The binding of human betacellulin to heparin, heparan sulfate and related polysaccharides

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.     

Distribution analysis of polysaccharides comprised of uronic acid-hexose/hexosamine repeating units in various shellfish species

Acidic polysaccharides are attractive functional ingredients in shellfish which are consumed as delicious and nutritious foods world widely. In the present study, acidic polysaccharides from 21 species of edible shellfish were characterized and quantified by analyzing their repeated disaccharides using the multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometer upon acid degradation and 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. A total of 6 glycosaminoglycans (GAGs) and 8 non-GAGs with repeated disaccharide units of a hexuronic acid linked to a hexosamine or a hexose were detected. Among them, chondroitin sulfate, heparin, →4)-β-GlcA-(1?→?2)-α-Man-(1?→?and →3)- β-GlcA-(1?→?3)-α-Gal-(1?→?were identified unambiguously by comparing with the references. The quantification results revealed that the contents of these polysaccharide varied greatly among shellfish species with a maximum over 100 mg/100 g. Furthermore, the dendrogram of hierarchical clustering analysis indicated that the composition of acidic polysaccharides in some shellfish species was related with the genetic relationship. Thus, the present study provides a more comprehensive knowledge about the distribution of acidic polysaccharides in various shellfish species.     

Macromolecular properties and end-group analysis of heparin isolated from bovine liver capsule.

Glycosaminoglycans were extracted from bovine liver capsule with 4 M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30% respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentation-equilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.     

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.     

Location of antithrombin-binding regions in rat skin heparin proteoglycans.

Rat skin heparin proteoglycan labelled biosynthetically with 35S was fractionated on a column of antithrombin-Sepharose into fractions with varying degrees of affinity for antithrombin. These were treated with NaOH to release heparin chains (Mr 60,000-100,000), by beta-elimination or incubated with serum to produce fragments of the same order of size as commercial heparin (Mr 5000-30,000), by endoglycosidase cleavage. Chains and fragments were then fractionated on antithrombin-Sepharose. The various fractions were deaminated with HNO2 at pH 1.5 followed by reduction with NaB3H4. Approx 90% of the incorporated 3H was associated with disaccharides. These were fractionated by high-performance ion-exchange chromatography. A unique minor component corresponding to the sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate) in the polysaccharide was found only in fractions with high affinity for antithrombin. The glucosamine residue linked to C-4 of this glucuronosyl unit was predominantly (or exclusively) N-sulphated rather than N-acetylated, pointing to a structural difference between the antithrombin-binding region of rat heparin and that of pig mucosal heparin. Calculations based on the distribution of the glucosaminyl 3-O-sulphate group showed that approximately two-thirds of the total antithrombin-binding regions present in the unfractionated material were accommodated by only 20% of the proteoglycan molecules, and by 10% of the polysaccharide chains. While most of the proteoglycan molecules thus lacked such regions (and hence affinity for antithrombin) a minor proportion of the polysaccharide chains contained on the average three binding regions per molecule. These findings support by direct chemical analysis an earlier proposal, based on anticoagulant activities of similar rat skin heparin fractions, that the distribution of antithrombin-binding sites in intact heparin proteoglycans is markedly non-random.     

Determination of the cross-linking effect of adipic acid dihydrazide on glycoconjugate preparation

The cross-linking effect of adipic acid dihydrazide (ADH) on polysaccharide derivatization can be evaluated by applying combination of elemental analysis and colorimetric assay. Elemental analysis is used for estimation of total ADH bound to polysaccharide and a colorimetric trinitrobenzene sulfonic acid assay is used to determine the part of ADH not involved in cross-linking. The difference of values expressed as molar ratios (per repeating unit) provides information on the amount of ADH involved in cross-linking the polysaccharides. Carboxymethylated polysaccharides were derivatized with different amounts of ADH to test the procedure. Analytical results showed that excess of ADH in the reaction only slightly decreased the cross-linking. The number of carboxyl groups remained unmodified even at high excess of ADH and high concentration of carbodiimide (EDC) coupling reagent.     

Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and Tivela mactroides

Heparin with high anticoagulant activity was isolated from the two marine clam species Anomalocardia brasiliana and Tivela mactroides. A large portion of the polysaccharide chains of both preparations bound with high affinity to immobilized antithrombin. Titrations monitored by tryptophan fluorescence showed that clam polysaccharide chains with Mr approximately 22,500 contained up to three binding sites for antithrombin and that the binding constants for the interaction of these chains with antithrombin were higher than those reported for mammalian heparin of comparable size. Structural analysis of clam heparin fractions and subfractions of clam heparin with differing affinity for immobilized antithrombin revealed the presence of large amounts (up to 25-30% of the total disaccharide units) of the 3-O-sulfated saccharide sequences (-GlcNSO3)-GlcA-GlcNSO3(3-OSO3)- and (-GlcNSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-, previously identified as unique markers for the antithrombin-binding region of heparin. The content of these saccharide sequences was found to increase with increasing affinity of the parent polysaccharide for antithrombin. Structural analysis of the clam heparins also demonstrated the occurrence of a novel saccharide sequence, tentatively identified as (-GlcNSO3)-IdA-GlcNSO3(3,6-di-OSO3)-, that has not previously been found in heparin or related polysaccharides. The contents of this latter sequence, at most 3-4% of the total disaccharide units, showed no correlation with the affinity for antithrombin.     

Controlled Photochemical Depolymerization of K5 Heparosan, a Bioengineered Heparin Precursor

Heparosan is a polysaccharide, which serves as the critical precursor in heparin biosynthesis and chemoenzymatic synthesis of bioengineered heparin. Because the molecular weight of microbial heparosan is considerably larger than heparin, the controlled depolymerization of microbial heparosan is necessary prior to its conversion to bioengineered heparin. We have previously reported that other acidic polysaccharides could be partially depolymerized with maintenance of their internal structure using a titanium dioxide-catalyzed photochemical reaction. This photolytic process is characterized by the generation of reactive oxygen species that oxidize individual saccharide residues within the polysaccharide chain. Using a similar approach, a microbial heparosan from Escherichia coli K5 of molecular weight >15,000 was depolymerized to a heparosan of molecular weight 8,000. The (1)H-NMR spectra obtained showed that the photolyzed heparosan maintained the same structure as the starting heparosan. The polysaccharide chains of the photochemically depolymerized heparosan were also characterized by electrospray ionization-Fourier-transform mass spectrometry. While the chain of K5 heparosan starting material contained primarily an even number of saccharide residues, as a result of coliphage K5 lyase processing, both odd and even chain numbers were detected in the photochemically-depolymerized heparosan. These results suggest that the photochemical depolymerization of heparosan was a random process that can take place at either the glucuronic acid or the N-acetylglucosamine residue within the heparosan polysaccharide.     

Collective sampling of intact anionic polysaccharide components and application in quantitative determination by LC-MS

Anionic polysaccharides, such as glycosaminoglycans (GAGs) and alginate, readily undergo source-induced fragmentation when analyzed by electrospray mass spectrometry with the use of high source cone voltage. The dissociation chemistry converts all components of a polysaccharide into a small set of structurally characteristic small saccharides. This chemistry enables the collective detection of a polysaccharide through the detection of one or more small saccharides. This ability, combined with the elution of polysaccharides as relatively compact bands using ion-pairing reverse phase liquid chromatography, created a unique opportunity for the development of LC–MS methods suitable for the quantitative analysis of intact anionic polysaccharides. Feasibility of this approach is demonstrated with a mixture of heparin, chondroitin sulfate A, and alginate.     

Interaction between cell-binding domain and extracellular matrix-binding domain of fibronectin determined by fluorescence depolarization

Interaction of domains in fibronectin was observed by photometry of fluorescence polarization of three kinds of dye; [N-(1-anilinonaphthyl-4)]maleimide (ANM tau = 5 ns), [N-(3-fluoranthyl)]maleimide (FAM tau = 20 ns), and [N-(3-pyrene)]maleimide (PRM tau = 100 ns). Each dye was labeled at a free sulfhydryl group in the cell-binding domain. Neither fluorescence of ANM with short fluorescent lifetime, FAM with long lifetime, nor PRM with longer fluorescent lifetime on fibronectin depolarized as much as the free dye. It was found that each dye was firmly fixed in the cell-binding domain. When heparin or gelatin was added in the solution of PRM-fibronectin complex, the fluorescence polarization tended to increase principally by combining heparin or gelatin to fibronectin. It was found that the rotation of whole or partial fibronectin containing the cell-binding domain through fluorescent lifetime of 100 ns was suppressed by combining of heparin or gelatin to fibronectin. When heparin or gelatin was added in the solution of ANM- or FAM-fibronectin complex, on the contrary, the fluorescence polarization tended to decrease, that is, slightly depolarize through the fluorescent lifetime of 5 or 20 ns, respectively. It was found that the rotation of the cell-binding domain, or of part of the fibronectin molecule containing the domain, was slightly promoted by combining heparin or gelatin to its domain. These results indicate that an interaction of the heparin- or gelatin-binding domain with the cell-binding domain was induced by the combining of heparin or gelatin to the respective domains.     

Properties of fucoidan from Cladosiphon okamuranus tokida in gastric mucosal protection

To elucidate the anti-ulcer potential of Cladosiphon fucoidan, anti-peptic activity, bFGF stabilizing activity and inflammatory properties of this and related substances were investigated. Anti-peptic activity was observed with this and other sulfated polysaccharides such as dextran sulfate, carrageenan, and Fucus fucoidan. However, non-sulfated polysaccharides such as mannan and dextran did not exert the anti-peptic activity. The loss of bFGF bioactivity was prevented by all sulfated polysaccharides tested except chondroitin sulfate, at pH 7.4 and at pH 4.0. At pH 2.0, only heparin protected the bFGF activity. The generation of superoxide by macrophages and PMNs was stimulated by dextran sulfate, carrageenan, and Fucus fucoidan, whereas Cladosiphon fucoidan, heparin and chondroitin did not. Dextran sulfate, carrageenan, and Fucus fucoidan also stimulated the secretion of TNFalpha from macrophages, while Cladosiphon fucoidan did not. Thus, Cladosiphon fucoidan is a sulfated polysaccharide without inflammatory action. These results suggest that Cladosiphon fucoidan is a safe substance with potential for gastric protection.     

Influences of anionic polysaccharides on DNA synthesis in isolated nuclei and by DNA polymerase α: Correlation of observed effects with properties of the polysaccharides

The basis of the differential effect of anionic polysaccharides on replicative DNA synthesis in liver and hepatoma cell nuclei was investigated. The differential effect of heparin was lost when more than 40% of its sulfate was removed. DNA synthesis in liver nuclei was optimally stimulated by heparin of molecular weight 22 600 and sulfate to hexosamine ratio 2.42, but inhibited by heparin of molecular weight 4300 and sulfate to hexosamine ratio 2.35. A heparin fragment (molecular weight 2800 and sulfate to hexosamine ratio 1.81), prepared by partial nitrous acid treatment was a potent inhibitor of DNA synthesis in hepatoma nuclei. There was no significant difference in the rate of entry of heparin or its subfractions into either liver or hepatoma nuclei. In both cases less than 15% of added polysaccharide entered the nuclei and only about 4.5% was found associated with the chromatin. The influence of the anionic polysaccharides on DNA synthesis was correlated with their ability to complex with histones as determined by relative light scattering in a laser nephelometer. The relative light scattered on mixing with histones (H1, H2A + H3, H4) was high for DNA synthesis stimulators (heparin, dextran sulfate); medium for DNA synthesis inhibitors (chondroitin 4- and 6-sulfates, heparan sulfate) and low for non-effectors (keratan sulfate, hyaluronic acid). Heparin and chondroitin sulfate H, which at low concentrations stimulate DNA synthesis in liver nuclei, inhibited DNA synthesis by calf thymus DNA polymerase α at all concentrations. This inhibition was not simply due to electrostatic interactions.     

白灵菇多糖对运动小鼠氧化应激的影响

体外研究表明,白灵菇多糖具有较高的清除自由基能力。然而,白灵菇多糖在体内对运动引起的氧化应激的影响尚不清楚。本研究将100只SPF级雄性昆明小鼠随机分为5组,低剂量组、中剂量组和高剂量组昆明小鼠分别按照50 mg·kg^-1·d^-1、100 mg·kg^-1·d^-1和200 mg·kg^-1·d^-1的剂量灌胃白灵菇多糖溶液,对照组和模型组昆明小鼠灌胃等体积的蒸馏水,连续灌胃4周。研究显示,白灵菇多糖溶液以浓度依赖性方式提高了小鼠的力竭游泳时间(p<0.05)。白灵菇多糖以浓度依赖性方式降低了小鼠血清AST、ALT和CK水平,并显著减少了骨骼肌的病理变化(p<0.05)。白灵菇多糖以剂量依赖方式升高力竭游泳小鼠体内SOD、CAT和GSH-PX水平,并降低了MDA水平(p<0.05)。此外,对小鼠灌胃白灵菇多糖可以剂量依赖方式提高小鼠血清总抗氧化活性(p<0.05)。上述研究表明,白灵菇多糖可有效提高力竭游泳小鼠的抗疲劳能力,减轻运动引起的心肌、肝脏、骨骼肌和氧化应激损伤,提高机体的抗氧化能力。     

Binding of heparin fractions and other polysulfated polysaccharides to plasma fibronectin: effects of molecular size and degree of sulfation of polysaccharides

Heparin was divided into four fractions on fibronectin-Sepharose. The higher affinity fraction for fibronectin was larger in molecular size, higher in sulfate content and higher in affinity for anti-thrombin III. Together with these heparin fractions, the following three series of heparin samples were examined to compare the affinity for fibronectin-Sepharose: four fractions separated on Sephadex G-100; five fractions separated on antithrombin III-Sepharose, and six partially and completely N-desulfated heparins. The result showed that the affinity of heparin for fibronectin was dependent exclusively on its molecular size, and that an appropriate level of sulfate content in heparin (1.9-2.4 mol/disaccharide) was essential for the affinity. The sulfated preparations of glycosaminoglycans (heparan sulfate, dermatan sulfate and chondroitin 4-sulfate) and neutral polysaccharides (amylose and dextran) having higher sulfate content than heparin were found to display higher affinity for fibronectin than heparin. This suggested that highly sulfated polysaccharides showed potent affinity irrespective of their polysaccharide structure. The sulfated chondroitin 4-sulfate having a sulfate content and molecular size comparable to those of heparin was inferior to heparin with respect to affinity. A competitive dissociation experiment indicated that heparin and other polysulfated polysaccharides share a common binding site on the fibronectin molecule.     

Oxidized chondroitin sulfate-cross-linked gelatin matrixes: a new class of hydrogels

A naturally occurring glycosaminoglycan such as chondroitin-6-sulfate was first converted in to its aldehyde derivative by periodate oxidation and used as a cross-linking agent for gelatin giving rise to a new class of hydrogels. Cross-linking was predominantly due to Schiff's base formation between the epsilon-amino groups of lysine or hydroxylysine side groups of gelatin and the aldehyde groups in oxidized chondroitin sulfate. The hydrogels were prepared from chondroitin sulfate with different degrees of oxidation and gelatin. They were characterized for degree of cross-linking, cross-linking density, equilibrium swelling, water vapor transmission rate, internal structure, and blood-compatibility. Degree of cross-linking of the gels determined by trinitrobenzene sulfonic acid assay showed that, the higher the degree of oxidation of the polysaccharide, the higher the degree of cross-linking. Examination of the internal structure by scanning electron microscopy showed that the hydrogels were highly porous in nature with interconnecting pores ranging from 50 to 200 mum. Equilibrium swelling showed that the gels retained about 90% water and did not undergo dehydration rapidly. The hydrogels were nontoxic and blood-compatible. Since an important phase of early wound healing has been shown to involve secretion of glycosaminoglycans such as chondroitin sulfate by fibroblasts which form a hydrophilic matrix suitable for remodeling during healing, this new class of hydrogels prepared from chondroitin sulfate and gelatin without employing any extraneous cross-linking agents are expected to have potential as wound dressing materials.     

巴氏蘑菇多糖对铅中毒大鼠脾脏细胞因子mRNA表达的影响

为了研究巴氏蘑菇Agaricus blazei多糖对铅中毒大鼠脾脏细胞因子mRNA表达的影响,进而探索巴氏蘑菇多糖对铅中毒大鼠的免疫调节作用。选用45日龄SD大鼠,随机分为6组,每组8只,雌雄各半。分别为正常对照组、多糖组、铅中毒模型组、铅+50mg/kg·d多糖试验组、铅+100mg/kg·d多糖试验组、铅+200mg/kg·d多糖试验组。铅中毒模型组、铅+多糖试验组分别给予含0.2%醋酸铅饮水,自由饮用。饲养60d后,采集脾脏,荧光定量RT-PCR对其细胞因子IL-1β、IL-2、IL-6、TNF-α     

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.     

Biosynthesis of heparin. Effects of n-butyrate on cultured mast cells

Murine mastocytoma cells were incubated in vitro with inorganic [35S]sulfate, in the absence or presence of 2.5 mM n-butyrate, and labeled heparin was isolated. The polysaccharide produced in the presence of butyrate showed a lower charge density on anion exchange chromatography than did the control material and a 3-fold increased proportion (54 versus 17% for the control) of components with high affinity for antithrombin. Structural analysis of heparin labeled with [3H] glucosamine in the presence of butyrate showed that approximately 35% of the glucosamine units were N-acetylated, as compared to approximately 10% in the control material; the nonacetylated glucosamine residues were N-sulfated. The presence of butyrate thus leads to an inhibition of the N-deacetylation/N-sulfation process in heparin biosynthesis, along with an augmented formation of molecules with high affinity for antithrombin. Preincubation of the mastocytoma cells with butyrate was required for manifestation of either effect; when the preincubation period was reduced from 24 to 10 h the effects of butyrate were no longer observed. Assays for microsomal N-acetylheparosan deacetylase activity failed to show any significant inhibition of the enzyme at butyrate concentrations well above those found to affect heparin biosynthesis in intact mastocytoma cells. Moreover, a polysaccharide formed on incubating mastocytoma microsomal fraction with UDP-[3H]glucuronic acid, UDP-N-acetylglucosamine, and 3'-phosphoadenylylsulfate in the presence of 5 mM butyrate showed the same N-acetyl/N-sulfate ratio as did the corresponding control polysaccharide, produced in the absence of butyrate. These findings suggest that the effect of butyrate on heparin biosynthesis depends on the integrity of the cell.     

Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides

A comprehensive study of the base hydrolysis of all phosphodiester bond-containing capsular polysaccharides of the 23-valent pneumococcal vaccine is described here. Capsular polysaccharides from serotypes 6B, 10A, 17F, 19A, 19F, and 20 contain a phosphodiester bond that connects the repeating units in these polysaccharides (also referred to as backbone phosphodiester bonds), and polysaccharides from serotypes 11A, 15B, 18C, and 23F contain a phosphodiester bond that links a side chain to their repeating units. Molecular weight measurements of the polysaccharides, using high performance size exclusion chromatography with tandem multiangle laser light scattering and refractive index detection, was used to evaluate the kinetics of hydrolysis. The measurement of molecular weight provides a high degree of sensitivity in the case of small extents of reaction, thus allowing reliable measurements of the kinetics over short times. Pseudo-first-order rate constants for these polysaccharides were estimated using a simple model that accounts for the polydispersity of the starting sample. It was found that the relative order of backbone phosphodiester bond instability due to base hydrolysis was 19A > 10A > 19F > 6B > 17F, 20. Degradation of side-chain phosphodiester bonds was not observed, although the high degree of sensitivity in measurements is lost in this case, due to the low contribution of the side chains to the total polysaccharide molecular weight. In comparison with literature data on pneumococcal polysaccharide 6A, 19A was found to be the more labile, and hence appears to be the most labile pneumococcal polysaccharide studied to date. The rate of hydrolysis increased at higher pH and in the presence of divalent cation, but the extent was lower than expected based on similar data on RNA. Finally, the differences in the phosphodiester bond stabilities were analyzed by considering stereochemical factors in these polysaccharides. These results also provide a framework for evaluation of molecular integrity of phosphodiester-bond-containing polysaccharides in different solution conditions.