Structures of five sulfated hexasaccharides prepared from porcine intestinal heparin using bacterial heparinase. Structural variants with apparent biosynthetic precursor-product relationships for the antithrombin III-binding site.

Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G. (1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.

internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasaccharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.
Heparin is a highly sulfated, linear polysaccharide that has various biological activities such as inhibition of blood coagulation (Marcum and Rosenberg, 1989), modulation of cellular proliferation (Clowes and Karnovsky, 1977;Thornton et al., 1983), potentiation of angiogenesis (Folkman and Ingber, 1989), and interactions with various growth factors (Maciag et al., 1984;Shing et al., 1984;Klagsbrun and Shing, 1985;Nakamura et al., 1986). The basic polymeric structure of heparin is an alternating repeat sequence of the disaccharide units 34IdoA␣/GlcA␤134GlcN␣13, which can be variably sulfated (for reviews see Rodén (1980), Gallagher and Lyon (1989), and Lindahl (1989)). It is synthesized on a serine residue of a protein core named "serglycin" through a specific structure, the so-called carbohydrate-protein linkage region: GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser (Lindahl and Rodén, 1965). Although the principal structure of the repeating disaccharide region, known as the regular region (Casu, 1985), is composed of the major trisulfated disaccharide unit, 34IdoA(2sulfate)␣134GlcN(N,6-disulfate)␣13, undersulfation and substantial structural variability are observed in the irregular region, which is distributed along the chain flanked by the regular region and accounts for approximately one quarter of the heparin polysaccharide chain. The structural variability is often the basis of a wide variety of domain structures with a number of biological activities ascribed to heparin.
Recent structural studies of the binding domains to ATIII (For review see Lindahl (1989)) and basic fibroblast growth factor  are the best known examples showing the relationships between the complicated fine structures and biological functions. The ATIII-binding site requires a minimal pentasaccharide sequence uniquely 3-O-sulfated on the central GlcN residue. This specific pentasaccharide has been shown to be primarily responsible for the anticoagulant activity of heparin (Lindahl et al., 1983). It has also been demonstrated that the binding domain to basic fibroblast growth factor requires a 2-O-sulfated IdoA residue and Nsulfated GlcN residue(s) for its specific interaction with the growth factor. Some structural variability has been observed within both binding sequences (Lindahl et al., 1984;Yamada et al., 1993;Maccarana et al., 1993).
We have been studying the basic primary structure of heparin to clarify the structural basis of its various biological activities. Previously, we demonstrated its structural variability by isolating six glycoserines from the carbohydrate-protein linkage region  and a number of tetrasaccharides from the repeating disaccharide region of porcine intestinal heparin after extensive digestion with bacterial heparin lyases (Yamada et al., 1993. In this study, we isolated and characterized five hexasaccharide structures from the repeating disaccharide region of the same heparin preparation after extensive enzymatic digestion to investigate the structure beyond the above tetrasaccharide sequences. These included three hitherto unreported hexasaccharide structures and structural variants with an apparent biosynthetic precursor-product relationship for the ATIII-binding site.
Preparation and Purification of Hexasaccharides-Stage 14 heparin was purified by anion exchange chromatography and digested with heparinase, and the digest was fractionated into fractions a-d by gel filtration as described previously . Fractions c and d contained tetra-and disaccharides, respectively, which were derived from the repeating disaccharide region as characterized by HPLC 1 (data not shown). Fraction a contained larger oligosaccharides and glycoserines/glycopeptides that were derived from the glycosaminoglycan-protein linkage region. Fraction b containing mainly hexasaccharides was subfractionated by HPLC on an amine-bound silica column  and structurally characterized in this study. Each peak was purified by rechromatography under the same conditions as the first step and desalted by gel filtration through a Sephadex G-25 column.
Digestion of Fraction b-19 with 2-Sulfatase and Subfractionation of the Digest-Fraction b-19 (170 nmol) was incubated with 60 mIU of 2-sulfatase in a total volume of 120 l of 6.7 mM CH 3 COONa, pH 6.5, containing 0.05% bovine serum albumin at 37°C for 140 min. The reaction was terminated by boiling for 1 min, and the digest was fractionated by HPLC on an amine-bound silica column as described below.
Digestion of the Isolated Hexasaccharides with Heparinase, Heparitinases, or 2-Sulfatase-Each isolated hexasaccharide (0.5-1.0 nmol) was digested using 1-5 mIU of heparinase, heparitinase I, II, or V, or 2-sulfatase as described previously . Successive enzymatic digestion of a given hexasaccharide with 2-sulfatase and then heparitinase I was also carried out as reported . Reactions were terminated by boiling for 1 min, and the reaction mixture was analyzed by HPLC as described below.
HPLC and Capillary Electrophoresis-Fractionation and analysis of unsaturated oligosaccharides were carried out by HPLC on an aminebound silica PA03 column using a linear gradient of NaH 2 PO 4 basically as described previously, except that a linear gradient of NaH 2 PO 4 was made from 16 -800 mM over 90 min . Eluates were monitored by absorption at 232 nm. Capillary electrophoresis was carried out to examine the purity of each isolated fraction in a Waters capillary ion analyzer as reported previously . The electrophoretic fractions were examined by absorption at 185 nm due to carbonyl groups because its sensitivity was higher than that of detection at 232 nm .
Nitrous Acid Degradation-Each heparin hexasaccharide (5.0 nmol) was treated at room temperature with HNO 2 at pH 1.5 for 30 min (Shively and Conrad, 1976a), and the resultant di-and/or tetrasaccharides were reduced under alkaline conditions with [ 3 H]sodium borohydride (0.50 mCi) as reported previously . Labeled oligosaccharides were separated by gel filtration chromatography on a column (1.0 ϫ 115 cm) of Bio-Gel P-2 using 0.25 M NH 4 HCO 3 /7% propanol as an eluent. Di-and tetrasaccharide fractions were separately pooled, lyophilized at least three consecutive times to ensure complete removal of the ammonium bicarbonate, and then reconstituted in water. These fractions were separated, respectively, by HPLC on an amine-bound silica column at a flow rate of 1 ml/min using a stepwise gradient of NaH 2 PO 4 . Samples were collected at 1.0-min intervals for radioactivity measurement in an Aloka LSC-700 liquid scintillation counter. Individual disaccharide peaks were identified by comparison with authentic heparin disaccharides as reported previously (Bienkowski and Conrad, 1985).
Other Analytical Methods-Uronic acid was determined by the carbazole method (Bitter and Muir, 1962). Unsaturated uronic acid was spectrophotometrically quantified based upon an average millimolar absorption coefficient of 5.5 at 232 nm (Yamagata et al., 1968). Amino sugars were quantified after acid hydrolysis in 3 M HCl at 100°C for 16 h using a Beckman 6300E amino acid analyzer (Sugahara et al., 1987).

RESULTS
Isolation of the Oligosaccharides-Purified stage 14 heparin from porcine intestine was exhaustively digested with heparinase and fractionated into fractions a-d by gel filtration on Cellulofine GCL-90 m . Amino sugar and uronic acid analyses showed that fraction b contained approximately 3 mol each of GlcN and HexA/mol of ⌬HexA (data not shown), i.e. hexasaccharides. Fraction b was subfractionated by HPLC on an amine-bound silica column into Fr. b-1 to b-26 . Nine major fractions, Fr. b-5, -6, -10, -15, -17, -19, -20, -22, and -24, were further purified by rechromatography. They altogether accounted for 77 mol% (as ⌬HexA) of the oligosaccharides obtained from fraction b. Fr. b-5, -6, and -10 contained glycoserines derived from the glycosaminoglycan-protein linkage region as reported previously . The other fractions, , were subjected to structural analysis below in this study. These individual fractions gave a single peak on HPLC but were approximately 95, 99, 54, 66, 96, and 75% pure, respectively, when examined by capillary electrophoresis (Fig.  1 (Linker and Hovingh, 1984) as confirmed by 1 H NMR spectroscopy, where 2S, 6S, and NS represent 2-O-, 6-O-, and 2-N-sulfation, respectively (data not shown). Apparently, this tetrasaccharide was partitioned into the hexasaccharide fraction due to its heavily sulfated structure. The amounts of the analyzed fractions isolated from 100 mg of the starting purified heparin are summarized in Table I.
Enzymatic Analysis-The disaccharide compositions of the isolated oligosaccharide fractions were determined by digestion with heparitinase(s) and/or heparinase, followed by HPLC on an amine-bound silica column. All the above oligosaccharide fractions except for Fr. b-17 were enzymatically degraded into approximately 3 mol of disaccharide units or 1 mol each of a diand a tetrasaccharide unit. As representative chromatograms, those obtained with Fr. b-15 and b-24 are shown in Figs. 2 and 3, respectively. Fr. b-15 yielded ⌬DiHS-6S, ⌬DiHS-diS 1 , and ⌬DiHS-triS upon heparitinase I digestion as shown in Fig. 2A; their recoveries were 113, 105, and 124%, respectively, when taking the UV absorbance of the parent oligosaccharide(s) in Fr. b-15 as 100% (Table I). Thus, the major component in this fraction was a hexasulfated hexasaccharide composed of a monosulfated, a disulfated, and a trisulfated disaccharide unit. Its sensitivity to 2-sulfatase was examined to localize the ⌬HexA(2S)-containing disaccharide unit ⌬DiHS-triS in the hexasaccharide sequence. The enzyme acts only on the ⌬HexA(2S) structure at the nonreducing end (McLean et al., 1984). When Fr. b-15 was successively digested with 2-sulfatase and then heparitinase I, it yielded ⌬DiHS-6S and ⌬DiHS-diS 1 with recoveries of 92 and 204%, respectively, indicating that ⌬DiHS-triS had been located at the nonreducing end and converted to ⌬DiHS-diS 1 by successive digestion (Fig. 2B). When digested with heparitinase V (Yoshida, et al., 1989), Fr. b-15 gave rise to equimolar amounts of ⌬DiHS-diS 1 and a presumable tetrasulfated tetrasaccharide (Fig. 2C), indicating that ⌬DiHS-diS 1 was derived from the other terminus, i.e. the reducing end. Therefore, the structure of the compound in Fr. The major component in Fr. b-24 accounted for 75% of the UV-absorbing materials in this fraction as judged by capillary electrophoresis (Fig. 1E). Exhaustive digestion of this fraction with a mixture of heparinase and heparitinase I yielded ⌬DiHS-triS, ⌬DiHS-diS 1 , and ⌬DiHS-diS 3 with recoveries of 158, 96, and 125%, respectively (Fig. 3A). Upon incubation with heparinase only, Fr. b-24 was degraded into two unsaturated components, ⌬DiHS-triS and a component that eluted near the

TABLE I Enzymatic analysis of the isolated oligosaccharide fractions
After each isolated oligosaccharide fraction was incubated with heparitinase I and/or heparinase, the reaction products were analyzed by HPLC. Recoveries of the di-and tetrasaccharides calculated based upon absorbance at 232 nm are shown in parentheses. The absorbances obtained with each intact compound were taken as 100%. c Identified based upon the co-chromatographic behavior with the previously isolated tri-or tetrasulfated tetrasaccharide structure, respectively, derived from the ATIII-binding site of heparin (Yamada et al., 1993).

FIG. 2. HPLC analysis of the enzyme digests of Fr. b-15.
Fr. b-15 (0.5 nmol) was digested with heparitinase I (A) and successively with 2-sulfatase and then heparitinase I (B) or with heparitinase V (C) as described under "Experimental Procedures." The digest was subjected to HPLC on an amine-bound silica column using a linear gradient of NaH 2 PO 4 from 16 to 800 mM over 90 min. Elution positions of the standard disaccharides isolated from heparin/heparan sulfate are indicated in A: 1, ⌬DiHS-0S; 2, ⌬DiHS-6S; 3, ⌬DiHS-NS; 4, ⌬DiHS-diS 1 ; 5, ⌬DiHS-diS 2 ; 6, ⌬DiHS-diS 3 ; 7, ⌬DiHS-triS. The peaks marked by asterisks are often observed between 35 and 40 min upon high sensitivity analysis and are due to an unknown substance eluted from the column resin. The broad peaks observed at around 5 and 10 min were derived from the incubation buffer or the enzyme preparation. elution position of a tetrasulfated tetrasaccharide, with recoveries of 171 and 110%, respectively (Fig. 3B), whereas it was degraded by heparitinase I into two unsaturated components, ⌬DiHS-diS 1 and a component that eluted near the elution position of a pentasulfated tetrasaccharide, with recoveries of 100 and 106%, respectively (Fig. 3C). These results together indicate that the major component in Fr. b-24 is a pentasulfated hexasaccharide composed of equimolar amounts of three disaccharide units corresponding to ⌬DiHS-triS, ⌬DiHS-diS 1 , and ⌬DiHS-diS 3 , and that the excess recovery of ⌬DiHS-triS upon heparinase or heparinase/heparitinase I digestion was probably due to degradation of contaminating oligosaccharide(s). The sequential arrangement of the three disaccharide units in the major hexasaccharide was determined based upon the sensitivity to 2-sulfatase. When digested successively with 2-sulfatase and then heparitinase I, the presumably pentasulfated tetrasaccharide peak shifted to a position corresponding to the loss of one sulfate group on HPLC, indicating that ⌬DiHS-triS had been located on the nonreducing terminal side of the major compound in Fr. b-24. Therefore, the structure of the major compound in this fraction is proposed as ⌬HexA- Fr. b-19 was resolved into several subcomponents by capillary electrophoresis, the major component accounting for only 54% of the UV-absorbing materials in this fraction (Fig. 1B), but it was not possible to fractionate it preparatively into its subcomponents. Therefore, it was first digested with 2-sulfatase and then the digest was analyzed by HPLC. The 2-sulfatase treatment resulted in a peak shift of 8 min of 61% of the parent compound on HPLC, indicating that the major product lost one sulfate group (data not shown). The major product, designated as Fr. b-19S, was isolated and subjected to structural analysis. The yield was 167 nmol/100 mg of the starting heparin. It was degraded by heparitinase I, yielding almost exclusively ⌬DiHS-diS 1 (Table I). Therefore, the major component in Fr. b-19S was deduced to be a hexasulfated hexasaccharide composed of 3 mol of the disulfated disaccharide unit corresponding to ⌬DiHS-diS 1 , i.e. ⌬HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S). Consequently, the structure of the major component in the parent fraction b-19 was ⌬HexA-(2S)-GlcN(NS,6S)-HexA-GlcN(NS,6S)-HexA-GlcN(NS,6S).
Heparitinase I digestion of both Fr. b-20 and -22 resulted in two unsaturated components, the trisulfated disaccharide ⌬DiHS-triS and a component that eluted near the elution position of the tri-or tetrasulfated tetrasaccharide (data not shown). Recoveries of the di-and tetrasaccharide components from Fr. b-20 or -22 were 109 and 62% or 122 and 106%, respectively ( Table I). The lower recoveries of the presumable tetrasaccharide components of these fractions compared with those of their counterpart disaccharides suggested that the excess disaccharides were derived from minor components in these fractions consistent with the results of capillary electrophoresis. The presumable tetrasaccharide components from both Fr. b-20 and -22 were resistant to heparinase and heparitinases I and II (data not shown), probably due to the 3-Osulfation of the reducing GlcN as reported previously for the ATIII-binding site-derived tetrasaccharides (Yamada et al., 1993). The presumable tetrasaccharides were co-chromatographed on HPLC with the authentic tetrasaccharides containing 3-O-sulfated GlcN residue (Yamada et al., 1993), demonstrating that the tri-and tetrasulfated tetrasaccharides derived from Fr. b-20 and -22 were identical to ⌬HexA-GlcNAc(6S)-GlcA-GlcN(NS, 3S) and ⌬HexA-GlcNAc(6S)-GlcA-GlcN(NS, 3S,6S), respectively.
Sensitivities of the compounds in Fr. b-20 and -22 to 2-sulfatase were examined to characterize the sequential arrangement of the constituent di-and tetrasaccharide units. After 2-sulfatase digestion, Fr. b-20 and -22 gave a single peak on HPLC, which eluted approximately 10 min earlier than the corresponding parent compound, indicating that the major compound in each fraction had a sulfate group on the C-2 position of the ⌬HexA residue at the nonreducing terminus. HPLC Analysis of the Di-and Tetrasaccharides Formed by HNO 2 /NaB 3 H 4 Treatment-To identify the internal uronic acid residues in the hexasaccharides of the isolated fractions, nitrous acid degradation products of each fraction were analyzed by HPLC. Bacterial lyase treatment converts the original structures of internal uronic acid, GlcA and IdoA in the oligosaccharides into the common 4,5-unsaturated, 4-deoxy-␣-Lthreo-hex-4-enepyranosyluronic acid. In contrast, nitrous acid treatment preserves the original uronic acid structures despite loss of an N-sulfate group and production of an artificial structure, anhydromannitol, at the reducing end of the resultant oligosaccharides Conrad, 1976a, 1976b).
The resultant di-and/or tetrasaccharide(s) from each hexasaccharide fraction were isolated by gel filtration on Bio-Gel P-2 and were analyzed by HPLC. These fractions obtained from  were confirmed to contain a disaccharide and a tetrasaccharide component as judged from their elution positions on HPLC (data not shown) (Bienkowski and Conrad, 1985). The tetrasaccharide component presumably derived from the reducing side of each of these original hexasaccharides was subjected to digestion with human liver ␣-iduronidase (Freeman and Hopwood, 1992) and Ampullaria ␤-glucuronidase (Tsukada and Yoshino, 1987) to identify the uronic acid residues at the nonreducing termini. After ␣-iduronidase digestion, a part (40, 57, or 60%) of the tetrasaccharide peak derived from Fr. b-15, -20, or -22 eluted 7-14 min earlier than the corresponding parent compound on HPLC. As representative chromatograms, those obtained with the tetrasaccharide from Fr. b-22 are shown in Fig. 4. Upon ␤-glucuronidase digestion, however, the tetrasaccharide fraction obtained from each of the three fractions was totally resistant to the action of the enzyme (data not shown). These results suggest that the nonreducing terminal uronic acid of the major tetrasaccharide obtained from Fr. b-15, -20, or -22 by nitrous acid treatment is not GlcA but rather IdoA. The reason for the partial insensitivity to the action of ␣-iduronidase is unclear at present but may have been due to side product(s) of the deamination reactions. It has been reported that the so-called ring contraction tetrasaccharides can be formed during deamination reactions (Bienkowski and Conrad, 1985). Based upon the above results, the structures of the major components in these fractions were deduced as follows: Fr Fr. b-24 was also degraded into a disaccharide and a tetrasaccharide component (data not shown) as judged from the gel filtration profiles of the deaminative cleavage products and the elution positions on the subsequent HPLC of the isolated di-and tetrasaccharides. The disaccharide eluted at around the position of ⌬HexA(2S)-anMan R (6S) on HPLC. Deaminative cleavage products of this fraction were not further analyzed because the internal uronic acid residues were easily identified as IdoA(2S) and nonsulfated GlcA by 500-MHz 1 H NMR analysis as described below. 500-MHz 1 H NMR Analysis-All the individual hexasaccharides were analyzed by 500-MHz 1 H NMR to confirm the structures proposed above. Chemical shifts were assigned by twodimensional homonuclear Hartmann-Hahn and correlation spectroscopy analyses (data not shown) as reported for the sulfated oligosaccharides isolated previously from heparin (Yamada et al., 1993) and heparan sulfate . The NMR data obtained in this study for the hexasaccharides are summarized in Table II.
The internal uronic acid residue of each isolated hexasaccharide was unambiguously identified by 500-MHz 1 H NMR spectroscopy based upon the chemical shifts of the anomeric proton signals and the coupling constants J 1,2 . Anomeric proton signals of an ␣IdoA and a ␤GlcA residue in heparin/heparan sulfate oligosaccharides are observed at around ␦ 5.2-5.0 and 4.7-4.5, respectively (Merchant et al., 1985;Yamada et al., 1995). The coupling constants J 1,2 of ␣IdoA and ␤GlcA in heparin/heparan sulfate oligosaccharides are approximately 3.0 and 8.0 Hz, respectively (Horne and Gettins, 1992;. In the spectrum of Fr. b-24, two internal uronic acid residues were identified as IdoA and GlcA based on the chemical shifts of the anomeric proton signals, at ␦ 5.184 and 4.576 (Fig. 5), and the coupling constants J 1,2 , 3.0 and 7.5 Hz, respectively. The chemical shifts of H-1 and H-2 of the IdoA residue were shifted downfield by approximately 0.2 and 0.6 ppm, respectively, when compared with those of the nonsulfated IdoA residue of the tetrasaccharides isolated from bovine kidney heparan sulfate , supporting the 2-sulfation of IdoA-4 of the compound in Fr. b-24 . Based upon these NMR data and the sequential arrangement of the disaccharide units determined by enzymatic analysis, the following structure is proposed for the major compound in this fraction: Fr. b-24, ⌬HexA(2S)-GlcN-(NS,6S)-IdoA(2S)-GlcNAc(6S)-GlcA-GlcN(NS,6S).
The two internal uronic acid residues of the hexasaccharide in Fr. b-15 were identified as IdoA and GlcA, based on the chemical shifts (␦ 5.024 and 4.566) of the anomeric proton signals, respectively. Analysis of the deamination products showed that the IdoA residue was located at position 4, suggesting in turn that the GlcA residue is located at position 2. Thus, the following structure is proposed for the major compound in this fraction: Fr. b-15, ⌬HexA(2S)-GlcN(NS,6S)-IdoA-GlcNAc(6S)-GlcA-GlcN(NS,6S).
Likewise, the structures of the major compounds in three other fractions were determined and summarized in Table III. Some of the chemical shifts of Fr. b-20 were not assigned due to the low quality of the spectra.

DISCUSSION
The five sulfated hexasaccharide structures isolated in this study share the pentasulfated hexasaccharide backbone ⌬HexA(2S)␣1-4GlcN(NS,6S)␣1-4IdoA␣/GlcA␤1-4GlcN(6S)-␣1-4GlcA␤1-4GlcN(NS) with one or two additional sulfate groups on GlcN-1, , have been isolated previously (Linhardt et al., 1992), whereas the other three  were isolated for the first time as discrete structures in this study. All the hexasaccharides contain the common trisulfated disaccharide unit on their nonreducing sides and the GlcN(NS) residue at their reducing termini, reflecting the substrate specificity of heparinase used for digestion of the starting heparin. These structural features are in good agreement with the established  Chemical shifts are given in ppm downfield from internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured indirectly relative to acetone (␦ 2.225 ppm) in 2 H 2 O at 26°C. The estimated error for the values to two decimal places was only Ϯ0.01 ppm because of partial overlap of signals, and that for the values to three decimal places was Ϯ0.002 ppm. Coupling constants J 1,2 (in Hz) of GlcA and IdoA residues are given in parentheses. specificity of heparinase that cleaves the glucosaminidic linkage in the GlcN(NS)␣1-4IdoA(2S) sequence in a polymer (Linker and Hovingh, 1984;Merchant et al., 1985) and that in the GlcN(NS)␣1-4IdoA(2S)␣1-4GlcN(NS,6S) sequence in small oligosaccharides . Heparin and heparan sulfate have been demonstrated to exhibit various biological activities (Kjellén and Lindahl, 1991). Especially their specific interactions with various growth factors have recently attracted much attention. However, the functional domain structures elucidated to date are limited to only a few examples, including the minimum pentasaccharide sequences for ATIII binding and basic fibroblast growth factor binding, which have been demonstrated as GlcN(6S)␣1-4GlcA␤1-4GlcN(NS,3S)␣1-4IdoA(2S)␣1-4GlcN(NS,6S) (Lindahl et al., 1983) and GlcA␤1-4GlcN(NS)␣1-4HexA1-4GlcN(NS)␣1-4IdoA(2S) , respectively. The oligosaccharide sequences for high affinity binding to acidic fibroblast growth factor, fibroblast growth factor 4, and hepatocyte growth factor have been partially characterized (Bâ rzu et al., 1989;Ishihara, 1994;Guimond et al., 1993;Lyon et al., 1994), but the essential sulfate groups in these sequences have not yet been identified.
The hexasaccharides isolated in this study appear to be large enough to potentially exhibit binding activities toward growth factors or other functional proteins. None of them, however, showed ATIII-mediated inhibition of factor Xa as examined according to Morita et al. (1977). Although the hexasaccharides in Fr. b-20 and -22 contain a 3-sulfated GlcN(NS) residue at their reducing termini, they lack a part of the minimum pentasaccharide sequence on their reducing sides, i.e. IdoA(2S)␣1-4GlcN(NS,6S). The isolated hexasaccharides are not expected to contain the functional domains for binding to basic fibroblast growth factor. The hexasaccharides, except for that in Fr. b-24, do not have an IdoA(2S) residue essential for binding to basic fibroblast growth factor. Although the hexasaccharide in Fr. b-24 contains an IdoA(2S) residue in its sequence, it lacks the disaccharide extension GlcA␤1-4GlcN(NS) of the basic fibroblast growth factor binding sequence on the nonreducing side. It remains to be determined whether the binding domains to the other growth factors or biologically active proteins are embedded in the isolated hexasaccharides.
The most interesting structural feature of the isolated hexasaccharides is that they include three overlapping pairs of structural variants with an apparent biosynthetic precursorproduct relationship for the ATIII-binding site. Structurally, the hexasaccharide in Fr. b-15 is a pro-form of that in Fr. b-22. The former lacks the 3-sulfate group on GlcN-1 of the latter. Likewise, the hexasaccharide in Fr. b-20 is a pro-form of that in Fr. b-22, the former lacking the 6-sulfate of GlcN-1 of the latter. The hexasaccharide in Fr. b-15 can be considered as a proform of that in Fr. b-24, where the former lacks the 2-sulfate on IdoA-4 of the latter. Among the above hexasaccharides, the previously isolated structures found in Fr. b-15 and -22 (Linhardt et al., 1992) have been subjects of much discussion as biosynthetic precursors and product, respectively, of the ATIIIbinding site as described below.
Only about one-third of chains of commercial porcine intestinal heparin contain an ATIII-binding site and have a high affinity for ATIII. The critical 3-O-sulfation of GlcN required for the ATIII-high affinity concludes the biosynthesis of the ATIII-binding site (Kusche et al., 1988). Based upon the isolation of a precursor tetrasaccharide sequence, which lacked the 3-O-sulfate group from heparin chains with ATIII-low affinity, Kusche et al. (1990) proposed that essentially each low affinity chain would contain a potential but not utilized 3-O-sulfation site. Linhardt et al. (1992) challenged this hypothesis, proposing that the existence of a low affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in    (Linhardt et al., 1992). the final step, but rather some earlier step involved in the formation of the precursor sites may be primarily responsible for high and low ATIII affinity heparins. Recently, Razi and Lindahl (1995) proposed an intriguing hypothesis that the 3-Osulfotransferase may be inhibited by a sulfated saccharide sequence outside the 3-O-sulfate acceptor region based upon the observation that an octasaccharide fraction isolated from ATIII-low affinity heparin, unlike low affinity heparin polysaccharide (Kusche et al., 1990), yielded high affinity components following incubation with a GlcN 3-O-sulfotransferase preparation.
The multiple pro-form structures demonstrated in this study probably do not represent precursors and products but rather reflect structural variants that have diverged during the modification reactions in the as yet unresolved but precisely programmed biosynthetic scheme. Practically, they will be valuable acceptor substrates for sulfotransferases to investigate such biosynthetic mechanisms by which the production of specific functional carbohydrate sequences is regulated. For example, it would be of interest to examine whether the hexasaccharide structures in Fr. b-15 and -20 serve as acceptor substrates for IdoA 2-O-sulfotransferase and GlcN 6-O-sulfotransferase (Habuchi et al., 1995) to produce the hexasaccharide structures found in Fr. b-24 and -22, respectively. It will be intriguing to evaluate these hexasaccharides as regulatory elements as well because they may control biosynthetic modifying enzymes.
They will also be useful for characterizing heparin/heparan sulfate-degrading enzymes including both the bacterial heparinase/heparitinases and mammalian heparanases. The former enzymes are essential tools for structural studies (Yoshida et al., 1989), whereas the latter have been implicated in tumor metastasis (for review see Nakajima et al. (1988)), T cell adhesion (Gilat et al., 1995), and chemoattractant functions (Hoogewerf et al., 1995).