Structure of the terminal reducing heptasaccharide of polysaccharide 1 isolated from the Bordetella pertussis endotoxin.

The tetrasaccharide beta-D-glucopyranosyl-(1,3)-beta-D-glucopyranuronyl-(1, 2)-L-glycero-alpha-D-manno-heptopyranosyl-(1,5)-3-deoxy-D-manno-2- octulosonic acid was isolated after treatment of polysaccharide 1 of Bordetella pertussis endotoxin with nitrous acid. Taking into account previously identified di- and trisaccharide fragments and analytical data obtained for the intact polysaccharide 1, we present the structure of a heptasaccharide that is thought to represent the region immediately adjacent to the hydrophobic (lipid A) moiety of lipopolysaccharide 1 of the B. pertussis endotoxin. This heptasaccharide represents 50 to 60% of the complete polysaccharide structure.     

Properties and participation of “free” endotoxin in the toxic activity ofMorganella morganii

"Free" and "bound" Morganella morganii endotoxin was characterized by chemical (determination of proteins, saccharides and 3-deoxy-2-octulosonic acid) and immunochemical (double-diffusion test, immunoelectrophoresis, tandem crossed immunoelectrophoresis) methods. Chemical analysis showed that "free" endotoxin contains more protein and phosphorus and less saccharides than bound endotoxin. Immunochemical tests revealed differences in the structure of polysaccharide portions of both endotoxins, and, on the other hand, identity of certain antigenic determinants. Free endotoxin possessed a higher biological activity.     

3-Deoxy-2-octulosonic acid 5-phosphate: a component of the endotoxin of Bordetella pertussis.

Upon hydrolysis with 2 N hydrochloric acid for 2 h, a 3-deoxy-octulosonic acid 5-phosphate was released from the endotoxin of Bordetella pertussis. The structure of the compound was established through chemical degradation. By periodate treatment of the intact endotoxin it was shown that positions 7 and 8 of the bound octulosonic acid phosphate were free, which, if present in a cyclic form, must be a pyranoside.     

Studies of polysaccharide fractions from the lipopolysaccharide of Pseudomonas aeruginosa N.C.T.C. 1999.

Two polymeric water-soluble fractions were isolated by gel filtration after mild acid hydrolysis of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 1999. The fraction of higher molecular weight retained the O-antigenic specificity of the lipopolysaccharide and may be 'side-chain' material. This fraction was rich in N (about 10%) and gave several basic amino compounds on acid hydrolysis; fucosamine (at least 2.8% w/w) was the only specifc component identified. The fraction of lower molecular weight was a phosphorylated polysaccharide apparently corresponding to 'core' material. The major components of this fraction and their approximate molar proportions were: glucose (3-4); rhamnose (1); heptose (2); 3-deoxy-2-octulonic acid (1); galactosamine (1); alanine (1-1.5); phosphorus (6-7). In the intact lipopolysaccharide this fraction was probably linked to lipid A via a second residue of 3-deoxy-2-octulonic acid, and probably also contained additional phosphate residues and ethanolamine. The residues of 3-deoxy-2-octulonic acid were apparently substituted in the C-4 or C-5 position, and the phosphorylated heptose residues in the C-3 position. The rhamnose was mainly 2-substituted, though a little 3-substitution was detected. The glucose residues were either unsubstituted or 6-substituted. Four neutral oligosaccharides were produced by partial acid hydrolysis and were characterized by chemical, enzymic, chromatographic and mass-spectrometric methods of analysis. The structures assigned were: Glcpalpha1-6Glc; Glcpbeta1-2Rha; Rhapalpha1-6Glc; Glcpbeta1-2Rhapalpha1-6Glc. The galactosamine was substituted in the C-3 or C-4 position, the attachment of alanine was indicated, and evidence that the amino sugar linked the glucose-rhamnose region to the 'inner core' was obtained.     

Studies on the chemical structure of the core-lipid A region of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305. Detection of a new 2-octulosonic acid interlinking the core oligosaccharide and lipid A component

Lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305 was treated with acid (0.1 M HCl, 100 degrees C, 1 h). The product obtained (LPSdegr) was subjected to various modification and degradation procedures including reduction, hydrazinolysis and strong acid hydrolysis. Methylation analysis of purified part structures revealed the presence of a 4'-phosphorylated (beta 1'-6)-linked D-glucosamine disaccharide (lipid A backbone), which carried in position 6' a hitherto unknown 2-octulosonic acid (OclA) in highly acid-stable linkage. It was further shown that OclA is substituted in position 5 by a glucose tetramer, the reducing residue of which is phosphorylated. The hydrophilic region of the LPSdegr could thus be characterized as a phosphorylated heptasaccharide of the following structure: (Formula: see text).     

Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin

Due to the formation of micelles, severance of the hydrophilic (poly- or oligosaccharide) and hydrophobic ("Lipid A") domains of bacterial lipopolysaccharides at pH 3.4 or 4.5 and 100 degrees is slow and sometimes does not proceed at all; partially degraded fragments are usually formed. At pH 3.4 (100 degrees) in aqueous 1% sodium dodecylsulphate (SDS), both lipopolysaccharides of the Bordetella pertussis endotoxin are cleaved within 20-30 min, but 80% of the glycosidically bound phosphate present in the hydrophobic domain is lost. Other endotoxins behave similarly. At pH 4.5 (100 degrees) and in the absence of detergent, hydrolysis of the glycosidic bonds of 3-deoxy-D-manno-2-octulosonic acid residues of the B. pertussis endotoxin is negligible but, in aqueous 1% SDS, severance of the two regions of LPS 1 is complete within 1 h (that of LPS-2 requires 3-4 h), and the glycosidically bound phosphate of the isolated hydrophobic region is preserved. Comparison of the rate of acid-catalysed hydrolysis of the glycosidically bound phosphate present in this "isolated Lipid A" preparation with that of 2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-alpha- and -beta-D-glucopyranose 1-phosphates established that the former 1-phosphate was the alpha anomer.     

Identification of 3-deoxy-manno-2-octulosonic acid, 3-deoxy-5-O-methyl-manno-2-octulosonic acid and 3-deoxy-lyxo-2-heptulosaric acid in the cell wall (theca) of the green alga Tetraselmis striata Butcher (Prasinophyceae)

The main constituent of the cell wall complex carbohydrate of the scaly green alga Tetraselmis striata Butcher is shown to be 3-deoxy-manno-2-octulosonic acid (42%). In addition two other 2-keto-sugar acids are present, namely, 3-deoxy-5-O-methyl-manno-2-octulosonic acid (7%), the first methylated derivative of 3-deoxy-manno-2-octulosonic acid found in nature, and 3-deoxy-lyxo-2-heptulosaric acid (11%). The characterization of the three 2-keto-sugar acids has been carried out on the corresponding methyl ester methyl glycosides using GLC-MS and 500-MHz 1H-NMR spectroscopy, and on the corresponding reduced alditol acetates using GLC-MS. Other monosaccharides occurring in the cell wall are D-galacturonic acid (14%), D-galactose (4%), D-gulose (2%), D-glucose (1%) and L-arabinose (1%).     

Isolation,purification, and characterization of flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae)

Summary Flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae) were isolated, purified by isopycnic cesium chloride-gradient and zonal sucrose gradient centrifugation and their structure and biochemical composition investigated. Three types of flagellar scales were purified to more than 90% purity, a fourth type up to 75% purity. In addition to the previously known types of flagellar scales (pentagonal scales, rod-shaped scales, hair-scales), a novel scale type (i.e., the knotted scales) was discovered. New information about the asymmetric structure of the rod-shaped scales is presented and consequently they are renamed man scales. Flagellar scales consist mainly of carbohydrate (50–70%), significant amounts of protein (11% of dry weight) were found only in pentagonal scales. The main sugars (90%) of the pentagonal and man scales are the unusual 2-keto-sugar acids 3-deoxy-5-O-methyl-2-octulosonic acid (5 OMeKDO), 3-deoxy-2-heptulosaric acid (DHA), and 3-deoxy-2-octulosonic acid (KDO), the knotted scales contain as major sugars galactose and arabinose in addition to KDO and 5 OMeKDO but lack DHA. 13 major polypeptides were identified in flagellar scales by one-dimensional SDS-PAGE, 11 of these are of high molecular mass (>116 kDa). While the majority of polypeptides was found associated with pentagonal scales, at least 4 polypeptides were tentatively assigned to the hair-scales and knotted scales.Abbreviations CSF crude scale fraction - PS pentagonal scales - MS man scales - HS hair-scales - KS knotted scales - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - DHA 3-deoxylyxo-2-heptulosaric acid - 5 OMeKDO 3-deoxy-5-O-methyl-manno-2-octulosonic acid - KDO 3-deoxy-manno-2-octulosonic acid - GA Golgi apparatus     

Induction by lipopolysaccharide of intracellular and extracellular interleukin 1 production: analysis with synthetic models

An attempt was made to identify the molecular structures that are present in bacterial LPS and induce the production of intracellular and extracellular pools of IL 1 by peritoneal macrophages of the mouse and by human monocytes. Activities of glycolipids and carbohydrates prepared by synthesis, and structurally related to the hydrophobic (Lipid A) and to the polysaccharide (PS) regions of LPS were compared with those induced by Bordetella pertussis endotoxin and by fragments derived therefrom. Both isolated regions of this LPS (PS and Lipid A) were able to induce IL 1 synthesis by monocytes and macrophages. Among the synthetic glycolipids employed, propyl-2-deoxy-2-[(3R)-3-hydroxytetrade-canamido]-4-O-pho sph ono-6-O-tetradecanoyl-beta-D-glucopyranoside (glycolipid M9) induced IL 1 secretion more efficiently than Lipid A and LPS, whereas the amounts of intracellular IL 1 produced upon induction by these three substances were comparable. Macrophages from C3H/HeJ mice were unresponsive to Lipid A and to glycolipid M9, but produced IL 1 when incubated with PS or with a hydrophilic fragment isolated after methanolysis of the endotoxin. However, all synthetic derivatives of 3-deoxy-D-manno-2-octulosonic acid (KDO) used in this study failed to induce IL 1 production by both mouse macrophages and human monocytes. The implications of these findings for a more precise comprehension of the molecular mechanism of LPS-induced activation of macrophages, and the relations between the molecular structures required for the induction of IL 1 production vs cytostatic activity in macrophages, are discussed.     

A novel glycosphingolipid from gram-negative aquatic bacteria.

The chloroform-methanol extractable lipids of the Gram-negative fresh-water bacteria Arcocella aquatica NO-502 and Flectobacillus major FM were found to contain an unusual ninhydrin-positive glycolipid. It was purified by two-stage silica gel-column chromatography. By the use of IR and (1)H-NMR spectroscopy, mass spectrometry and chemical-degradation experiment, the lipid was established to be 1-O-monoglycosyl ceramide, the carbohydrate moiety of which was the alpha-pyranose-ring form of 7-desoxy-7-amino-D-manno-heptulosonic acid, or 1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranose. The ceramide portion consisted mainly (by 95% in the A. aquatica glycolipid and 80% in the F. major glycolipid) of 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenine. The minor molecular species differed from the major one only in fatty acid structure. The glycolipid accounted for 8 and 11% of the total lipids extracted from A. aquatica NO-502 and F. major FM cells, respectively.     

Structural studies on the O-antigen of Aeromonas salmonicida

Lipopolysaccharide from a strain of Aeromonas salmonicida salmonicida was isolated from cells by the aqueous phenol method in 2.3% yield (based on dry weight of bacteria). Hydrolysis of the lipopolysaccharide in 1% acetic acid afforded O-polysaccharide (19% by weight), core-oligosaccharide (12.2%) and lipid A (44.6%). Analysis indicated that 3-deoxy-D-manno-2-octulosonic acid was absent from the lipopolysaccharide and that no low-molecular-weight compounds were released by the mild hydrolysis. The O-polysaccharide had the monosaccharide composition of rhamnose, glucose and N-acetylmannosamine in molar ratio of 1.0:1.58:0.83. 75% of the N-acetylmannosamine residues were substituted at position 4 by O-acetyl groups. Hydrolysis of the methylated polysaccharide proved to be both difficult and dependent on the method of hydrolysis chosen, in all cases a partially methylated disaccharide of rhamnose and N-acetylmannosamine was identified in the hydrolysate. Methylation analysis, periodate oxidation and proton magnetic resonance analysis were used to confirm the structure of the repeating unit as: (formula; see text).     

Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. III. Reappraisal of established methods

Established methods for analysis of components of lipopolysaccharides were assessed. Optimal release of glucosamine from lipopolysaccharide occurs after hydrolysis in 6 M hydrochloric acid at 100°C for 4 h and fatty acids are best released by treatment with boron trifluoride/methanol at 100°C for 6 h. The semicarbazide assay for 3-deoxy-d-manno-octulosonic acid was modified to give results comparable to those from the periodate/thiobarbituric acid method. It was concluded that each molecule of lipopolysaccharide from Salmonella minnesota R595 contains two octulosonic acid residues and only four fatty acids, on average. There are two amide-linked hydroxyacids, together with, on average, 0.5 residues of ester hydroxyacid and a total of 1.5 residues of ester-linked normal fatty acids. This conclusion differs from the accepted view of Salmonella lipid A, but is supported by NMR results.     

Studies of the polysaccharide fraction from the lipopolysaccharide of Pseudomonas alcaligenes

Studies of the lipopolysaccharide of Pseudomonas alcaligenes strain BR 1/2 were extended to the polysaccharide moiety. The crude polysaccharide, obtained by mild acid hydrolysis of the lipopolysaccharide, was fractionated by gel filtration. The major fraction was the phosphorylated polysaccharide, for which the approximate proportions of residues were; glucose (2), rhamnose (0.7), heptose (2-3), galactosamine (1), alanine (1), 3-deoxy-2-octulonic acid (1), phosphorus (5-6). The heptose was l-glycero-d-manno-heptose. The minor fractions from gel filtration contained free 3-deoxy-2-octulonic acid, P(i) and PP(i). The purified polysaccharide was studied by periodate oxidation, methylation analysis, partial hydrolysis, and dephosphorylation. All the rhamnose and part of the glucose and heptose occur as non-reducing terminal residues. Other glucose residues are 3-substituted, and most heptose residues are esterified with condensed phosphate residues, possibly in the C-4 position. Free heptose and a heptosylglucose were isolated from a partial hydrolysate of the polysaccharide. The location of galactosamine in the polysaccharide was not established, but either the C-3 or C-4 position appears to be substituted and a linkage to alanine was indicated. In its composition, the polysaccharide from Ps. alcaligenes resembles core polysaccharides from other pseudomonads: no possible side-chain polysaccharide was detected.     

Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Location of acyl groups at the lipid A backbone

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12 degrees C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetra-decanoic acid at GlcN II is positionally replaced by delta 9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to GlcN II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to GlcN I are free or only partially substituted.     

Lipopolysaccharide in the outer membrane of the filamentous prochlorophyte Prochlorothrix hollandica

Abstract A lipopolysaccharide (LPS) fraction was isolated from Prochlorothrix hollandica by hot phenol/water extraction. Negatively stained preparations of an aqueous LPS dispersion showed the triple-layered appearance of the LPS aggregates. Glucose (main sugar), rhamnose, fucose, galactose, mannose, xylose, and 3- O -methyl-xylose were found as the constituents of the polysaccharide moiety. Glucosamine and the 3-hydroxy fatty acids, 3-OH-16:0, 3-OH-14:0, and the rarely detected iso-3-OH-15:0, constitute the lipid A of the LPS. l -glycero- d -manno-heptose and 3-deoxy- d -manno-2-octulosonic acid (dOclA), typical components of inner core oligosaccharides from enterobacterial LPS, were lacking in the isolated LPS fraction from Prochlorothrix hollandica .     

Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021

K polysaccharides (KPSs) of Sinorhizobium meliloti strains are strain-specific surface polysaccharides analogous to the group II K antigens of Escherichia coli. The K(R)5 antigen of strain AK631 is a highly polymerized disaccharide of pseudaminic and glucuronic acids. During invasion of host plants, this K antigen is able to replace the structurally different exopolysaccharide succinoglycan (EPS I) and promotes the formation of a nitrogen-fixing (Fix(+)) symbiosis. The KPS of strain Rm1021 is a homopolymer of 3-deoxy-D-manno-2 octulosonic acid (Kdo). The Kdo polysaccharide is covalently linked to the lipid anchor, has a low molecular weight (LMW), and is symbiotically inactive. On introduction of the Rm41-specific rkpZ gene into strain Rm1021, a modified KPS is expressed that is able to substitute EPS I during symbiosis with the host plant. To better understand the nature of modification conferred by rkpZ, we performed a structural analysis of the KPS using nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS), and gas chromatography (GC-MS). The modified KPS retained primary polyKdo structure, but its degree of polymerization (DP) and level of production were increased significantly. In contrast to the wild-type polyKdo, only a part of polyKdo was lipidated. Shorter polysaccharide chains were lipid-free, whereas longer polysaccharide chains were lipidated. Sinorhizobium meliloti Rm1021 was found to carry two paralogs of rkpZ. Both genes are involved in polyKdo production, but they only show partial functional activity as compared with the rkpZ of Rm41.     

Structural analysis of the lipid A component of Campylobacter jejuni CCUG 10936 (serotype O:2) lipopolysaccharide. Description of a lipid A containing a hybrid backbone of 2-amino-2-deoxy-D-glucose and 2,3-diamino-2,3-dideoxy-D-glucose

The chemical structure of Campylobacter jejuni CCUG 10936 lipid A was elucidated. The hydrophilic backbone of the lipid A was shown to consist of three (1----6)-linked bisphosphorylated hexosamine disaccharides. Neglecting the phosphorylation pattern, a D-glucosamine (2-amino-2-deoxy-D-glucose) disaccharide [beta-D-glucosaminyl-(1----6)-D-glucosamine], a hybrid disaccharide of 2,3-diamino-2,3-dideoxy-D-glucose and D-glucosamine [2,3-diamino-2,3-dideoxy-beta-D-glucopyranosyl-(1----6)-D-glucosamine], and a 2,3-diamino-2,3-dideoxy-D-glucose disaccharide were present in a molar ratio of 1:6:1.2. Although the backbones are bisphosphorylated, heterogeneity exists in the substitution of the polar head groups. Phosphorylethanolamine is alpha-glycosidically bound to the reducing sugar residue of the backbone, though C-1 is also non-stoichiometrically substituted by diphosphorylethanolamine. Position 4' of the non-reducing sugar residue carries an ester-bound phosphate group or is non-stoichiometrically substituted by diphosphorylethanolamine. By methylation analysis it was shown that position 6' is the attachment site for the polysaccharide moiety in lipopolysaccharide. These backbone species carry up to six molecules of ester- and amide-bound fatty acids. Four molecules of (R)-3-hydroxytetradecanoic acid are linked directly to the lipid A backbone (at positions 2, 3, 2', and 3'). Laser desorption mass spectrometry showed that both (R)-3-hydroxytetradecanoic acids linked to the non-reducing sugar unit carry, at their 3-hydroxyl group, either two molecules of hexadecanoic acid or one molecule of tetradecanoic and one of hexadecanoic acid. It also suggested that the (R)-3-(tetradecanoyloxy)-tetradecanoic acid was attached at position 2', whereas (R)-3-(hexadecanoyloxy)-tetradecanoic acid was attached at position 3', or at positions 2' and 3'. Therefore, the occurrence of three backbone disaccharides differing in amino sugar composition and presence of a hybrid disaccharide differentiate the lipid A of this C. jejuni strain from enterobacterial and other lipids A described previously.     

Identification of 2-amino-6-O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-D-glucose as a major constituent of the hydrophobic region of the Bordetella pertussis endotoxin

2-Amino-6-O-(2-amino-2-deoxy-β- d-glucopyranosyl)-2-deoxy- d-glucose substituted on the amino group of the reducing 2-amino-2-deoxy- d-glucose unit by a 3-hydroxytetradecanoyl group was shown to be a major constituent of the “Lipid A” fragment obtained by acid hydrolysis of the Bordetella pertussis endotoxin.     

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli. and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.