ARCHIVES

OF BIOCHEMISTRY

Vol. 293, No. 1, February

AND BIOPHYSICS

14, pp. 54-60,1992

Depolymerization Ferrous Ions’

of Heparin by Complexed

Biswajit

Lai, Manuel Pousada, Douglas Stanton, and Isidore Danishefsky2

Department

Lahiri,

Pi-Shiang

of Biochemistry

and Molecular Biology, New York Medical College, Valhalla, New York 10595

Received July 29, 1991, and in revised form October 14, 1991

Treatment of porcine heparin with the ferrous-EDTA complex and ascorbic acid for 24 h at 37°C results in the degradation of most of the glycosaminoglycan to smaller fragments. About 65% of the products comprise oligosaccharides composed of less than 30 sugar units. The extent of depolymerization is decreased significantly if ascorbate or EDTA is not included in the reaction mixture. Gel filtration of the reaction products yielded fractions with narrow chain length ranges. The sulfate content of the fractions and their electrophoretic mobilities on cellulose acetate indicate that the components have equivalent charge densities. Depolymerization products with 20 or more sugar units retain significant anticoagulant potencies as measured by their effect in accelerating the neutralization of factor Xa by antithrombin. 0 1992 Academic Press, Inc.

Although heparin can be described as a polymer of alternating units of sulfated glucosamine and hexuronic acid, its fine structure is highly complex due to numerous variations along the chain. These microheterogeneities include differences in the degree of sulfation and positions of sulfates, the occurrence of small amounts of N-acetylglucosamine instead of the more common sulfoaminoglucosamine units, the presence or absence of a unique oligosaccharide sequence that is crucial for anticoagulant activity, and variations in the nature of the uranic acid, i.e., glucuronic or iduronic acid (1). Most of the investigations to define the structure of oligosaccharide segments involve degradation of heparin followed by characterization of the isolated products. The depolymerization reagents generally employed are nitrous acid and flavobacterium heparinase, both of which cleave glucosaminidic bonds between sulfoaminoglucosamine and uranic acid. Heparinase is specific for iduronic acid (2, 3), whereas 1 This research was supported by NIH Grant HL 16955. ’ To whom correspondence should be addressed.

54

the action of nitrous acid is independent of the nature of the uranic acid (4). Current studies in our laboratory involve the determination of the sequence of individual units or groups of oligosaccharides in heparin and related glycosaminoglycans with respect to distance from the protein linkage region or the reducing terminus (5). Although various aspects of the sequence have been clarified by use of the above mentioned depolymerization methods (6-8) certain details require the availability of additional reagents with different specificities. One of the systems which we have investigated involves the degradation of heparin by the ferrous-EDTA complex in the presence or absence of ascorbic acid. This complex has been shown to effect depolymerization of hyaluronic acid and nucleic acids by free radical oxidation of the respective sugar moieties (9, 10). In the present report, we demonstrate the degradation of heparin by the iron-EDTA complex and describe the products that are generated. EXPERIMENTAL

PROCEDURES

Materials and methods. Porcine mucosal heparin, supplied by the Cohelfred Co., was fractionated according to size by gel filtration on Sephadex G-200 as described previously (11). The high molecular weight fraction with an average M, 18000 was employed in the present studies. Disaccharides and tetrasaccharides generated by digestion of heparin with flavobacterium heparinase were isolated in our laboratory by preparative HPLC? (12). Heparin oligosaccharides with 10,16, and 22 sugar units were kindly supplied by Drs. II. Lindahl (Uppsala), J. Choay (Paris), and R. D. Rosenberg (Boston). Disaccharides derived from chondroitin sulfate were purchased from Seikagaku, Inc. Preparations of heparin with different molecular weights employed for standardization of Bio-Gel P-100 columns were described in a previous report (13). Analyses of heparin and degradation products for uranic acid and glucosamine were performed as reported previously (8). Sulfate was determined by the method of Silvestri et al. (14). Anticoagulant activities were measured by the effect of specific concentrations of heparin in

s Abbreviations used: EDTA, ethylenediaminetetraacetati GlcN, 2amino-2-deoxyglucose; HPLC, high-performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Tris, tris(hydroxymethyl)aminomethane. 0003-9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

OXIDATIVE

DEGRADATION

accelerating the neutralization of coagulation factor Xa by antithrombin (8). Reaction and fructionations. A total of 20 mg of the high molecular weight heparin fraction from porcine mucosa was dissolved in 20 ml of a solution composed of 1.75 mM FeSO,, EDTA, and ascorbic acid in 0.13 M sodium phosphate, pH 7.2, and kept at 37°C for 24 h. The reaction mixture was then concentrated to 2 ml by flash evaporation below 37”C, and the insoluble material consisting of iron salts and ascorbate was removed by centrifugation. The precipitate did not contain any detectable heparin or fragments when assayed with Alcian blue and carbazole (8). Controls in which FeSO, and ascorbate were omitted did not result in any precipitation under similar conditions. The concentrated solutions were desalted by passage through a column of Sephadex G-25 (92 X 2.5 cm) or Bio-Gel P-2 (86 X 2 cm) and eluted with water. Effluents were monitored by conductivity measurements and assays for uranic acid. Fractions containing heparin or oligosaccharides were combined and concentrated. Samples of the concentrates were applied to a column of Bio-Gel P-100 (106 X 0.95 cm) and eluted with 0.5 M NH,HCOs at a flow rate of 3.4 ml/h. Appropriate fractions were pooled and lyophilized. The experiments in which ascorbate was omitted were performed by the same procedures as described above. Subfractionation of the low molecular weight materials was performed on Bio-Gel P-4 (196 X 1.5 cm) equilibrated and eluted with 0.5 M NHIHCOB at a flow rate of 4 ml/h. Gel electrophoresis. Electrophoretic separations were performed on vertical slab gels 44 X 37 cm and 7 mm in thickness. The resolving gel was prepared from 20% acrylamide and 2% bisacrylamide in 0.1 M borate-Tris-0.01 M EDTA, pH 8.3, as defined by Rice et al. (15). A stacking gel formed with 5% acrylamide and 0.65% of the cross-linking agent in the same buffer components adjusted to pH 6.3 was also employed. Polymerization reagents and electrophoretic buffers were the same as detailed previously (15). Then lo-r1 solutions of lo-30 pg of samples were mixed with 5 ~1 of 0.1% bromophenol blue and 50% sucrose in stacking gel buffer and applied to the gel. Phenol red (0.1%) was also used as a marker. Electrophoresis was carried out at 300 V for 16 h. The gel was stained for 4 h with a solution of 0.5% Alcian blue in 40% methanol containing 5% acetic acid-3% glycerol and then destained with 40% methanol in aqueous 5% acetic acid. The gel was then washed, sequentially, with solutions containing increasing concentrations of ethanol, and air dried. Cellulose acetate electrophoresk. Electrophoretic mobilities on cellulose acetate were determined in a buffer consisting of 1.0 M pyridine0.566 M formic acid, pH 3 (16). A Beckman Model R-100 microzone electrophoresis cell and Duostat Model RD-2 power supply were employed for these experiments. The separation was performed at 140 V for 20 min. Glycosaminoglycans were stained with 0.5% Alcian blue in 5% acetic acid and destained with 7.5% acetic acid in 5% methanol. High-performance liquid chromatography. Analyses by HPLC were performed with a Waters M-6000 solvent delivery system, U6K injector, R401 differential refractometer, and M-730 data module. Samples were chromatographed on an Altex Spherogel TSK 3OOOSW-10 p column (600 X 7.5 mm.) that included a TSK GSWP (75 X 7.5 mm) precolumn. The eluting solvent was 0.5 M NaCl in 0.05 M sodium phosphate, pH 7.3. The flow rate was 1 ml/min and the chart speed of the recorder was 0.5 cm/min; V, and VT were determined with dextran, M, 500,000, and glucose, respectively. Paper chromatography. Chromatography on Whatman No. 1 paper was performed utilizing isobutyric acid-l.25 M NH,OH (5:3.6, v/v) for 40 h (17). The paper was stained with alkaline AgNO, (18). RESULTS

Porcine heparin preparations consist of a heterodisperse mixture of glucosaminoglycans with molecular weights ranging from approximately 6000 to 30,000. Since the present investigations involved depolymerization of

55

OF HEPARIN

the heparin we conducted our experiments with a relatively narrow high molecular weight fraction, M, 16-18 X 103, obtained by gel filtration of the commercial product (7). In initial studies, the heparin was treated with solutions containing various concentrations of ferrous sulfate, EDTA, and ascorbate for different time intervals and the reaction was monitored by changes in the HPLC patterns. Figure 1 shows the results of representative experiments and also demonstrates the applicability of HPLC for de-

B

0

15

25

MN

FIG. 1. HPLC profile of heparin depolymerization. (A) Intact heparin. (B) Solution of heparin (9.6 mg/ml) in 1 pM FeSO,, EDTA, and ascorbic acid immediately after mixing. (C) Reaction mixture after 24 h at room temperature. (D) Solution of heparin in 2.5 mM FeS04, EDTA, and ascorbate incubated for 3 h at room temperature. Each injection contained 288 pg of heparin. VOand VT were 10.1 and 26.0 ml, respectively, corresponding to the same values in minutes in the diagram.

56

LAHIRI

tecting the depolymerization process. The conditions adopted for most of the subsequent experiments (see Experimental Procedures) were those which provided significant degradation when low concentrations of reagents were employed. After the reaction and desalting by gel filtration, the oligosaccharide mixture was passed through a column of Bio-Gel P-100. The elution profile of the treated material as compared with that of the original heparin is shown in Fig. 2A. The fractions did not show any characteristic absorbance peak at 235 nm, which indicates the absence of any significant amounts of products with 4,5-unsaturated uranic acids at the nonreducing terminus. The possible presence of such oligomers was considered in view of the report that degradation of hyaluranic acid by ferrous sulfate yields products with unsaturated uranic acids and the suggestion that the free radical reaction proceeds via the abstraction of the hydrogen from the carbon-5 of a uranic acid unit (19). The fractions were pooled as shown in Fig. 2A, lyophilized, and redissolved to provide solutions with similar carbohydrate concentrations. Polyacrylamide gel electrophoresis of aliquots from each pool revealed that the reaction generated a wide range of oligosaccharides (Fig. 3A). Pool I is composed of material that underwent relativety little degradation and most of pool II consists of materials with an average M, of 8000 on the basis of gel filtration or 22 sugar units by PAGE. The other pools comprise mixtures of lower molecular weight oligosaccharides; e.g., pools IV and V are composed mainly of 6 to 12 sugar units. The elution volumes of the components in pool V correspond to oligosaccharides with less than 6 units. It should be noted that the reducing termini in the oligosaccharides employed as standards consist of anhydromannose units rather than sulfoaminoglucosamine. The size of the products in the pools may, therefore, differ to some extent from those indicated by the standards. Other experiments were performed with the same reagents except that ascorbate was not included in the reaction mixtures. Fractionation of the desalted solution of the glycans on Bio-Gel P-100 showed that although considerable degradation of the heparin had occurred, the degree of the reaction was much less than that in the presence of ascorbate (Fig. 2B). Polyacrylamide gel electrophoresis of the pooled fractions (Fig. 3B) shows the extent of degradation and the sizes of the products that were formed. The relative amount in each pool for both reaction conditions is indicated in Table I. Although each pool consists of a mixture of oligosaccharides the molecular weight ranges of the major components differ significantly. Analyses of the materials for glucosamine and sulfate (Table I) show that the surviving oligosaccharides are similar to each other and to the original heparin with respect to the overall molar proportion of sulfate. These data suggest that the degradative cleavages are random with some preference for nonsulfated or low sulfated sugars. The results of electrophoresis on cel-

ET AL.

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ML FIG. 2. Fractionation of the desalted reaction mixture on Bio-Gel P100. The column was monitored by absorbance at 530 nm by the carbaxole assay of aliquots for uranic acid. (A) The solid line shows the results for the reaction in the presence of ascorbate. The pattern for the original undegraded heparin is shown by the dashed lime. (B) Elution pattern of products for the reaction in the absence of ascorbate. The vertical lines indicate the fractions that were combined in each pool.

lulose acetate, which show that the components in pools I through IV have higher mobilities than the major component in the original high molecular weight porcine heparin, are also consistent with a greater cleavage susceptibility by chains or segments with less sulfations (Fig. 4). Pool V which is composed mainly of smaller oligosaccharides did not adhere to the cellulose acetate and was washed off during the staining. Although not directly relevant to the present experiments, it is noteworthy that bovine heparin shows a higher electrophoretic mobility and exhibits a sharper band than porcine heparin. This has been a consistent finding throughout our studies.

OXIDATIVE

DEGRADATION

57

OF HEPARIN

16

16-

-

lo-

6-

a-

4-

61

6

5

4

3

2

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23

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56

169

FIG. 3. Gel electrophoresis of pooled fractions. (A) Reaction in the presence of ascorbate. Lane 1, original unreacted heparin; lanes 2-5, pools I, II, III, and IV plus V, respectively. Lanes 6-8 consist of oligosaccharide standards which have the number of sugar units shown on the left side. (B) Reaction performed in the absence of ascorbate. Lane 1, original heparin; lane 2, total reaction mixture at zero-time; lanes 3-6, pools I, II, III, and IV plus V, respectively; lanes 7-9, oligosaccharide standards.

The heparin fragments were found to have considerable anticoagulant activity (Table I). Since this activity requires the presence in the chain of a unique oligosaccharide sequence (reviewed in Ref. (20)) it appears that this sequence is refractory or less susceptible to degradation by the ferrous-EDTA, even in the presence of ascorbic acid. Another characteristic of the fractions (not shown) is a relative increase in the ratio of uranic acid to glucosamine with decreasing oligosaccharide chain lengths. These findings might suggest that the glucosamine moieties are more susceptible to oxidative degradation than the uranic acid units. However, such a conclusion is not definitive since the absorbance values in the carbazole method for determination of uranic acids are known to vary for different glycosaminoglycans (21) and with the degree of sulfation (22). Since pools IV and V contained low molecular weight oligosaccharides, they were combined and subfractionated on Bio-Gel P-4 (Fig. 5 and Table II). Gel electrophoresis of the fractions together with oligosaccharide standards demonstrated that fraction A consists primarily of octaand decasaccharides while fraction D consists mainly of tetrasaccharides (Fig. 6). The size assignments for the fractions in fractions D and E were also confirmed by

paper chromatography. The system employed (see Experimental Procedures) gives distinct differences in mobilities for a number of heparin-derived, structurally defined di- and tetrasaccharides (Table III). Higher oligosaccharides do not migrate from the origin. The relative mobility of the primary component in fraction D confirms its assignment by PAGE as a tetrasaccharide. Additionally, the paper chromatographic mobility of fraction E is consistent with that of a disaccharide. Fractions A through D have similar mobilities on cellulose acetate electrophoresis (Fig. 7). Fraction E and part of fraction D did not bind to the cellulose acetate and were washed off during the staining. Since these fractions arise from the combination of pools IV and V, the stained components in lanes 3-6 must have been derived primarily from pool IV. The primary staining components in fractions A through D have an apparently higher charge density than the original undegraded porcine heparin as was found upon electrophoresis of pool I through IV (Fig. 4). The diffuse or dual bands exhibited by the porcine heparin preparation (e.g., lane 3 in Fig. 4 and lane 2 in Fig. 7) may correspond to the slow-moving and fast-moving heparin components described by Bianchini et al. (24, 25). They presented evidence that the two heparin species

58

LAHIRI TABLE Characteristics

ET AL.

I

of Pooled

Fractions

0.6

Percentage of total Pool No.O

+ Ascb

- Ascb

DP”

I II III IV V

13 22 29 24 12

41 22 14 11 12

>35 20-40 8-20 6-12 43

SO,/GlcNd (molar ratio) 2.4 2.6 2.8 2.9

Activitye (units/mg) 95 45 41

Depolymerization of heparin by complexed ferrous ions.

Treatment of porcine heparin with the ferrous-EDTA complex and ascorbic acid for 24 h at 37 degrees C results in the degradation of most of the glycos...
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