Heparin-derived Oligosaccharides Research Articles

Glycosaminoglycans Facilitate Procathepsin B Activation through Disruption of Propeptide-Mature Enzyme Interactions

Lysosomal cysteine cathepsin B participates in numerous diverse cellular processes. In acquiring its activity, the proregion, which blocks the substrate-binding site in the proenzyme, needs to be cleaved off. Here we demonstrate that polyanionic polysaccharides, glycosaminoglycans (GAGs), can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B. We show that naturally occurring GAGs such as chondroitin sulfates and heparin, as well as the synthetic analog dextran sulfate, accelerate the processing in a concentration-dependent manner. Heparin oligosaccharides down to the size of tetrasaccharides were efficient in accelerating the procathepsin B processing, whereas disaccharides were without effect. Further, the ability of the GAGs to accelerate procathepsin B processing was sensitive to increasing NaCl concentrations, indicating that electrostatic interaction between the GAGs and procathepsin B are operative in the accelerating effect. Also the processing of the catalytic procathepsin B mutant by wild type cathepsin B was enhanced in the presence of GAGs, suggesting that GAGs induce a conformational change in procathepsin B, converting it into a better substrate. Site-directed mutagenesis showed that His(28), Lys(39), and Arg(40), located within the procathepsin B propeptide, have significant roles in the acceleration of procathepsin B activation induced by short GAGs. Because procathepsin B and GAGs often co-localize in vivo, we propose that GAGs may play a physiological role in the activation of procathepsin B.

Design, synthesis, FGF-1 binding, and molecular modeling studies of conformationally flexible heparin mimetic disaccharides

Disaccharide mimetics of a heparin sequence that binds to fibroblast growth factors were prepared by coupling a D-galactose donor with a methyl beta-D-gluco- or xylopyranoside acceptor. When fully sulfated, the glucose or xylose moieties exist in solution in equilibrium between the (4)C1 and (1)C4 conformers, as confirmed by 1H NMR spectroscopy, thus mimicking the conformationally flexible L-iduronic acid found in heparin. Docking calculations showed that the predicted locations of disaccharide sulfo groups in the binding site of FGF-1 are consistent with the positions observed for co-crystallized heparin-derived oligosaccharides. Predicted binding affinities are in accord with experimental Kd values obtained from binding assays and are similar to the predicted values for a model heparin disaccharide.

Modulatory effects of heparin and short-length oligosaccharides of heparin on the metastasis and growth of LMD MDA-MB 231 breast cancer cells in vivo

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I125 CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases.

The Heparin/Heparan Sulfate Sequence That Interacts with Cyclophilin B Contains a 3-O-Sulfated N-Unsubstituted Glucosamine Residue

Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH2), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH2-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.

On-line NMR detection of microgram quantities of heparin-derived oligosaccharides and their structure elucidation by microcoil NMR

The isolation and purification of sufficient quantities of heparin-derived oligosaccharides for characterization by NMR is a tedious and time-consuming process. In addition, the structural complexity and microheterogeneity of heparin makes its characterization a challenging task. The improved mass-sensitivity of microcoil NMR probe technology makes this technique well suited for characterization of mass-limited heparin-derived oligosaccharides. Although microcoil probes have poorer concentration sensitivity than conventional NMR probes, this limitation can be overcome by coupling capillary isotachophoresis (cITP) with on-line microcoil NMR detection (cITP-NMR). Strategies to improve the sensitivity of on-line NMR detection through changes in probe design and in the cITP-NMR experimental protocol are discussed. These improvements in sensitivity allow acquisition of cITP-NMR survey spectra facilitating tentative identification of unknown oligosaccharides. Complete structure elucidation for microgram quantities of the purified material can be carried out through acquisition of 2D NMR spectra using a CapNMR microcoil probe.

Heparin Binding Induces a Conformational Change in Pigment Epithelium-derived Factor

Pigment epithelium-derived factor (PEDF) is a noninhibitory serpin found in plasma and in the extracellular space. The protein is involved in different biological processes including cell differentiation and survival. In addition, it is a potent inhibitor of angiogenesis. The function is likely associated with binding to cell surface receptors in a heparin-dependent way (Alberdi, E. M., Weldon, J. E., and Becerra, S. P. (2003) BMC Biochem. 4, 1). We have investigated the structural basis for this observation and show that heparin induces a conformational change in the vicinity of Lys(178). This structural change was evident both when binding to intact heparin and specific heparin-derived oligosaccharides at physiological conditions or simply when exposing PEDF to low ionic strength. Binding to other glycosaminoglycans, heparin-derived oligosaccharides smaller than hexadecasaccharides (dp16), or type I collagen did not affect the structure of PEDF. The conformational change is likely to expose the epitope involved in binding to the receptor and thus regulates the interactions with cell surface receptors.

Stimulation of Hepatocyte Growth Factor Production by Heparin-derived Oligosaccharides

We previously reported that heparin post-transcriptionally stimulates the production of hepatocyte growth factor (HGF). In this study, we addressed the size-dependency of heparin fragments on the HGF-inducing activity aiming to obtain fragments without antiblood coagulant activity. Heparin fragments, produced by digestion with heparinase, were size-fractionated and tested for HGF-inducing activity in cultured human fibroblasts. The HGF-inducing activity deceased with the reduction in oligosaccharide size. Decasaccharides exerted an activity comparable with undigested heparin, while smaller oligosaccharides showed lesser activities. The anticoagulant activity of heparin fragments also decreased with size and anticoagulant activity of decasaccharides was <13% that of undigested heparin. Further fractionation of decasaccharides by anion-exchange chromatography revealed that most of the decasaccharides had HGF-inducing activity and the extent of sulfation was roughly related to the activity. The lack of N-sulfation in heparin markedly reduced HGF-inducing activity, whereas 2-O-desulfation or 6-O-desulation had a lesser influence. Moreover, an N-sulfated disaccharide showed significant HGF-inducing activity, suggesting the involvement of N-sulfation in HGF-inducing activity. Because of the much reduced anticoagulant activity, potential applications of heparin-derived oligosaccharides such as decasaccharides is considerable as a therapeutic agent for many diseases.

A Molecular Mechanism for the Heparan Sulfate Dependence of Slit-Robo Signaling

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.

Antiangiogenic Antithrombin Blocks the Heparan Sulfate-dependent Binding of Proangiogenic Growth Factors to Their Endothelial Cell Receptors

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic growth factors fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on endothelial cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit growth factor-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(121). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on endothelial cells by competing with the growth factors for binding the heparan sulfate co-receptor, which mediates growth factor-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

N-Glycosylation of Fibroblast Growth Factor Receptor 1 Regulates Ligand and Heparan Sulfate Co-receptor Binding

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.

Crystallographic analysis of calcium-dependent heparin binding to annexin A2.

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.

New Insights into the Inhibition of Human Neutrophil Elastase by Heparin

In the normal feedback mechanism of injury and repair in the lung, fragmented heparan sulfate proteoglycans (HSPGs) from damaged extracellular matrix and cells are believed to interact with elastases to limit their activity. An imbalance in the HSPG-elastase response may play an important role in situations where uncontrolled lung injury leads to diseases such as emphysema. To gain insight into this complex process of heparin and heparan sulfate regulation of elastases, an experimental study was undertaken to resolve the mechanism and structural requirements of heparin inhibition of human neutrophil elastase (HNE). Kinetic analyses were completed using in vitro assays with synthetic and insoluble elastin substrates in the presence of HNE and various heparin preparations (14-15 kDa; 17-19 kDa), heparin-derived oligosaccharides (4-22 saccharides), and chemically modified heparins (2-O-, 6-O-, O-, and N-desulfated). Results showed that heparin inhibits HNE by a tight-binding, hyperbolic, competitive mechanism, contrary to previous reports in the literature. A minimum length of at least 12-14 saccharides is required for inhibition, after which inhibitory activity increases with chain length (or molecular mass). Although all N- and O-sulfate groups contribute to inhibition, 2-O-sulfate groups are less critical than either N- or 6-O-sulfate groups, indicating that inhibitory activity is dependent upon the heparin fine structure. Molecular-docking simulations support the kinetic results and provide a plausible model for the size requirement, whereby positively charged, clamp-like regions at the ends of the interdomain crevice (elastase fold) are used by heparin to bridge the active site and inhibit activity.

Molecular weight dependent tissue factor pathway inhibitor release by heparin and heparin oligosaccharides

Heparin and low molecular weight heparins exert their vascular effects by mobilizing tissue factor pathway inhibitor (TFPI) from the vascular endothelium into the blood circulation. We compared the influence of molecular weight on the TFPI release by heparin and its fractions in a non-human primate model. Primates were treated with unfractionated heparin, a low molecular weight heparin (gammaparin), or a heparin-derived oligosaccharide mixture (C3). Endothelial TFPI release was determined using both immunologic and functional assays. After intravenous administration, all agents significantly increased TFPI levels ( p < 0.05) in a dose dependent manner. The increase produced by unfractionated heparin and gammaparin was greater than that by C3 at an equal dosage ( p < 0.05). With subcutaneous injection, all agents produced less TFPI release. Repeated administration of heparin-derived oligosaccharides gradually increased TFPI release. A 1.89 fold increase in TFPI levels was observed 4 days after C3 treatment (2.5 mg/kg). Our findings indicated that TFPI release is dependent on the molecular weight of heparin and its derivatives. Heparin oligosaccharides exert their vascular effects through increased TFPI release after long-term repeated administration.

Disease-associated Sequence Variations Congregate in a Polyanion Recognition Patch on Human Factor H Revealed in Three-dimensional Structure

Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site.

Gas-Phase and Solution Conformations of Selected Dimeric Structural Units of Heparin

The molecular structure of four dimeric units (D-E, E-F, F-G, and G-H) of the DEFGH structural unit of heparin, their anionic forms, and their sodium salts have been studied using the B3LYP/6-31+G(d) method. The optimized geometries indicate that the most stable structure of these dimeric units in neutral state is stabilized via "bifurcated" sodium bonds. The equilibrium structure of the biologically active anionic forms of the glycosaminoglycans studied changed considerably in a water solution. The stable-energy conformations around glysosidic bonds found for the individual dimeric species investigated are in agreement with the available experimental structural data for the structurally related heparin-derived oligosaccharides.

NMR characterization of the interaction between the C-terminal domain of interferon-gamma and heparin-derived oligosaccharides.

Interferons are cytokines that play a complex role in the resistance of mammalian hosts to pathogens. IFNgamma (interferon-gamma) is secreted by activated T-cells and natural killer cells. IFNgamma is involved in a wide range of physiological processes, including antiviral activity, immune response, cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes. IFNgamma activity is modulated by the binding of its C-terminal domain to HS (heparan sulphate), a glycosaminoglycan found in the extracellular matrix and at the cell surface. In the present study, we analysed the interaction of isolated heparin-derived oligosaccharides with the C-terminal peptide of IFNgamma by NMR, in aqueous solution. We observed marked changes in the chemical shifts of both peptide and oligosaccharide compared with the free state. Our results provide evidence of a binding through electrostatic interactions between the charged side chains of the protein and the sulphate groups of heparin that does not induce specific conformation of the C-terminal part of IFNgamma. Our data also indicate that an oligosaccharide size of at least eight residues displays the most efficient binding.

Structural determinants of heparan sulfate interactions with Slit proteins

We have previously demonstrated that the Slit proteins, which are involved in axonal guidance and related processes, are high-affinity ligands of the heparan sulfate proteoglycan glypican-1. Glypican–Slit protein interactions have now been characterized in greater detail using two approaches. The ability of heparin oligosaccharides of defined structure (ranging in size from disaccharide to tetradeccasaccharide) to inhibit binding of a glypican-Fc fusion protein to recombinant human Slit-2 was determined using an ELISA. Surface plasmon resonance (SPR) spectroscopy, which measures the interactions in real time, was applied for quantitative modeling of heparin–Slit binding on heparin biochips. Heparin was covalently immobilized on these chips through a pre-formed albumin–heparin conjugate, and the inhibition of Slit binding by heparin, LMW heparin, and heparin-derived oligosaccharides (di-, tetra-, hexa-, and octa-) was examined utilizing solution competition SPR. These competition studies demonstrate that the smallest heparin oligosaccharide competing with heparin binding to Slit was a tetrasaccharide, and that in the ELISA maximum inhibition (∼60% at 2 μM concentration) was attained with a dodecasaccharide.

Liquid Chromatography/Mass Spectrometry Sequencing Approach for Highly Sulfated Heparin-derived Oligosaccharides

Liquid chromatography/mass spectrometry (LC/MS) is applied to the analysis of complex mixtures of oligosaccharides obtained through the controlled, heparinase-catalyzed depolymerization of heparin. Reversed-phase ion-pairing chromatography, utilizing a volatile mobile phase, results in the high resolution separation of highly sulfated, heparin-derived oligosaccharides. Simultaneous detection by UV absorbance and electrospray ionization-mass spectrometry (ESI-MS) provides important structural information on the oligosaccharide components of this mixture. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. UV in combination with MS detection led to the identification of oligosaccharides arising from the original non-reducing end (NRE) of the heparin chain. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. Interestingly, 16 NRE oligosaccharides are observed, having both an even and an odd number of saccharide residues, most of which are not predicted based on biosynthesis or known pathways of heparin catabolism. Quantification of these NRE oligosaccharides afforded a number-averaged molecular weight consistent with that expected for the pharmaceutical heparin used in this analysis. Molecular ions could be assigned for oligosaccharides as large as a tetradecasaccharide, having a mass of 4625 Da and a net charge of -32. Furthermore, MS detection was demonstrated for oligosaccharides with up to 30 saccharide units having a mass of >10000 Da and a net charge of -60.

Inhibitory effect of heparin-derived oligosaccharides on secretion of interleukin-4 and interleukin-5 from human peripheral blood T lymphocytes

To investigate the inhibitory effect of heparin-derived oligosaccharides (Oligs) on secretion of interleukin-4 (IL-4) and interleukin-5 (IL-5) from human peripheral blood T lymphocytes (PBTLs). Oligs were prepared by three different heparin depolymerization methods and separated by gel filtration chromatography. PBTLs from ten adult patients with allergic eosinophilic gastroenteritis were treated with phytahematoagglutinin (PHA) and Oligs. The supernatants from the cell culture of PBTLs were harvested and subjected to the determination of IL-4 and IL-5 contents by ELISA method. At the concentration of 5 microg/mL, Oligs with different Mr had different effects on the secretion of IL-4 and IL-5. The tetrasaccharide with Mr of 1,142, produced by depolymerizing heparin with hydrogen peroxide, had the strongest inhibitory effect on the secretion of IL-4. It decreased the IL-4 content from 375.6+/-39.2 ng/L (PHA group) to 12.5+/-5.7 ng/L (P<0.01). The hexasaccharide with Mr of 1,806, produced by depolymerizing heparin with beta-elimination method, had the strongest inhibitory effect on the secretion of IL-5. It decreased the IL-5 content from 289.2+/-33.4 ng/L (PHA group) to 22.0+/-5.2 ng/L (P<0.01). The inhibitory activity of Oligs on the secretion of IL-4 and IL-5 from human PBTLs closely depends on their molecular structure, and there may be an essential structure to act as an inhibitor. The most effective inhibitors of IL-4 and IL-5 secretion are tetrasaccharides and hexasaccharides, respectively.

Controlled γ-ray irradiation of heparin generates oligosaccharides enriched in highly sulfated sequences

Controlled physical depolymerization of heparin was performed in aqueous solution in the presence of isopropanol by γ-irradiation. Isopropanol both makes selective the radical-induced scission of the glycosidic linkage involved in the depolymerization and acts as hydrogen donor scavenger of heparin intermediate radicals that usually result in UV-absorbing by-products. Several preparations of heparin-derived oligosaccharides (HDO) were reproducibly obtained from different unfractionated heparins (UFH). From each preparation, an intermediate fraction with an average molecular weight of about 2200 Da (γ-HDO) was isolated by gel permeation chromatography and its sulfation pattern was characterized by nuclear magnetic resonance spectroscopy. The comparison of the sulfation pattern of HDO with that of parent UFHs revealed a significant decrease in the relative content of d-glucuronic acid with respect to the total amount of uronic acid. Further γ-ray treatments of heparin chains that survived the first depolymerization procedure led to a further decrease of d-glucuronic acid and also slightly reduced the content of N-acetyl-glucosamine residues, thus enriching oligosaccharide chains in the highly sulfated sequences. MALDI mass spectrometric analysis of oligomeric components indicated that γ-HDO prevalently contains highly sulfated chains (tetra to decasaccharides, including both odd and even number of monosaccharidic units) chiefly constituted of repeating sequences of the trisulfated disaccharide l-iduronic acid 2-sulfate (1→4) d-glucosamine- N,6 disulphate. γ-Irradiation in the presence of isopropanol thus generated heparin fragments largely preserving the prevalent structure of the parent heparin and involved glucuronic acid as the major site of cleavage.