THE c,OURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 17, Issue of September 10, pp. 6841-6846, 1975

Printed in U.S.A.

Structure of Heparin CHARACTERIZATION OF THE PRODUCTS FORMED FROM HEPARIN BY THE ACTION OF A HEPARINASE AND A HEPARITINASE FROM FLA VOBA CTERIUM HEPARINUM*

(Received for publication, November 11, 1974)

MARIA E. SILVA AND CARL P. DIETRICH

From the Departamento de Bioquimica e Farmacologia, Escola Paul&a de Medicina, C.P. 20372,OlOOO Sa”o Paul0 - S.P. - Brazil

The total degradation of heparin by the joint action of a purified heparinase and a heparitinase from Flavobacterium heparinum is reported. The heparinase acts directly upon heparin, yielding 52% of a trisulfated disaccharide (0-(LX-L-ido-4-enepyranosyluronic acid 2-sulfate)-(l-4)-2-sulfoamino-2-deoxy- n-glucose 6-sulfate) and 40% of a tetrasaccharide besides small amounts of hexa- and disaccharides.

The tetrasaccharide is in turn completely degraded by the heparitinase, forming trisulfated disaccha- ride and disulfated disaccharide (0-(oc-D-glyco-4-enepyranosyluronic acid)-( l-4)-2-sulfoamino-2-deoxy- D-glucose g-sulfate) in equal amounts. These and other results indicate that the tri- and disulfated disac- charides are linked alternately, in a proportion of 3:1, respectively. The primary structure of heparin and the mode of action of the heparinase and the heparitinase are proposed based on the analysis of the different products formed by the action of the enzymes.

Heparin is degraded by a group of induced enzymes from Flavobacterium heparinum to oligo-, di-, and monosaccharides (l-4). A partially purified heparinase’ preparation degrades heparin mainly to a trisulfated disaccharide in 70 to 80% yield (2, 3). Physical and chemical analysis of this trisulfated disaccharide led to the conclusion that heparin structure is largely a repeating sequence of 0-(a-L-idopyranosyluronic acid 2-sulfate)-(l-4)-2-sulfoamino-2-deoxy-n-glucose 6-sulfate (1, 5).

Besides the trisulfated disaccharide, tetra- and hexasaccha- ride as well as a disulfated disaccharide are formed from heparin by the action of the heparinase (4). Since the crude extracts of F. heparinum are able to degrade heparin com- pletely to monosaccharides (l), other enzymes have to be present in these crude extracts in order to degrade the oligosaccharides formed by the action of the heparinase.

Two heparitinases which are able to degrade heparitin sulfate have been recently isolated from F. heparinum (4). One of the heparitinases (heparitinase I) produces N-acetylated disaccharides as major products while the other (heparitinase II) produces disulfated disaccharides from heparitin sulfate B.

* This work was aided by Grants from the Funda@o de Amparo & Pesquisa do Estado de SBo Paulo (FAPESP), Conselho National de Pesauisas (CNPo.) and Financiadora de Estudos e Proietos (FINEP). ‘ 1,

’ The abbreviations used are: heparinase, heparin lyase; hepariti- nase II, heparitin sulfate B, lyase; trisulfated disaccharide, o-(01-~- ido-4-enepyranosyluronic acid 2-sulfate)-(l-4).2-sulfoamino-2-deoxy- n-glucose B-sulfate; disulfated disaccharide, 0-(cu-D-glyco-4- enepyranosyluronic acid)-( l-4).2-sulfoamino-2-deoxy-n-glucose 6-sul- fate; glucosamine 2,6-disulfate, 2-deoxy-2-sulfoamino-o-glucose-6-O. sulfate. Heparitin sulfate is also known as heparan sulfate and heparin monosulfuric acid.

Since heparin also contains disulfated disaccharides it was found important to test the action of heparitinase II upon heparin and its products formed by the action of the hepari- nase

The present paper reports the complete degradation of heparin to disaccharides by the joint action of a heparinase and a heparitinase from F. heparinum. The probable structure of heparin based on the analysis of the degradation products formed by the enzymes is reported. Preliminary accounts of parts of this work have appeared (4-6).

EXPERIMENTAL PROCEDURE

Materials-Commercial heparin preparations were kindly supplied by Lederle Laboratories (Pearl River, N.Y.), Upjohn Co. (Kalamazoo, Mich.), and Roche Laboratories (Slo Paulo, Brazil). Heparin was also purchased from Fischer Scientific Co. (Fairlawn, N.J.). Heparitin sulfate B was prepared from crude heparitin sulfate (kindly supplied by the Upjohn Co.) by methods already described (7). For most of the studies heparin from the Upjohn Co. was used. The anticoagulant activity of this preparation measured by the U.S.P. assay was 167 i.u./mg and the elementary analysis was as follows: C, 20.4; H, 4.1; N, 2.2; S, 11.9; Na, 12.3; sulfated ash, 34.7%.

Preparation of Enzymes-The 100,000 x g supernatant of extracts from induced cells of Flauobacterium heparinum was prepared as previously described (4). Two hundred milligrams (dry weight) of this supernatant in 2 ml of 0.1 M ethylenediamineacetate buffer, pH 7.0, were subjected to a large scale agarose gel electrophoresis in the same buffer as previously described (3) except that the electrophoresis was run at 5 volts/cm for about 54 hours at 5O. After the run the enzymes were eluted from the gel as previously reported (8) and assayed as described below.

Assay of Enzymes-A typical incubation mixture contained 1 to 10 fig of enzyme protein, 100 pg of heparin or other substrates and other additions as indicated, and 0.05 M ethylenediamineacetate buffer, pH

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7.0, in a final volume of 20 ~1. After inactivation by heating, the reaction mixtures were spotted on Whatman No. 1 paper and chro- matographed in Solvent A (isobutyric acid/l M NH,, 5/3 v/v) for 48 hours or in Solvent B (butanol/acetic acid/H,O, 10/3/7 v/v/v) for 48 hours. In some instances electrophoresis also was used as already described (1). The products were quantitated by densitometry after silver nitrate staining or toluidine blue staining as previously described (1, 9). In some instances quantitation was performed (after location of the products with guide strips and elution with water) by hexosamine determinations as described below.

The large scale preparation of degradation products was performed as follows: 100 mg of heparin or other heparin products were incubated with 1 to 2 mg of heparinase or heparitinase II in 0.05 M ethylenediamineacetate, pH 7.0, for 36 hours in a final volume of 20 ml at 30”. When the heparinase was used the incubation mixtures also contained 0.02 M MnCl,. After the incubation, the mixtures were concentrated and applied as a 40-cm band on Whatman No. 3MM paper and chromatographed in Solvent A for 72 hours. The products were located in the paper with the aid of a short wave ultraviolet lamp and eluted with water. The eluted materials were purified by chroma- tography in Solvent B or electrophoresis until one single spot was obtained in the two solvent systems and electrophoresis. The products were then further purified by precipitation with methanol-acetone mixtures as previously described (1).

Analytical Procedures-Amino sugars were measured after acid hydrolysis in 4 N HCl for 6 hours at 100” in sealed tubes, by a modified Elson-Morgan reaction (10). Reducing power was measured by the Somogyi-Nelson procedure (11). Labile and total sulfate was measured after acid hydrolysis in 0.05 N HCl for 2 hours at 100” and 8 N HCl for 6 hours at loo”, respectively, by a method previously described (7). Uranic acid was measured by the Dische carbazole reaction (12).

To identify the reducing end of the di- and tetrasaccharides, 0.2 pmol of the compounds were incubated with 5 pmol of NaBH, for 18 hours at room temperature. After incubation, uranic acid and hexosa- mine were determined as described above. Molecular weight determi- nations were performed according to Hilborn and Anastassiadis (13) except that gel slabs instead of gel cylinders were used (7). The products also were characterized with the aid of enzymes of the sequential degradation of heparin as previously described (3).

RESULTS

Purification of Heparinase and Heparitinases-In the con- ventional fractionation by agarose gel electrophoresis reported

previously (3, 4) the heparitinase II is not completely resolved

from an a-glycuronidase as well as the heparinase. In order to

obtain some heparitinase fractions free of glycuronidase the

agarose gel electrophoresis was run for 54 hours at 5 volts/cm.

Fig. 1 shows the results of this experiment. Some heparitinase

FRACTION NUMBER

FIG. 1. Fractionation of the enzymes in agarose gel electrophoresis. Heparin or heparitin sulfate B (100 pg) or an unsaturated N-acetylated nonsulfated disaccharide from heparitin sulfate B (25 pg) were incubated with 10 ~1 of the agarose gel fractions for 16 hours at 30” in a final volume of 20 ~1. The disaccharide products formed, by the action of the heparinase, from heparin (A-A), and heparitin sulfate B by the action of heparitinase II (m----m) and heparitinase I (A-A), and N-acetylglucosamine from the N-acetylated disaccharide by the action of the glycuronidase (O---O) were quantified by densitometry after chromatography and silver nitrate staining as described under “Experimental Procedure.”

fractions are apparently free of glycuronidase. The agarose

fractions containing only the activity of heparitinase II and

heparinase were pooled and assayed for purity after long

incubation periods. The results of this experiment are shown in

Table I. Only negligible amounts of a glycuronidase activity

were found in the selected fractions of heparitinase II. Likewise

the heparinase was free of contamination with heparitinase II

and a-glycuronidase.

End Products Formed from Heparin by Action of Heparin- use-The products formed from heparin by the action of the heparinase after different periods of incubation are shown in Figs. 2 and 3. After 6 hours two main products accumulate, namely a tetrasaccharide and a trisulfated disaccharide. The trisulfated disaccharide and the tetrasaccharide are both formed in equivalent amounts during the different periods of incubation. The hexasaccharide formed initially decreases

slowly after long incubation periods. Addition of more enzyme or incubation for longer periods of time did not change this pattern. The amount of products formed from heparin is shown

TABLE I

Substrate specificity of heparinase and heparitinase I1 One hundred micrograms of heparin and heparitin sulfate B and 25

pg of an unsaturated N-acetylated nonsulfated disaccharide (A Di- GLcNAc), unsaturated disulfated, and trisulfated disaccharides (A Di-Di S and A Di-Tri S, respectively) as well as glucosamine 2,6-disul- fate (GlucN-2,6-disulfate) were incubated with 0.2 ~g of selected fractions of heparinase and heparitinase II in a final volume of 20 ~1 at

30” for 30 hours. After the incubation the products formed were

quantified as described in Fig. 1.

Products formed

Substrate Heparinase Heparitinase

II

!A?

Heparin 50 0 Heparitin sulfate B 0 25

A Di-GlcNAc <0.5 <0.5 A Di-Di S <0.5 <0.5 A Di-Tri S 0 0 GlucN-2,6-disulfate 0 0

j -ADi-TRI S

-TETRASACCHARIDE

’ v.: -HEXASACCHARIDE

-ORIGIN

015 6 4 6 -TIME (HR.)

FIG. 2. Products formed from heparin by action of heparinase at different times. Heparin (100 bg) was incubated with 2 fig of hepari- nase at 30” in 0.05 M ethylenediamine acetate, pH 7.0, containing 0.02 M MnCl, in a final volume of 20 ~1 for the times indicated. The products were stained with toluidine blue reagent after chromatogra- phy for 72 hours in Solvent A as described under “Experimental Procedure,” A Di-Tri S, trisulfated disaccharide.

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100

TIME(HOURS)

FIG. 3. Time-dependent formation of heparin degradation products by action of heparinase. These data are the quantitative results obtained from the experiment described in Fig. 2. The trisulfated disaccharide (A-A), the tetrasaccharide (A-A), and the hex- asaccharide ( n -m) were quantified by densitometry after toluidine blue staining. The extinction coefficient of the trisulfated disaccharide and the tetrasaccharide by total hexosamine is approximately the same.

TABLE II

Amount of products formed from heparin by action of heparinase

Heparin (1 mg) was incubated with 5 pg of heparinase in 0.05 M

ethylenediamineacetate buffer, pH 7.0, containing 0.02 M MnCl, in a final volume of 100 ~1 for 30 hours at 30”. After incubation the mixture was spotted on Whatman No. 1 paper as &cm bands and chromato-

graphed in Solvent A. The products formed were located wih the aid of an ultraviolet lamp and eluted with water. Hexosamine determina-

tions were then performed on the eluates. The relative concentrations

of the products were expressed in per cent of total hexosamine present in the incubated heparin. A Di-Di S and A Di-Tri S, unsaturated di- and trisulfated disaccharides, respectively.

Product Amount

9c

A Di-Di S 3 A Di-Tri S 52 Tetrasaccharide 40 Hexasaccharide 3 Oligosaccharide 2

in Table II. About 2 mol of trisulfated disaccharide and 0.8 mol of tetrasaccharide (52 and 40% of total hexosamine present in the incubated heparin, respectively) are produced from hepa- rin by the action of the heparinase. Only a small amount of hexa- and oligosaccharides as well as a disulfated disaccharide are formed after degradation (8%). Each one of these products were prepared in large scale and reincubated with the heparin- ase. Only the hexasaccharide was still degraded by the enzyme forming the tetrasaccharide and the trisulfated disac- charide. The tetrasaccharide was completely resistant to the action of the heparinase. Several commercial preparations were incubated with the heparinase. Fig. 4 shows that all the heparin preparations are degraded by the heparinase forming the two major end products in similar amounts.

End Products Formed from Heparin and Heparin Degrada-

tion Products by Action of Heparitinase II-The heparitinase II does not act upon heparin (Table III). Nevertheless, when the heparitinase II is combined with the heparinase in the incubation, different amounts of degradation products are formed from heparin. There is a decrease in the amount of tetrasaccharide formed with an increase of tri- and disulfated disaccharides. It appears from this experiment that only the tetrasaccharide is the product susceptible to the action of the heparitinase II. In order to substantiate this hypothesis and also to obtain a better stoichiometry of the reaction, the

-aDI-TRI S

TETRA.

ORIGIN

1 2 3 4 5 6 7 a 9 10 11 12 13 14 -HEPARINS

FIG. 4. Products formed from 14 commercial heparin preparations by action of heparinase. Different commercial heparins (50 pg) were incubated with 0.1 wg of heparinase at 30” in 0.05 M ethylenediamine- acetate buffer, pH 7.0, containing 0.02 M MnCl, in a final volume of 20 ~1 for 12 hours. The mixture was chromatographed in Solvent A for 48 hours and the products were stained with toluidine blue reagent as described under “Experimental Procedure.” The 14 commercial hepa- i-ins were: 1, 4, 5, 7, 8, 9, 10 (The Upjohn Co., different batches); 3, 6, 13 (Lederle); 2 (Fischer); 11 (Ricker); 12 (B. D. H.); 14 (Wilson). A Di-Z’ri S, trisulfated disaccharide; tetru, tetrasaccharide.

TABLE III Amount of products formed from heparin by action of heparinase and

heparitinase II

Heparin (1 mg) was incubated with 5 pg of heparitinase II, heparinase or mixture of the two enzymes. The incubation as well as

the hexosamine determinations were performed as described in Table

II. A Di-Di S and A Di-Tri S, unsaturated di- and trisulfated disaccharides, respectively.

Product

Hepari- Hepari-

tinase Hepari- nase +

(t”:e 1)

nase hepariti- (tube 2) nase II

(tube 3)

A Di-Di S

A Di-Tri S Tetrasaccharide

Hexa- and oligosaccharide

‘3% pnol hevosamuze

3 12 +9

52 73 t21 40 10 -30

5 5

TABLE IV

Amount ofproducts formed from heparin tetrasaccharide by action of

heparitinase II

Heparin tetrasaccharide (250 fig) was incubated with 2 fig of heparitinase II in 0.05 M ethylenediamine acetate buffer, pH 7.0, at 30”

in a final volume of 100 ~1. The hexosamine determination of the products was performed as described in Table II. The results are expressed as per cent of total hexosamine of the incubated tetrasaccha-

ride. A Di-Tri S and A Di-Di S, unsaturated tri- and disulfated disaccharides, respectively.

Compound Products

0 hr 30 hr A

% Tetrasaccharide 100 4.5 -95.5

A Di-Tri S 0 50.1 +50.1 A Di-Di S 0 45.4 +45.4

isolated tetrasaccharide was incubated with heparitinase II. The result of this experiment is shown in Table IV. From each mole of tetrasaccharide about 1 mol of trisulfated disaccharide and 1 mol of disulfated disaccharide are formed by the action of the enzyme. A complete conversion of the tetrasaccharide to the di- and trisulfated disaccharides was obtained in this

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experiment. Fig. 5 shows the increase of absorbance at 230 nm of the tetrasaccharide when incubated with the heparitinase II. The sum of the absdrbances of the unsaturated disaccharides formed is almost twice the absorbance of the tetrasaccharide. This enzyme does not act upon the hexa- and the disaccha- rides. The heparitinase I (Fig. 1) had no activity toward heparin or its degradation products.

Product Identification-The chemical analysis of the prod- ucts formed from heparin by the action of the heparinase and heparitinase II is shown in Table V. The molar ratios of sulfate to hexosamine and uranic acid agrees with the proposed structures (Fig. 6). The disulfated disaccharide as well as the tetrasaccharide are unsaturated as it has been previously

reported for the trisulfated disaccharide (14). This indicates that the heparinase and heparitinase II act as eliminases producing an unsaturation at the uranic acid moiety. The evidences for the major products of heparin formed by the action of the heparinase being a trisulfated disaccharide and a tetrasaccharide are as follows: after reduction with borohy- dride there is a disappearance of 90% of hexosamine from the trisulfated disaccharide and about 50% from the tetrasaccha- ride. This result also shows that hexosamine is the reducing end of both compounds. The tetrasaccharide has only 50% of the reducing activity of the trisulfated disaccharide. The molar extinction coefficient of the tetrasaccharide is about the same as that of the trisulfated disaccharide. The tetrasaccharide is transformed in two disaccharide units upon the action of heparitmase II. The sulfate to hexosamine ratio being around 2.5 indicates that it is composed of a tri- and a disulfated disaccharide. The molecular weight of these two compounds obtained by polyacrylamide gel electrophoresis (Table V) also agrees with the proposed structures.

The enzymes from F. heparinum involved in the sequential degradation of heparin also were used to confirm the proposed structures (3). The trisulfated disaccharide is transformed to disulfated disaccharide by the action of a purified disaccharide sulfoesterase recently described (3). The disulfated disaccha- rides obtained either from the trisulfated disaccharide by the action of the disaccharide sulfoesterase, or by the action of heparitinase II upon the tetrasaccharide are degraded by a glycuronidase (also isolated from F. hepnrinum (3)) to a compound identified as glucosamine 2,6-disulfate (l), and an (Y, /3-keto acid. The tetrasaccharide is not susceptible to the action of the glycuronidase or the disaccharide sulfoesterase. Other analyses of these fragments have already been reported (1, 14).

0310-

/ --+’

OZ40- ,-r Oj70-

TIME( HOURS)

FIG. 5. Formation of unsaturated products from heparin tetrasac- charide by action of heparitinase II. Heparin tetrasaccharide (500 fig) was incubated with 2 Fg of heparitinase II in 0.05 M ethylenediamine- acetate buffer, pH 7.0, at 30” in a final volume of 100 ~1. Aliquots of 10 ~1 were taken at the times indicated, diluted with 1 ml of water, and measured spectrophotometrically at 230 nm. W-W, tetrasaccharide plus heparitinase II; q PO, control, tetrasaccharide with inactivated enzyme.

TABLE V Analytical data for heparin degradation products -

I’ RELATIVE CHROMATOGRAPHYC MIGRATION

MOLAR PROPORTIONS % OF REMAINING SUGARS AFTER DROHYDRIDE REDUCTION

.EDUCING ;UGAR LOLES/MOLEL

OF IEXOSAMINE

R s

I M

H

COMPOUND

A I .ABILE EXOSAMINE : E ;ULFATE

1.05

1.42

0.97

1.04

0.93

IOLECULAR WEIGHT E 230 ml in II20

B

n: T

1 s

1 OTAL URONII ULFATI ACID

2.61 2.00

3.03 1.60

2.46 1.40

3.08 1.03

1.82 0.95

EXOSAMINEURONICACID SOLVENT P i I iOLVENT I

0.08

0.32

0.90

1.00

L

HEPARIN

HEXASACCHARIDE

TETRASACCHARIDI

A Di-Tri S

A Di-Di S

Glucosamine

0.07

0.21

0.71

1.00

0.19

0.36

0.67

0.84

1.00

-2,000 k 6,000

1,800

1,200

600

500

k.33.103

3.71.103

3.45.103

3.32.103

90 105

53 1 90

12 96

a 95

* Only glucosamine was detected after acid hydrolysis and chromatography.

The standards used for the determination of the molecular weights by polyacrylamide were: Chondroitin

Sulfate B, Miles Lab., M.W. 19.000; Heparitin Sulfate C, M.W. 9,300 (see ref. 8) and Hyaluronic acid

from Sigma Co., M.W. > 170,000 (limit of penetration in 6% polyacrylamide gel).

**The carbazole reaction gives an anomalous high value for uranic acid in heparin (24).The disaccharide

degradation products, on the other hand, give the expected ratio to hexosamine. It is noteworthy to

mention that the uranic acid/hexosamine ratio increases with increasing chain lenght of the heparin

fragments. This could be used as an independent cryterion to determine the approximate number of

residues of the fragment.

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HEPARIN

/ /ITI\ 1

ADI-TRI S ADI-DIS FIG. 6. Proposed structure of heparin and mode of action of heparinase and heparitinase II in the degradation of heparin to disaccharides.

T&a, tetrasaccharide; A Di-Tri S and A Di-Di S; tri- and disulfated disaccharides.

DISCUSSION

The structure of heparin and the mode of action of the heparinase and heparitinase II shown in Fig. 6 are compatible with the data reported in this paper. Heparin is a linear chain composed of basic hexa- or octasaccharide repeating units. The evidence for absence of branchings in the structure is the finding that at least 95% of the degradation products formed from heparin by the action of the enzymes are unsaturated. If some trisulfated disaccharides or disulfated disaccharides were forming branched chains in the heparin molecule we would expect either lack of degradation by the enzymes or the formation of saturated disaccharide units by the enzymatic degradation of heparin.

We have previously shown that the trisulfated disaccharide is the major disaccharide unit of heparin comprising about 70 to 80%’ of the total structure (2-4) and that iduronic acid was the uranic acid of this disaccharide (14). We now report the presence of disulfated disaccharide units in about 20% of the total compound. These disulfated disaccharide units do not seem to be clustered in a special region of the heparin molecule but dispersed alternating with the trisulfated disaccharides. The evidence for this is the isolation of a tetrasaccharide by the action of the heparinase in high yield and the presence of both tri- and disulfated disaccharides in its structure. Also the kinetics of formation of the trisulfated disaccharide and tetrasaccharide favours this alternating structure. The finding

2The high yield of trisulfated disaccharide obtained by the action of heparinase upon heparin reported previously was probably due to contamination of this enzyme with heparitinase II.

that the tetrasaccharide is not susceptible to the glycuronidase suggests that the trisulfated disaccharide is at the nonreducing end, as shown in Fig. 6.

No gross differences were observed among 14 commercial heparin preparations degraded with the heparinase. All these heparins were degraded by the heparinase to tetrasaccharide and trisulfated disaccharide to about the same extent. This suggests that these compounds are the major units present in the commercially available heparins tested and not due to the differences of sulfate content of these different preparations.

We recently have shown that commercial heparin prepara- tions are composed of at least 21 molecular species (15). These species have molecular weights from 3,000 to 37,500 with intervals of about 1,500 to 2,000 which are the weight of an hexa- or octasaccharide. These results also suggest that one hexa- or octasaccharide repeating unit is the most probable fundamental unit of the heparin molecule.

No evidence for the nature of the uranic acid residue of the disulfated disaccharide units is at present available. Neverthe- less, since heparin contains also glucuronic acid in its structure (16), it is conceivable that the disulfated disaccharide units are composed of this type of uranic acid residue. Evidence for the presence of an iduronic acid-containing trisulfated disaccha- ride unit in heparin molecule has been provided by Perlin et al. (14) and Lindahl and Axelsson (17). The latter authors were also able to demonstrate that the disulfated disaccharide contain glucuronic acid in its structure. Nevertheless the yield of the total uranic acid content obtained from heparin by the chemical methods employed were rather low (17). Recently

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Hook et al. have improved these yields to 70% and have shown that iduronic acid constitutes 77% of the total uranic acid residues of heparin (18), thus approaching the results reported previously for the trisulfated disaccharide (14). The disaccha- ride products obtained by the enzymatic degradation, as described in the present paper, account for 95% of the heparin molecule.

incorporated in the /? configuration are epimerized to iduronic acid upon sulfation.

Acknowledgment-We wish to express our appreciation to Dr. Sonia M. C. Dietrich for help in the preparation of this manuscript.

Both disulfated disaccharides formed from heparin either by the combined action of the heparinase and the disaccharide sulfoesterase or by the action of heparitinase II upon the tetrasaccharide are degraded by an a-glycuronidase also iso- lated from extracts of induced F. heparinum cells (3). These disulfated disaccharides on the other hand were not hydrolysed by a P-glycuronidase also present in noninduced extracts.3 These results indicate that most of the glycuronidic linkages of heparin have (Y configuration and are in agreement with those obtained by Warnick and Linker (19) who purified the induced a-glycuronidase and studied its specificity, together with the P-glycuronidase, towards different substrates. Furthermore Perlin et al. (20) were unable to find any proton magnetic resonance spectral evidence for the presence of p glycuronidic linkages in heparin. These and other results (21) are in contrast to a report by Helting and Lindahl(22) who isolated glucuronic acid by the action of a p-glucuronidase upon heparin fragments obtained by nitrous acid treatment. Furthermore other results obtained by Hijok et al. in studies of incorporation of radioac- tive glucuronic acid in mouse mastocytoma particulate prepa- rations indicate that the incorporated uranic acid is epimerized at C” to a-L-iduronic acid upon sulfation (23). According to the authors this transformation can only occur if the glucuronic acid was incorporated in the polymer in a P-D configuration. These results, nevertheless, do not rule out the possibility that glucuronic acid is also incorporated in the a-~ configuration since only one-third of glucuronic acids were epimerized to iduronic acids at the polymer level in the experiments reported by Hijok et al. (23). All these apparent contradictory results could be reconciled if we assume that the glucuronic acids are incorporated in both configurations and most of the ones

1. 2. 3.

4.

5.

6.

7.

8. 9.

10.

11. 12. 13.

14.

15.

16.

17. 18.

19. 20.

21. Kiss, J. (1974) Advan. Carbohyd. Chem. Biochem. 29,229-303 22. Helting, T., and Lindahl. U. (1971) J. Biol. Chem. 246,5442-5447 23. Hook, M., Lindahl, U., BHckstrom, G., Malmstrom, A., and

3 Unpublished data. 24.

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M E Silva and C P Dietrichaction of a heparinase and a heparitinase from Flavobacterium heparinum.

Structure of heparin. Characterization of the products formed from heparin by the

1975, 250:6841-6846.J. Biol. Chem. 

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