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THE Jonxivnr, OF BIOLOGICAL CHEMISTRY Vol. 244, No. 18, Issue of September 25, pp. 4852-4863, 1969

Printed in U.S.A.

The Composition and Structure of the Carbohydrate of

Pineapple Stem Bromelain*

(Received for publication, April 25,1969)

JANE SCOCCA$ AND Y. C. LEE

Prom the Department of Biology and McCollum-Pratt Institute, the Johns Hopkins University, Baltimore, Maryland 21218

SUMMARY

Two major components of pineapple bromelain, purified to electrophoretical homogeneity, appeared to have the same oligosaccharide group consisting of n-glucosamine, n-mannose, D-xylose, and L-fucose in ratios of 2:2: 1: 1. By exhaustive proteolysis of the bromelains, glycopeptides containing only Asx, Glx, and Ser were obtained. Periodate oxidation, methylation, and glycosidase digestion showed that the oligosaccharide chain has a highly branched structure in which all the neutral sugars are in nonreducing terminal positions and both N-acetyl-D-glucosamine residues occur in internal positions.

In contrast to the rapidly growing store of information on the composition, structure, and synthesis of glycoproteins of animal origin, there is little information on the occurrence and nature of glycoproteins of plant origin (1). Pineapple stem bromelain is one of the few plant glycoproteins which has been investigated with respect to the composition and structure of the carbohydrate prosthetic group.

Heinicke and Gortner (2) first reported the preparation and preliminary characterization of pineapple stem bromelain. Three separate laboratories studied the properties of stem bromelain and found that the enzyme contained a small amount of carbohydrate. Murachi, Yasui, and Yasuda (3) reported a carbohydrate content of 2% in the purified enzyme, whereas Ota, Moore, and Stein (4) reported 1.46% carbohydrate. Feinstein and Whitaker (5), who isolated several proteolytically active components from bromelain, found 2 to 4 moles of carbohydrate per mole of the purified components.

In agreement with several earlier studies on bromelain (2, 6, 7), the present studies show that crude stem bromelain contains several separable enzymes that exhibit similar proteolytic ac- tivities. The two most abundant proteolytically active components have been purified to electrophoretic homogeneity. The isolated bromelain components and the glycopeptides derived

* This work has been supported by Research Grant AM09976 from the National Institutes of Health. Contribution 569 from McCollum-Pratt Institute, the Johns Honkins Universitv.

$ Recipient of United States Public Health Service Preioctoral Fellowship Fl-GM-33, 882.

therefrom are identical in carbohydrate composition. Struc- tural studies of the glycopeptide carbohydrate by chemical and enzymatic techniques indicate a highly branched carbohydrate moiety.

MATERIALS

Bromelain powder prepared by acetone precipitation of pineapple stem juice (2) (Dole, Lot 498) was the generous gift of Dr. R. M. Heinicke, Dole Company, Hawaii. Casein was purchased from Mann. Bovine serum albumin was from Armour.

Hexoses, pentoses, hexosamines, and glycosides other than those described below were commercially obtained from Pfan- stiehl Laboratories (Waukegan, Illinois), Sigma, and Pierce Chemical Company (Rockford, Illinois). Methyl-cr+fucopyranoside was prepared from L-fucose by refluxing with metha- nolic HCI and was purified on Dowex l-X2, 200 to 400 mesh (OH- form) (8). n-Galactosamine was the gift of Dr. S. Rose- man.

The following ion exchange resins and chromatography media were obtained from commercial sources: Dowex 5OW-X2 and -X8, 200 to 400 mesh (H+ form), and Dowex l-X8, 200 to 400 mesh (Cl- form) (J. T. Baker Chemical Company, Phillipsburg, New Jersey); AG 5OW-X8, 200 to 400 mesh (H+ form) (Bio- Rad Laboratories, Richmond, California); Amberlite MB-3 (Mallinckrodt Chemical Works, St. Louis, Missouri); Sephadex G-25 (fine), Sephadex G-50 (fine), Sephadex G-100, and sulfoethyl-Sephadex C-50 (Pharmacia); and Avicel SF (F.M.C. Corporation, Marcus Hook, Pennsylvania). Dowex 1 was converted to bicarbonate form by washing with 1 M ammonium bicarbonate until the washes contained no chloride.

Hexokinase (type C-130, from yeast), lactic dehydrogenase (type I, from rabbit muscle), and papain were from Sigma. Carboxypeptidase A and pepsin were from Worthington. Mol- lusk ,&glucuronidase was from the Pierce Chemical Company. L-Fucose dehydrogenasel was the generous gift of Dr. Harry Schachter, and jack bean cr-n-mannosidase (9) was kindly do- nated to us by Dr. Y. T. Li. Almond emulsin a-mannosidase2 was prepared from almond emulsin (/3-glucosidase from Worth- ington). Pronase, Grade B, from Calbiochem was purified by gel filtration on Sephadex G-100 to remove carbohydrate material prior to use.

/3-Diphosphopyridine nucleotide, ,&diphosphopyridine nucleo-

1 H. Schachter and S. Roseman, unpublished results. 2 Y. C. Lee, unpublished results.

4852

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Issue of September 25, 1969 J. Scocca and Y. C. Lee 4853

tide (reduced form), phosphoenolpyruvic acid, p-hydroxymercuribenzoate, and Tris were purchased from the Sigma. Adeno- sine triphosphate was purchased from P-L Biochemicals.

Trimethylchlorosilane and hexamethyldisilazane were from K and K Laboratories. Neopentylglycolsuccinate (HiEFF-3BP, 3% on Gas Chrom Q, 100 to 120 mesh) and ECNSS-M (3% on Gas Chrom Q, 100 to 120 mesh) for gas chromatography were purchased from the Applied Science Laboratories, Inc.

METHODS

Unless otherwise mentioned, all evaporations were at 35-40” under reduced pressure in a rotary evaporator. All pH values indicated were the values measured at room temperature. Spec- trophotometric data were obtained in cells with l-cm light path.

Enzymatic Activity

A modification of Kunitz’s assay described by Laskowski (10) was used to measure proteolytic activity. To 0.25 ml of enzyme solution was added 0.25 ml of 0.05 M Na phosphate buffer, pH 7.6, containing 20 mM mercaptoethanol. After 1 min of incubation at 37”, 0.5 ml of 3% casein in 0.025 M sodium phosphate buffer was added. The reaction was stopped after 18 min by adding 1.5 ml of 10% trichloracetic acid. The precipitated protein was removed by centrifugation, and the absorbance of the supernatant at 280 rnp was measured. Zero time controls and blanks without enzyme were included in each incubation. These modifications made the assay linear in the range of Azao values between 0.25 and 0.50. Assays of activity were conducted within this range. One unit of enzyme was defined as an increase in Atso of 0.100 for an 18-min incubation under the conditions described.

Determination of Protein, Amino Acids, and Amino Groups

Absorbance at 280 rnp was used to monitor protein during purification; At:m = 19.0 (3) was used to calculate protein content. Protein was also measured by the biuret reaction (11) with bovine serum albumin as standard. Amino acid content of samples was determined with a Beckman amino acid analyzer, model 120 A, B, or C. Samples were hydrolyzed in 6 N HCl at 110” for 24 hours in sealed, evacuated ampoules. Hydrolysates were evaporated to dryness immediately after hydrolysis. COOH-terminal amino acids liberated after hydrazinolysis (12) were also determined by the amino acid analyzer. Free amino groups were measured by Rosen’s ninhydrin assay (13) with alanine as a standard.3

Neutral Sugar Hydrolysis

For determination of individual neutral sugars, samples of glycopeptide or glycoprotein were hydrolyzed in 1 N H&04 at 100” for 6 hours. Hydrolysates were deionized with Dowex l-X8, 200 to 400 mesh (bicarbonate form) and Dowex 50-X2 or -X8,200 to 400 mesh (H+ form) added batchwise to the hydrolysate. The neutral hydrolysates were filtered through Millipore filters on Buchner funnels, dried, and analyzed.

Calorimetric and Enzymatic Determination of Neutral &ugars

Total neutral sugar content was measured by a modified procedure (14) of the phenol-sulfuric acid method (15) with n-man-

s Unless otherwise specified, amino group content of samples is expressed as micromoles of amino group as alanine.

nose as standard.4 Fucoee was measured by the cysteine-sulfuric acid method (16). The assay was scaled down to a final volume of 1.2 ml, and it could be used to measure 1 to 16 pg of fucose. The color yields exhibited by mannose and xylose in this reaction were only 5% of that of fucose, on a weight basis. Xylose was measured by an orcinol reaction (17). The reaction was scaled down to a final volume of either 1.0 or 2.0 ml, and it could be used to measure 1 to 6 pg of xylose. Samples of mannose and fucose showed only 5% of the absorbance of equivalent weights of xylose in the assay. Mannose was measured wit,h a hexokinase reaction by determining the product, ADP, in the coupled enzyme system described by Kornberg and Pricer (18). The assay was stoichiometric for mannose over the range employed, 2 to 20

I% The configuration of fucose was determined with L-fucose de-

hydrogenase? The enzyme is specific for L-fucose, and DPNH formed in the reaction is measured at 340 rnp.

Chromatographic Determination of Neutral Sugars

Gas Chromatography-Alditol acetates of neutral sugars were prepared and chromatographed as described by Sawardecker, Sloneker, and Jeans (19).

Thin Layer Chromatoraphy-Neutral sugars were chromatographed on glass plates coated with a 0.25.mm layer of sodium borohydride-reduced crystalline cellulose (Avicel SF) (20). Samples of neutral sugars, released by acid hydrolysis, were spotted in four lanes on a plate. Each spot contained approximately 8 pg of neutral sugar. Plates were developed three times in a mixture of l-butanol-pyridine-acetic acid-ethyl acetate-H*0 (10:3:3:3:4). After development, the inner two lanes were protected with aluminum foil and cooled with a damp sponge, while the sugars in the outer lanes were visualized by spraying with a 2-aminobiphenyl reagent (21) followed by heating with a heat gun. Areas of the inner lanes corresponding to t,hose containing reducing sugar were scraped loose with a scalpel and collected by suction into glass wool-plugged Pasteur pipettes attached to a water aspirator. The collected cellulose was eluted with distilled water, and the eluted reducing sugars were assayed by the Park-Johnson method (22). Areas between sugar-containing spots were also collected from each plate and analyzed for reducing value; these served as blanks.

Automated Borate Chromatography-Free neutral sugars were also measured by automated borate-complex anion exchange chromatography as described by Lee, McKelvy, and Lang (23).

Determination of Amino Sugars

Amino Sugar Hydrolysis-Amino sugars were released from the protein or glycopeptide by hydrolysis in 4 N HCl at 100’ for 6+ to 9 hours. After hydrolysis, samples were evaporated to dryness under reduced pressure at 40” to remove HCl. To correct for glucosamine destruction, known amounts of standard N-acetyl-D-glucosamine were added to samples prior to hydrolysis, or standard D-glucosamine was exposed to the same conditions of hydrolysis in a separate ampoule.

Elson-Morgan Reaction-Glucosamine was measured by the Elson-Morgan reaction as described by Gatt and Berman (24) after separation from neutral sugars on an AG 5OW-X8 column

(25).

4 Carbohydrate content (i.e. neutral sugar content) of samples is expressed as equivalent weight of mannose unless otherwise specified.


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4854 Carbohydrate of Pineapple Bromelain Vol. 244, No. 18

Amino Acid Analyzer-The glucosamine content of glycopeptide or glycoprotein hydrolysates was measured on the short column of the amino acid analyzer. Irregularities of the base line observed with whole protein hydrolysates were reduced by isolating glucosamine on an AG 5OW-X8 column (25) prior to analysis.

Ion Exchange-Neocuproine Method-Glucosamine in hydrolysates was also measured without prior separation from neutral sugars by an automated procedure (26), with a column and buffer similar to that of the amino acid analyzer but employing the neocuproine method (27) for sugar detection. Routine glucosamine measurements were performed on the 3-mm column (Proce- dure 1 in Reference 26). This method will be referred to as the “neocuproine method.” Glucosamine was distinguished from galactosamine on the g-mm column (Procedure II in Reference 26), and from mannosamine on the 3-mm column using a dilute sodium citrate buffer (pH 5.29, 0.15 M in Na+) as eluent.

Acrylamide Gel Electrophoresis

Gel and buffer systems recommended by Canalco were prepared for analytical gel electrophoresis at pH 4.3 (28) and pH 9.5 (29). Upper gels were omitted. At both pH levels bromelain components migrated toward the cathode. Gels were stained for protein either with 0.1% Amido schwarz in 7.5% acetic acid or with 0.05% Coomassie brilliant blue in 12.5% trichloracetic acid (30).

Preparative acrylamide gel electrophoresis was performed on a Buchler model 3-1700 preparative polyacrylamide gel electrophoresis apparatus. The standard 7.5% running gel and the standard lower and eluting buffers for electrophoresis at pH 8.9 described by Buchler Instruments5 were used. Stacking and spacer gels were omitted. When the recommended upper buffer was used, excessively high voltage was encountered when electrophoresis was run toward the cathode. Replacement of this buffer by the more concentrated Tris-glycine buffer used in the analytical gel electrophoresis at pH 9.5 described above reduced the voltage.

Periodate Oxidation

GP II6 and GP III were oxidized with 0.01 or 0.05 M sodium periodate, at an estimated 4-fold molar excess of periodate, at room temperature in the dark. The reaction was followed by measuring the decrease in absorbance at 240 rnp and terminated by adding a large excess of ethylene glycol. In one experiment, GP II and GP III oxidized with 0.01 M periodate were desalted on a Sephadex G-25 column (1 x 50 cm) with 0.1 M ammonium carbonate as eluent. The desalted glycopeptides were evaporated to dryness, dissolved in water, and hydrolyzed to determine neutral and amino sugar content. In a second experiment, GP II was N-acetylated with 2y0 acetic anhydride in acetone (31) prior to periodate oxidation. After oxidation in 0.05 M

periodate, the glycopeptide was reduced with sodium borohydride; sodium was removed with Dowex 5OW-X8, 200 to 400 mesh (H+ form) ; and borate was removed by repeated evaporation with methanol. The borohydride-reduced glycopeptide was isolated on a Sephadex G-25 column (1 x 110 cm), with 0.1 M

trimethylammonium acetate buffer, pH 5.0, as the eluent. The

5 Operation instructions for Buchler preparative polyacrylamide gel electrophoresis apparatus, Poly-Prep.

6 GP II and GP III are glycopeptides prepared from bromelains II and III, respectively.

glycopeptide fractions were evaporated to dryness to remove the volatile buffer, and the sample was dissolved in water; aliquots were hydrolyzed to determine neutral sugar, amino sugar, and amino acid composition.

Methylation

Samples were methylated by the method of Hakomori (32) with sodium methylsulfinyl methide and methyl iodide. Before methylation samples were dried overnight over NaOH in an evacuated desiccator at 40”. The carbanion was prepared by the method of Corey and Chaykovsky (33) and titrated with HCl to determine anion concentration. Although in Hakomori’s procedure (32) carbanion is added to the sample in amounts equimolar to the free hydroxyl content of the sample, in our experiments a 5- to lo-fold excess of anion to free hydroxyl groups resulted in more complete methylation. Fresh carbanion was added to the sample in anhydrous dimethylsulfoxide; and the sample was flushed with nitrogen, tightly capped, and shaken at room temperature for 1 hour. Methyl iodide was added, and the shaking continued for 2 hours. A solution of 5 M NaCl in either 0.05 N HCl (34) or 2.0 N acetic acid was then added to the sample; the mixture was extracted three times with CHCl,; and the combined CHC13 extracts were washed five times with water. After methylation, glycopeptides were dried, methanolyzed in 0.85 N HCl in dry methanol in sealed ampoules at 100” for 12 hours, dried rapidly under reduced pressure at room temperature, and dissolved in CSZ for gas chromatography.

N-Acetylated GP II and GP III, each containing 1 mg of carbohydrate, were methylated as described above, using a 1:l ratio of the carbanion to estimated free hydroxyls. After ex- traction, the carbohydrate remained in the aqueous layer. The glycopeptides were recovered from the reaction mixture by gel filtration on a Sephadex G-25 column (1 x 110 cm) with 1 N

acetic acid as eluent (35), freeze-dried, and dried under reduced pressure at 40” prior to a second methylation. Methylation was repeated with a 7: 1 ratio of anion to hydroxyl groups. The carbohydrate was found in the CHC13 extract at this stage. One-fifth of the CHC13 extract was methanolyzed for gas chromatographic analysis; the remaining extract was dried; and methylation was repeated twice more. Half of the remaining sample was then methanolyaed for gas chromatographic analysis.

Gas chromatography was carried out on a Packard model 7800 gas chromatography apparatus on columns (6 ft X 4 mm) packed with neopentylglycolsuccinate (3% on Gas Chrom Q, 100 to 200 mesh). Chromatography was carried out at 178” with a carrier Nz flow rate of 35 ml per min.

RESULTS

Purijkation of Brometain Components-The buffer used throughout the purification was 0.1 M Tris chloride, pH 8.7, at 3”, containing 0.5 mM p-hydroxymercuribenzoate. All purification steps were performed at 3-6”. Eight grams of stem bromelain were stirred in 80 ml of the buffer for 3 hours. The extract was centrifuged at 3200 x g for 10 min. The clear brown supernatant solution was applied to a Sephadex G-100 column and eluted with the buffer. Larger and smaller molecular weight carbohydrates were separated from the bulk of the protein by the gel filtration step (Fig. 1). The fractions indicated by the horizontal bar were pooled. Pooled fractions from six such gel filtrations were combined and applied to an SE-Sephadex C-50


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25 20

/ 1.0 2'0 3:o

EFFLUENT CL)

FIG. 1. Gel filtration of crude extract of bromelain on f G-100. Crude extract (77 ml) with 3.37 g of protein was a a column (6 X 96 cm) and eluted with 0.1 M Tris-HCl, pH

800

000 2

2 600 f

3

200 ?i L

800 if,

lhadex lied to 7, con-

taining 0.5 rnM p-hydroxymercuribeneoate. The flow rate was 54 ml per hour, and 20-ml fractions were collected. 0, absorbance at 280 mp; 0, carbohydrate as mannose by the phenol-sulfuric acid assay; A, enzyme units determined by the casein assay as described in “Methods.”

column, and the column was eluted with a linear gradient of KC1 in the buffer. The bromelain components were separated by this gradient elution (Fig. Z), and the components were designated in order of elution from the column. The diffuse leading edge was termed bromelain I and the two major peaks bromelains II and III. An additional component, bromelain IV, could be eluted when the salt concentration in the eluting buffer was increased to 0.5 M. Analytical polyacrylamide gel electrophoresis at pH 9.5 showed at least three distinct protein bands in the samples from the leading edge of the first SE-Sephadex peak (bromelain I) but showed only one protein band in samples from each of the fractions in the major peaks. The indicated fractions in these principal enzyme peaks were pooled and designated bromelain II and bromelain III. Bromelain II was precipitated by adding ammonium sulfate to give 0.8 saturation, and bromelain III was precipitated by adding solid ammonium sulfate to give 0.6 saturation. Ammonium sulfate saturation of 0.6 was sufficient to precipitate either component. Three hours after the ammonium sulfate addition to the pooled fractions, the precipitates were centrifuged at 8000 x g for 20 min, dissolved in 0.05 M sodium phosphate buffer, pH 7.0, and dialyzed against the same buffer. A small amount of precipitate which formed during dialysis was removed by centrifugation at 6300 X g for 30 min.

The purification procedure for bromelain is summarized in Table I. Purification was approximately 2-fold with respect to enzyme activity and 25-fold with respect to carbohydrate content. The carbohydrate content of the samples decreased in both gel filtration and ion exchange steps but did not decrease significantly in the ammonium sulfate precipitation step. The proteins obtained after ammonium sulfate precipitation were used for determinations of the neutral and amino sugar and amino acid composition of bromelain and for preparation of glycopeptides.

By polyacrylamide disc gel electrophoresis, purified bromelains II and III appeared as single bands, at both pH 4.3 and pH 9.5 (Fig. 3). The components exhibited distinctly different mo-

1:o 2.0 30 40 50

EFFLUENT CL)

‘500

?coO

z 1500 ;,

t f

,000 Y iz 5

500

FIG. 2. Ion exchange chromatography of pooled bromelain G-100 fractions on SE-Sephadex C-50. Pooled enzyme (3120 ml) with 10.6 g of protein was applied to a column (5 X 43 cm) and eluted with a linear gradient of KC1 in 0.1 M Tris-HCl, pH 8.7, containing 0.5 mM p-hydroxymercuribenzoate. The mixing chamber contained 4 liters of 0.025 M KC1 in buffer, and the reser- voir contained 4 liters of 0.15 M KC1 in buffer. The flow rate was 53 ml per hour, and 20-ml fractions were collected. l , absorbance at 280 rnp; 0, neutral sugars by the phenol-sulfuric a say; A, enzyme units determined by the casein assay.

tcid as-

TABLE I Purification of bmnelahr

Fraction Volumt

ml

Crude extractd. . . . 463 G-100. . . . 3120 Sulfoethyl-Sepha-

dex Bromelain II. . 545 Bromelain III. . 735

Ammonium sulfate Bromelain II 84 Bromelain III. 53

Total yzz Protein

-- x 10-s n&g/ml

39.9 39.2 31.9 3.39

9.15 3.88 5.70 1.99

8.20 17.4 5.99 24.2

*

-.

Specific activity YieldC

--

units/?ng 70

220 100 301 80

Neutral sugars

432 23 2.41 390 14 2.12

561 21 2.48 467 15 2.05

a Measured by the casein assay. b Determined from Am. 0 Based on protease activity units. d Data for the crude extract represent the sum and averages

for six separate preparations of crude extracts.

bilities at pH 9.5, whereas at pH 4.3, a mixture of the two components appeared as a single band.

When samples of the purified components were subjected to preparative acrylamide gel electrophoresis at pH 8.9, each component was eluted as a single peak in which protein, carbohydrate, and enzymatic activity coincided (Fig. 4).

Preparation of Glycopeptides-Glycopeptides were prepared from bromelains II and III by digesting the proteins with Pro- nase. Bromelains II (1.27 g) and III (1.11 g), in 0.05 M phosphate buffer, were adjusted to pH 7.8 with 2 N NaOH. Pronase (0.5% of bromelain, w/w) was added, and the mixtures were incubated at 50” with thymol crystals added to prevent microbial growth. As the pH dropped during digestion, it was periodically readjusted with 2 N NaOH to pH 7.6 to 7.8. After 24 hours the same quantity of fresh Pronase was added to the digests and


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FIG. 3. Acrylamide gel electrophoresis of purified bromelain components. Samples of bromelains II and III (25 to 50 rg) from the final ammonium sulfate step of purification (see Table I) and a mixture of the two components were subjected to acrylamide gel electrophoresis. Left, electrophoresis at pH 9.5 in 7.5% gels (0.5 X 6 cm) was run for 90 min at 5 ma per gel with migration toward the cathode. Right, electrophoresis at pH 4.3 in 7.5% gels (0.5 X 6 cm) was run for 75 min at 5 ma per gel with migration toward the cathode. Gels were stained for 60 min with 0.1% Amido schwarz in 7.5y0 acetic acid.

0 100 200 300 ML EFFLUENT

FIG. 4. Preparative acrylamide gel electrophoresis of purified bromelain III. Bromelain III (31.5 mg) from the final step of purification (see Table I) was subjected to electrophoresis at pH 8.9 in a 7.5% running gel of 2-cm height on a Buchler preparative acrylamide gel apparatus. The protein was electrophoresed toward the cathode for approximately 18 hours. The gel column was eluted at a flow rate of 0.4 ml per min, and fractions of 6.2-ml were collected. 0, Absorbance at 280 mp; 0, enzyme units per ml measured by the casein assay; A, neutral sugars measured by the phenol-sulfuric acid assay.

incubation was continued for 72 hours, at which time the rate of release of amino groups had decreased substantially. The ex- tent of digestion was estimated from the release of free amino groups measured by the ninhydrin assay. The total increase in free amino groups was 98 and 132 pmoles per mg of carbohydrate from bromelains II and III, respectively. These values repre- sented 22 and 25% of the estimated values for complete digestion of the proteins to free amino acids. The digests were filtered to remove insoluble material which had formed during digestion,

a.

FRACTION NUMBER VOLUME (ml)

FIG. 5. a, gel filtration of the first Pronase digest of bromelain II The concentrated digest (18 ml) containing 32.3 mg of neutral sugar as mannose was applied to a Sephadex G-50 column (3 X 110 cm) and eluted with water. Fractions of 10 ml were collected. 0, amino groups as alanine measured by Rosen’s ninhydrin assay; l , neutral sugars as mannose measured by the phenol-sulfuric acid assay. b, gel filtration of the pepsin and carboxypeptidase digest of glycopeptide from bromelain II on Sephadex G-50. The digest (5.0 ml) containing 31.5 mg of neutral sugars as mannose was fractionated as in a. o, neutral sugars as mannose by the phenol-sulfuric acid assay.

concentrated, and fractionated on a Sephadex G-50 column (3 x 110 cm) eluted with water. The column effluent was analyzed by the ninhydrin and the phenol-sulfuric acid color reactions. After the first digestion, in both samples, the carbohydrate was eluted from the G-50 column in a single peak emerging ahead of the bulk of the free amino acids and peptides (Fig. 5a). The recovery of carbohydrate after the first digestion and isolation was 100 and 96% for bromelains II and III, respectively. Fractions containing carbohydrate were pooled, concentrated, and adjusted to pH 7.4. A second digestion with Pronase (Pronase- carbohydrate, 1: 5, w/w) continued for 2 days with bromelain III and 3 days with bromelain II. The increase in free amino groups was 25 and 11 pmoles per mg of carbohydrate for bromelains II and III, respectively. The digests were filtered, concentrated, and purified on Sephadex G-50. Again, the carbohydrate from each sample was eluted as a single peak followed by a peak of peptides and amino acids. Recoveries of carbohydrate from the preceding step were 99 and 89% for bromelains II and III, respectively. Fractions containing carbohydrate were pooled and freeze-dried.

The glycopeptide from bromelain III containing 19.3 mg of carbohydrate was dissolved in a small volume of water, adjusted to pH 6.2, and incubated with 0.76 mg of papain in the presence of 1 mM dithiothreitol and 2 InM EDTA. After 2 days of incubation at 50”, the pH was adjusted to pH 7.5, 0.35 mg of carboxypeptidase A was added, and incubation was continued at 37” for 24 hours. After the papain-carboxypeptidase A treatment, the increase in amino groups was only 0.37 pmole per mg of carbohydrate.

The glycopeptide from bromelain II, containing 34.2 mg of carbohydrate, was dissolved in water, adjusted to 0.05 M in sodium citrate buffer, pH 4.4, and incubated with 1 mg of pepsin

I I I , I

20 40 60 SO I00 200 400 600 800


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2 4 6 8

HOURS OF HYDROLYSIS

FIG. 6. The release of neutral sugars from bromelain II glycopeptide as a function of hydrolysis time. Samples were hydrolyzed in 1 N HzSOI at 100” at a neutral sugar concentration of 0.2 mg per ml for the times indicated. Hydrolysates were deionized with Dowex l-X8, 200 to 400 mesh (bicarbonate form) and AG 5OW-X8, 200 to 400 mesh (Hf form). After deionization, neutral sugar content of the hydrolysates was measured by automated borate chromatography. Man, mannose; Fuc, fucose; Xyl, xylose.

for 48 hours at 37”. The solution was then adjusted to pH 7.5, the solution was adjusted to 0.05 M NaCl by adding solid NaCl, and then 0.41 mg of carboxypeptidase A was added. After incubation for an additional 48 hours at 37”, the observed increase in free amino groups was 1.2 pmoles per mg of carbohydrate.

The small increases in amino group observed in the third di- gestions of both samples were considered to lie within the error of the measurements. The samples were filtered, concentrated, and purified on Sephadex G-50 in yields of 92 and 91 y. from the preceding step. The fractions indicated by the horizontal bar in Fig. 5b were pooled and used for all further studies of the glycopeptides. The material in the leading edge of the peaks, which was not combined with the peaks, was found to contain the same neutral sugars in the same proportions as the peak fractions. Glycopeptides from bromelains II and III were designated GP II and GP III, respectively.

IdentQication of Neutral Sugar Components-Neutral sugars liberated by acid hydrolysis of a glycopeptide prepared by Pro- nase digestion of unfractionated bromelain components were converted to their alditol acetates. On gas chromatography (19) only three peaks corresponding to acetates of fucitol, xylitol, and mannitol were detected.

Mannose released from bromelain by acid hydrolysis was quantitatively phosphorylated by hexokinase. This established that the mannose in bromelain was n-mannose. More than 90% of the fucose released from GP II by mild acid hydrolysis was oxidized by L-fucose dehydrogenase, which indicated that the fucose in bromelain is of L configuration. The configurational specificity of these enzymes has been well established. Al- though the configuration of xylose was not determined in this study, release of xylose from bromelain glycopeptide by a P-D-

xylosidase (36) indicates D configuration for xylose. The glycopeptide from bromelain II was hydrolyzed in 1 N

TABLE II

Carbohydrate composition of brornelain components

Method

Automated borate chromatography

Thin layer chromatography

Hexokinase Cystein-H&SO* Orcinol

Elson-Morgan Amino acid analyzer Neocuproine method

Sugar

Mannose Fucose Xylose



GlcN GlcN GlcN

- I Molar ratios relative to

mannose as 2.00

2.09 2.00 1.07 1.12 0.88 0.95

2.00” 2.00 1.00” 1.03 1.00” 0.93

2.00 2.00 1.33 1.21 1.33 1.18

2.49a.b 2.586 2.3Ob

1.89” 1.96c

a Analyses were performed on a preparation containing a trace of bromelain I.

b Molar ratios are expressed relative to mannose as 2.00 with mannose content measured by the hexokinase assay.

c Molar ratios are expressed relative to mannose as 2.00 with mannose content measured by automated borate chroma.tography.

H&04 at 100” for 2, 4, 6, and 8 hours and deionized; the free neutral sugar content of the hydrolysates was determined by automated borate chromatography. The release of neutral sugars during acid hydrolysis is shown in Fig. 6; the maximum yield of neutral sugars was obtained at 4 to 6 hours of hydrolysis. Since 4-hour hydrolysates from bromelain proteins had been found by paper chromatography to contain small amounts of oligosaccharides in addition to mannose, fucose, and xylose, 6 hours was selected as the optimum hydrolysis time. The recovery of the neutral sugars under these hydrolysis conditions was determined by adding known amounts of mannose, fucose, and xylose to GP II prior to hydrolysis. Galactose was added immediately after hydrolysis to serve as an internal standard to correct for any mechanical losses during the deionization procedure. The recovery of the internal standard galactose was 100%. Recoveries of neutral sugars subjected to hydrolysis with the glycopeptide in 1 N H&Oh at 100.’ for 6 hours were 82oj, for xylose, 89% for fucose, and 93% for mannose, and these were considered to be minimum recovery factors.

Protein samples were hydrolyzed as described above. Re- coveries of sugars after hydrolysis, deacidification, and concentration were at least 90% as determined by phenol-sulfuric acid assay. Table II lists the molar ratios of the neutral sugars determined by three separate techniques described above. Molar ratios of the neutral sugars were the same for each bromelain component and were reproducible from batch to batch of the purified bromelain. Mannose, fucose, and xylose occurred in a ratio of 2: 1: 1. Assuming a molecular weight of 33,000 (3)) and using the data obtained by the borate chromatography, the num- bers of sugar residues per mole of protein were: mannose (2.24), fucose (l.lO), xylose (0.98) for bromelain II, and mannose (1.98), fucose (0.95), xylose (0.90) for bromelain III. The molar ratios


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TABLE III

Composition of glycopeplides obtained by proteolytic digestion of bromelain

Neutral sugars were measured by automated borate chromatography, amino sugars by the neocuproine method, and amino acids on a Beckman amino acid analyzer. Hydrolysis conditions for release of neutral sugars, amino sugars, and amino acids from the glycopeptides are described in “Methods.” Molar ratios are expressed relative to mannose as 2.00.

Residues Bromelain II glycopeptide

Bromelain III glycopeptide

Mannose 2.00 2.00 Fucose 1.03 1.05 Xylose 1.01 1.01

GlcN 2.02 2.08

ASP 2.07 2.08 Thl- 0.19 0.10 Ser 1.16 1.22

Glu 1.22 1.16

Pro 0.28 0.18

GUY 0.50 0.38 Ala 0.20 0.13 Val 0.21 0.12 Ile 0.14 0.07 Leu 0.08 Trace

Tyr Trace Trace

- I

-

Molar ratios

+ L ORIGIN,

3 2 I

FIG. 7. Paper electrophoresis of GP III. GP III containing 20 rg of neutral sugar as mannose was streaked on strips of Whatman No. 1 for electrophoresis at 46 volts per cm for 45 min in pH 2.37 pyridine formate buffer or in pH 6.46 pyridine acetate buffer. Strips were split lengthwise and stained for carbohydrate with periodate benzidine reagent and for amino acids with ninhydrin reagents.

of sugar to protein were slightly but consistently higher (about 10%) for bromelain II.

Identi$cation of Amino Sugar Components-By the neocuproine method, the amino sugar released by acid hydrolysis of glycopeptides was identified as glucosamine. On the g-mm column, elution time of the amino sugar (70 min) corresponded to that of glucosamine (70 min) rather than galactosamine (77 min), while on the 3-mm column eluted with the diluted buffer (0.15 N Na+), the elution time (43 min) corresponded to that of glucosamine (43 min) rather than that of mannosamine (48 min).

Glucosamine was separated from any neutral sugars in the acid hydrolysates on an AG 5OW-X8 column (25) and then identified as n-glucosamine by its quantitative phosphorylation by hexokinase.

Bromelain glycopeptide and whole bromelain III were hydrolyzed in 4 N HCl at 100” for 5 to 93 hours, HCl was removed

by evaporation under reduced pressure, and the glucosamine content of the hydrolysates was measured on the amino acid analyzer as described in “Methods.” Glucosamine release reached a plateau during 5 to 9+ hours of hydrolysis.

In the glucosamine determination by the Elson-Morgan reaction or by the amino acid analyzer, recoveries of glucosamine were estimated by treating glucosamine alone under the same conditions of hydrolysis and isolation. Recoveries varied from 80 to 93%. In the glucosamine determination by the neocuproine method, recoveries of glucosamine were estimated from the recovery of known amounts of standard N-acetyl-n-glucosamine added to the samples prior to hydrolysis. In this case, recoveries varied between 90 and 100%.

The molar ratios of glucosamine to mannose in bromelains II and III are listed in Table II. By the Elson-Morgan procedure, and on the amino acid analyzer, the glucosamine content appeared to be either 2 or 3 moles per mole of protein, but the data obtained by the neocuproine method suggested that the value is 2 rather than 3.

Composition of Glycopeptides-Amino acid, amino sugar, and neutral sugar composition of GP II and GP III are listed in Table III. Molar ratios of the neutral and amino sugars were the same as those found for the intact proteins. The nonintegral values for the amino acids suggested that the glycopeptides were heterogeneous in the peptide region. As will be shown below, the glycopeptides could be further fractionated by paper electrophoresis.

Electrophoretic Separation of Glycopeptides-Paper electrophoresis of glycopeptides on Whatman No. 1 paper strips was performed at 46 volts per cm for 45 min in pH 2.37 acetic acid- formic acid buffer (5.75 ml of formic acid + 18.8 ml of acetic acid per liter) and in pH 6.46 pyridinium acetate buffer (37). Strips were dried, cut in half longitudinally, and stained for carbohydrate with periodate-benzidine (38) and for peptide with 1% ninhydrin in acetone or with the cadmium-ninhydrin reagent

(39). Glycopeptides II and III exhibited the same behavior on paper

electrophoresis (Fig. 7). After electrophoresis at both pH 2.37 and 6.46, and staining with the periodate-benzidine reagents, two strongly positive bands and one faintly positive band were observed. The electrophoretogram at pH 2.37 stained with 1% ninhydrin in acetone showed only two bands corresponding to the two heaviest periodate-positive bands. The electrophoretogram at pH 6.46 stained with the more sensitive cadmium- ninhydrin reagent showed three bands, corresponding to each periodate-positive band. The three bands exhibited striking color differences with the cadmium-ninhydrin reagent. Band 1 was pink, Band 2 yellow, and Band 3 brick red. Three other faint bands appeared in the cadmium-ninhydrin-dipped strips. The color differences diminished and all bands turned reddish pink after several days at room temperature.

For preparative electrophoresis GP III containing 2.08 mg of neutral sugars as mannose was concentrated to 50 ~1 and streaked on Whatman No. 3 paper. The sample was subjected to electrophoresis in the pH 6.46 pyridinium acetate buffer at 54 volts per cm for 45 min. After drying, guide strips were cut from both sides of the sheet and stained with cadmium-ninhydrin reagent to locate the glycopeptides. Areas of the electrophoretogram which corresponded to the three ninhydrin bands were cut out and eluted with water. ,After elution the samples were concentrated to 0.2 to 0.5 ml, applied to a Sephadex G-25 column


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Issue of September 25, 1969 J. Xcocca and Y. C. Lee 4859

TAULIX IV

Composition of glycopeplides from GP III separated by paper electrophoresis (Fig. ?‘)

Neutral sugars were measured by automated borate chromatography, amino sugars by the neocuproine method, and amino acids on a Beckman amino acid analyzer. Hydrolysis conditions for the release of neutral sugars, amino sugars, and amino acids are described in “Methods.” Molar ratios are expressed relative to mannose as 2.00. Composition of unfractionated bromelain III glycopeptides is also listed.

Residues

Mannose Fucose Xylose GlcN ASP Ser Glu GUY Others

Molar ratios relative to mannose

Unfractionated Band 2 Band 3

2.00 2.00 I 2.00 1.05 1.04 1.06 1.01 1.02 0.93 2.08 2.12 2.07 2.08 1.17 2.13 1.22 1.18 1.12 1.16 1.18 1.07 0.38 0.14 0.06

iee Table III Negligible Negligible

(1 x 115 cm), and eluted with 0.1 N acetic acid. Purification of the glycopeptides on Sephadex G-25 removed small amounts of large molecular weight carbohydrat.es derived from the paper, and residual pyridine from the buffer. Fractions containing the glycopeptide were pooled and freeze-dried. Recovery of carbohydrate from the starting material in the purified bands was 2.2% in Band 1, 29.8% in Band 2, and 49% in Band 3. These and an estimated 7% consumed in staining of the guide strips accounted for 88% of the applied neutral sugars.

The compositions of unfractionated GP III and of the isolated glycopeptides are listed in Table IV. Distinct differences in the amino acid composition of the glycopeptides were found; Band 3 contained 1 more aspartic acid or asparagine residue than Band 2. The neutral and amino sugar compositions of Bands 2 and 3 were identical and were unchanged from that of the unfractionated glycopeptide.

Upon hydrazinolysis (12) of the unfractionated glycopeptide for 24 hours at 80”, the seryl residue was quantitatively released. This indicates that both Bands 2 and 3 have serine as a COOH- t,erminal amino acid.

Deamination with Nitrous Acid-To determine whet,her any of the glucosamine residues of the glycopeptide contained unsubstituted amino groups, the glycopeptide was treated with nitrous acid under conditions described by Lee and Montgomery (41). The results are given in Table V. Recovery of the glycopeptide after nitrous acid treatment and isolation by gel filtration was 89 y0 based on the neutral sugar content. Glucosamine was not lost during the treatment, and 1 residue of aspartic acid was lost.

Period&e Oxidation-When GP II and GP III were treated with 0.01 or 0.05 M periodate as described above, consumption of periodate, measured by decrease in absorbance at 240 rnp, occurred rapidly during the first few hours and continued more slowly throughout the remainder of the react.ion. Oxidations of GP II and GP III in 0.01 M periodate were terminated after 42.5 and 47 hours, respectively. Neutral sugar chromatograms of hvdrolvzed glycopeptides showed only traces of mannose. The

Tnu~s V Composition of GP II before and after nitrous acid treutment

GP II containing 1.28 @moles of glucosamine in 0.4 ml of water was treated with 50~1 of 5% sodium nitrite and 50~1 of 40% acetic acid. The sample was gently mixed, allowed to stand at room temperature for 10 min, and evacuated in a desiccator attached to a water aspirator to remove nitrous acid. The glycopeptide W:LS isolated from the reaction mixture on a Sephadex G-25 column (1 X 100 cm) eluted with 0.1 N acetic acid. Portions of the treated glycopeptide were analyzed for the amino sugar, amino acid, and neutral sugar content. Neutral sugars were measured by automated borate chromatography, amino sugars by the neocuproine method, and amino acids on a Beckman amino acid analyzer. Molar ratios are expressed relative to mannose as 2.00.

Residue

Molar ratios

Intact GP II Nitrous acid- treated GP II

Mannose 2.00 2.00 Fucose 1.01 1.01 Xylose 0.91 0.94 GlcN 2.04 1.96 Asp 2.24 1.12

TABLE VI

Anal&s of periodate-oxidized GP II

N-Acetylated GP II (0.75 rmole) was oxidized with 0.48 ml of 0.04 M sodium periodate for 32 hours at room temperature in the dark. The oxidation mixture was quenched with a large excess of ethylene glycol, and the sample was reduced with 4.5 ml of sodium borohydride (10 mg per ml). Sodium ion was removed with AG 5OW-X8, and borate was removed by repeated evaporation with methanol. The oxidized glycopeptide was isolated from the other components of the reaction mixture by gel filtration on Sephadex G-25. Aliquots of the isolated glycopeptide were analyzed. Neutral sugars were measured by automated borate chromatography, amino sugars by the neocuproine method, and amino acids on a Beckman amino acid analyzer.

Residue

Mannose Fucose Xylose GlcN Asp

Molar ratios

Intact GP II Period$gidized

2.00 0.03 1.01 <O.Ol 0.91 <O.Ol 2.04 2.13 2.24 2.24

-

glucosamine content of the samples indicated that at least 70 to 80% of the glycopeptide was recovered after oxidation and gel filtration.

Oxidation of GP II in 0.05 M sodium periodate was terminated after 32 hours. Recovery of the glycopeptide after isolation by gel filtration was 82% based on aspartic and glutamic acid content. As had been observed with 0.01 M periodate, all of the neutral sugars were oxidized, but no glucosamine had been destroyed by the periodate oxidation; the ratio of aspartic acid to glucosamine of oxidized GP II was the same as that of intact GP II (Table VI).

Mild Acid HydrolysisSamples of GP II were heated in 1 N

acetic acid at 100” for 2 to 10 hours, dried under reduced pres-


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sure, dissolved in water, and analyzed for free neutral sugars. Preliminary experiments indicated that sugars were not released from the glycopeptide during the removal of acetic acid by evaporation at 40”; removal of acetic acid with Dowex l-X8 (bicarbonate) gave the same results.

The rate of release of fucose and xylose from GP II by mild acid hydrolysis is shown in Fig. 8. Fucose was hydrolyzed rapidly; the release was reaching a maximum at 10 hours when 90% of the total fucose was liberated. Xylose liberation occurred more slowly, and the rate of release did not decrease substantially during 10 hours of hydrolysis, at which time 20% of the total xylose had been hydrolyzed. Only traces of free mannose were found after 10 hours of hydrolysis.

Digestion with Glycosidases-Specific incubation conditions for the digestion of GP II with glycosidasea are given in Table VII. Glycosidase preparations often contain monosaccharides, pre- sumably bound to the enzymes. For this reason, a control containing only the glycosidase was incubated under the same conditions in each of the experiments listed. The free neutral sugar content of the controls ranged from 0 to 30% of the total free sugar content of the complete incubation mixtures, and the latter values have been corrected for the controls in Table VIII. The results can be summarized as follows. (a) Digestion with jack

bean Lu-n-mannosidase released 1.2 residues of mannose. (6) The mollusk @-glucuronidase preparation, which showed activity against p-nitrophenyl glycosides of cr-n-mannose, a-L-fucose, and /3-n-xylose, released 1 residue of mannose and smaller amounts of

fucose and xylose. (c) The emulsin a-mannosidase preparation exhibited little P-xylosidase compared to cY-mannosidase activity (about 0.3%) with the p-nitrophenyl glycosides as substrates; however, digestion of the glycopeptide under the described conditions liberated 1 mannose and 0.8 xylose residues. (d) Dou- bling the quantity of almond emulsin cY-mannosidase did not’ increase the amount of mannose released. (e) When fucose-free glycopeptide (prepared by mild acid hydrolysis) was used as a substrate, the release of mannose and xylose was essentially the

same as that obtained from the intact glycopeptide. In one experiment, GP II was partially hydrolyzed (Fig. 8) to

release fucose, digested with almond emulsin ac-mannosidase (as described in Table VII), and fractionated on a Sephadex G-25 column. A glycopeptide core containing 2 moles of glucosamine and 1 mole of mannose was isolated. This indicated that the recovery of only 1 residue of mannose monosaccharide after the a-mannosidase digestion was not due to loss of the liberated mannose.

Methylation Studies-GP II and GP III were methylated and methanolyzed, and the methyl ethers of neutral sugars were

TABLE VIII

Comparison of glucosamine anal,yses

60 Tempera-

ture GlU-

cosamine

Extrap- olated

34 34

90-100

Reports HCI Time

Murachi et al.

(42) Murachi (43)

Ota et al. (4) Feinstein and

Whitaker (5) This study

N hrs

5.7 20

5.7 12, 22

6 20 6 16

4 6 4 6

4a

6a 2-4~

2-3" 2"

loo”

110

110 115

100 100

0 2 4 6 6 IO

HOURS OF HYDROLYSIS

FIG. 8. Release of sugars from GP II by mild acid hydrolysis Samples of GP II (0.04 pmole per sample) were heated in 1 N acetic acid at 100” for the times indicated. After removal of the acid by flash evaporation, neutral sugar content of the hydrolysates was measured by the automated borate chromatography. Fuc, fu- case; Xyl, xylose.

a Determined with an amino acid analyzer. a Determined with the neocuproine method (26)

TABLE VII

Release of neutral sugars from GP II by digestion with glycosidases

Substrate glycopeptide was incubated with glycosidases under the conditions listed in the table, for 2 days at 37”, with toluene added as a preservative. After the incubation, toluene was removed by evaporation, and the released monosaccharides were measured directly by automated borate chromatography.

Substrate

?nM

0.4 0.4 0.67 0.53 0.33

Sugars released

ManIX%? FUCW2 Xylose

moles/?nole glycopeglide

1.20 <0.05 <0.05 1.03 0.27 0.81 1.12 0.30 0.92

0.98 1.02* 0.86 1.13 0.31 0.12

Enzyme Source Units” PH

.-

cY-Mannosidase . Jack bean a-Mannosidase Almond emulsin a-Mannosidase Almond emulsin or-Mannosidase Almond emulsin &Glucuronidase. Mollusk

2.3 4.3 0.6 4.8 1.2 4.8 1.06 4.8 O.OBc 4.3

* One unit of glycosidase hydrolyzes 1 rmole of an appropriate p-nitrophenyl glycoside per min at 37”. b The substrate for this incubation was GP II hydrolyzed in 1 N acetic acid at 100” for 6 hours to release fucose. Free fucose was

not removed prior to enzymatic digestion. c Units 0fPucosidase activity. This enzyme prepara.tion conta,ih a negligible level-of or-mannosidase activity.


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I I I I I I I I

0 I 2 3 4 5 6 7 6 9

MINUTES

FIG. 9. Gas chromatography of methyl ethers of neutral sugars from methglated and methanolized GP II. Glycopeptide was methylated four times by Hakomori’s procedure as described in “Methods.” Permethvlated glvcopeptide was methanolized in 0.85 N HCl in methanolfor 12 hi;rs at iO0’. Met,hanolysates were dried rapidly at room temperature by flash evaporation, dissolved in carbon disulfide, and analyzed by gas chromatography. Chro- matographic conditions are described in “Methods.” Peaks 1, 2, 3, and 4 are identified as methyl 2,3,4-tri-O-met,hyl-@-n-xylopyranoside, its or-anomer, methyl 2,3,4-tri-O-methyl-o-L-fucopyranoside, andmet,hyl2,3,4-tri-O-methyl-a-n-mannopyranoside, respectively.

identified by gas chromatography. After two methylations of the glycopeptide, gas chromatography of the sugars obtained from methanolysates indicated the presence of sugars which corresponded in position of elution to the following standard sugars: methyl tri-O-methyl-fi-n-xylopyranoside, methyl tri-O-methyl- a-n-xylopyranoside, methyl tri-O-methyl-a-L-fucopyranoside, and methyl tetra-o-methyl-cr-n-mannopyranoside. In addition, small amounts of several other sugars not identifiable with the available standards and a substantial amount of a sugar thought to represent methyl di-O-methyl-a-n-mannopyranoside were present. Fig. 9 shows a gas chromatogram of the methylated sugars obtained from GP II after methylation of the glycopeptide had been repeated for a total of four times. Only four methylated sugars were detected; these were identified as methyl tri-o-methyl-&-n-xylopyranoside and its anomer, methyl tri-O- methyl-Lu-L-fucopyranoside, and methyl tet’ra-o-methyl-&D-mannopyranoside. The dimethylmannose peak had virtually disap- peared, and peak areas were approximately 1:1:2 for the fully methylated derivatives of xylose, fucose, and mannose, respectively. The recoveries of methylated material were estimated by the phenol-sulfuric acid assay. The color yields of the methyl ethers are less than those of the unsubstituted neutral sugars in this assay (15). The color yield of 2,3,4,6-tetra-o-methyl-n- mannose was found to be about 50y0 of that of mannose. The exact values for the color yields of xylose and fucose were not determined. The recovery of t’he glycopeptides after four methylations was 35% and 24% for GP II and GP III, respectively, in the phenol-sulfuric acid assay with mannose as standard. If the color yields of fully methylated fucose and xylose are approximately the same as that of fully methylated mannose, then the over-all recovery of methylated glycopeptides would be 70 and 48%, respectively.

DISCUSSION

Proteolytically active components of similar size and charge from pineapple stem bromelain were separated on SE-Sephadex C-50 at pH 8.7. The presence of more than one proteolytically act’ive component in commercial bromelain was originally suggested by Heinicke (2) on a basis of electrophoretic separations of crude bromelain at pH 6.5, which showed four distinct components with proteolytic activity. Heinicke’s suggestion was confirmed both by El-Gharbawi and Whitaker (7), who separated five proteolyticallg active components by chromatography of commercial bromelain on Bio-Rex 70 at pH 6.10, and by Murachi and Neurath (6), who separated two proteolytically active components by chromatography of bromelain on Duolite CS 101 resin at pH 6.05. Later publications from two laboratories reported the purification of a homogeneous enzyme from crude stem bromelain (3,4).

Under conditions similar to those described by Ota et al. (4), we also obtained a single peak by cation exchange chromatography. However, this material was heterogeneous by polyacrylamide gel electrophoresis. By carefully controlling the elution conditions as described in the experimental section, we were able to separate these components.

Multiple components may have been present in the pineapple plant stem; alternatively, they may have been formed by autodigestion during purification of the enzyme. Ota (41) demon- strated recently that extensive autodigestion of bromelain will occur under appropriate conditions. Although we included a mercurial compound at all stages of t,he purification to inhibit autodigestion, such precautions were probably not followed in the initial preparation of bromelain acetone powder.

In the absence of evidence that the isolated bromelain components originated from a single protein precursor, the carbohydrate moieties of the purified components were characterized separately. The carbohydrate composition of the separate bromelain components appeared to be identical, however. Both bromelain II and bromelain III contained 2 moles of mannose, 1 of fucose, 1 of xylose, and 2 of glucosamine per mole of protein. This composition differed from the compositions reported by other laboratories. Murachi, Suzuki, and Takahashi (42) found that a glycopeptide, prepared by digestion of bromelain with Pronase, contained mannose, fucose, xylose, and glucosamine in the ratio 3 : 1: 1:4. Other workers reported different values for glucosamine (Table VIII). In view of these discrepancies, an evaluation of the methods used to measure the neutral and amino sugar composition is necessary.

The molar ratios of the neutral sugars, as determined by three independent methods, agreed well (Table JI). The ratios of fucose and xylose, determined by the specific calorimetric methods, to mannose, determined by the hexokinase method, were somewhat higher than the molar ratios determined by the other two methods. The former determinations were performed on unfractionated hydrolysates, and it seemed probable that one or more of the calorimetric reactions was not specific enough to per- mit accurate measurements on unfractionated sugar mixtures. Uoth the thin layer chromatography method and the automated borate chromatography method of neutral sugar determination were considered superior to the calorimetric-enzymatic techniques used, because the neutral sugars were separated prior to determination of the individual components. In particular, automated borate chromatography was, in our experience, highly


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Carbohydrate of Pineapple Brolnelain Vol. 244, No. 18

accurate and reliable (23). When ahquots of a hydrolysate were used both for specific calorimetric reactions and for automated borate chromatography and the results were compared, mannose values averaged 6% lower, fucose values 12oj, higher, and xylose values 32 ‘% higher in the calorimetric reactions.

In the report by Murachi et al. (42), the neutral sugars were determined by a combination of paper chromatography and specific calorimetric reactions, and the maximum release of neutral sugars occurred after hydrolysis in 1 N HCI at 100” for 3 hours. In our hands, however, at least 4 hours of hydrolysis wit,h 1 N H&O4 at 100” were required for maximum liberation of xylose or mannose; the mannose to xylose ratio would be greater than 2 : 1 at times earlier than 4 hours (Fig. 6).

The molar ratios of glucosamine determined by the Elson- Morgan procedure or by the amino acid analyzer indicated a glucosamine content of 2 to 3 moles/2 moles of mannose while the analyses by the neocuproine method suggested 2 moles (Table II). The amino acid analyzer measurements of glucosamine are subject to interference by peptides present in hydrolysates, and the Elson-Morgan measurements are subject to interference by the presence of salts. Neither type of interference can occur when glucosamine is measured by the neocuproine method. For this reason, glucosamine values determined by the neocuproine method were considered more reliable. In addition, the recoveries of glucosamine standards were consistently higher in glucosamine determinations by the neocuproine method, possibly because the separation of glucosamine on an AG 5OW-X8 column prior to analysis was not required in this method and was not used.

The previously reported values for glucosamine (Table VIII) were obtained with the hydrolytic conditions suitable for amino acid analysis, but much too drastic for amino sugar determination. The necessity for the use of large correction factors in these reports often invites gross errors in hexosamine determination. The conditions for maximum liberation of glucosamine were opti- mized in this work and lie within the range that is usually employed by investigators of complex carbohydrates.

GP II and GP III were identical in carbohydrate composition to each other and to the whole proteins from which they were prepared, indicating that no selective loss of carbohydrate residues occurred during the preparation of the glycopeptides. The two principal glycopeptides present in GP III were separated by paper electrophoresis; amino acid analyses indicated that these peptides (Bands 2 and 3 of Fig. 7) differed by the presence of 1 additional aspartic acid or asparagine residue in the Band 3 glycopeptide. The greater mobility of this peptide toward the anode suggested that the additional residue was aspartic acid rather than asparagine. The third glycopeptide isolated from GP III (Band 1 of Fig. 7) was present in minor amounts; it contained substantial amounts of amino acids not found in the other two glycopeptides.

M M X F A Y U

: N L C

I I I 1

GIcNAc+GlcNAc----+Peptide

FIG. 10. A possible structure of the carbohydrate unit of pineapple stem bromelain. 2Mun, mannose; XyZ, xylose; Fuc, fucose.

Ota (41) also isolated a tripeptide (Asp, Glu, Ser) containing carbohydrate from autodigestion products of pineapple bromelain. Edman degradation of this glycopeptide showed that neither a glutamyl nor a seryl residue was involved in the peptide- oligosaccharide linkage.

Glucosamine was not destroyed in samples of GP II treat’ed with nitrous acid, indicating that the amino groups of the glucosamine residues were substit,uted. The loss of aspartic acid in these samples indicated that aspsrtic acid was t,he NHz-terminal residue of the peptide.

The quantitative recovery of glucosamine in periodate-oxidized glycopeptides indicated that the glucosamine residues were not located at nonreducing terminal positions of the carbohydrate moiety, and that the glucosamine residues were substituted at, the 3 or 4 position, or both. The destruction of all neutral sugars in the same oxidized samples suggested that neutral sugars occupied nonreducing ends of the carbohydrate unit or that the neutral sugars were not substituted at positions which would render them periodate-resistant.

The release of large quantities of fucose unaccompanied by equimolar amounts of the other neutral sugars during mild acid hydrolysis indicated that fucose occupied a nonreducing terminal position in the glycopeptide. Virtually all of the fucose-containing glycoproteins which have been characterized contain fucose at a nonreducing terminus. The release of mannose alone in the digestion of GP II with jack bean a-n-mannosidase suggested that a mannose residue was present in a nonreducing terminal position. None of the mannosidase preparations tested were able to remove 2 mannose residues; t’he 2nd mannose residue seemed to be inaccessible to the enzyme, and it remained inaccessible when fucose-free GP II was used as a substrate. This suggested that the unhydrolyzed mannose residue was not blocked by fucosa. The release of more xylose than fucose by the almond emulsin mannosidase indicated that the xylose residue was not located internal to t’he fucose residue in the carbohydrate unit, while the release of xylose unaccompanied by equimolar amounts of mannose, observed in mild acid hydrolysis, indicated that xylose was not internal to a mannose residue. These results suggested that xylose occurred at a nonreducing terminus of the carbohydrate moiety. This is in agreement with the report of release of xyloee by fi-n-xylosidase (36).

Exhaustive methylation of the glycopeptide gave a permethyl- ated product from which only the fully methylated neutral sugar derivatives were obt.ained. Since fully methylated sugars are formed only from nonreducing terminal sugars, it appeared that all of the neutral sugar residues occupied terminal positions in a highly branched carbohydrate moiety.

Embracing all the results obtained, it was possible to postulate a structure of t,he type indicated in Fig. 10 in which all of the neutral sugar residues occur in nonreducing terminal positions attached to a central glucosamine core. However, in the absence of a conclusive demonstration that the carbohydrate occurs in a single unit attached to one amino acid, other st,ructural models are also possible. The fine structure of this oligosaccharide side chain is being further investigated.

Acknowledgments-We are greatly indebted to Dr. S. Roseman for his imerest and assistance in the pursuit of this work, Thanks are due to Dr. R. M. .Heinicke for a generous gift of bromelain, to Dr. Y. T. Li for jack bean ol-mannosidase, and to Dr. H. Schachter for L-fucose dehydrogenase.


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REFERENCES

1. GOTTSCHALK, A., in A. GOTTSCHALK (Editor), Glycoproteins, their composition, structure and function, Vol. 6, BBA Li- brary, American Elsevier Publishing Company, New York, 1966, p. 20.

2. HEINICKE, R. M., AND GORTNER, W. A., Econ. Botany, 11, 225 (1957).

3. MURACHI, T., YASUI, M., AND YASUDA, Y., Biochemistry, 3, 48 (1964).

4. OTA, S., MOORE, S., AND STEIN, W. H., Biochemistry, 3, 180 (1964).

5. FEINSTEIN, G., AND WHITAKER, J. R., Biochemistry, 3, 1050 (1964).

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Jane Scocca and Y. C. LeeBromelain

The Composition and Structure of the Carbohydrate of Pineapple Stem

1969, 244:4852-4863.J. Biol. Chem.

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