The Antithrombin-binding Sequence in Heparin

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 258, No. 16, Issue of August 25, pp. 9826-9830,1983 Printed in U.S.A.

The Antithrombin-binding Sequence inHeparin IDENTIFICATION OF AN ESSENTIAL 6-0-SULFATE GROUP* (Received for publication, December 21, 1982)

Ulf Lindahl, Gudrun Backstrom, and Lennart Thunberg From the DeDartment of Medical and Phvsioloeical Chemistry, Swedish University of Agricultural Sciences, The Biomedical “ Center, S-75123 Uppsala, Sweden

Commercially available heparin preparations areheterogenous in that only a fraction, generally less than half, of the molecules are capable of binding with high affinity to the protease inhibitor antithrombin (1-3). These high affinity components, which account for most of the blood anticoagulant activityof the unfractionated material, contain a specific oligosaccharide sequence which mediates the binding to the inhibitor. A tetrasaccharide structure, -1dUA-GlcNAc(6OS03)-GlcUA-GlcNS03(6-OS03)-,’ foundto be overrepre-

sented in heparinmolecules with high affinity for antithrombin (as compared to the corresponding low affinity species) (4) could be recovered in heparin oligosaccharides isolated by affinity chromatography on antithrombin-Sepharose(5).The smallest such antithrombin-binding oligosaccharide, formed by deaminative cleavage of heparin with nitrous acid, was an octasaccharide, the nonreducinghalf of which (units 1-4 in Fig. 1) consisted of the tetrasaccharide sequence described (Ref. 6 and references therein). Units 7 and 8 of this octasaccharide displayed extensive structural variability and were therefore considered to fall outside the actual antithrombinbinding sequence (6). The extension of the binding region toward the nonreducing terminuswas established by affinity chromatography following chemical and/or enzymatic modification of the octasaccharide (6). N-Deacetylation of the N acetylglucosamine unit 2 had no significant effect on the affinity of the octasaccharide for antithrombin, whereas removal of the entire nonreducing terminal disaccharide unit (1-2)was accompanied by a drastic loss of binding affinity. On the other hand, release of only the terminal iduronosyl unit 1 by digestion of the octasaccharide with a-L-iduronidase hadno effect onthebinding properties. It was therefore concluded that the actual bindingregion is comprised of the pentasaccharide sequence 2-6 (within brackets in Fig. 1). Further characterization of the antithrombin-binding octasaccharide revealed a novel structural feature,a 3-0-sulfate group located at the glucosamine unit 4 (7). Since this substituent appeared to be unique to the antithrombin-binding region of the heparinmolecule (8,9), it ispresumably essential to the interaction with the inhibitor. The functional roles of various other sulfate groups have been amenable to experimentalevaluation. Selective N-desulfation of the octasaccharide thus indicated that the N-sulfategroups of residues 4 and 6 are both required for binding to antithrombin (10). Furthermore, theloss of bioaffinity caused by removal of the nonreducing terminal disaccharide (units 1 and 2), as contrasted to the retention of affinity afterrelease of unit 1 only, tentatively implicated the 6-sulfate group on unit 2 (6). In the present study, this conclusion was verified by selective removal of the 6-sulfate group, using a specific N-acetylglucosamine-6-sulfate sulfatase.

* This work was supported by Grant 2309 from the Swedish Medical Research Council and Grant A5861 from the Swedish Council for Forestry and AgriculturalResearch and by KabiVitrum AB, Stockholm, the National Swedish Board for Technical Development, and the Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ The abbreviationsused are: UA, unspecified hexuronic acid EXPERIMENTALPROCEDURES GlcUA, D-glUCUr0niC acid IdUA, L-iduronic acid; GlcNAc, 2-deoxyMaterials-Heparin from pig intestinal mucosa (Stage 14 material) 2-acetamido-~-glucose(N-acetyl-D-glucosamine); aMan, 2,5-anhywas obtained and purified as described (6). dro-D-mannitol; N-sulfate, sulfamino group; 0-sulfate, ester sulfate A heparin octasaccharide with high affinity for antithrombin was group. The location of 0-sulfate groups is indicated in parentheses. Specific residues within the antithrombin-bindingoctasaccharide will isolated, reduced (unit 8 ) with unlabeled NaBH4, andN-deacetylated be referred to by the numbers shown in Fig. 1. The same mode of (unit 2) by hydrazinolysis (6). The N-deacetylated octasaccharide designation will also be applied to derivatives, including oligosaccha- was radiolabeled by treatment with [“Clacetic anhydride (6),yielding ride fragments, that contain terminal 2,5-anhydro-~-mannitol resi- [2-’4C](1-8) (Fig. 1) with a specific activity of 55 X lo3 cpm of l4C/ dues. For example, the trisaccharide (2-4) is composed of N-acetyl- pg of uronic acid. UA-[3H]aMan disaccharides were prepared from heparin as deD-glucosamine, D-gh“onic acid, and 2,5-anhydro-~-mannitol, arscribed and separated into mono-0- and di-0-sulfated species by ranged in the sequence GlcNAc-GlcUA-aMan.

9826

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An octasaccharide with high affinity for antithrombin, isolated after partial deaminative cleavage of heparin, waspreviously found to have anL-iduronosyl-Nacetylglucosaminyl-6-0-sulfate nonreducing terminal disaccharide unit. After digestion of this octasaccharide with a-L-iduronidase and N-acetylglucosamine-6sulfatesulfatase,twofractions,withhigh and low affinity for antithrombin, respectively, were isolated by affinity chromatography on antithrombin-sepharose. Structural analysis showed that the high affinity fraction contained intact octasaccharide, whereas the low affinity fraction consisted of the expected heptasaccharide, lacking a 6-sulfate group on the terminal N-acetylglucosamine residue. Digestion of the octasaccharide with a-L-iduronidase only yielded heptasaccharide which was identical with the low affinity species except for the presence of this 6-sulfategroup. This less degraded heptasaccharide retained high affinity for antithrombin. It is concluded that the 6sulfate group on the N-acetylglucosamineresidue is of critical importance to the interaction between heparin and antithrombin.

9827

The Antithrombin-binding Sequence in Heparin

r 2

3

4

5

4

7

8

OH

FIG. 1. Structure of the antithrombin-binding octasaccharide isolated after partial deaminative cleavage of heparin with nitrous acid. The 2,5-anhydro-~-mannitolunit 8 corresponds to an N-sulfated Dglucosamine residue in the intact polysaccharide. The pentasaccharide extending from position 2 to position 6 represents the actual binding sequence (within brackets). Structural variants are indicated by X (H or SO;) and by the sugar residue shown below the main structure (6). The3-0-sulfate group marked by an asterisk is unique to the antithrombin-binding region and has not been found elsewhere in the heparin molecule (7-9). The sulfate groups marked with an ( e ) were previously shown to be essential for high affinity binding of the octasaccharide to antithrombin (10); the present report deals with the role of the 6-0-sulfate group in position 2. A radioactive "C label (circled) was introduced as indicated. For additional information. see the text.

RESULTS AND DISCUSSION

so:

COO-

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preparative paper electrophoresis (11). Tetrasaccharides with the general structure U A - G ~ C N A C - G ~ C U A -were [~H obtained ] ~ M ~by ~a similar procedure, involving HN02-NaB3H4 treatment of heparin, followed by isolation of labeled oligosaccharides by gel chromatography (6).Unlabeled reference oligosaccharideswere prepared following partial random depolymerization of heparin with nitrous acid (6). a-L-Iduronidase from human kidney (12) was a gift from Dr. Leonard H. Rome, University of California, Los Angeles, CA. a-NAcetylglucosaminidase (13) and N-acetylglucosamine-6-sulfatesulfatase (14), both isolated from human urine, were gifts from Dr. Kurt von Figura, University of Miinster, Miinster, West Germany. Analytical Methods-Hexuronic acid was determined by the carbazole reaction with ~-glucurono-6,3-lactoneas standard (15). The methods used to determine radioactivity are described in Ref. 16. Gel chromatography was performed with columns of Sephadex G-15 (1X 175 cm) or Sephadex G-50 (1 X 200 cm, superfine grade), both eluted with 1 M NaCl a t -3.5 ml/h. High voltage paper electrophoresis (40 V/cm) was conducted on Whatman No. 3MM paper in 1.6 M formic acid (pH 1.7). Affinity chromatography on antithrombin-Sepharose was performed as described (6). Chemical and Enzymatic Modification Procedures-Treatment of saccharides with nitrous acid (pH 1.5) followed by reduction of the deamination products was performed as described (6), except that unlabeled NaBH4 (10 mM) was substituted for NaB3H4in the reduction step. The reduced oligosaccharides were isolated by gel chromatography on Sephadex G-15 in 0.2 M H4NHC03 and were then desalted by lyophilization. For enzymatic degradation, oligosaccharides (30 pg or less of hexuronic acid) were digested with either 1 unit of a-L-iduronidase in 250 pl of 0.05 M acetate buffer (pH 4.3) containing 0.1 M NaC1, 3 mM NaN3, and 0.05% Triton X-100; 20 microunits of N-acetylglucosamine-6-sulfate sulfatase in 100 plof 0.05 M acetate buffer (pH 4.0) containing 20 pg of bovine serum albumin; or 750 microunits of a-Nacetylglucosaminidase in 50 p1 of 0.05 M citrate buffer (pH 4.5). In each case, 1 unit of enzyme activity was defined as the amount of enzyme required to catalyze the splitting of 1 pmol of substrate/min at 37 "C. The digestions were continued for about 20 h at 37 "C.

HLOX

@J&J,

HO OH

b i t $ so; (3-8)

FIG. 2. Enzymatic modification procedures involving units 1 and 2 of the antithrombin-binding heparin oetasaccharide. The sequence shown comprises units 1-4. R, the tetrasaccharide sequence extending from unit 5 to unit 8; X , H or SO5 (see Refs. 6 and 7). In addition, the points of cleavage on treatment with nitrous acid, followed by reduction (HN02-NaBH4),are indicated. The glucosaminidic linkage between units 4 and 5 willbe split, and the internal N-sulfatedglucosamine unit 4 converted into a terminal2,5anhydromannitol residue; the structure of the tetrasaccharide fragment (1-4) will thus be IdUA-GlcN['4C]Ac(6-OS03)-GlcUA-aMa~(3,(6-di-)OS03).

A sample (1.5 x lo6 cpm) of the antithrombin-binding N ['4C]acetyl-labeled heparin octasaccharide [Z-l4C](1-8)was digested with a-L-iduronidase as described under "Experimental Procedures." After desalting by passage through a column (1X 70 cm) of Sephadex G-15 equilibrated with 10% aqueous ethanol, a portion (1 x lo6 cpm) of the digested material was incubated further in the presence of N-acetylglucosamine-6-sulfate sulfatase. The final product, expected tocontaintheheptasaccharide[2-'4C](2-8)lacking a 6- unretarded by the immobilized protein. In contrast,digestion sulfate group on unit 2 (Fig. Z), was subjected to analytical with a-L-iduronidase only (expected to release the nonreducaffinitychromatographyonantithrombin-Sepharose.Two ing terminal iduronicacid unit 1) did not significantly affect distinctfractions were observed (Fig. 3 8 ) , onewith high the affinity properties of the antithrombin-binding octasacaffinity for antithrombin, similar to the intact octasaccharidecharide (Fig. 3A; see also Ref. 6). The formation of a low (not shown), and one withlow affinity which appeared to be affinity component on consecutive digestions of the octasac-

9828

The Antithrombin-bindingSequence in Heparin 1.5

A

0.6

LA

HA

3

30

2

20

1

10

2

40

4

A

e ._

1.a

0.4

0.c

3.2 CI

m

0

z

0

m

E

V

20

1

u)

4

3

1.5

0

0.4

2

Y

40

1.o

I (3

20

0.5

1

z E, V

Y

2

0

x

1.6 D.6

B9

.. (3

CI

x

A

D.2

FRACTIONNUMBER

FIG.3. Affinity chromatography on antithrombin-sepharose of products (-10 X 10’ cpm) obtained on digesting the

40

60

80

100

ELUTION VOLUME ( m l )

FIG.4. Gel chromatography on Sephadex G-15 of ‘“C-la“C-labeled antithrombin-binding octasaccharide [2-14C](18) with a-L-iduronidase (A) (0)or a-L-iduronidase and N- beled oligosaccharides (0)obtained by HN02-NaBH, treatacetylglucosamine-6-sulfatesulfatase (B) (0).Samples (2 mg) ment of intact antithrombin-binding heparin octasaccharide of commercial heparin were included as internal standard (0,hexu- ([2-’“C](l-S) ( A ) ,product obtained on digesting this octasacronic acid determinedby the carbazole reaction). The separation into charide with a-L-iduronidase ( B ) ,product obtained on digestfractions of high ( H A ) and low ( L A ) affinity for antithrombin is ing thisoctasaccharide with a-L-iduronidaseand N-acetylgluindicated by the horizontal bars in A . The arrow indicates the start cosamine-6-sulfate sulfatase, fraction with high affinity for antithrombin (0,and product obtained on digesting this ocof the NaCl gradient. - - -, NaCl concentration (M), shown only in tasaccharide with a-L-iduronidase and N-acetylglucosamineB. 6-sulfate sulfatase, fraction with low affinity for antithrombin (D). Each sample (-20 X lo3 cpm of 14C) was mixed with charide with a-L-iduronidase andN-acetylglucosamine-6-sul- reference di- and tetrasaccharides (0,-200 X lo3 cpm of 3H altoby gel chromatography on fate sulfatase should therefore presumably be due to release gether) derived from heparin and analyzed of the 6-sulfate group from the N-acetylglucosamine 6-0- Sephadex G-15 in 1 M NaCl (see “Experimental Procedures.”

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20

sulfateunit 2 , exposedby theaction of a-L-iduronidase. Conversely, the high affinity fraction would consist either of charide recovered from the intact octasaccharide separated intact octasaccharide (remaining in the digested sample due into two components (Fig. 5A, a and 6) which were previously to incomplete action of the a-L-iduronidase) orof the hepta- (6) ascribed thestructures IdUA-GlcNAc(6-OS03)-GlcUAsaccharide [2-14C](2-8), still carrying a terminal 6-0-sulfate aMan(3-OS03)and IdUA-GlcNAc(6-OSOB)-GlcUA-aMangroup (due toincomplete action of the sulfatase). In order to (3,6-di-OSO,), respectively; the difference in migration rate confirm and differentiate these alternative interpretations, thus reflects the presenceor absence of the variable 6-sulfate the products of enzymatic modification were subjected to group of unit 4 (indicated by X in Fig. 1).The two peaks, a structural characterization. Such studies were facilitated by and b, were observed also with the a-L-iduronidase-digested material (Fig. 5A), in accord withthe conclusion that a isolation of the labeled fragments obtained on deaminative cleavage of the glucosaminidic linkage between units 4 and 5 fraction of the octasaccharide molecules had escaped degra(indicated in Fig. 2 for the initial octasaccharide [2-14C](1- dation by the exoglycosidase. However, in addition,two novel 8) and for its heptasaccharide derivative obtained by a - ~ - major components, a, and bl, were detected, which had migrated somewhat faster than compounds a and b, respectively iduronidase digestion). Gel chromatographyonSephadex G-15 of the tetrasac(Fig. 5A). The migration positionsof these components were charide [2-14C](1-4) formed by deamination of the intact, as expected, assuming that compoundsa, and bl were trisac14C-labeledoctasaccharide [2-14C](1-8) showed a single peak charides, differing from the corresponding tetrasaccharides, a which coincided with that of the corresponding tetrasaccha- and b, respectively, by the absence of a nonreducing terminal ride [4-3H](1-4) isolated (6) after HN02-NaB3H4 treatment iduronic acid unit. The increase in migration rate resulting of the unlabeled octasaccharide (Fig. 4A). Similar treatment from the loss of this residue is readily explained considering of the a-L-iduronidase-digestedoctasaccharide yielded, as ex- theconditions of electrophoresis; at pH 1.7, the carboxyl pected, a trisaccharide, [2-14C](2-4) but, in addition,signifi- groups of hexuronic acid units are essentially undissociated cant amounts of labeled tetrasaccharide, indicating incom- and will therefore not contribute to theoverall negative charge plete removal of the terminal iduronic acid unit 1 (Fig. 4B). of the oligosaccharides. The amounts of components al and This conclusion was corroborated by high voltage paper elec- bl, in relation to those of a and b, supported the conclusion trophoresis (pH 1.7) of the same components. The tetrasac- from gel chromatography that most, but notall, of the octa-

The Antithrombin-binding Sequence in Heparin

9829

0 10 20 MIGRATION DISTANCE (cm)

FIG. 5. Paper electrophoresis at pH 1.7 of "C-labeled oligosaccharides obtained by HNOa-NaBH4 treatment of antithrombin-binding heparin octasaccharide [Z-"C](l-S) before (0)and after (0)digestion with a-L-iduronidase (A) or obtained by HNOa-NaB& treatment of fractions with high (0) and with low (0)affinity for antithrombin recovered after digesting the octasaccharidewith a-L-iduronidase and N-acetylglucosamine-6-sulfate sulfatase (B). The standards shown below the tracings are monosulfated (0and disulfated (ZI)UA-[3H] aMan disaccharides. The designations of the various peaks in A are used in the text.

saccharide molecules had been attacked by the a-L-iduronidase. In order to characterize the components with high and low affinity for antithrombin detected after digestion of the octasaccharide with both a-L-iduronidase and N-acetylglucosamine-6-sulfate sulfatase, respectively, the affinity chromatography illustrated in Fig. 3B was reproduced on a preparative scale (-800 X lo3 cpm of I4C).The resulting fractions, 40% high affinity and 60% low affinity material, were desalted by passage through Sephadex G-15. Since characterization of the material digested only with a-L-iduronidase indicated that a portion of the octasaccharide molecules had remained intact, these molecules would beexpected to resist also the action of the sulfatase and would therefore be recovered in the final high affinity fraction. Accordingly, deamination of this fraction yielded a labeled tetrasaccharide (Fig. 4C), which separated on paper electrophoresis (Fig. 5B) into the same two peaks, a and b, that were observed after deamination of the intact octasaccharide (Fig. 5A). It is noted that peak bl, corresponding tothestructure GlcNAc(6-OS03)-GlcUAaMan(3,6-di-OS03),is essentially absent in Fig. 523, suggesting that removal of the iduronic acid unit 1 by the iduronidase was generally followed by release of the exposed 6-sulfate group in the subsequent sulfatase digestion. Deamination of the low affinity fraction produced a labeled product

"t

ELUTION VOLUME (ml )

FIG. 6. Gel chromatographyon Sephadex 6-50 of products obtained on digesting the antithrombin-binding octasaccharide [2-"C](1-8) with a-L-iduronidase andN-acetylglucosamine-6-sulfate sulfatase: fraction with high affinity for antithrombin ( A and B ) , fraction with low affinity for antithrombin (C and D ) , before furtherincubation with a-Nacetylglucosaminidase ( A and 0, and after incubation with a-N-acetylglucosaminidase( B and D ) . Before chromatography, each sample (-15 X lo3 cpm of 'IC) wasmixed with unlabeled reference oligosaccharides (7 mg of hexuronic acid) prepared by partial deamination of heparin. Eluate fractions were analyzed for radioactivity (0) and for hexuronic acid (-). The number of monosaccharide residues in the various reference oligosaccharides is indicated above the appropriate peaks.

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which behaved like a trisaccharide on gel chromatography (Fig. 40). This trisaccharide peak was slightly but significantly shifted in relation to that derived from the octasaccharide after digestion with a-L-iduronidase only (Fig. 4B); the difference in size would be due to the 6-sulfate group (on unit 2) released by the sulfatase (Fig. 2). The electrophoretic properties of the trisaccharide obtained from the low affinity fraction conformed to the postulated distribution of sulfate groups (Fig. 5B). Again, two peaks were observed, but now with migration positions corresponding to those expected for mono- and disulfated trisaccharides, respectively. The structures assigned to these components would thus be GlcNAcGlcUA-aMan(3-OSOJ for the slower migrating and GlcNAcG1cUA-aMan(3,6-di-OSO3) forthe faster-migrating species. The experiments described lead to the tentativeconclusion that the low affinity fraction consisted of heptasaccharide which lacked not only the iduronic acid unit 1 but also the 6sulfate group an the N-acetylglucosamine unit 2. Since the heptasaccharide still carrying a 6-sulfate group on unit 2 (obtained by digesting the octasaccharide with a-L-iduronidase only) retained high affinity for antithrombin, the loss of bioaffinity must be explicitly attributed to the loss of this particular sulfate group. In order to conclusively establish this relationship, it was essential to confirm the absence of a 6sulfate group on the terminal N-acetylglucosamine residue in the low affinity heptasaccharide. To thisend, the low affinity

9830

The Antithrombin-bindingSequence in Heparin

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fraction wasdigested withan a-N-acetylglucosaminidase ric techniques indicated affinities differing by about a 1000known to act only on nonsulfated terminal N-acetylglucosa- fold (18, 19). mine units (17). Since thesingle N-acetylglucosamine residue Acknowledgments-We thank Dr. L. H. Rome for providing the ain the potential substrate was N-['*C]acetyl-labeled, an effect L-iduronidase and Dr. K. von Figura for the gifts of N-acetylglucosof the enzyme would be readily manifested on subsequentgel amine-6-sulfate sulfatase and a-N-acetylglucosaminidase. chromatography by the appearance of a labeled monosacchaREFERENCES ride (Fig. 2). Before digestion with the a-N-acetylglucosaminidase, the low affinity fraction appeared ongel chromatogra1. Lam, L. H., Silbert, J. E., and Rosenberg, R. D. (1976)Biochem. phy (Sephadex G-50) as a distinct peak of 14C activity, corBiophys. Res. Commun. 69,570-577 2. Hook, M., Bjork, I., Hopwood, J., and Lindahl, U. (1976)FEBS responding to the elution positionof a heptasaccharide (Fig. Lett. 66, 90-93 6C). After incubation with the enzyme, most of the radioac3. Anderson, L.-O., Barrowcliffe, T. W., Holmer, E., Johnson, E. tivity appeared in the elution position of a monosaccharide A., and Sims, G . E. C. (1976)Thromb. Res. 9,575-583 N-[14C] 4. Rosenberg, R. D., and Lam, L. H. (1979)Proc. Natl. Acad. Sci. (Fig. 6D). This result demonstrated that the terminal acetylglucosamine unit in the low affinity heptasaccharide U. S. A. 76,1218-1222 5. Lindahl, U., Backstrom, G., Hook, M., Thunberg, L., Fransson, was nonsulfated. The occurrence of significant N-acetylgluL.-A,, and Linker, A. (1979)Proc. Natl. Aced. Sci. U. S. A. 76, cosamine-6-sulfate sulfatase activity in the a-N-acetylglucos3198-3202 aminidase preparationwas excluded, since the enzyme failed 6. Thunberg, L., Backstrom, G., and Lindahl, U. (1982)Carbohydr. to release any labeledmonosaccharidefrom thematerial Res. 100,393-410 recovered after digesting the high affinity octasaccharide with 7. Lindahl, U., Backstrom, G., Thunberg, L., and Leder, I. G . (1980) a-L-iduronidase only (not shown). Thehigh affinity fraction Proc. Natl. Acad. Sci. U. S. A. 77,6551-6555 8. Meyer, B., Thunberg, L., Lindahl, U., Larm, O., and Leder, I. G. recovered after iduronidase and sulfatase digestion of the (1981)Carbohydr. Res. 88,cl-c4 octasaccharide apparently consisted of intact molecules that 9. Casu, B., Oreste, P., Torri, G., Zopetti, G., Choay, J., Lormeau, had escaped the initial degradation by the iduronidase. As J.-C., Petitou, M., and Sinay, P. (1981)Biochem. J. 197,599expected, these components were unaffected by treatment 609 with a-N-acetylglucosaminidase, but emerged as labeled oc- 10. Riesenfeld, J., Thunberg, L., Hook, M., and Lindahl, U. (1981) tasaccharide after (Fig. 6B) as well as before (Fig. 6A) incuJ. Eiol. Chem. 256,2389-2394 bation with the enzyme. Due to lack of enzyme, the reason 11. Jacobsson, I., Hook, M., Pettersson, I., Lindahl, U., Larm, O., Wiren, E., and von Figura, K. (1979)Biochem. J. 179, 77-87 for the partial resistance of the octasaccharide preparation 12. Rome, L. H., Garvin, A. J., and Neufeld, E. F. (1978)Arch. against iduronidase digestion was not further investigated. Biochem. Biophys. 189, 344-353 Taken together, the results obtained in the present study 13. von Figura, K. (1977)Eur. J. Biochem. 80,525-533 indicate that the 6-sulfategroup on the N-acetylglucosamine 14. Basner, R., Kresse, H., and von Figura, K. (1979)J. Biol. Chem. unit 2 of the antithrombin-binding octasaccharide, i.e. the 254,1151-1158 nonreducing terminal unitof the actual bindingsequence (6), 15. Bitter, T., and Muir, H. M.(1962)Anal. Biochem. 4,330-334 is of crucial importance to the polysaccharide-protein inter- 16. Lindahl, U., Jacobsson, I., Hook, M., Backstrom, G., and Feingold, D. S. (1976)Biochem. Biophys. Res. Commun. 70, 492action. The difference in elution position on antithrombin499 Sepharose caused by removal of this sulfate group is of the 17. von Figura, K. (1977)Eur. J. Biochem. 80,535-542 same magnitude as that observed for native heparin species 18. Nordenman, B., and Bjork, I. (1978)Biochemistry 17,3339-3344 with high or low affinity for antithrombin. Determination of 19. Nordenman, B., Danielsson, A., and Bjork, I. (1978)Eur. J. Biochem. 90,l-6 the binding constantsfor such fractionsby spectrophotomet-