Heparinase-II-catalyzed degradation of N-propionylated heparin.

Currently there is great interest in the preparation of modified heparins and heparin-like polymers that possess specific and useful bioactivities. This paper demonstrates the potential of a particularly versatile endopolysaccharide lyase (heparinase II) as an analytical tool with which to assess both the chemical modification occurring during synthesis of such polymers and the actual primary structure of the final product of the enzyme activity. Additionally, the work widens our knowledge of the specificity range of this enzyme. The study involved a novel derivative of heparin containing the unnatural N-propionyl group, which was prepared from de-N-sulfated heparin. The extent of the chemical modification was followed throughout the preparation process by incubating samples with heparinase II and analyzing, with HPLC, the products of degradation catalyzed by the enzyme.

Heparinase II from Flavobacterium heparinum. HPLC analysis of the saccharides generated from chemically modified heparins.

Saccharides produced by the action of heparinase II on native pig mucosal heparin (heparin IS), 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-acetylheparin (heparin IVA) were analysed by reversed-phase HPLC using Spherisorb ODS2. Fractions obtained by gel filtration with Bio-Gel P-4 were similarly examined. Heparin IS gave delta UA-2S----GlcNS-6S (IS) as the major unsaturated disaccharide and lesser amounts of delta UA----GlcNS-6S (IIS), delta UA-2S----GlcNS (IIIS), delta UA----GlcNS (IVS), delta UA-2S----GlcNAc-6S (IA), delta UA----GlcNAc-6S (IIA), delta UA-2S----GlcNAc (IIIA) and delta UA----GlcNAc (IVA). Heparins IA, IVA and IVS gave as the predominant unsaturated disaccharide that corresponding to the major repeat structure of the polymer. These were respectively delta UA-2S----GlcNAc-6S (IA), delta UA-GlcNAc (IVA) and delta UA----GlcNS (IVS). Minor disaccharides from the heterogeneous structure in native pig heparin and from residual O-sulphates after the de-O-sulphating process were detected. Heparin IH was degraded more slowly than any of the N-substituted heparins. The predominant unsaturated disaccharide was IH, which was derived from the major repeating unit. In addition, disaccharides IIH, IIIH, IA, IIA and IVA were detected. Heparin IVH showed little degradation, the unsaturated disaccharide IVH not being detected after 24 h. Disaccharide IVA was obtained from the heterogeneous sequence in heparin IVH. Several higher oligosaccharides were identified in the gel-filtration fractions including saccharides from the linkage region (for heparin IS and IVA) and the anti-thrombin binding site (for heparin IS only). A tetrasaccharide and hexasaccharide, with the structures delta UA----GlcNAc----UA----GlcNAc and delta UA----GlcNAc----UA----GlcNAc----UA----GlcNAc, were present in the HPLC profiles of heparins IA and IVA.

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.

Flavobacterium heparinum sulphamidase for D-glucosamine sulphamate. Purification and characterisation.

A novel assay has been developed for 2-deoxy-2-sulphamido-D-glucose (GlcNS) sulphamidase from Flavobacterium heparinum. This has enabled the 1930-fold purification of the enzyme from a soluble fraction of bacterial homogenate. From SDS/polyacrylamide gel electrophoresis the enzyme was shown to have a relative molecular mass of 81,500. Ca2+ was essential for enzyme activity. Inorganic phosphate and sulphate inhibited activity by 28% and 29% respectively at 5 mmol dm-3. The purified sulphamidase had a pH optimum of 7.0 and a Km of 8.32 mumol dm-3 for GlcNS. The degradation of 2-deoxy-2-sulphamido-6-O-sulpho-D-glucose (GlcNS-6S) was also re-investigated. The two sulphate groups were hydrolysed sequentially in a single non-bifurcate manner, in contrast to previous reports [Dietrich, C.P., Silva, M.E. and Michelacci, Y.M. (1973) J. Biol. Chem. 248, 6408-6415].

Flavobacterium heparinum 6-O-sulphatase for N-substituted glucosamine 6-O-sulphate.

A specific glyco-6-O-sulphatase has been purified to homogeneity from Flavobacterium heparinum. The enzyme hydrolyses the 6-O-sulphates of 2-deoxy-2-sulphamido-6-O-sulpho-D-glucose (GlcNS-6S), 2-acetamido-2-deoxy-6-O-sulpho-D-glucose (GlcNAc-6S) and 2-amino-2-deoxy-6-O-sulphato-D-glucose (GlcN-6S). The activity was purified 2100-fold by successive chromatography on CM-Sepharose CL-6B, Sepharose CL-4B, hydroxyapatite and blue-Sepharose CL-6B. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed a protein of relative molecular mass 64000. Four novel assays were developed using 35S-labelled and 14C-labelled monosaccharides. The purified enzyme was free of all other known heparin-degrading enzymes. In particular this was the first resolution of the 6-O-sulphatase from the sulphamidase. Optimal activity was at pH 7.5. Enzyme activity was virtually unaffected by Na+ and K+ ions. Enhancements of activity of 12% and 30% were effected by Mg2+ and Ca2+ ions respectively. Inorganic phosphate and sulphate (both 0.005 mol dm-3) inhibited activity by 48% and 50% respectively. The Km value for the free amino substrate GlcN-6S was 1.35 mmol dm-3. In contrast the Km values for the GlcNAc-6S and GlcNS-6S were 54 mumol dm-3 and 16 mumol dm-3 respectively.

Flavobacterium heparinum 3-O-sulphatase for N-substituted glucosamine 3-O-sulphate.

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3-O-sulphate from 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose and 2-acetamido-2-deoxy-3-O-sulpho-D-glucose. The activity was purified 10 800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecylsulphate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56 000. Two novel assays were developed using 2-[14C]acetamido-2-deoxy-3-O-sulpho-D-glucose and 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose as respective substrates. The purified 3-O-sulphatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N-acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2-fold enhancement of activity was effected by 0.002 mol dm-3 Ca2+. Inorganic phosphate and sulphate inhibited 3-O-sulphatase activity. The Km value of the N-acetylated substrate was determined to be 42 mumol dm-3. No activity was detected with 2-amino-2-deoxy-3-O-sulpho-D-glucose.

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].

Beta-agarases I and II from Pseudomonas atlantica. Substrate specificities.

Beta-Agarase I and II were characterised by their action on agar-type polysaccharides and oligosaccharides. Beta-Agarase I, an endo-enzyme, was specific for regions containing a minimum of one unsubstituted neoagarobiose unit [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose], hydrolysing at the reducing side of this moiety. Yaphe demonstrated that agar was degraded by this enzyme to neoagaro-oligosaccharides limited by the disaccharide but with a predominance of the tetramer [Yaphe, W. (1957) Can. J. Microbiol. 3, 987-993]. Beta-Agarase I slowly degraded neoagarohexaose but not the homologous tetrasaccharide. [1-3H]Neoagarohexaitol was cleaved to neoagarotetraose and [1-3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to methylated, sulphated and unsubstituted neoagaro-oligosaccharides which were invariably terminated at the reducing end by unsubstituted neoagarobiose. The novel enzyme, beta-agarase II, was shown to be an endo-enzyme. Preliminary evidence indicated this enzyme was specific for sequences containing neoagarobiose and/or 6(1)-O-methyl-neoagarobiose. It degraded agar to neoagaro-oligosaccharides of which the disaccharide was limiting and predominant. Beta-Agarase II rapidly degraded isolated neogarotetraose and neoagarohexaose to the disaccharide. With [1-3H]neoagarohexaitol, exo-action was observed, the alditol being cleaved to neoagarobiose and [1-3H]neoagarotetraitol. Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol. Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24% of the starting carbohydrate. This fraction was almost exclusively disaccharides (22.4%) containing neoagarobiose (7.4%) and 6(1)-O-methyl-neoagarobiose (15%). Beta-Agarase II is probably the 'beta-neoagarotetraose hydrolase' reported by Groleau and Yaphe as an exoenzyme against neoagaro-oligosaccharides [Groleau, D. and Yaphe, W. (1977) Can. J. Microbiol. 23, 672-679].

beta-agarases I and II from Pseudomonas atlantica. Purifications and some properties.

The agarose-degrading system of Pseudomonas atlantica has been re-examined. In addition to the previously reported extracellular endo-beta-agarase [Yaphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333-335] a second, membrane-bound endo-enzyme activity, beta-agarase II has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose] and also to degrade partially 6-O-methylated agarose to neoagarobiose and 6(1)-O-methyl-neoagarbiose. Novel assays were devised for beta-agarase II and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of beta-agarase I and II. beta-Agarase I was purified 670-fold from the bacterial medium by a new method using ammonium sulphate precipitation and gel filtration on Sephadex G-100. The enzyme was resolved from the small amount of extracellular beta-agarase II. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32000. Activity was observed against agar over the pH range 3.0-9.0 and optimally at pH 7.0. The enzyme could be used indefinitely at 30 degrees C but only for up to 2 h at 40 degrees C. beta-Agarase II was partially purified (5-fold) from the soluble fraction of disrupted cells by chromatography on Sephadex G-100, hydroxyapatite and DEAE-Sepharose CL-6B. This preparation was free of beta-agarase I and disaccharidase. beta-Agarase II was stimulated by NaCl, optimally in the range 0.10-0.20 mol dm-3 (2.4-fold the activity at 0.010 mol dm-3 NaCl). Alkali earth metal (0.002 mol dm-3 CaCl2 or 0.005 mol dm-3 MgCl2) gave 1.2-fold the normal activity. Optimum activity was over pH 6.5-7.5.

Porphyran primary structure. An investigation using beta-agarase I from Pseudomonas atlantica and 13C-NMR spectroscopy.

Porphyran, a highly substituted agarose from Porphyra umbilicalis was degraded by highly purified beta-agarase I from Pseudomonas atlantica. This enzyme cleaved at the reducing side of units of beta-neoagarobiose (3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-beta-D-galactopyranose). The oligosaccharides were divided into fractions of low and high molecular weight by dialysis. The permeate (23% of total starting carbohydrate) was separated by ion-exchange into neutral and anionic fractions. Gel filtration of the neutral fraction (19%) resolved two major oligosaccharides. These were shown by 13C-NMR spectroscopy to be 6(3)-O-methyl-neoagarotetraose and 6(3),6(5)-di-O-methyl-neoagarohexaose. Gel filtration of the anionic oligosaccharides (3.3%) revealed two novel monosulphated tetrasaccharides, 6-O-sulphato-alpha-L-galacto-pyranosyl-(1 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactopyranose and its 6(3)-O-methylated derivative. The 13C-NMR data from the sulphated tetrasaccharides provided a novel reference which was used to characterise higher, partially sulphated fragments in the dialysis permeate. The fraction retained on dialysis (77%) had an average degree of polymerisation of 40 and was homologous with the high-molecular-weight anionic permeate. From 13C-NMR spectroscopy porphyran was found to comprise 49% sulphated disaccharide units and these were calculated to occur in stretches averaging 2.0-2.5 contiguous units.

Glycosulphatase from Pseudomonas carrageenovora. Purification and some properties.

A glycosulphatase present in the soluble fraction of disrupted Pseudomonas carrageenovora has been purified 500-fold by gel filtration on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose CL-6B. By dodecylsulphate/polyacrylamide gel electrophoresis the enzyme is practically homogeneous and has a molecular weight of 55 000. Conditions of optimal sodium chloride concentration and pH at 25 degrees C were 0.25--0.50 mol dm-3 and pH 7.0 respectively. The purified enzyme was inhibited by inorganic phosphate. Preparation is described of neocarrabiose 4-O-[35S]sulphate and neocarratetraose 4-O-[35S]sulphate from labelled Chondrus crispus. The purified glycosulphatase is active against both these substrates although only one of the two sulphate esters in the tetrasaccharide is hydrolysed. Analysis of the reaction products was by gel filtration, electrophoresis and 13C nuclear magnetic resonance spectroscopy. The results are consistent with the products of desulphation being respectively neocarrabiose and neocarratetraose 4-O-monosulphate with the sulphate ester proximal to the reducing end [3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-D-galactose 4-O-sulphate].

kappa-Carrageenase from Pseudomonas carrageenovora.

A kappa-carrageenase was isolated from the cell-free medium of cultured Pseudomonas carrageenovora. From dodecylsulphate/polyacrylamide gel electrophoresis, a single protein (identified as the kappa-carrageenase) was detected in the medium. Activity against nominal carrageenan types and inspection of the products indicate the enzyme to be a kappa-carrageenase. Purification is described here by ammonium sulphate precipitation and subsequent CM-Sepharose CL-6B ion-exchange chromatography. Molecular weight was estimated as 35,000 by dodecylsulphate/polyacrylamide gel electrophoresis. Products of degradation were analysed by gel filtration, spectrophotometric assays and 13C nuclear magnetic resonance. These results are consistent with the product of limit digest being neocarrabiose 4-O-sulphate.