30 cm while r-Di* migrated 25.5 cm.

The paper electrophoretic mobilities in sodium molybdate buffer of the reduced disaccharides of glucose have been tabulated (13). The disaccharides containing the (1 -+ 3) linkage do not move, while those having (1 + 2) or (1 + 6) linkages migrate about twice as far as do those having the (1 -+ 4) linkage. In this electrophoretic system, r-Di* migrated almost twice as far as maltitol, suggesting that it had a (1 + 2) or (1 + 6) linkage.

Periodate oxidation of a reduced (1 + 6)-linked disaccharide, labeled with tritium in position 1 of the glucitol moiety should release all of the label as formaldehyde. Periodate oxidation of the corresponding (1 --f 2)-linked disaccharide should produce a nonvolatile radioactive fragment which, after acid hydrolysis and sodium borohydride reduction, would yield radioactive glycerol. r-Di* (10 pg) was treated with 2 ml of 0.1 InM sodium periodate for 20 hours at room temperature, during which 4.5 eq of periodate were consumed. Ethyleneglycol was added to reduce excess periodate to iodate. In the control experiment, the sodium periodate was destroyed by the addition of ethyleneglycol before the same amount of r-Di* was added. Both solutions were then evaporated, after which they were dried by the addition and evaporation of benzene and then methanol. The residue was taken up in 0.2 ml of water, and the radioactivity was deter-

mined by scintillation counting in 10 ml of Bray’s solution. The amount of nonvolatile tritium in the periodate-oxidized sample was 6% of that in the control. In a parallel experiment, radioactive glycerol could not be detected after acid hydrolysis and sodium borohydride reduction of periodate-oxidized r-Di*.

r-Di* cochromatographed with isomaltitol but not with gentiobiitol (Table I, Solvent B), suggesting that it contained an 01 linkage. In a total volume of 50 ~1 of neutral phosphate buffer, r-Di* (5 pg) and 100 pg of either isomaltitol or gentiobiitol were incubated for 20 hours at room temperature with 0.2 mg of p- glucosidase. The products were chromatographed in Solvent B. No reaction occurred in the tube containing isomaltitol and r-Di*. In the tube containing gentiobiitol and r-Di*, glucitol and glucose were produced, but radioactive glucitol was not observed. The identity of r-Di* as isomaltitol was confirmed by its co- chromatography in Solvent A and coelectrophoresis in borate and molybdate buffers.

Radioactive 1,2,3, .J, 5-Penta-0-methylglucitol from Methylated r-MGP*-Fully methylated r-MGP* was hydrolyzed at 100” for 4 hours in 90% formic acid. Methylated maltitol and isomaltitol, both labeled with tritium in position 1 of the glucitol moiety, were hydrolyzed similarly for standards. The three radioactive samples, together with hexamethylglucitol and 2,3,4,6-tetramethylglucitol were chromatographed on a thin layer plate of Silica Gel H (Solvent E). The distribution of radioactivity on the plate was determined as described under “Experimental Procedure.” The single radioactive compound from methylated r-MGP* migrated with the radioactive compound from isomaltitol (Fig. 2, B and C, dashed lines). Sur- prisingly, two peaks of radioactivity were obtained from the methylated maltitol sample, both of which migrated between hexamethylglucitol and tetramethylglucitol (Fig. 2A). Further hydrolysis in 90% formic acid did not alter the relative amounts

TABLE I Chromatographic properties of di- and trisaccharides

of glucose

Compound Linkage

Solvent A Solvent B

Sophorose. PU -+ 2) 1.00 0.95 Sophoritol. . . au + 2) 0.96 1.13 Nigerose................ or(l + 3) 1.25 1.13 Nigeritol.. a(1 + 3) 0.98 1.01 Laminaribiose. a0 + 3) 1.42 1.23 Laminaribiitol. so --+ 3) 1.24 1.17 Maltose. . cx(1 -+ 4) l.OOa 1.000 Maltitol cY(l -+ 4) 0.91 1.12 Cellobiose.. 8(1 --f 4) 0.92 0.87 Cellobiitol B(l + 4) 0.80 0.93 Isomaltose cy(l + 6) 0.76 0.77 Isomaltitol. o/(1 + 6) 0.78 0.93 Gentiobiose 8U + 6) 0.70 0.68 Gentiobiitol. SO + 6) 0.67 0.78

Maltotriose ol(l --f 4) 0.49 0.34 Maltotriitol. cx(1 + 4) 0.43 0.40 Isomaltotriose a(1 --f 6) 0.22 0.22 Isomaltotriitol. cy(1 + 6) 0.23 0.29

0 The I&r0 of maltose is 0.44 in Solvent A and 0.41 in Solvent B. It moved about 20 cm after 4 days in both solvents.


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Issue of August 25, 1968 M. H. Saier, Jr. and C. E. Balk 4323

of these compounds, but hydrolysis in 1 N HCl at 100” for 4 hours or in 0.2 N NaOH for 30 min resulted in the complete disappearance of the faster of the two compounds, leaving the slower. It was concluded that the compound with the greater migratory rate was the formate ester of 1,2,3,5,6pentamethylglucitol. Treatment of the formic acid hydrolysates of methylated isomaltitol and methylated r-MGP* with 1 N HCI at 100” for 4 hours resulted in the disappearance of the fast radioactive compound and the appearance of another compound of smaller RF (Fig. 2, B and C, solid lines). Thin layer chromatography on Silica Gel G (Solvent G) also distinguished between the two pentamethylglucitols of interest, and again the radioactive product from r-MGP * chromatographed with 1,2,3,4,5-pentamethylglucitol.

Determination of Number of (1 -+ 6) Linkages in MGP-a- Amylase-digested MGP was methylated, and the product was purified and methanolyzed as before (3). Gas liquid chromatography showed major peaks for methyl 2,3,4,6-tetra-O- methylglucoside and methyl 2,3,6-tri-0-methylglucoside, as well as several minor peaks which were thought to be trace impurities. Passage of methylated cr-amylase-digested MGP in methanol’through a Sephadex LH-20 column (1.5 x 110 cm), before methanolysis, removed these impurities. The gas chro-

J

0 5 10 15 20

DISTANCE ALONG CHROMATOGRAM (cm) FIG. 2. Thin layer chromatography of methylglucitol from

methylated r-MGP*. Peaks correspond to labeled glucitol derivatives, the solid lines being pentamethylglucitols and the dashed lines being the corresponding monoformate esters. A, standard 1,2,3,5,6-pentamethylglucitol prepared from maltitol; B, standard 1,2,3,4,5-pentamethylglucitol prepared from isomaltitol; C, the pentamethylglucitol from r-MGP*. Bars indicate the areas on the chromatogram occupied by standard 2,3,4,6- tetramethylglucitol (I) and hexamethylglucitol (II).

I / I I

C

1 E

z 3 5:

: :;

B F

A A

TIME (Minutes)

FIG. 3. Tracing of the gas chromatogram of methanolyzed methylated MGP. Peak A, methyl 3-O-methylglycerate; B and C, the /3- and a-anomers of methyl 2,3,4,6-tetramethylglucoside; D and F (shoulder), the p- and or-anomers of methyl 2,3,4-trimethylglucoside; E and G, the p- and cu-anomers of methyl 2,3,6- trimethylglucoside.

matographic pattern is shown in Fig. 3. Peaks were observed corresponding to methyl 3-0-methylglycerate and to the cr- and p-anomers of methyl 2,3,4,6-tetra-0-methylglucoside, methyl 2,3,4-tri-0-methylglucoside, and methyl 2,3,6-tri-o-methylglucoside. The methyl 2,4,6-tri-0-methylglucosides chromatographed with the methyl 2,3,6-tri-0-methylglucosides.

Pure samples of maltose and isomaltose were methylated, purified, and methanolyeed. HCl and methanol were removed from the methanolysate in a desiccator (not evacuated) over CaClz and KOH pellets to insure that methyl tetra-o-methylglucoside was not lost by evaporation (14). The ratio of the area under the peak corresponding to methyl 2,3,4-tri-O-methyl-P- glucoside to that under the peak corresponding to methyl 2,3,4,6-tetra-0-methyl-P-glucoside in the methanolysates of methylated isomaltose and of methylated cY-amylase-digested MGP were determined. These ratios were 0.77 (average of four determinations) for isomaltose, and 0.38 (average of six determinations) for cr-amylase-digested MGP. Since there are known to be two nonreducing termini in cY-amylase-digested MGP (3), this result suggests that there is a single (1 --f 6) linkage in the polysaccharide.

Radioactive Tri-, Tetra-, and Pen&saccharides from r-MGP*

Isolation of Oligosaccharides-Chromatograms of partial acid hydrolysates of r-MGP* showed two areas of radioactivity between the origin and glucitol (Fig. 1, Peaks II and III). The material in Peak II was isolated and rechromatographed for 9 days in Solvent B. The distribution of radioactivity on the chromatogram suggested that at least six labeled compounds were present. Chromatography of the total hydrolysate for 4 days (Solvent F) revealed a peak with Risomaititoi = 0.79.

cY-Amylase-digested r-MGP* (10 pmoles, specific activity, 600,000 scintillation cpm per pmole) was subjected to hydrolysis for 20 hours at 63” in 1 N HCl. The products were chromatographed for 4 days with Solvent F, and the radioactive peak

(Risomaltitol = 0.79) was cut from the chromatogram and eluted with water. The product (250,000 cpm) was passed through a


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Sephadex G-25 column. A broad peak of radioactivity eluted in a position characteristic of oligosaccharides with 3 to 5 sugar residues. Some material, which eluted as a mixture of larger oligosaccharides, was discarded. The major products were chromatographed on paper for 9 days (Solvent B). The distribution of radioactivity on the chromatogram is shown in Fig. 4. Radioactive material from each of the three major peaks (A, B, and C) was passed through a Sephadex G-25 column; in each case, the majority of the radioactivity was eluted as a single compound. Material from Peak A had the elution volume of isomaltotriitol. This unknown was designated r-Tri*. Peak B eluted as a tetrasaccharide and was designated r-Tetra*. Peak C eluted just ahead of r-Tetra* and was called r-Penta*. Analyses described below for the purified compounds suggested that r-Tri* and r-Tetra* were free of contaminating radioactive and nonradioactive oligosaccharides, but that r-Penta* contained nonreducing impurities. This compound was further purified by paper chromatography, developing for 2 weeks with Solvent A and by a subsequent gel filtration. The yields of the three oligosaccharides are given in Table II.

Purity and Composition 0.f Oligosaccharides-Chromatography

I I I I I -A

DISTANCE ALONG CHROMATOGRAM (cm)

FIG. 4. Separation by paper chromatography of labeled oligosaccharides obtained by partial acid hydrolysis of r-MGP*. Material corresponding to Pea/c II in Fig. 1 was resolved into three components, each containing labeled glucitol. A, r-Tri*; C, r-Penta*; B, r-Tetra*. Standard maltotriitol migrated in the area marked by the bar, just behind Peak A. The faster migra- tion of B and C reflects the presence of 6-O-methylglucose in these two compounds.

TABLE II Yields of tri-, fetra, and pentasaccharides from Fig. 4

Compound RGIC (Solvent B)

Yield of pure oligosaccharide’”

Fraction of r-MGP*

subjected to hydrolysis

m~moles CPm %

r-Tri* (Peak A) 0.18 103 62,000 1.0 r-Tetra* (Peak B) 0.25 55 33,000 0.5 r-Penta* (Peak C). 0.22 40 24,000 0.4

a From 10 rmoles of r-MGP* with a specific activity of 600,000 cpm per pmole.

TABLE III Test for presence of reducing contaminants in oligosaccharides

Compound

-

._

r-Tri*. r-Tetra*. r-Penta*.

from Fig. 4

Radioactivity of sample Radioactivity in 6.O-methyl lucitol

Before NaBTa After NaBTa after Na B T4

treatment treatment treatment

cPm

1600 1800 0 850 850 0

1200 1100 0 -

of the oligosaccharides (Solvent B, 9 days) showed that only one radioactive component was present in each preparation. Acid hydrolysis of each revealed that all of the activity was present as tritiated glucitol. The possibility that the preparations were contaminated by reducing nonradioactive oligosaccharides wa.s eliminated by the following experiment. An aliquot of each preparation, containing a known amount of radioactivity, was exposed to excess sodium borotritide (25 pg) at 60” for 10 min. Cations were removed with Dowex 50 (H+) resin, and the solution was evaporated repeatedly with methanol. The residue was hydrolyzed in 1 N HCl, and the products were chromatographed (Solvent B). Because the specific activity of the sodium borotritide was about 8 times that of the r-1MGP* from which the oligosaccharides were derived, a 25010 contamination with reducible sugars would have doubled the radioactivity of the product. The results (Table III) show that oligosaccharides containing reducing terminal glucose or 6-0-methylglucose residues were essentially absent from all preparations.

The sugar composition of the oligosaccharides was determined as follows. An aliquot of each preparation, containing a known amount of radioactivity, was hydrolyzed in 1 N HCl for 4 hours at 100”. HCl was removed, the solution was neutralized with 1 M ammonium carbonate, and sodium borotritide (25 pg) was added. The mixture was kept at 60” for 10 min, after which cations were removed with Dowex 50 (H+) resin and the solution was evaporated repeatedly with methanol. The products were chromatographed (solvent B), and when the solvent front reached the end of the chromatogram, development was termi- nated and the distribution of radioactivity on the chromatogram was determined. Glucitol and 6-0-methylglucitol were the only radioactive products (Fig. 5). The counts associated with each polyol, per 1000 cpm present in the starting oligosaccharide preparations, are tabulated in Table IV (Columns 2 and 3). Because the initial specific activities of the three oligosaccharides were identical, the relative amounts of radioactive glucitol derived from glucose residues in each oligosaccharide can be calculated by subtracting the amount of radioactivity in the oligosaccharide originally subjected to hydrolysis (1000 counts) from the values listed in Column 2 (Table IV, Column 3). The relative amounts of 6-O-methylglucose in the hydrolysates can also be calculated, since the specific activity of the 6-0-methylglucitol must equal that of the glucitol which was derived from glucose residues (Table IV, Column 4). The results confirmed the con- clusions, based on gel filtration, regarding the molecular sizes of the oligosaccharides.

r-MGP* CLS Source of Three Ol@osaccharides-Since each of the oligosaccharides was obtained in low yield from the starting material (Table II), it was important to establish that they were


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Issue of August 25, 1968 M. H. Xaier, Jr. and C. E. Ballou 4325

derived from r-MGP* rather than from trace impurities. The presence of 6-O-methylglucose in r-Tetra* and r-Penta* provided evidence that these oligosaccharides were derived from MGP. That all of the radioactivity associated with the intact oligosaccharides was present as glucitol suggested that they were derived from what had been the reducing end of MGP. Conclusive evidence for the latter conclusion was provided by partial acid hydrolysis. A time study of the acid hydrolysis of r-Di* showed that after 20 min in 1 N HCl at 100” (sealed tube), approximately one-half of the reduced disaccharide had been cleaved. The three oligosaccharides were subjected to the same acid treatment for 25 min. In each case, the two major products chromatographed with glucitol and isomaltitol, while a smaller amount of radioactivity migrated with the higher oligosaccharides. The high percentage of radioactive isomaltitol obtained from each oligosaccharide (Table IV, Columns 9 and 10) shows that this unit is at the “reducing” end of all of the oligosaccharides.

Identificution of a p-(1 + 3) Linkage in Main Chain of MGP

Structure of r-!fri*-r-Tri*, 2.5 mpmoles (1500 cpm), was oxidized by 0.05 M sodium periodate (50 11) for 40 hours at room temperature. Ethyleneglycol was then added to decompose excess periodate. Dowex 1 (Cl-) resin was added to remove anions, and the product was hydrolyzed in 1 N HCl at 100” for 3 hours. HCl was removed and the hydrolyzed products were reduced with excess sodium borotritide (25 pg). After removal of boric acid by repeated evaporation of the cation-free solution from methanol, the residue was chromatographed in Solvent B. Two radioactive products, glucitol and glycerol, were present in equimolar amounts (Fig. 6A). Isomaltotriitol (25 mpmoles) was oxidized and analyzed by the same procedure. The presence of glycerol as the only radioactive product (Fig. 6D) showed that oxidation of the oligosaccharide had been complete. Therefore, the isolation of glycerol and glucitol in equimolar amounts from the oxidation products of r-Tri* suggested the presence of a (1 -+ 3) linkage.

The reduced disaccharides of glucose, containing the cr-(1 4 3) linkage (nigeritol) and the p-(1 + 3) linkage (laminaribiitol), can be distinguished by paper chromatography (Table I). Removal of the glucitol moiety of r-Tri* and reduction of the resultant disaccharide should produce one of these compounds. Control experiments established that 0.1 mM sodium periodate, at room temperature, quickly oxidizes polyols such as glucitol but does not oxidize glucopyranose residues involved in glycosidic linkage.

01 0 2 4 6 8 10

DISTANCE ALONG CHROMATOGRAM (Inches) FIG. 5. Radioactive products obtained upon reduction with

sodium borotritide of the total acid hydrolysates of the three oligosaccharides from Fig. 4. A, r-Tri*; B, r-Tetra*; C, r-Penta*. The quantitative results are given in Table IV. The bars indicate the areas on the chromatogram occupied by the standard glucitol (1) and 6-0-methylglucitol (II).

Under these conditions, 4 moles of periodate were consumed per mole of isomaltotriitol, and further oxidation did not occur. The major product, after sodium borohydride reduction, was characterized as isomaltosyl-(1 -+ 1)-ethyleneglycol. Small aglycons are cleaved by acetolysis much more readily than are large ones (15). We found that methyl maltoside was cleaved almost quantitatively to maltose; isomaltosyl-(1 + l)-ethyleneglycol was cleaved to isomaltose; and glyceric acid could be cleaved from the rest of MGP leaving the entire polysaccharide portion of the molecu’e intact. These observations led to a procedure (described below for isomaltotriitol) for the selective removal of the reducing glycose residue from an oligosaccharide.

TABLE IV Composition of oligosaccharides obtained by partial acid hydrolysis of r-MGP*

1. Compound

Radioactivity after partial acid

3. Radioactivity 4. Radioactivity 6. 7. No. of 6-O- 8. Ratio of

2. Radioactivity in gluci toP in newly formed in newly formed

5. No. of Radioactivity hydrolysis” in

glucitol glucitol glucose residues in 6.O-methyl- g,ucose to c&O- glucitol”

methylglucose residues methylglucose

residues 9. Iso- 10.

m&it01 Glucitol

CPnz Mm cpn/unit % %

r-Tri*. 8,500 7,500 3,75@ 2.00 500 0.13 40 53 r-Tetra*. 8,600 7,600 2.02 4,100 1.09 1.85 31 48 r-Penta*. 12,500 11,500 3.07 3,700 0.99 3.1 33 47

0 Per 1000 cpm in starting oligosaccharide. b In 1 N HCl at 100” for 25 min. c Assuming a reduced trisaccharide structure, which is consistent with the results from the gel filtration pattern and from paper

chromatography.


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4326 Lipopolysaccharide of Mycobaderium phlei Vol. 243, No. 16

Isomaltotriitol (3 pmoles) was oxidized in 200 ml of 0.1 mM

sodium periodate for 6 hours at room temperature. The de- crease in the absorbance at 224 rnp was used as a measure of the extent of the reaction. Ethyleneglycol was added to destroy excess periodate, and the solution was evaporated to dryness. Sodium borohydride (5 mg in 2 ml of water) was added, and after 20 min at room temperature, cations were removed with Dowex 50 (H+) resin. The solution was concentrated and boric acid was removed by repeated evaporation with methanol. The dry residue was acetylated by heating it at 100” for 2 hours with 1 ml each of acetic anhydride and pyridine. Excess reagents were removed at 100” in a stream of air, and glacial acetic acid (1 ml), acetic anhydride (1 ml), and concentrated sulfuric acid (50 ~1) were added. The solution was mixed and left at room temperature for 4 hours. Pyridine (3 ml) was then added and solvents

A

;:I o 2 4 6 8 10 12 14 16 18

DISTANCE ALONG CHROMATOGRAM (Inches)

FIG. 6. Identification of products obtained in the periodate oxidation of the oligosaccharides from Fig. 4. Each substance (A, r-Tri* ; B, r-Tetra*; C, r-Penta*; D, isomaltotriitol) was oxidized with sodium periodate, the product was hydrolyzed with acid, and the fragments were reduced with sodium borotritide. The chromatograms show the radioactive products which represent the fragments that resisted oxidation by periodate. For Fig. 6E, r-Penta* was oxidized with periodate and reduced with sodium borohydride. The product was hydrolyzed completely and the sugars were reduced with sodium borotritide. This yielded labeled hexitols, uncontaminated with labeled glycerol and erythritol, representing the hexose units that survived periodate oxidation. The numbers indicate the total radioactivity of each of the peaks. The bars indicate the areas on the chromatograms occupied by the different standards.

I I I I I I A Nigeritol

lsomaltitol I

Laminaribiitol \\ / -II- ,

I s

I I I I I

f$ 6000 lB -

ii2 -mm

3000 -

0 0 2 4 6 8 10

J


FIG. 7. Stepwise degradation of oligosaccharides. A, isomaltotriitol was oxidized selectively with sodium periodate, and the product was reduced with sodium borohydride and subjected to a controlled acetolysis. This sequence of steps selectively removed the original glucitol moiety to yield isomaltose, which was reduced with sodium borotritide and identified by chromatography. B, the same sequence of reactions, when applied to r-Tri*, yielded a substance with the chromatographic property of laminaribiitol. The bars indicate the areas occupied by the standard compounds.

were removed at 100” in a stream of air. The dry residue was dissolved in methanol (1 ml), and an equal volume of 0.5 N

aqueous sodium hydroxide was added. After 20 min, the basic solution was acidified with Dowex 50 (H+) resin. The cation-free solution was treated with Dowex 1 (Cl-) ,resin to remove anions. After evaporation, dilute aqueous ammonium carbonate and sodium borotritide (0.1 mg) were added. After 20 min at room temperature, cations were removed with Dowex 50 (Hf) resin, and the residue was evaporated repeatedly with methanol. The chromatogram, reproduced in Fig. 7A, shows that isomaltitol was the only radioactive product.

The same procedure was applied to r-Tri* (2.5 mpmoles, 1500 cpm). Periodate oxidation was carried out in 1 ml of 0.1 IIIM

sodium periodate, and acetolysis was carried out for 4 hours at room temperature in 0.5 ml of the same acetolysis mixture. Other volumes were reduced appropriately. The single radioactive product had the chromatographic properties of laminaribiitol with Solvents A and B (Fig. 7B).

The P-configuration of the nonreducing terminal glucose residue in r-Tri* was confirmed by enzymatic studies. Under the conditions described in the legend to Fig. 8, nigerose (100 pg) was hydrolyzed to glucose by glucoamylase but was not digested by P-glucosidase. The reverse was true for laminaribiose. Isomaltitol was not cleaved by either enzyme preparation. r-Tri* was not digested by glucoamylase (Fig. SA), but was converted to r-Di* by /I-glucosidase (Fig. 8B).

Determination of Number of (1 4 3) Linkages in MGP-Fully


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methylated, a-amylase-digested MGP (14 mg) was hydrolyzed in 90% formic acid at 100” for 3 hours, and then in 1 N HCl at 100” for 3 hours. After removal of acid, the solution was neutralized with ammonium carbonate, and sodium borotritide (50 mg, specific activity 0.1 mC per mg) was added. Reduction was carried out at 60” for 20 min. The color yield, by the phenol- sulfuric acid assay, decreased to less than one-tenth of its original value. Dowex 50 (H+) resin was added and removed by filtration, and the resin was washed several times with methanol. The combined filtrate was taken to dryness and thrice evaporated with methanol. Periodate oxidation of the partially methylated radioactive polyols was carried out in 3 ml of 0.05 M

sodium periodate. After 24 hours, the oxidation was complete with consumption of 71 pmoles of periodate. The assumption being that the partially methylated polyols listed in Table V (Column 1) were produced in the amounts listed in Table V (Column 2), the predicted uptake of periodate is 67 pmoles. Ethyleneglycol was added to destroy excess periodate, followed by sodium borohydride, and reduction was carried out at 60” for 20 min. Cations were removed with Dowex 50 (H+) resin, and boric acid was removed by evaporation with methanol. The radioactive products (Table V, Column 4)) all of which were nonvolatile (with the exception of 2-0-methylglycerol), were analyzed by paper chromatography (Solvent A). Two peaks of radioactivity resulted. The slower migrated with all of the standard tri-O-methylglucitol derivatives; the faster migrated with 2,3- di-O-methylthreitol and 2,3,4,6-tetra-0-methylglucitol which were not separated. The ratio of counts in the two peaks was 1: 11. On the assumption that there was only one nonoxidizable trimethylglucitol per molecule of MGP and that all of the nonvolatile oxidized products were in the second peak, the predicted ratio is 1: 12. With Solvent B, three peaks of radioactivity resulted. The slowest peak migrated with standard 2,4,6-tri-O- methylglucitol. The two faster overlapping peaks corresponded to 2,3-di-0-methylthreitol and 2,3,4, R-tetra-0-methylglucitol. Again the ratio of the counts in the slowest peak to those in the two faster peaks was 1:ll. With Solvent E, the three tri-O- methylglucitol derivatives could be distinguished, and 2,3,4,6- tetra-O-methylglucitol and 2,3,4-tri-0-methylxylitol migrate well ahead of 2,3-di-O-methylthreitol. The pattern of radioactivity produced by the mixture of partially methylated polyols derived from methylated cr-amylase-digested MGP, together with the positions of relevant standard compounds, is shown in Fig. 9. The relative amounts of radioactive 2,4,6-trimethylglucitol, 2,3-di-0-methylthreitol, 2,3,4-tri-o-methylxylitol, and 2,3,4,6-tetra-0-methylglucitol (Table V, Column 5) are in good agreement with the predicted ratios.

The mixture of partially methylated polyols was exposed to

400 -

z- E - 200 -

: E OL

I

2 g 400 a 02

200

0 L

- B

0 2 4 6 8 10


FIG. 8. Evidence for the P-configuration of the terminal glucosyl unit of r-Tri*. The incubation with glucoamylase was carried out in 0.1 ml of 0.1 M acetate buffer, pH 5.0, containing glucoamylase (100 units) and r-Tri* (5 mpmoles). The incubation with p- glucosidase was performed in 0.1 ml of 0.1 M phosphate buffer, pH 6.9, containing fl-glucosidase (1 mg of protein) and r-Tri* (5 mpmoles). After 12 hours at room temperature, 1 ml of methanol was added, the solutions were boiled, and denatured protein was removed by centrifugation. The solutions were concentrated and the products were chromatographed (Solvent B). A, the distribution of radioactivity on the chromatogram after exposure to glucoamylase; B, the distribution of radioactivity on the chromatogram after digestion byp-glucosidase. The bars indicate the positions of standard r-Tri* (I) and r-Di* (II).

0.05 M sodium periodate as before. The ratios of the three radioactive peaks remained the same, demonstrating that oxidation had been complete.

Structure of Branch Point in MGP

Structure of r-!!‘etra*-Partial acid hydrolysis of r-Tetra* (1 N

HCl at 100” for 25 min) resulted in the formation of radioactive isomaltitol (Table IV, Columns 9 and 10) showing that this frag-

ment was at the reducing end. When r-Tetra* was hydrolyzed for 6 min and the products were subjected to chromatography for 9 days (Solvent B), a compound with the chromatographic properties of r-Tri* was observed.

r-Tetra* (1.5 mpmoles) was oxidized by 0.05 M sodium periodate for 40 hours at room temperature (as for r-Tri* above) and the products were similarly hydrolyzed, reduced with sodium

TABLE V

Analysis of methylated polyols expected and obtained from methylated or-amylase-digested MGP

1. Radioactive glucitol derivative 2. Amount expected

per mole of 3. Expected periodate consumption per mole 4. Radioactive product after periodate oxidation 5. Molar ratios calcuated

polysaccharide of polysaccharide and borohydride reduction from Fig. 9

2,6-Dimethylglucitol 2,4,6-Trimethylglucitol

2,3,6-Trimethylglucitol 2,3-4-Trimethylglucitol 2,3,4,6-Tetramethylglucitol

moles moles

1 2

1 0 9 9 1 1 2 0

2-Methylglycerol

2,4,6-Trimethylglucitol 2,3-Dimethylthreitol 2,3,4-Trimethylxyli to1 2,3,4,6-Tetramethylglucitol

1.0 7.9

2.9


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borotritide, and chromatographed. Glucitol and erythritol were obtained in equimolar amounts, while l-O-methylerythritol was not detected (Fig. 6B). The amounts of radioactive ethyleneglycol and l-O-methylglycerol could not be estimated quantitatively because of their volatility. The presence of glucitol demonstrated that a (1 + 3) linkage was present in the oligosaccharide, and the formation of erythritol showed that the second glucose unit in the oligosaccharide was substituted in position 4. Therefore, the 6-O-methylglucose residue must be at the nonreducing terminus, attached by a (1 + 4) linkage to the second glucose residue.

Confirmation that the 6-0-methylglucose was in the terminal position in the reduced tetrasaccharide and that it had the ac- configuration was obtained by partial acid hydrolysis. r-Tetra* was hydrolyzed for 6 min in 1 N HCl at 100”. Because the (1 + 6) linkage is much more stable to acid than are the (1 --t 3) and (1 -+ 4) linkages, only a small amount of laminaribiitol should be produced. Cleavage of the (1 -+ 3) linkage should produce a disaccharide containing 6-0-methylglucose and glucose. The products of partial acid hydrolysis were reduced with sodium borotritide and chromatographed in Solvents A, B, and D. A radioactive compound migrated with 6-O-methyl-a-glucopyranosyl-(1 + 4)glucitol but not with oc-glucopyranosyl-(1 --f 4)6-0- methylglucitol (Fig. 10). Because these solvents distinguish the two anomeric forms of reduced disaccharides in which the glycosidic linkages involve the same hydroxyl of glucitol (Table I), these results provide convincing evidence for the LY configuration.

Struclure of r-P&a*-Partial acid hydrolysis of r-Penta* (1 N HCl at 100” for 25 min) yielded radioactive isomaltitol (Table

I I I I B E

- I

A C D -- m

DISTANCE ALONG CHROMATOGRAM (cm) FIG. 9. Determination of the number of (1 -+ 3) linkages in

MGP. ol-Amylase-digested MGP was methylated and hydrolyzed, and the methylglucoses were reduced with sodium borotritide. The resulting methylglucitols were oxidized with sodium periodate and the products reduced with sodium borohydride. These steps yielded tritium-labeled 2,3-dimethylthreitol from the 2,3,6-trimethylglucose; 2,3,4-trimethylxylitol from the 2,3,4-trimethylglucose; 2,4,6trimethylglucitol from the 2,4,6- trimethylglucose; and 2,3,4,6-tetramethylglucitol from the 2,3,4,6-tetramethylglucose. A chromatogram of the products and the radioactivities associated with each are shown, along with bars which indicate the areas on the chromatogram occupied by the standard compounds. A, 2,4,6-trimethylglucitol; B, 2,3,6- trimethylglucitol; C, 2,3-dimethylthreitol; D, 2,3,4-trimethylxylitol; E, 2,3,4,6tetramethylglucitol. Quantitative results are given in Table V.

I I I I I I I I I

z E A B C D E c - - - - 5 -

5 d 80-

E

I 0 2 4 6 8 10 12 14 16 18 20


FIG. 10. A paper chromatogram (4 days with Solvent A) showing the labeled products formed by the partial acid hydrolysis of r-Tetra* followed by reduction with sodium borotritide. The bars outline the areas on the chromatogram occupied by the standards. A, isomaltitol; B, laminaribiitol; C, or-glucosyl-(1 + 4).6-O-methylglucitol; D, 6-0-methyl-a-glucosyl-(1 + 4)-glucitol; E, glucitol.

I I I I I I I I I N 5 A B C 8-

I-m

“E 2 6-

: 5 4- F

2 0 2- 0 d _ ____ __ _.

‘0 2 4 6 8 10 12 14 16 18 20


FIG. 11. A paper chromatogram (9 days with Solvent B) showing the labeled products formed by partial acid hydrolysis of r-Penta*. The bars represent the areas on the chromatogram occupied by the standards. A, r-Tri*; B, r-Penta*; C, r-Tetra*.

IV, Columns 9 and 10). r-Penta* was hydrolyzed for 6 min under the same conditions, and the products were chromatographed for 9 days (Solvent B). The distribution of radioactivity on the chromatogram is reproduced in Fig. 11. Com- pounds with the properties of r-Tri* and r-Tetra* were observed, which suggested that r-Penta* was a derivative of r-Tetra*.

This was confirmed, and the linkage of the nonreducing terminal glucose residue was established by periodate oxidation. r-Penta* was oxidized by 0.05 M sodium periodate for 40 hours at room temperature (see above). The oxidized product was di- vided into three parts. One part was hydrolyzed completely (2 N HCl at 100” for 2 hours), and the products were reduced with sodium borotritide and chromatographed. Approximately equimolar amounts of glucitol, 6-0-methylglucitol, erythritol, and glycerol were produced (Fig. 6C). The second part of the periodate-oxidized r-Penta* was reduced with sodium borohydride. The acetal linkages were then hydrolyzed (2 N HCl at 100” for 2 hours) and the products were reduced with sodium borotritide. The radioactive polyols were glucitol and 6-O- methylglucitol, in about equimolar amounts (Fig. 6~5’). The third fraction of periodate-oxidized r-Penta* was partially hydrolyzed (0.25 N HCl at room temperature for 24 hours) and the products were reduced with sodium borotritide. Two of the radioactive products had the chromatographic properties of


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6-0-methylglucopyranosyl-(1 --) 2)-erythritol (RGic = 1.60 with Solvent B and 1.50 with Solvent D) and a-glucopyranosyl- (1 + I)-ethyleneglycol (RGic = 1.75 with Solvent B and 1.84 with Solvent D). These results established that the nonreducing terminal glucose unit in r-Penta* was linked to the 6-O-methylglucose residue by a (1 + 3) linkage.

In a preceding section it was shown that P-glucosidase cleaved laminaribiose, but not nigerose, while glucoamylase was active on the latter. The anomeric configuration of the terminal glucose unit in r-Penta* was investigated with these enzyme preparations and with the yeast ol-glucosidase preparation. r-Penta* was subjected to digestion by /3-glucosidase under the conditions described in the legend to Fig. 8. After removal of salts with a mixed bed resin, half of the sample (2.5 mpmoles) was exposed to sodium borotritide (25 pg), and radioactive impurities were removed. Chromatography (Solvent B) showed that no radioactive glucitol was produced. The other half of the sample was chromatographed for 9 days in Solvent B. The radioactive oligosaccharide had the chromatographic properties of r-Penta*. Glucoamylase also left r-Penta* intact. However under the conditions described under “Experimental Procedure” the yeast a-glucosidase preparation completely digested r-Penta* to r- Tetra*. These results are in agreement with those of Lee (2) who isolated the disaccharide, glucopyranosyl-(1 ---f 3) -6-O- methylglucose, from an acetolysate of MGP. Although the optical rotation suggested the cr-anomeric configuration, the disaccharide was not digested by glucoamylase.

The structures of the radioactive nonreducing oligosaccharides obtained from partial acid hydrolysates of r-MGP* are shown in Fig. 12. The procedures for the analyses of the products of periodate-oxidized r-Penta* are summarized below the structure.

Anomeric Con$guration of the Glucose Residue Linked to Glyceric Acid

The following procedure, based on the findings of Brown and Subba Rao (16), resulted in complete reduction of the aglycon to radioactive glycerol. MGP (10 mg) was converted to the methyl ester in 1 ml of 5% methanolic HCl at room temperature for 20 min. Methanol (5 ml) and excess Amberlite CG-45 (type 1) were added, and after shaking the mixture for 1 hour, water (0.5 ml) was added. Resin was filtered from the neutral solution which was evaporated to dryness. The polysaccharide was dissolved in 1.5 ml of 70% aqueous bis(2methoxyethyl)ether. This was added to a suspension of 30 mg of sodium borotritide (specific activity, 0.1 mC per mg) and 10 mg of ammonium bicarbonate in 0.5 ml of bis(2-methoxyethyl)ether. The solution was warmed gradually to 80” over a lo-min interval, and after 10 min at this temperature, the solution was cooled to room temperature, and Dowex 50 (H+) and water (1.5 ml) were added. The resin was filtered off and solvents were removed under vacuum. Boric acid was eliminated from the cation-free solution by repeated evaporat,ion of methanol. Two ml of 0.5 N NaOH were added to the dry residue, and after 20 min the solution was neutralized with Dowex 50 (H+). Salts and radioactive impurities were removed by passage of the polysaccharide through a Sephadex G-25 column. The polysaccharide eluted from the column as a sharp peak. The amount of radioactivity in tubes containing the reduced MGP was proportional to the concentration of carbohydrate. The salt-free product was applied to a small DEAE- Sephadex column (carbonate form) in water (3). More than 95% of the carbohydrate and radioactivity passed through the

r Penta’ , r.letra’ I

r-Tr” m

I’

GlyCerOl-JH

ErythrlM3H

(Ethyleneglycol-3H.

Glycolaldehyde-‘H)

GlyCtYOl-3H

6 0.Methylglucosyl-

(1-Z) erythr,tolL3H

Glucosyl-‘l-l)-

ethyleneglycol- 3”

(Glycolaldehyde- 3H)

FIG. 12. The structures of the reduced oligosaccharides isolated from partial acid hydrolysates of r-MGP* (under the brackets). The reactions illustrated at the bottom of the figure show the procedures used for the characterization of r-Penta*. 0, O- methyl group; *, position of labeling with tritium.

column, showing that the product was neutral. Paper chromatography of a total acid hydrolysate (Solvents A and B) showed that glycerol was the only radioactive product.

A portion of this form of MGP (4 mg, lo7 cpm) in which the glyceric acid was reduced to radioactive glycerol (r-glycerol- MGP*) was hydrolyzed in 1 N HCI for 15 min at IOO”, and the products were chromatographed (40 hours, Solvent B). Two peaks of radioactivity migrated between glycerol and the origin. The faster had RGlc = 1 .OO in this solvent and chromatographed with standard a!- or P-glucopyranosyl-(1 --+ 2)-glycerol in Sol- vents A, B, and D. The slower compound had RGlc = 0.36 and was presumed to be cu-glucopyranosyl-(1 + 6)-glucopyranosyl- (1 + 2)-glycerol-l-3H. Both were isolated free of radioactive impurities by preparative paper chromatography. Glucose and radioactive glycerol were the only products after total acid hydrolysis of both compounds.

Under the conditions described in the legend to Fig. 8, glucoamylase was inactive on authentic CX- and P-glucopyranosyl-

(1 --) 2)-glycerol. P-Glucosidase readily and completely hydrolyzed /3-glucopyranosyl-(1 -+ 2)-glycerol, but not ar-glucopyranosyl-( 1 -+ 2)-glycerol. The glucopyranosyl-(1 --) 2)- glycerol from r-glycerol-MGP* was not hydrolyzed by @-glucosidase, suggesting that its configuration was (Y. To establish that the enzyme was active, radioactive fl-glucopyranosyl-(1 4 2)-erythritol and the radioactive cu-glucopyranosyl-(1 --) 2)- glycerol from r-glycerol-MGP* were mixed and incubated with /3-glucosidase as before. The former glycoside was completely hydrolyzed to glucose and radioactive erythritol, but the latter was not affected. Under the conditions described under “Ex- perimental Procedure” the cu-glucosidase from D. vunryi completely hydrolyzed standard a-n-glucopyranosyl-(1 + 2)-glycerol and the radioactive glucosyl-glycerol from r-glycerol-MGP*, but not P-n-glucopyranosyl-(1 + 2)-glycerol (Fig. 13). The slight activity of the enzyme preparation against the /3 anomer may have been due to contamination with the Q: anomer.

Glucoamylase hydrolyzes isomaltose at a slow rate (Table VI). The compound with RGlo = 0.36, formed by partial acid hydroly-


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sis of r-glycerol-MGP*, was hydrolyzed by this enzyme to glucose and a radioactive compound with the properties of Lu-glucopyranosyl-(1 --) 2)-glycerol. The rate of hydrolysis was about that observed for isomaltose. These results show that the compound of RGle = 1.00 (Solvent B) was cu-glucopyranosyl-(1 + 2)-

0 4 8 12

INCHES ALONG CHROMATOGRAM FIG. 13. Determination of the anomeric configuration of the

glucosyl residue linked to glyceric acid in MGP. Standard and unknown glucosides were incubated with the yeast or-glucosidase preparation as described under “Experimental Procedure,” and the products were analyzed by paper chromatography. A, standard a-o-glucopyranosyl-(1 + 2)-glycerol; B, standard P-D- glucopyranosyl-(1 --t 2)-glycerol; C, glucopyranosylL(1 -+ 2)- glycerol from r-glycerol-MGP*. Bars indicate the areas on the chromatogram occupied by standard w or fl-n-glucopyranosyl- (1 -+ 2) -glycerol (I) and glycerol (11).

TABLE VI Substrate specijkity of glucoamylase

Incubations with glucoamylase were for 1 to 5 hours at room temperature in 0.2 ml of 0.1 M sodium acetate buffer, pH 5, and 1.0 mM substrate (Column 2) or variable substrate concentrations (Columns 3 and 4). The assay tubes were heated at 100” for 1 min to inactivate the enzyme; and, after cooling, the Glucostat reagent (2 ml) was added. After 15 min, 4 N HCl (25~1) was added and the absorbance at 400 ml* was determined. Appropriate con- trols lacking only glucoamylase and containing known amounts of glucose were included with each series of determinations.

1. Compound

Maltose....................... a-Glucopyranosyl-(1 --) 4)-6-

0-methylglucose Maltitol Nigerose..................... a-Glucopyranosyl- (1 + 3) -6-

0-methylglucose Nigeritol.. Isomaltose.................... Isomaltitol. . a-Glucopyranosyl-(1 -+ l)-

glycerol a-Glucopyranosyl-(1 + 2)-

glycerol

-

i

-

!. Relative rate of hydrolysis

100

10 0.1 0.3

<0.05 <0.05

0.4 <0.05

<0.05

<0.05

3. Michaeli! constant

9n.w

0.8

20

4

-

j

--

-

L Relative maximum velocity of hydrolysis

100

5

1

COOH FIG. 14. Structure of the reducing end of the polysaccharide.

l , O-methyl group.

glycerol-l-3H, and the compound of RGls = 0.36 was a- glucopyranosyl-(1 ----f 6) -cr-glucopyranosyl- (1 + 2) -glycerol-l-3H.

DISCUSSION

Structure of Reducing End of MGP--We have shown previously (3) that the D-glyceric acid, which is linked glycosidically to the reducing end of the polysaccharide, can be removed by a Lossen degradation. If the resulting polysaccharide is reduced with sodium borotritide and then hydrolyzed, radioactive glucitol is obtained. This established that the first hexose at the reducing end of the polymer was glucose. We have extended this study by isolating larger fragments containing the labeled glucitol, which are produced by controlled acid hydrolysis of r- MGP*. Their characterization has allowed us to define the order and configurations of the first five hexoses that follow the glyceric acid moiety. The anomeric configuration of the glucose attached to glyceric acid was determined by reducing the acid to glycerol, with sodium borotritide, and characterizing the glucosyl- glycerol fragment that was produced by controlled acid hydrolysis. From these studies, we assign the structure in Fig. 14 to the reducing end of the polysaccharide.

Analytical Technigues-Because of the small amount of material available for study, special techniques were required for much of the characterization. The use of sodium borotritide, of high specific activity, to label reducible fragments permitted several analyses which otherwise would have been unfeasible. Complete quantitative analyses of the products of periodate oxidation of oligosaccharides were performed with nanomolar amounts of the compounds. When coupled with a selective acid hydrolysis of linkages involving periodate-oxidizedsugar residues, either the oxidized fragments or the unoxidized sugars could be labeled with tritium. The analysis of acidic and enzymic hydrolysates of oligosaccharides was also facilitated. It was possible to label and identify the products which contained the sugar residue that came from the reducing terminus by reducing before hydrolysis or to label all products by reducing after hydrolysis. Coupling several analytical techniques, such as methylation, sodium borotritide reduction, and periodate oxidation (see Table V), permitted the quantitative analysis of mixtures of closely related compounds.

General procedures for the stepwise degradation of an oligosaccharide from the reducing end are not well developed (7). Mild periodate oxidation, followed by acetolysis of the reduced product, proved to be a simple and effective route to this goal. By this method, isomaltotriitol and r-Tri* yielded isomaltose and laminaribose, respectively. The small aglycon cleaved from the remainder of these molecules was ethylene glycol because the sugar moiety had been attached to the primary hydroxyl of glucitol. Attachment to a secondary hydroxyl would result in


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Issue of August 25, 1968 M. H. Xaier, Jr. and 6. E. Ballou 4331

the production of a glycoside with glycerol or erythritol at there- ducing end. We have found that a-glucopyranosyl-(1 + 2)- glycerol is easily cleaved by acetolysis, although ac-glucopyranosyl-(1 + 2)-erythritol is more resistant to this treatment. The procedure may therefore have general applicability. Since removal of the terminal residue frees the aldehyde function of the next sugar unit, the reactions can be applied several times in suc- cession to the ‘same oligosaccharide, thus providing a general method for the sequential degradation of oligosaccharides com- parable to the Edman technique for degrading peptides.

Chromatographic Analyses-On chromatography both isomers of 6-0-methylmaltose migrate more slowly than glucose (3). However, the tetrasaccharide (r-Tetra*), consisting of three glucose units (r-Tri*) and a terminal 6-0-methylglucose residue, migrated ahead of r-Tri*. Even r-Penta*, containing an addi- tional glucose unit, migrated more rapidly than r-Tri*. Thus, the presence of the single O-methyl ether increased the chromatographic mobilities of the larger oligosaccharides far more than it increased those of the disaccharides.

The reduced disaccharides of glucose had characteristic chromatographic properties (Table I), on the basis of which we could distinguish between the anomers. Thus, it was surprising that the anomers of the glucosides of ethyleneglycol, glycerol, and erythritol were not separated with the same systems. These observations suggest that differential interactions between the two moieties of the anomeric forms of glycosides, causing mark- edly different chromatographic properties, are dependent on the size of the aglycon.

The chromatographic mobilities of higher oligosaccharides of glucose depend on the linkages. r-Tri*, containing an a-(1 + 6) and a p-(1 4 3) linkage migrated with maltotriitol, between isomaltotriitol and laminaritriose. Lee characterized an oligosaccharide, produced by acetolysis of MGP, as maltotriose on the basis of its composition, size, and chromatographic properties (2). We note that these properties do not distinguish maltotriose and P-glucopyranosyl-(1 + 3)-ac-glucopyranosyl-(1 + R)-glucose. The substance Lee isolated may have been the latter or a mixture of the two.

Specificities of a- and ,&Glucosidases-The specificity of ac- glucoamylase has been studied by Pazur and Kleppe (17). At 0.2 M substrate concentrations, the rates of hydrolysis of nigerose and isomaltose were reported to be 0.34 and 0.06, respectively, relative to that of maltose. While Lee (2) concluded that the branching disaccharide from MGP, glucopyranosyl-(1 + 3)-6-0- methylglucose, had the a! configuration, it was not hydrolyzed by glucoamylase. The nonreducing terminal glucose residue in r- Penta*, which is attached to the 6-0-methylglucose unit by a (1 + 3) linkage, should also have the a! configuration since these two sugars correspond to Lee’s disaccharide. We confirmed this conclusion employing specific cy- and /3-glucosidases. Lee as- sumed that glucoamylase could not act on substrates in which the primary hydroxyl group of the reducing terminal glucose unit was substituted. However, we have found that this enzyme will hydrolyze ol-glucopyranosyl-(1 --f 4) -6-O-methylglucose (3). Therefore, we reinvestigated the substrate specificity of the enzyme, and found (Table VI, Column 2) that the enzyme has a

much narrower specificity at low than at high substrate concentrations. This suggested that the affinity of the enzyme for maltose must be greater than that for the other substrates, a prediction which was verified (Table VI, Columns 3 and 4). These results also explain our observations that cr-glucopyranosyl-(1 + 1) -glycerol and cr-glucopyranosyl-(1 -+ 2) -glycerol were not hydrolyzed by the enzyme under the conditions we employed. Since the concentrations in vivo of substrates are likely to be at the millimolar level, glucoamylase probably is highly specific for the a-(1 --t 4).linked substrates in the intact cell. In contrast to the above results, P-glucosidase was active on all standard fi-glucosides we investigated, including cellobiitol, laminaribiitol, gentiobiitol, and the B-D-glucopyranosides of ethyleneglycol, glycerol, and erythritol. Similarly the yeast Lu-glucosidase hydrolyzed every cu-glucoside tested. These included maltose, nigerose, isomaltose, a-glucopyranosyl-(1 + l)- glycerol, cr-glucopyranosyl-(1 --f 2)-glycerol, cr-glucopyranosyl- (1 + 2)-erythritol, isomaltitol, and methyl a-D-glucoside. It was inactive on the P-glucosides tested, including cellobiose, gentiobiose, and p-glucopyranosyl-(1 + 2)-glycerol. Malto- oligosaccharides in which the nonreducing terminal glucosyl residue is 0-methylated in positions 3 or 6 were not hydrolyzed by the enzyme.

Acknowledgments-We are grateful to Dr. J. H. Pazur for the preparations of glucoamylase and nigeran and to Dr. H. J. Phaff for the strain of Debaryomyces vanryi. We also thank Dr. J. M. Keller for reading the manuscript.

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LEE, Y. C., AND BALLOU, C. E., J. Biol. Chem., 239, PC3602 (1964).

LEE, Y. C., J. Biol. Chem., 241, 1899 (1966). SAIER, M. H., AND BALLOU, C. E., J. Biol. Chem., 243, 992

(1968). PAZUR, J. H., KLEPPE, K., AND BALL, E. M., Arch. Biochem.

Biophys., 103, 515 (1963). STEWART, T. S., AND BALLOU, C. E., Biochemistry, 7, 1855

(1968). STOREY, J., AND WEBBER, J. M., in R. L. WHISTLER AND M. L.

WOLFROM (Editors), Methods in carbohydrate chemistry, Vol. I. Academic Press. New York. 1962. n. 339.

BO~ENG, H. O., A&D LINDBE~, B:,- Advance. Carbohyd. Chem., 16, 77 (1960).

DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F., Anal. Chem., 28, 350 (1966).

BRAY, G. A., Anal. Biochem., 1, 279 (1960). PARTRIDGE. S. M.. Nature, 164, 443 (1949). ANET, E. I@. L. J:, AND I&Y~OLDS; T. ‘M., Nature, 174, 930

(1954). GORDON, H. T., THORNBURG, W. T., AND WERUM, L. H.,

Anal. Chem., 28, 849 (1956). FOSTER, A, B., in R. L. WHISTLER AND M. L. WOLFROM

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STEWART, T. S., MENDERSHAUSEN, P. B., AND BALLOU, C. E., Biochemistry, 7, 1843 (1968).

GUTHRIE, R. D., AND MCCARTHY, J. F., Advance. Carbohyd. Chem., 22, 11 (1967).

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PAZUR, J. H., AND KLEPPE, K., J. Biol. Chem., 237, 1002 (1962).


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Milton H. Saier, Jr. and Clinton E. Ballou: STRUCTURE OF THE REDUCING END OF THE POLYSACCHARIDE

Mycobacterium phlei-Methylglucose-containing Lipopolysaccharide of OThe 6-

1968, 243:4319-4331.J. Biol. Chem.

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