Heparinase II from Flavobacterium heparinum. Action on chemically modified heparins

Five chemically modified heparins were derived from native pig mucosal heparin (pig heparin Is). These were de-N-sulphated heparin (heparin IH), N-acetylheparin (heparin IA), de-N/O-sulphated heparin (heparin IVH), de-O-sulphated heparin (heparin IVs) and de-O-sulphated N-acetyl-heparin (heparin IVA). Their structures were studied by 13C-NMR spectroscopy at 90.56 MHz. Native heparin and the derivatives were incubated with Flavobacterium heparinase II at 25 degrees C. The progress of degradation was followed by the delta A235 and the final composition examined by gel filtration with Bio-Gel P-4. Native heparin (Is) was readily degraded by heparinase II and, with the exception of heparin IVH for which degradation was negligible, the chemically modified derivatives were also degraded. Approximately 90% of the saccharides from heparins Is, IA, IVs and IVA were disaccharides and tetrasaccharides. For heparin IH, which was degraded more slowly, the proportion was 65%. Heparins Is, IVs and IVA underwent initial rapid degradation. The digestion of heparin Ia proceeded rapidly after an initial lag phase. The undegraded polymers produced similar elution profiles from Bio-Gel P-4. Following the action of heparinase II on heparins Is, IA, IVs and IVA, the elution profiles revealed a major peak of disaccharides and minor peaks of higher oligomers. The profile of heparin IH revealed a greater proportion of intermediate-molecular-mass saccharides. Our results demonstrate a broad specificity for heparinase II. It is capable of lysing both N-acetylated and N-sulphated heparins independent of O-sulphation. Heparinase II will also degrade heparin derivatives that are non-N-substituted provided that they are O-sulphated.     

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.     

Chemical Microdetermination of Heparin in Plasma

A new, simple, and highly sensitive method for the determination of heparin has been established. Heparin was first converted into unsaturated disaccharides through the action of heparin lyases I, II, and III. A major trisulfated unsaturated disaccharide product results, consistent with structural analysis of a number of pharmaceutical heparins using one- and two-dimensional¹H NMR spectroscopy. This disaccharide was analyzed by HPLC using fluorometric postcolumn derivatization. The correlation between the amount of this trisulfated unsaturated disaccharide and anticoagulant activity of heparin as measured by anti-IIa was determined. The analysis of these pharmaceutical heparins showed a linear correlation between both HPLC and bioassay. This HPLC method was then applied to a pharmacokinetic study of heparin intravenously administered to rabbits.     

Flavobacterium heparinum 2-O-sulphatase for 2-O-sulphato-delta 4,5-glycuronate-terminated oligosaccharides from heparin

The glycosulphatase which hydrolyses the 2-O-sulphate of the disaccharide, 4-deoxy-2-O-sulphato-alpha-L-threohex-4-enopyranosyl uronic acid-(1----4)-2-deoxy-2-sulphamido-6-O-sulphato-D-glucose (delta UA-2S----GlcNS-6S), has been isolated from the soluble fraction of disrupted Flavobacterium heparinum. The activity was purified 3300-fold by chromatography on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and blue-Sepharose CL-6B. From sodium dodecylsulphate/polyacrylamide gel electrophoresis, the enzyme was homogeneous and of 62000 Mr. A novel assay was devised using the de-N-sulphonated [1-3H]alditol, 4-deoxy-2-O-sulphato-alpha-L-threo-hex-4-enopyranosyl uronic acid-(1----4)-2-amino-2-deoxy-6-O-sulphato-D-[1-3H]glucitol (delta UA-2S----[1-3H]GlcNH2-ol-6S). This alditol was shown by 13C-NMR to be desulphated in the analogous manner to the original reducing trisulphated disaccharide. The purified 2-O-sulphatase was completely free of heparinase I, heparinase II (heparitinase), chondroitinases AC, chondroitinase B, the delta 4,5-glycuronidase for heparin delta 4,5-disaccharides, the 6-O-sulphatase and the 2-sulphamidase. It was optimally active over the range pH 5.5-6.5 and was practically unaffected by Na, K, Ca or Mg ions. Inorganic phosphate inhibited the activity. The Km value for the alditol substrate was 1.22 mmol dm-3. Using 13C-NMR, the 2-O-sulphatase was found to hydrolyse the analogous esters of higher delta 4,5-oligosaccharides from heparin. This contrasts with the findings of other authors [Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973) J. Biol. Chem. 248, 6408-6415].     

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound ah-sepha-rose 4b

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono(“phospho”)-cellulose. The heparinase preparation thus obtained was free from sulfoamidase for 2-deoxy-2-sulfoamino-D-glucose (GlcN-2S), sulfatase for 2-amino-2-deoxy-6-O-sulfo D-glucose (GlcN-6S), as well as (δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin. The remaining sulfatase for 4-deoxy-α-L-thero-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) in the heparinase preparation was removed by affinity chromatography with dermatan sulfate-bound AH-Sepharose 4B coated with dermatan sulfate. The heparitinase preparation separated by column chromatography on hydroxyla patite was purified by affinity chromatography with heparin-bound AH-Sepharose 4B coated with heparin. Sulfatase for 2-amino-2-deoxy-6-O-sulfo-D-glucose (GlcN-6S) and δ_4,5glycosiduronase for the unsaturated disaccharides obtained from heparin were removed by this chromatography. Sulfatase for 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid 2-sulfate (δUA-2S) remaining in the heparitinase preparation was finally removed by column chromatography on hydroxylapatite. The recoveries of the purified preparations of heparinase and heparitinase were estimated to be 39 and 50%, respectively, from the crude enzyme fractions obtained by the first column chromatography on hydroxyl- patite. The purified heparinase and heparitinase were free from all enzymes that could degrade the sulfated unsaturated disaccharides produced from heparin with heparinase.     

Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.     

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/l, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.     

Unsaturated disaccharides in the enzymatic digests of heparan sulfates

High performance liquid chromatography was performed by an ion-pair reversed-phase method of six standard unsaturated disaccharides derived from heparan sulfate and heparin. Separation of delta Di-GlcNAc, delta Di-GlcN(2S), delta Di-GlcNAc(6S), delta Di-GlcN(2,6- or 2,2'-diS) and delta Di-GlcN(2,6,2'-triS) was achieved on a column of Jasco SC-02 with 10 mM tetrabutylammonium phosphate (pH 7.0) containing 30 or 47% methanol as a mobile phase. delta Di-GlcN(2,6-diS) and delta Di-GlcN(2,2'-diS) were separated on the same column with 35 mM triethylamine phosphate (pH 5.3). Four preparations (BL-1.0-1, BL-1.0-2, BL-1.0-3, and BL-1.25-1) separated from crude bovine lung heparan sulfate, a standard bovine lung heparan sulfate (BL-ST), bovine kidney heparan sulfate 1.0 M Fr and 1.25 M Fr (BK-1.0 and BK-1.25), and porcine kidney heparan sulfate 1.0 M Fr (PK-1.0) were digested with a mixture of heparinase, and heparitinases 1 and 2. The resulting foregoing unsaturated disaccharides in the digests were analyzed by the above HPLC procedures. The proportions of the unsaturated disaccharides in the digests of BL-1.25-1 and BL-ST were similar, but those of the others differed from each other. It is noteworthy that delta Di-GlcNAc plus delta Di-GlcNAc(6S) in the digest of BL-1.0-1 was approximately 95% of the total unsaturated disaccharides. Small amounts of delta Di-GlcN (2,6,2'-triS) were found in all the samples. It was found that delta Di-GlcN(2,2'-diS) was a prominent component in the disulfated unsaturated disaccharides from BL-1.25-1 and BK-1.25.     

Enzymatic preparation of heparin disaccharides as building blocks in glycosaminoglycan synthesis.

Pharmaceutical heparin and heparan sulfate, isolated from a side-stream of a commercial heparin manufacturing process, have been enzymatically depolymerzed with heparin lyases obtained from Flavobacterium heparinun. Heparin afforded a trisulfated disaccharide product that was recovered from the reaction mixture using gel permeation chromatography. Heparan sulfate afforded unsulfated disaccharide that was conveniently recovered from the product mixture by ion exchange chromatography. Both disaccharides were obtained in gram amounts at 90% or higher purity. Both enzymatically prepared disaccharides were chemically protected to prepare building blocks required for the future chemical synthesis of therapeutically valuable heparin oligosaccharides.     

Synthesis of chondroitin sulfate D and heparin proteoglycans in murine lymph node-derived mast cells. The dependence on fibroblasts

Proteoglycans synthesized in cultured mast cells derived from horse serum-immunized lymph node cells were analyzed. Treatment of the 35S-proteoglycans extracted from these cells with either chondroitinase ABC or AC resulted in 95% +/- 7% and 84% +/- 7%, respectively (mean +/- S.E., n = 3), of the radioactivity associated with disaccharides eluting in the included volume of PD-10. The 35S-proteoglycans were not hydrolyzed by nitrous acid elimination treatment. The chondroitinase ABC-generated disaccharides were analyzed by aminocyano high performance liquid chromatography. 35S-Disaccharides eluted in a major peak at a retention time of 8.1 min, corresponding to the disaccharide of chondroitin 4-sulfate proteoglycan (delta Di-4S), and a second peak at 12 min, corresponding to the disaccharide of chondroitin sulfate D proteoglycan (delta Di-diSD). Further treatment with chondro-4-sulfatase did not affect the retention time of the disaccharide corresponding to delta Di-diSD whereas this peak disappeared after the digested proteoglycan was treated either by chondro-6-sulfatase or by both sulfatases. Therefore, this disaccharide was identified as chondroitin sulfate D. Quantification of the radiolabeled disaccharides showed that delta Di-diSD contributed 20% +/- 2% (n = 3) of the total sulfated disaccharides of the chondroitin sulfate of these cultured cells. The role of fibroblasts in inducing the shift of chondroitin sulfate D into heparin proteoglycan in these mast cells was also investigated by using three types of monolayers: mouse embryonic skin fibroblasts (MESF), rat embryonic skin fibroblasts (RESF), and 3T3 fibroblasts. 35S-Proteoglycans that were extracted from the lymph node-derived mast cells cultured for 30 days on MESF and on 3T3 fibroblast monolayers were 93% +/- 4% and 30% +/- 7% (n = 3) susceptible to nitrous acid elimination, respectively. No degradation by nitrous acid was observed in 35S-proteoglycans extracted from cells cultured on RESF monolayer. Since the MESF was found to be the most potent monolayer in the induction of heparin synthesis, the kinetics of changes in the synthesis of proteoglycan types were determined in lymph node-derived mast cells cultured on MESF for up to 30 days. It was found that the synthesis of chondroitin sulfate gradually declined whereas that of heparin starting between 4 and 7 days after plating gradually increased. From the 17th day on, only the synthesis of heparin was detected.     

Glycosaminoglycan composition of the large freshwater mollusc bivalve Anodonta anodonta

In this paper, glycosaminoglycans from the body of the large freshwater mollusc bivalve Anodonta anodonta were recovered at about 0.6 mg/g of dry tissue, composed of chondroitin sulfate (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of a fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of a more sulfated, slow-moving component having a greater molecular mass. The chondroitin sulfate was composed of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74. Heparin was subjected to the oligosaccharide mapping after treatment with heparinase and then separation of the resulting unsaturated oligosaccharides by SAX-HPLC. A heparin sample from Anodonta anodonta showed a degree of sulfation similar to that of bovine mucosal heparin because of the presence of approximately the same mol % of the trisulfated disaccharide (DeltaUA2S(1-->4)-alpha-D-GlcN2S6S), a slight modification of the other oligosaccharides, and a significant increase of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (-->4)-beta-D-GlcA(1-->4)-alpha-D-GlcN2S3S6S(1-->) part of the ATIII-binding region. However, the anticoagulant activity of mollusc heparin was quite similar to that of pharmaceutical grade heparin. The data obtained again emphasize the heterogeneity of GAGs from molluscs.     

Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum: evidence for the presence of a high content of antithrombin III binding site

Heparin with high anticoagulant activity (activated partial thromboplastin time of 347 +/- 56.4 and anti-Xa activity of 317 +/- 48.3) was isolated from the marine clam species Tapes phylippinarum in an amount of approximately 2.1 mg/g dry animals. Agarose-gel electrophoresis showed a high content of the slow-moving heparin component (22 +/- 6.8%) and 78 +/- 5.4% of the fast-moving species. An average molecular mass of 13,600 was calculated by PAGE analysis, whereas a number average molecular weight Mn value of 10,700, a weight average molecular weight Mw of 14,900, and a dispersity index Mn/Mw of 1.386 were obtained by high-performance size-exclusion chromatography. Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by strong anion exchange-HPLC revealed the presence of large amounts (more than 130% than standard pharmaceutical heparin obtained from bovine intestine) of the oligosaccharide sequence bearing part of the ATIII-binding region, DeltaUA2S (1-->4)-alpha-D-GlcN2S6S (1-->4)-alpha-L-IdoA (1-->4)-alpha-D-GlcNAc6S (1-->4)-beta-D-GlcA (1-->4)-alpha-D-GlcN2S3S6S in the T. phylippinarum heparin, in comparison with bovine mucosal heparin and a sample of porcine mucosal heparin previously published. Furthermore, as expected from the oligosaccharide compositional analysis, due to the presence of a great mol % (80.6%) of the trisulfated disaccharide DeltaUA2S(1-->4)-alpha-D-GlcN2S6S, mollusc heparin is a more sulfated polysaccharide than bovine mucosal heparin (73.5%) and a sample of porcine mucosal (72.8%) heparin previously reported. To our knowledge, this is the first article describing a clam heparin having the ATIII binding site mainly identical to that of human and porcine intestinal mucosal heparins and bovine intestinal mucosal heparin but different from that found in beef lung heparin.     

Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.     

Heparin and heparan sulfate disaccharides bind to the exchanger inhibitor peptide region of Na+/Ca2+ exchanger and reduce the cytosolic calcium of smooth muscle cell lines. Requirement of C4-C5 unsaturation and 1--> 4 glycosidic linkage for activity

Heparin and heparan sulfate fragments, obtained by bacterial heparinase and heparitinases, bearing an unsaturation at C4-C5 of the uronic acid moiety, are able to produce up to 80% reduction of the cytosolic calcium of smooth muscle cell lines. Unsaturated disaccharides from chondroitin sulfate, dermatan sulfate, and hyaluronic acid are inactive, indicating that, besides the unsaturation of the uronic acid, a vicinal 1 --> 4 glycosidic linkage is needed. An inverse correlation between the molecular weight and activity is observed. Thus, the ED(50) of the N-acetylated disaccharide derived from heparan sulfate (430 Da) is 88 microm compared with 250 microm of the trisulfated disaccharide (650 Da) derived from heparin. Except for enoxaparin (which contains an unsaturation at the non-reducing end and 1 --> 4 glycosidic linkage), other low molecular weight heparins and native heparin are practically inactive in reducing the cytosolic calcium levels. Thapsigargin (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor), vanadate (cytoplasmic membrane Ca(2+)-ATPase inhibitor), and nifedipine and verapamil (Ca(2+) channel antagonists) do not interfere with the effect of the trisulfated disaccharide upon the decrease of the intracellular calcium. A significant decrease of the activity of the trisulfated disaccharide is observed by reducing extracellular sodium, suggesting that the fragments might act upon the Na(+)/Ca(2+) exchanger promoting the extrusion of Ca(2+). This was further substantiated by binding experiments and circular dichroism analysis with the exchanger inhibitor peptide.     

Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz 1H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as delta UAp2S (1----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D-GlcNp2S3S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid). The 1H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D- GlcNp2S3S6S(1----4)-alpha-L-IdoAp2S(1----4)-alpha-D-GlcNp 2S6S(1----. This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.     

Mass spectrometric evidence of heparin disaccharides for the catalytic characterization of a novel endolytic heparinase

Heparin like glycosaminoglycans (HLGAGs) are struc-turally complex linear polysaccharides composed of re-peating disaccharide unit of uronic (α-L-iduronic or β-D-glucuronic) acid linked 1→4 to α-D-glucosamine, whichis a highly variable sulfation pattern and ascribes to eachglycosaminoglycan (GAG) chain a unique structuralsignature. This signature dictates specific the GAG-pro-tein interactions underlying critical biological processesrelated to cell and tissue functions [1]. Only in fe…     

Chondroitinase C from Flavobacterium heparinum.

A chondroitinase that acts upon chondroitin sulfate C and hyaluronic acid was isolated from Flavobacterium heparinum. This enzyme was seperated from constitutional chondroitinase AC and an induced chondroitinase B also present in extracts of F. heparinum previously grown in the presence of chondroitin sulfates A, B or C. The enzyme acts upon chondroitin sulfate C producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-6S), and upon hyaluronic acid producing unsaturated nonsulfated disaccharide (delta Di-OS). Chondroitin sulfate A is also degraded producing oligosaccharides and delta Di-6S but not delta Di-4S. The chondroitinase C is also distinguished from the chondroitinases B and AC by several properties, such as effect of ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The substrate specificity of the chondroitinase C is different from that of any other chondroitinase or hyaluronidase described so far.     

Novel heparin degradation products. Isolation and characterization of novel disaccharides and oligosaccharides produced from heparin by bacterial degradation

1. Heparin was degraded by enzymes of adapted Flavobacterium heparinum. Several degradation products were separated by combined Sephadex-gel filtration and paper chromatography, and chemically analysed. 2. These products were identified as glucosamine 2,6-disulphate, saturated disaccharides constituted of uronic acid and glucosamine and containing two and three sulphate residues, and tetra- and hexa-saccharides with the same basic disaccharide units. 3. The implications of these findings with respect to the present knowledge of heparin structure and its enzymic degradation are discussed.     

Analysis of unsaturated disaccharides from glycosaminoglycuronan by high-performance liquid chromatography

A rapid and simple analytical method for unsaturated disaccharide isomers formed by enzymatic digestion from hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin by high-performance liquid chromatography using an amine-bound silica column with a linear gradient of sodium dihydrogen phosphate was developed. The analyses were performed on isomers of two groups belonging to the chondroitin sulfate family and the heparin sulfate family. In both families, disaccharide isomers eluted in the order non-, mono-, di-, and trisulfated disaccharides by elevating salt concentrations. The method was applied to the analysis of constituent disaccharides of representative sulfated glycosaminoglycans, which proved that most constituents could be quantified separately. This method is advantageous in that enzymatic digests can be applied directly on a column without any pretreatment and good resolution of several disaccharides can be obtained by one chromatography.     

Structure of heparin. Characterization of the products formed from heparin by the action of a heparinase and a heparitinase from Flavobacterium heparinum.

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 (O-(alpha-L-ido-4-enepyranosyluronic acid 2-sulfate)-(1leads to 4)-2sulfoamino-2-deoxy-D-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 disaccharide and disulfated disaccharide (O-(alpha-D-glyco-4-enepyranosyluronic acid)-(1leads to 4)-2-sulfoamino-2-deoxy-D-glucose 6-sulfate) in equal amounts. These and other results indicate that the tri- and disulfated disaccharides 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.