The influence of corticosteroids on the release of novel biomarkers in human endotoxemia Objective Sepsis intervention studies need better patient stratification methods , and one way to realize this is the introduction of stable biomarkers . A set of recently developed novel biomarkers , based upon precursor-fragments of short-lived hormones , was previously shown to be increased during sepsis . However , it is not known whether these biomarkers are influenced by sepsis intervention strategies . Therefore we investigated the markers in a model of human endotoxemia intervened by increasing doses of prednisolone . Introduction During the past decade numerous sepsis intervention strategies have been introduced ; only few of them , however , have shown beneficial effects . Major problems in sepsis intervention trials were mixed results and unclear risk stratification methods . The lack of clear stratification methods was especially disturbing , since the effectiveness of intervention in these studies correlated closely with disease severity and risk of mortality -LSB- 1 -RSB- . Within this context , there is a need for biomarkers to predict disease severity and mortality risk . During sepsis a wide range of inflammatory proteins is released and in theory all could be used as a biomarker . The vast majority of these proteins is biologically active though and as a consequence rapidly cleared from the circulation . A promising new approach is the measurement of stable prohormone fragments of bioactive peptide-hormones -LSB- 2 -- 4 -RSB- . In theory , these fragments often have no known biological function during sepsis and are therefore less rapidly cleared and less sensitive to small homeostatic changes -LSB- 5 -- 7 -RSB- . The most prominent prohormone sepsis marker is procalcitonin -LRB- PCT -RRB- , which represents the precursor hormone of calcitonin . Its diagnostic and prognostic properties were superior to C-reactive protein -LRB- CRP -RRB- in multiple studies -LSB- 8 -RSB- . In addition , PCT responded to sepsis intervention strategies and represented a promising marker for monitoring of treatment effect -LSB- 9 , 10 -RSB- . In recent years , additional assays have been developed for measurement of stable prohormone fragments , such as mid-regional pro-adrenomedullin -LRB- MR-proADM -RRB- -LSB- 5 -RSB- , derived from the precursor of adrenomedullin -LRB- ADM -RRB- ; mid-regional pro-atrial natriuretic peptide -LRB- MR-proANP -RRB- , derived from the precursor of atrial natriuretic peptide -LRB- ANP -RRB- -LSB- 6 -RSB- ; and C-terminal proAVP -LRB- CT-proAVP -RRB- , derived from the precursor of arginine -- vasopressin -LRB- AVP ; also known as the antidiuretic hormone -RRB- -LSB- 7 -RSB- . Levels of these biomarkers were higher in samples from patients with sepsis -LSB- 2 -- 4 -RSB- . Yet it is not known whether these levels are influenced by a potential therapeutic sepsis intervention . In order to investigate the response of the novel biomarkers to a sepsis intervention strategy we applied the model of human endotoxemia and pre-treated subjects by increasing doses of corticosteroids , which is one of the few relatively successful , and among the most cost-effective , septic shock intervention strategies to date -LSB- 11 -RSB- . Methods Subjects and study design The study was designed according to the requirements of the Declaration of Helsinki and approved by the institutional scientific and ethics committees , and written informed consent was obtained from all subjects . The study was performed simultaneously with an investigation of the effects of prednisolone on activation of the cytokine network , coagulation and hemodynamic variables in the same cohort -LSB- 12 -RSB- . Thirty-two healthy male volunteers -LSB- mean -LRB- ± SE -RRB- age 23.9 ± 0.7 years -RSB- were admitted to a hospital clinical research unit . Screening tests were all normal . Prednisolone -LRB- prednisolone sodium phosphate oral solution 5 mg/ml ; prepared by institutional pharmacy -RRB- was administered orally at a dose of 0 , 3 , 10 or 30 mg -LRB- n = 8 per group -RRB- at 2 h prior to LPS injection . All participants were challenged at t = 0 h with LPS -LRB- Escherichia coli lipopolysaccharide , lot G ; US Pharmacopeia , Rockville , MD -RRB- as a bolus intravenous injection at a dose of 4 ng/kg , which is a standardized dose -LSB- 13 -RSB- . Assays Blood was collected at intervals from 2 h before LPS injection to 24 h thereafter . Blood was immediately centrifuged -LRB- 4 °C , 10 min , 3000 rpm -RRB- and plasma was stored at -- 20 °C until assayed . Uptake of orally administered prednisolone was measured by HPLC-MS/MS technology -LRB- CombinatoRx , Inc. , Boston , MA , USA -RRB- . MR-proADM , MR-proANP , CT-proAVP and PCT were measured by chemiluminescence label-coated tube-based sandwich immunoassays -LRB- Brahms Diagnostica , Berlin , Germany -RRB- as previously described -LSB- 5 -- 7 , 14 -RSB- . Statistical analysis Differences between treatment groups were analyzed using a non-parametric mixed model approach -LRB- repeated-measures ANOVA -RRB- . The effects reported are treatment effects adjusted for time effects . p values < 0.05 were considered statistically significant . Values presented are given as mean ± SE . Results Pharmacokinetic data As shown in Fig . 1 , uptake of orally administered prednisolone was demonstrated by a dose-dependent increase in plasma levels during endotoxemia . The levels peaked from 1 h before LPS administration to 1 h after LPS administration -LRB- peak levels 302 ± 34 ng/ml , 190 ± 19 ng/ml and 71 ± 5 ng/ml in the groups receiving prednisolone 30 , 10 and 3 mg respectively ; t = -- 1 h -RRB- . Fig. 1Prednisolone . Mean -LRB- ± SE -RRB- values of prednisolone concentrations after LPS administration -LRB- 4 ng/kg IV , t = 0 h -RRB- to healthy male volunteers , preceded by oral administration of prednisolone 3 mg -LRB- circles -RRB- , 10 mg -LRB- triangles -RRB- , or 30 mg -LRB- squares -RRB- at t = -- 2.5 h Novel prohormone biomarkers MR-proADM levels increased after LPS administration from 0.31 ± 0.02 nmol/l at baseline to a peak value of 1.05 ± 0.11 nmol/l in the control group at t = 4 h -LRB- Fig . 2 -RRB- . The rise was inhibited after prednisolone 30 mg and 10 mg pre-treatment -LRB- levels at t = 4 h : 0.65 ± 0.06 nmol/l and 0.65 ± 0.1 nmol/l respectively ; p < 0.05 -RRB- , but not after the low dose of prednisolone 3 mg . MR-proANP levels rose from 42.4 ± 2.2 pmol/l at baseline to 118.4 ± 17.2 pmol/l in the control group at t = 4 h . These levels were reduced after prednisolone 30 mg and 10 mg -LRB- peak levels : 91.5 ± 9.9 pmol/l and 64.8 ± 5.6 pmol/l at t = 2 h and t = 4 h , respectively ; p < 0.05 -RRB- . CT-proAVP levels showed a modest increase from 12.5 ± 4.0 pmol/l to 18.0 ± 4.8 pmol/l in the control group at t = 4 h . The rise was inhibited after prednisolone 30 mg -LRB- peak level : 6.1 ± 1.0 pmol/l ; p < 0.05 -RRB- and not significantly changed by 10 mg -LRB- peak level : 10.6 ± 3.4 pmol/l ; p < 0.1 -RRB- . In contrast , prednisolone 3 mg pre-treatment was associated with enhanced CT-proAVP release -LRB- peak level 34.7 ± 11.5 pmol/l ; p < 0.05 -RRB- . PCT levels increased from a baseline level of 0.04 ± 0.02 ng/ml to a peak value of 16.7 ± 2.6 ng/ml in the control group . Peak PCT levels all occurred at t = 24 h and were not significantly affected by prednisolone pre-treatment -LRB- 11.9 ± 3.0 ng/ml , 13.1 ± 2.5 ng/ml and 17.6 ± 3.5 ng/ml in the 30 , 10 and 3 mg group , respectively -RRB- . Fig. 2Novel prohormone biomarkers . Mean -LRB- ± SE -RRB- values of MR-proADM , MR-proANP , CT-proAVP and PCT after LPS administration -LRB- 4 ng/kg IV , t = 0 h -RRB- to healthy male volunteers , preceded by oral administration of prednisolone 3 mg -LRB- open circles -RRB- , 10 mg -LRB- triangles -RRB- , 30 mg -LRB- squares -RRB- or , in the control group , 0 mg -LRB- filled circles -RRB- at t = -- 2.5 h. * p < 0.05 vs. control group . NS , non significant Discussion The primary objective of this study was to determine the effect of increasing doses of prednisolone on the release of novel biomarkers during human endotoxemia . We show here that prednisolone pharmacologic concentrations peaked at the time of LPS infusion and that the release of MR-proADM , MR-proANP and CT-proAVP was subsequently inhibited in a dose-dependent way . The levels of PCT were not significantly inhibited within the study period of 24 h. However , previously inhibition of PCT was shown by anti-inflammatory agents within a period of 168 h -LSB- 9 -RSB- . The levels of CT-proAVP were , remarkably , increased after prednisolone 3 mg , yet the reason for this remains unclear because the pattern was unique to this biomarker . The increased levels of MR-proANP , MR-proADM and CT-proAVP reflect activation of their mature counterparts . Difficulties in measurement of the mature hormones made it difficult to determine their roles during sepsis -LSB- 5 -- 7 -RSB- . The currently investigated biomarkers are produced after proteolytic processing of the same prohormone precursors as the mature hormones ; thus , they reflect production levels of mature hormones in stoichiometric concentrations -LSB- 7 , 15 -RSB- . In theory , the biomarker assays may measure levels of both cut prohormone fragments and full-length prohormones . However , the meaning of this relationship remains unclear in plasma because no well-defined methods are currently available for measurement of full-length prohormone plasma levels . The biomarker levels in this study were dose-dependently influenced by corticosteroid intervention , which is important for treatment effect monitoring . The development of new biomarkers is needed for improvement of sepsis stratification methods , which influence trial outcomes . Several sepsis intervention strategies have shown promise -LSB- 16 -RSB- ; in this study corticosteroids were selected because they aim primarily at inhibition of the immune response , and for this intervention in particular , effects are dose-dependent and improvement of patient stratification methods is urgently needed -LSB- 11 , 17 -RSB- . Prednisolone is among the most frequently applied corticosteroids -LSB- 18 -RSB- ; the oral route is convenient and potentially safer than intravenous administration -LSB- 19 -RSB- . The doses of prednisolone used in our model were chosen according to common dose regimens for prednisolone -LSB- 18 -RSB- . These doses were relatively low -LRB- equivalent to 12 , 40 and 120 mg hydrocortisone -RRB- in comparison to doses used for sepsis intervention -LRB- hydrocortisone 200 -- 300 mg per day -RRB- , yet sufficient to reduce the release of the investigated biomarkers during endotoxemia . Of note , it has been demonstrated that the effects of orally administered doses of prednisolone do not differ from those of comparable doses of intravenously administered hydrocortisone -LSB- 20 -RSB- . The human endotoxemia model differs from sepsis in many aspects : it uses a single-dose drug intervention and bolus intravenous challenge of LPS , it is self-limiting , and it lacks an infectious source -LSB- 13 -RSB- . The study subjects are healthy young people not using any co-medication . The intervention must be administered before LPS injection in order to reach clinically relevant drug concentrations during endotoxemia because LPS is extremely quickly cleared from the circulation . Due to these differences , data can not be directly extrapolated to the sepsis field and must always be confirmed in patient studies . Nonetheless , the model is the best available in healthy humans and has proven useful to obtain a proof of principle of the actions of drugs and/or to study mechanisms that contribute to the activation of inflammatory pathways -LSB- 13 -RSB- . With these limitations in mind , we demonstrate here that the levels of the novel biomarkers MR-proADM , MR-proANP , CT-proAVP and PCT are all increased after LPS injection , and that the levels of MR-proADM , MR-proANP and CT-proAVP can be inhibited by prednisolone . These results add to their potential as biomarkers in sepsis .

O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O


The structural plasticity of heparan sulfate NA-domains and hence their role in mediating multivalent interactions is confirmed by high-accuracy 15N-NMR relaxation studies Considering the biological importance of heparan sulfate -LRB- HS -RRB- and the significant activity of its highly-sulfated regions -LRB- S-domains -RRB- , the paucity of known functions for the non-sulfated NA-domains is somewhat puzzling . It has been suggested that chain dynamics within the NA-domains are the key to their functional role in HS . In this study , we investigate this hypothesis using state-of-the-art nuclear magnetic resonance -LRB- NMR -RRB- experiments at multiple frequencies . To resolve the problem of severe overlap in 1H-NMR spectra of repetitive polysaccharides from proteoglycans , we have prepared oligosaccharides with the chemical structure of HS NA-domains containing the 15N nucleus , which has enough chemical shift dispersion to probe the central residues of octasaccharides at atomic resolution using 600 MHz NMR . By performing NMR relaxation experiments at three magnetic-field strengths , high quality data on internal dynamics and rotational diffusion was obtained . Furthermore , translational diffusion could also be measured by NMR using pulse field gradients . These experimental data were used , in concert with molecular dynamics simulations , to provide information on local molecular shape , greatly aiding our relaxation analyses . Our results , which are more accurate than those presented previously , confirm the higher flexibility of the NA-domains as compared with reported data on S-domains . It is proposed that this flexibility has two functional roles . First , it confers a greater area of interaction from the anchoring point on the core protein for the bioactive S-domains . Secondly , it allows multiple interactions along the same HS chain that are dynamically independent of each other . Introduction Heparan sulfate -LRB- HS -RRB- , a member of the glycosaminoglycan -LRB- GAG -RRB- family , is thought to have appeared early in metazoan evolution -LSB- 1 , 2 -RSB- . It is ubiquitous in vertebrate extracellular matrices as a constituent of proteoglycans and its extensive repertoire of biological roles , via interactions with hundreds of proteins , make it the most intensely studied of the GAGs . In particular , HS has been implicated in several important biological functions that involve binding to anti-thrombin -LRB- AT -RRB- , fibroblast growth factors -LRB- FGFs -RRB- , cytokines and chemokines -LSB- 3 -RSB- . Chemically , the HS biopolymer consists of repeating disaccharide units of variably-sulfated uronic acid -LRB- d-glucuronic or l-iduronic acid -RRB- and glucosamine -LRB- N-acetylated or N-sulfated -RRB- monosaccharides . It is this variability in chain epimerization and sulfation -LRB- see Fig. 1 -RRB- that appears to be responsible for the specificity of interactions between HS and proteins -LSB- 4 -RSB- , rather than the basic linkage pattern of the polymer , which is conserved in all forms . Fig. 1The basic disaccharide repeating unit of heparan sulfate . In the compounds studied here , the number of repeats -LRB- n -RRB- is 2 , 3 and 4 , for compounds 1 , 2 and 3 respectively . In these compounds , R1 = COCH3 and R2 = R3 = R4 = OH , the uronic acid residue is glucuronic acid and the terminal uronic acid residue is 4 -- 5 unsaturated In some cases , such as the interaction between anti-thrombin -LRB- AT -RRB- and a HS pentasaccharide sequence -LRB- which includes the rare 3-O-sulfated glucosamine -RRB- -LSB- 5 -RSB- , the local spatial arrangement of sulfo groups is postulated to be the major driver of specificity . Furthermore , the internal and global conformation and dynamics of the chains also play important roles in HS bioactivity -LSB- 6 -- 8 -RSB- . These conformational contributions to function are not easy to separate from the others , since the density and arrangement of the opposing sulfo groups may profoundly influence the local dynamics of HS-chains , creating an ambiguity in relating HS structure to function . However , solution-state nuclear magnetic resonance -LRB- NMR -RRB- studies can be used to resolve this ambiguity by directly measuring molecular conformation and dynamics on the nanosecond time scale with atomic resolution -LSB- 9 , 10 -RSB- . Numerous NMR studies have been conducted to gain insight into the 3D-structure of HS chains . Much of the early literature was concerned with the conformation and dynamics of the l-iduronate residues -LSB- 7 , 11 -- 13 -RSB- , and not without controversy -LSB- 14 -RSB- . From these studies , which used NMR coupling-constants and basic relaxation data , a picture emerged of iduronic acid residues that have high internal flexibility , but their dynamic segmental motions are decoupled from neighboring residues -LSB- 6 -RSB- . More recent studies of HS chain dynamics using model-free analysis -LSB- 15 -RSB- of NMR relaxation data have attempted to describe the internal and overall motion of the various residues of HS -LSB- 16 -- 18 -RSB- . Interpretation of these data has not been without controversy either . Hricovini et al. -LSB- 18 -RSB- compared the dynamics of the non-sulfated region of HS -LRB- NA-domain -RRB- with those from a highly sulfated HS fragment -LRB- S-domain -RRB- using the model-free analysis and found that the NA-domain was more rigid . This was ascribed to the lack of flexible iduronic acid residues in the NA-domains . However , a more recent publication by Angulo et al. -LSB- 16 -RSB- -LRB- also using the model-free approach -RRB- showed very restricted motion of the internal iduronic acid residues , in contrast to earlier investigations . At the heart of these conflicting results lies : -LRB- a -RRB- the difficulty in separation of internal motion due to inter-converting ring conformations of the flexible iduronic acid residue and motion due to flexibility of the glycosidic linkages , -LRB- b -RRB- problems associated with gathering of high quality data for these nearly linear biopolymers , and -LRB- c -RRB- interpretation of NMR data from polydisperse samples -LSB- 11 -RSB- . These difficulties can be overcome by : -LRB- a -RRB- restricting the analysis to rigid glucosamine rings -LRB- which are not epimerized -RRB- , -LRB- b -RRB- increasing the NMR chemical shift dispersion using isotopic enrichment , and -LRB- c -RRB- improved purification protocols . In protein NMR , isotopic labeling in conjunction with modern heteronuclear filtering experiments -LRB- which remove all proton resonances not correlated to a labeled nucleus -RRB- are used to overcome issues of signal overlap . In particular , 15N-enrichment is frequently used because the nitrogen atom is highly sensitive to changes in its local environment and consequently has a large range of NMR chemical shifts . The fact that there is often only one nitrogen atom per residue , as opposed to several protons , further adds to spectral simplicity . For dynamic molecular studies , the 15N-nucleus is also desirable since the 1H-15N system within an amide moiety can be considered as an isolated spin-pair for relaxation purposes , simplifying the analysis . Furthermore , 15N has a small negative gyromagnetic ratio , making it more sensitive to dynamics compared to the 13C nucleus -LRB- which has a larger and positive gyromagnetic ratio -RRB- . For example , recent use of 15N-labeling in the GAG hyaluronan has permitted full residue-specific NMR signal assignment and dynamical studies of oligosaccharides of up to eight residues , with unprecedented accuracy -LSB- 19 -RSB- . In this study , isotopic 15N-labeling of the glucosamine residues was used to overcome the difficulties associated with previous NMR relaxation analyses of HS dynamics . The polymer used here was the capsular polysaccharide from E. coli K5 bacteria , which is chemically identical to the NA-domains from HS . By growing the bacteria in 15N-enriched media , quantitative isotopic labeling of the polysaccharide can be achieved on the milligram scale , as described previously -LSB- 20 -RSB- . The polysaccharide was degraded using K5-lyase enzymatic digestion and separated into pure oligosaccharides using large-scale ion-exchange chromatography to facilitate the study of dynamics at the atomic level . Three 15N-labeled oligosaccharides -LRB- see Fig. 1 -RRB- were purified to homogeneity and used for NMR studies : a tetrasaccharide -LRB- 1 -RRB- , a hexasaccharide -LRB- 2 -RRB- and an octasaccharide -LRB- 3 -RRB- . 1H-15N NMR relaxation experiments -LRB- T1 and heteronuclear nOe experiments -RRB- were fitted to both isotropic and anisotropic models of molecular diffusion to assess the validity of assuming isotropic overall motion in these non-sulfated molecules . The libration of the amide group -LRB- which contributes to the measured dynamics -RRB- was taken into consideration using experimental J-coupling data . Furthermore , diffusion ordered spectroscopy -LRB- DOSY -RRB- -LSB- 21 , 22 -RSB- and molecular dynamics -LRB- MD -RRB- simulations were used to assist in evaluating the models of molecular shape . The approach presented here can easily be extended to the various modified forms of HS , as these can be produced by chemical and enzymatic modification of the K5 capsular polysaccharide -LSB- 23 -RSB- . Our novel findings shed light upon the role of dynamics in HS , and it is intended that these approaches will be adopted more generally , leading to higher quality dynamic data with atomic precision for these most interesting and important biomolecules . Theory 15N NMR relaxation and rotational diffusion The dynamic behavior of biomolecules on the sub-nanosecond timescale can be probed by NMR relaxation , by measuring T1 - and T2 - relaxation rates and the nOe enhancement -LRB- η -RRB- at multiple magnetic fields -LSB- 9 -RSB- and interpreting the results using the Lipari -- Szabo model-free approach -LSB- 15 -RSB- . For small molecules -LRB- as described here -RRB- the T1-and T2-relaxation rates are similar for the 1H-15N system , thus in this case only T1 and η are considered -LSB- 19 -RSB- . Several -LRB- often confusing -RRB- treatises of these equations are present in the literature , thus the basic theory is briefly reconsidered here . Relaxation rates for an isolated N -- H spin-pair are related to the spectral density function , J -LRB- ω -RRB- , through Eqs . 1 and 2 , where γ is the gyromagnetic ratio , ħ is Planck 's constant over 2π , ω is the relevant nuclear Larmor frequency , ΔσN is the difference between the axial symmetric 15N chemical shift tensor -LRB- estimated to be − 135 ppm for N-acetylglucosamine see also methods section -RRB- and rNH is the N -- H inter-nuclear distance -LRB- a value of 1.01 Å is used here , see also methods section -RRB- . For an isotropic -LRB- spherical -RRB- model of molecular diffusion the spectral density function is given by Eq . 3 , where τ is the average rotational correlation time -LRB- radians s − 1 -RRB- and D is the rotational diffusion coefficient . In the case of a symmetric top -LRB- cylindrical -RRB- model of molecular diffusion the anisotropic spectral density function is given by the Eqs . 4 and 5 , where and are the correlation time and rotational diffusion coefficient perpendicular to the symmetry axis , and are the corresponding variables parallel to the symmetry axis -LRB- by this definition -RRB- , and α is the angle between the N -- H vector and the cylindrical axis of symmetry . When the internal motion -LRB- τe -RRB- is much faster than the global rotational correlation time -LRB- τM -RRB- , these two phenomena can be separated using the model free approach of Lipari and Szabo -LSB- 15 -RSB- . In their treatment , if it is assumed that molecular tumbling is isotropic , the spectral density function is represented by Eqs . 6 and 7 , where S2 is the generalized order parameter . The corresponding anisotropic relationship is shown in Eq . 8 , where Janiso -LRB- ω -RRB- is described by Eq . 4 and the are given in Eq . 9 . It should be noted that this derivation -LRB- resulting in Eq . 8 -RRB- is consistent with the original proposal of Lipari and Szabo -LSB- 15 -RSB- and slightly different to a recent and broadly-used implementation of that theory -LSB- 24 -RSB- . Diffusion measurements The average translational diffusion rate can be measured by pulsed-field-gradient -LRB- PFG -RRB- NMR -LSB- 25 -RSB- , often referred to as diffusion ordered spectroscopy -LRB- DOSY -RRB- -LSB- 21 , 22 -RSB- . In this experiment , the diffusion coefficients can be estimated by varying the gradient amplitude -LRB- g -RRB- and fitting the experimental data to the theoretical Stejskal -- Tanner Eq . 10 -LSB- 26 -RSB- . In Eq . 10 , S is the signal amplitude , S0 is the echo amplitude that would have resulted had there been no diffusion , Dt is the translational diffusion coefficient -LRB- not to be confused with the rotational diffusion rates described above -RRB- , δ is the gradient pulse width and Δ ′ is the diffusion time corrected for the effects of a finite gradient pulse width . Often the relative diffusion coefficient is sought , but when the absolute value is of interest , a more correct value of Dt can be obtained by taking the systematic errors -LRB- due to non-uniform field gradients -RRB- into account by fitting to a different expression , which compensates for this , Eqs . 11 and 12 -LSB- 27 -RSB- , where the coefficients cn are probe and pulse-sequence dependent . The hydrodynamic radius of a molecule can be shown to be related to the translational diffusion coefficient , Dt . This radius , and thus Dt for an isolated polymer in solution , scales -LRB- via an exponent , v -RRB- with molecular mass -LRB- M -RRB- , Eq . 13 . The value of v can be shown to be 3/5 and 1/3 for fully solvated and collapsed polymer chains , respectively -LSB- 28 -RSB- . A polymer is fully solvated in a solvent where its interactions with the solvent are favored over self-association -- the converse is true for a collapsed polymer . In a so-called Flory or θ solvent , the polymer behaves as a random coil , where v = 1/2 -LSB- 28 -RSB- . Results 15N-labeled polymer with a chemical structure identical to the non-sulfated domains of heparan sulfate -LRB- HS -RRB- was prepared from E. coli . K5 capsular polysaccharide and enzymatically digested . The resulting oligomers were purified and three samples were prepared that had 4 , 6 and 8 sugar units -LRB- see experimental section for details -RRB- . 1D 1H-NMR spectra and mass spectrometry -LRB- not shown -RRB- determined that the oligosaccharides were uniformly 15N-labeled and 1H-15N-HSQC spectra -LRB- see Fig. 2 -RRB- confirmed that the samples contained no major contaminants . Fig. 215N-1H HSQC spectrum of the tetra - , hexa - and octa-saccharides -LRB- from top to bottom -RRB- of the NA-domain of heparan sulfate at a proton-resonant frequency of 900 MHz . The abscissa shows 1H chemical shift -LSB- ppm -RSB- . Labels on the spectrum correspond to amide groups , labeled according to their position within the oligosaccharide , using Greek nomenclature . Variability in the line shapes are due to acquisition and processing of the NMR spectra Relaxation experiments -LRB- 1H-15N T1 and heteronuclear nOe -RRB- were measured for each of the oligosaccharides -LRB- 1 , 2 and 3 -RRB- at three magnetic field strengths -LRB- 14.1 , 18.1 and 21.1 T -RRB- . In each case the 1H-15N HSQC-based T1-relaxation experiments were repeated 12 times with a variable relaxation delay and the resultant peak heights were fitted to exponential curves -LRB- relaxation curves for the octasaccharide at 14.1 T are shown in Fig. 3 -RRB- . The heteronuclear nOes were calculated from the ratio of peak heights in 1H-15N spectra , recorded with and without 1H-presaturation . Fig. 3T1-relaxation curves for an octasaccharide unit from the NA-domain of heparan sulfate at the lowest magnetic field strength used in this study -LRB- 14.1 T -RRB- . The resonances from the amide groups of the central residues -LRB- ψ and γ -RRB- are resolved and have slightly different relaxation times . For the specific assignments see Fig. 2 Table 1 shows the calculated exponential decay constants -LRB- T1 -RRB- and the corresponding nOe ratios -LRB- η -RRB- . The errors in the tabulated T1 values were taken to be twice the exponential curve fitting errors , while the errors in the nOe ratios were estimated by calculating the ratio of the sum of the amplitudes -LRB- A -RRB- of the two HN signals from the anomeric amide group -LRB- α/β -RRB- and the same signal in the ω-amide group -LRB- clearly resolved in all spectra , see Fig. 2 -RRB- , according to Eq . 14 . Table 1Fitted 1H-15N T1-relaxation data and measured nOe -LRB- η -RRB- ratios for three lengths -LRB- 4 - , 6 - and 8-saccharide units denoted 1 , 2 and 3 respectively -RRB- of the NA-domain from heparan sulfate at three magnetic-field strengths -LRB- 14.1 , 18.1 and 21.1 T -RRB- 14.1 T18 .1 T21 .1 TηT1 -LSB- ms -RSB- ηT1 -LSB- ms -RSB- ηT1 -LSB- ms -RSB- 1α − 1.351,790 − 1.231,625 − 0.421,526 β − 1.631,614 − 1.221,524 − 0.561,445 ω − 1.891,255 − 0.951,183 − 0.391,1162 α − 0.961,613 − 0.851,473 − 0.281,400 β − 1.121,456 − 0.791,368 − 0.341,310 γ − 1.07995 − 0.339420.14889 ω − 1.171,050 − 0.38978 − 0.019393 α − 0.811,437 − 0.841,362 − 0.141,342 β − 0.991,318 − 0.751,278 − 0.331,252 γ − 0.587550.047970.50803 aψ − 0.77831 − 0.178240.40803 aω − 0.92946 − 0.189100.25908 aHigh resolution spectrum not collected -- value estimated from a low-resolution spectrum -LRB- see experimental section -RRB- . The heteronuclear experiments for the octasaccharide -LRB- 3 -RRB- were collected using two different sweep-widths . In the first experiment a larger sweep width was used , where all nuclei were present and unfolded , but in which the γ - and ψ-amide groups could not be resolved . These resonances were resolved using a second , narrower spectral width -LRB- not shown -RRB- . The spectral width of the second experiment was chosen so that aliased peaks did not interfere with the resonances of interest . Conversion of the resultant relaxation measurements into 3D-molecular information was performed via the model-free method for calculating the spectral density function -LRB- as needed by relaxation Eqs . 1 and 2 -RRB- . Due to the potential shape anisotropy of the oligosaccharides , two methods were used to estimate the spectral density functions , one that assumed spherical symmetry and one that incorporated cylindrical symmetry , see Eqs . 6 and 8 . Fitting the relaxation data to these equations resulted in order parameters , overall and internal tumbling times , and in the case of anisotropy , a set of angles . It was found that the relaxation data was not sensitive to the internal correlation time -LRB- τe -RRB- and thus it varied significantly when allowed to change during the fitting process . However , this parameter is of secondary interest , and by fixing the internal correlation time at 30 ps -LRB- consistent with molecular dynamics simulations -RRB- , higher quality and more robust fits to the experimental data could be produced , as noted previously -LSB- 29 -RSB- . The uncertainty in this variable is reflected in previous literature data for similar compounds , where τe ranges from a few picoseconds to several hundred picoseconds in similar compounds -LRB- 15 -- 50 ps for a HS pentasaccharide and 110 -- 290 ps for a HS hexasaccharide -RRB- and with large errors -LRB- ± 25 -- 85 ps -RRB- -LSB- 16 -- 19 , 30 -RSB- . The results , given in Table 2 , therefore assume a constant internal correlation time of 30 ps and clearly show the expected trend in generalized order parameter -LRB- S2 -RRB- , where the amide groups at the central residues are more ordered -LRB- higher S2 values -RRB- than those at the terminal residues . Also , the overall correlation times increase as the oligosaccharides get longer , as expected a priori . Table 2Calculated model-free parameters for the tetra - , hexa - and octa-saccharides -LRB- 1 , 2 and 3 respectively -RRB- of the NA-domain of heparan sulfate , using a constant internal correlation time of 30 ps , fitted to isotropic and anisotropic models of rotational diffusionModels of rotational diffusionIsotropicS2S2calc1τM -LSB- ns -RSB- 0.57 α0 .320.46 τe -LSB- ns -RSB- 0.03 β0 .350.59 ω0 .480.622 τM -LSB- ns -RSB- 0.71 α0 .350.49 τe -LSB- ns -RSB- 0.03 β0 .36 γ0 .600.70 ω0 .550.613 τM -LSB- ns -RSB- 0.74 α0 .32 τe -LSB- ns -RSB- 0.03 β0 .34 γ0 .87 Ψ0 .82 ω0 .67 AnisotropicS2α -LSB- ° -RSB- S2calcαcalc -LSB- ° -RSB- 1 -LSB- ns -RSB- 0.35 α0 .3315.40.4664.2 -LSB- ns -RSB- 0.65 β0 .3640.10.5962.11.88 ω0 .5658.60.6263.1 τe -LSB- ns -RSB- 0.03 2 -LSB- ns -RSB- 0.57 α0 .313.80.4962.8 -LSB- ns -RSB- 0.79 β0 .3649.5 1.39 γ0 .6258.60.7064.4 τe -LSB- ns -RSB- 0.03 ω0 .6184.70.6163.33 -LSB- ns -RSB- 0.39 α0 .3975.0 -LSB- ns -RSB- 1.00 β0 .4166.7 2.59 γ0 .652.2 τe -LSB- ns -RSB- 0.03 Ψ0 .6623.2 ω0 .5431.1 These values were calculated from experimental data collected at three magnetic-field strengths -LRB- 14.1 , 18.1 and 21.1 T -RRB- . The S2calc and αcalc values were calculated by averaging data extracted from 50 ns molecular dynamics simulations Molecular shape was probed using the DOSY experiment , applied to the three samples . The diffusion coefficients were estimated from their respective methyl peaks at 2.03 ppm -LRB- Table 3 -RRB- using the Stejskal -- Tanner Eq . 10 . The resulting diffusion values were used to calculate the scaling-factor exponent -LRB- ν -RRB- in Eq . 13 , which provides information about the overall molecular shape . Figure 4 shows a plot of log -LRB- Dt -RRB- against log -LRB- M -RRB- , showing the slope of the fitted line -LRB- ν -RRB- to be 0.48 . Fig. 4Plot of log -LRB- Dt -RRB- vs. log -LRB- M -RRB- for three different lengths of the NA-domain of heparan sulfate , consisting of 4 , 6 and 8 sugar units -LRB- Dt is the translational diffusion time and M the weight-averaged molecular mass -RRB- . The dotted line is the best-fit to a straight line -LRB- as described in the theory section , Eq . 13 -RRB- Table 3Calculated translational diffusion times using the methyl resonance of the various oligomers -LRB- tetra - , hexa - and octasaccharides labeled in table as 1 , 2 and 3 respectively -RRB- of the NA-domain from heparan sulfate at a proton frequency of 400 MHzCompoundD -LSB- 10 − 6 cm2/s -RSB- 13.08 ± 0.000422.55 ± 0.000732.22 ± 0.0007 Molecular dynamics -LRB- MD -RRB- simulations were performed on the tetra - and hexa-saccharides in water boxes . Figure 5 shows 40 snapshots from the simulation of the hexasaccharide -LRB- stripped of ions and water molecules -RRB- , superimposed on the atoms from the central linkage . The simulation data were analyzed to give theoretical values of both S2 and the angle -LRB- α -RRB- , defined as the angle between the molecular axis and the N -- H vector -LRB- see Table 2 -RRB- . The calculated S2 values show similar trends to the experimental data , but are generally higher . The α values for both the tetra - and hexa-saccharide show nearly random behavior , consistent with an isotropic model . Fig. 5Overlay of 40 snapshots from 50 ns of molecular dynamics simulations of the hexasaccharide of unsulfated heparan sulfate . The molecules are superimposed using the atoms in the adjacent linkages . The position of the nitrogen atom -LRB- blue sphere -RRB- in each structure is highlighted The coupling constants of 1 and 2 were measured from 1D 1H spectra except for the central residues of 2 where there was overlap in the 1D spectrum . In this case , cross-peaks in the 1H-15N HSQC were fitted to 2D Lorentzian line-shapes using a non-linear fitting scheme -LRB- these data are given in Table 4 -RRB- . Table 43J -LRB- H2 − HN -RRB- - coupling constant -LRB- in Hz -RRB- of the tetra - and hexa-saccharides -LRB- 1 and 2 respectively -RRB- from the NA-domains of heparan sulfate 3J -LRB- α − -LSB- H2 − HN -RSB- -RRB- -LSB- Hz -RSB- 3J -LRB- β − -LSB- H2 − HN -RSB- -RRB- -LSB- Hz -RSB- 3J -LRB- γ − -LSB- H2 − HN -RSB- -RRB- -LSB- Hz -RSB- 3J -LRB- ω − -LSB- H2 − HN -RSB- -RRB- -LSB- Hz -RSB- 18.438.559.1428.308.659.149.24 All values have an estimated error of ± 0.05 Hz . Discussion Analysis of the NMR relaxation data The results clearly show the advantages of 15N-labeling , where even the amide groups in the central residues of an octasaccharide are well resolved -LRB- Fig. 2 -RRB- . In addition to the gain resolution , an excellent spread of relaxation data is found due to the use of the 15N-nucleus -LRB- Table 1 -RRB- . The average change in nOe enhancement -LRB- η -RRB- over the different fields is 1 unit , with some of the η-values passing through zero . In addition , much more precise measurements can be made of the longer T1-relaxation times in the 1H-15N system -LRB- compared to 1H-13C -RRB- . The data follows a clear trend and even without further analysis the difference in relaxation properties of the central and terminal residues of the octasaccharide can be appreciated , as well as the difference between the various lengths of the polymer . The experimentally measured relaxation datasets were interpreted using the model-free approach -LSB- 15 -RSB- , which relates the measured relaxation values at multiple fields to dynamic conformational properties . In the model-free approximation it is assumed that internal motion -LRB- libration -RRB- and global motion -LRB- diffusion -RRB- are uncoupled and on significantly different timescales . Furthermore , it is assumed that molecular dynamics occurs with respect to a rigid diffusing molecular frame . While neither of these assumptions are entirely true for biomolecules , they yield good results if an appropriate model for molecular diffusion is chosen . The two simplest models for diffusion are based on the isotropic sphere and the anisotropic symmetric top -LRB- rod or cylindrical shape -RRB- -LSB- 15 -RSB- . In proteins , for example , the simpler isotropic spherical shape usually suffices , but this model may not be accurate for rod-shaped carbohydrate polymers -LSB- 18 -RSB- , which are expected to become more anisotropic -LRB- rod shaped -RRB- with increasing length of the polymer chain . Previous studies on HS analogues have modeled diffusion using an anisotropic model , improving the agreement with experimental data -LSB- 6 , 11 , 16 -- 18 , 30 , 31 -RSB- . In such a symmetric-top model , the correlation time about the symmetry axis of the rod shape is separated from that perpendicular to the symmetry axis . Values of the parallel correlation time tend to be similar in linear carbohydrates -LRB- ~ 0.5 ns -RRB- and -LRB- by definition -RRB- are always smaller than the perpendicular correlation time ; the ratio of the two correlation times can be used as a measure of anisotropy . Other parameters in the model-free approach are the internal correlation time -LRB- τe -RRB- , the generalized order parameter -LRB- S2 -RRB- and , in the anisotropic model , an angle -LRB- α -RRB- . Isotropic or anisotropic model of rotational diffusion ? From the diffusion ordered spectroscopy -LRB- DOSY -RRB- data in Table 3 and Fig. 4 it is apparent that the overall polymeric shape of the NA-domain of HS is not consistent with a rigid rod . In fact , the slope of the line in Fig. 4 is almost equal to the theoretical value for a random coil -LRB- ν = 1/2 -RRB- , which indicates that even the oligosaccharides have significant segmental motion . This is supported by the model-free analysis of the tetra - and hexa-saccharides -LRB- using an isotropic model of diffusion -RRB- , where relatively low values of S2 are found . However , when similar analysis was used to interpret relaxation measurement from the octasaccharide , relatively higher S2-values were found at the central amide groups . In contrast , the corresponding parameters , calculated using the anisotropic model of diffusion , are consistent with a random coil . These data therefore suggest that the tetra - and hexa-saccharides undergo nearly isotropic diffusion , whereas the octasaccharide diffuses anisotropically -LRB- all with high internal and segmental motion -RRB- . It is likely that it is the α-linkages within the oligosaccharides that are responsible for a compact dynamic shape at shorter lengths , which only starts to behave anisotropically as an octasaccharide . The lower order parameters calculated using an anisotropic diffusional model are due to the introduction of an additional parameter -LRB- α -RRB- that is the angle between the inter-nuclear N -- H vector and the molecular cylindrical-symmetry axis . This parameter adds another dimension of complexity , providing a more accurate interpretation of the experimental data than using isotropic diffusion . For example , in the isotropic diffusional model , if there is a large amplitude motion along an N -- H vector that is coincident with the long axis of the molecule , the T1 and η values will be interpreted as low amplitude motion , which will be compensated for by an increase in the overall rotational diffusion . However , in the anisotropic model , the angular dependence of the internal motion with respect to the molecular frame is taken into account , and the data fitted so that the angle -LRB- α -RRB- is also optimized . Inspection of Table 2 reveals that the amide groups at the central residues in the octasaccharide are indeed coincident with long axis of the molecule -LRB- small α -RRB- , explaining the decrease in order parameter compared to the isotropic model . Further inspection shows that whenever the N -- H vector is along the long axis , the anisotropic model predicts more motion and , when the vector is pointing along the short axis -LRB- large α -RRB- , the converse is true . More importantly , the anisotropic interpretation indicates that the two amide groups in the central residues of the octasaccharide have similar dynamics , as inferred from their S2 values -LRB- Table 1 -RRB- , in contrast to the results from the isotropic analysis , where the γ-amide group appears to be more ordered -LRB- higher S2 -RRB- than the more central ψ-amide group . Furthermore , naïve interpretation of the raw experimental relaxation values -LRB- see Fig. 3 , for example -RRB- would agree with the conclusion of the analysis via the isotropic model . However , this initial inspection is likely to be erroneous , because one would logically expect that the amide group in the most central residue -LRB- i.e. , ψ in Fig. 2 -RRB- would be more ordered -LRB- or at least similarly ordered -RRB- than that in a neighboring residue , which is in agreement with the initially counterintuitive results from the anisotropic diffusional model . Therefore , these findings -LRB- in addition to the DOSY data -RRB- substantiate the use of an anisotropic model in the analysis of octasaccharide dynamics . Considering now analysis of the shorter oligosaccharides using an anisotropic model of diffusion , it is found that both the parallel and perpendicular diffusional rates increase , whereas one would expect only to increase . This suggests that there is no well-defined anisotropic interpretation and the simpler isotropic model is preferable . Such an observation is further substantiated by molecular dynamics simulations performed for the tetra - and hexa-saccharide -LRB- see Fig. 5 and Table 2 -RRB- . In these simulations it was found that the angle α is nearly random for all amides , consistent with an isotropic model . Microscopic interpretation of S2 NMR scalar coupling constants -LRB- J-couplings -RRB- -LSB- 32 -RSB- can also be used to measure dynamics due to bond librations -LRB- via a suitable empirical equation -RRB- and the 3J -LRB- H2 − HN -RRB- coupling constant is a direct reporter of amide dynamics in these oligosaccharides . Theoretical calculations have shown that , given the same chemical environment , a decrease in the coupling constant is associated with increased amide librations . From Table 4 , it can be seen that in 2 the central γ-amide group has a higher coupling constant compared to that of the terminal ω-amide group . This is contrary to expectation , as the central amide group is regarded to be more rigid . In this case , it appears that the change in chemical environment causes the unexpected observation . This is not expected to be the case when comparing coupling constants measured in 1 and 2 from amides in similar chemical environments , i.e. , those at the α - , β - and ω-positions , where a variability of 0.1 Hz is observed in the coupling constants . This can be attributed to slight changes in amide libration of a few degrees -LRB- using a previously derived relationship between coupling constants and torsional libration -LSB- 32 -RSB- -RRB- . A similar relationship between dihedral angle and S2 indicated that these angular variabilities would correspond to deviations in S2 of less than 0.01 -LSB- 15 -RSB- . Furthermore , the expected differences in amide group librations across individual molecules are less than 5 ° -LRB- using the above mentioned relationship -RRB- , which would result in small changes in S2 of less than 0.05 . Therefore , differences in amide libration are not responsible for the more dramatic change in S2 observed at specific amide positions -LRB- Table 2 -RRB- . The major remaining site-specific contributor to S2 in the NA-domains from HS -LRB- which contain relatively rigid 4C1-puckered sugar rings -RRB- must thus be due to segmental motions induced by linkage librations . The S2-values reported here can therefore be seen as direct reporters of the chain dynamics in these polymers . Role of dynamics in HS The S-domains of HS have previously been investigated using relaxation data from C -- H vectors and an interpretation based on an anisotropic diffusional model -LRB- see Angulo -LSB- 16 -RSB- and references therein -RRB- . The values reported for of such domains are 1.6 , 1.7 and 2.6 ns for penta - , hexa - and hepta-saccharide fragments , respectively . In addition , these fragments show a much higher degree of anisotropy compared to the NA-domain analyzed here . This would suggest that the S-domains are comparatively more rigid , as reflected in the higher S2 values reported : 0.91 , 0.91 and 0.93 for central residues of the penta - , hexa - and hepta-saccharide fragments , respectively -LRB- cf. ~ 0.65 for the NA-domains found here -RRB- . From the findings presented here it thus appears that the NA-domains of HS are much more dynamic than the S-domains . At the molecular level , the only chemical difference between the NA - and S-domains of HS is sulfation and epimerization , and it has been suggested previously that both share a similar local 3D-chain conformation -LSB- 33 -RSB- . Therefore , it is more likely that the major contributor to chain rigidity of S-domains is the repulsion of multiple negative charges , rather than specific cross-residue intramolecular interactions , but this remains to be validated by further experimentation . From a biological viewpoint this confirms the hypothesis that the primary function of the NA-domains is to give the HS chains flexibility , since the S-domains are significantly stiffer . This would give proteoglycan HS chains the ability to rapidly and effectively explore space around their tethering points on the core protein -LRB- Fig. 6 -RRB- . It would also allow them to dynamically adapt their shapes and present themselves effectively to a range of protein targets . In addition , this would permit HS chains to interact in a multivalent fashion , without the dynamics of one interaction greatly affecting others . This may explain the omnipresence of functional S-domains in HS , which are separated by completely unsulfated NA-domains -LSB- 34 -RSB- . Supporting evidence is provided by Stringer and Gallagher -LSB- 8 -RSB- , who found an HS fragment with specifically high affinity for the platelet factor 4 -LRB- PF4 -RRB- tetramer . This HS fragment consisted of two S-domains separated by an NA-domain long enough to allow for interaction of the two S-domains with each side of the PF4 tetramer . This argument is also consistent with the diversity of molecules that HS interacts with and the picture that its primary role is in regulation and control -LSB- 2 , 4 , 35 -RSB- . Fig. 6Schematic diagram illustrating the proposed increase in flexibility due to the NA-domains of heparan sulfate and the concomitant increase in spatial range that can be covered together with the potential for mediating multivalent interactions Conversely , the current study would suggest that the S-domain rich heparin proteoglycans do not have the local plasticity of HS proteoglycans . Therefore , in order to provide the same effective local concentration of sulfo-groups , heparin may be required at higher concentrations than HS . This is supported by the fact that when mast cells are activated their cytoplasmic granules -LRB- where heparin is thought to act as a depository for cationic molecules i.e. histamines , serotonin , etc. -RRB- are released into the extracellular matrix resulting in a high concentration of highly active -LRB- S-domain rich -RRB- heparin molecules -LSB- 36 -RSB- . Such action would allow heparin to quickly interact with proteins at the site of release , a response which is important in inflammatory processes , where heparin acts as an anticoagulant -LSB- 36 -RSB- . However , as its concentration falls due to diffusion from the point of release its highly potent effects would drop-off rapidly . Conclusions Heparan sulfate -LRB- HS -RRB- NA-domains have been investigated using NMR and computational methods , to determine whether their dynamic properties are consistent with previous hypotheses , suggesting that NA-domains add flexibility to HS . For these nearly-linear biomolecules conventional 1H-NMR methods yield poor spectra with overlapping resonances that are difficult to interpret . We have demonstrated that these difficulties can be overcome by use of 15N-labeling and efficient purification protocols . This approach , which could easily be extended to sulfated domains , allows the dynamical properties of NA-domains of HS to be studied with unprecedented accuracy . In this study , three lengths of the enzymatically-digested polymer -LRB- consisting of 4 , 6 and 8 sugar units , respectively -RRB- were isolated , purified and high-quality 15N-NMR relaxation data was collected for each at three magnetic-field strengths . The resultant data was analyzed using the model-free approach using both isotropic and anisotropic models of rotational diffusion . To complement these studies , and to ease their interpretation , molecular shape was further probed by measuring translational diffusion -LRB- using NMR pulse field gradients -RRB- and by performing solvated molecular dynamics simulations . The resultant order parameters -LRB- S2 -RRB- from the model-free analysis reveal a high degree of disorder in the oligosaccharide chains , with S2-values that are similar in the hexa - and octa-saccharide , suggesting that these values represent polymer behavior . Following these analyses , it was determined that the NA-domains of HS behave as random coils and that for shorter oligosaccharides their rotational diffusion can be approximated to be isotropic , whereas for the octasaccharide the results are more consistent with anisotropic diffusion . The flexibility exhibited by the NA-domains is contrary to literature data on the sulfated S-domains of HS , which appear to be much more rigid -LSB- 16 -RSB- . It is proposed that the NA-domains primarily serve a motional role in HS , consistent with reports of the domain structure of HS . It is therefore argued that the motional properties of the NA-domains increase the efficiency of HS in control and regulation . These findings also suggest that heparin does not have this flexibility , a view consistent with the known roles of heparin , which include storage of ionic molecules and involvement in processes requiring a fast , local response . Methods and materials Biochemical preparation of oligosaccharides K5 capsular polysaccharide was prepared from cultures of Escherichia coli Bi8337/41 -LSB- O10 : K5 : H4 -RSB- , originally supplied by Professor Ian S. Roberts . Cells were grown in a starter culture of 50 ml of LB broth , 1 ml of that media was pelleted and resuspended in 500 ml of M9-minimal media containing 1g/l 15N-ammonium chloride -LRB- 15N > 99 % -RRB- as the only nitrogen source . The polysaccharide purification procedure was similar to that described previously and is summarized here -LSB- 37 -RSB- . After 16 h of growth the cells were harvested -LRB- 5,000 g , 10 min -RRB- and washed once with 50 ml of phosphate-buffered saline -LRB- pH 7.2 -RRB- . Cell pellets were resuspended and incubated -LRB- 30 min , 37 °C -RRB- in 50 ml extraction buffer -LRB- 50 mM Tris -- Cl , 5 mM EDTA , pH 7.3 -RRB- four times , retaining the supernatants . Polysaccharide was precipitated from the pooled supernatants by addition of sodium cetyl-3-ethyl ammonium bromide to 0.1 % -LRB- w/v -RRB- followed by a 16 h incubation at room temperature . The precipitate was recovered by centrifugation -LRB- 10,000 g , 20 min -RRB- , re-suspended in 1 M NaCl , precipitated by addition of ethanol to 80 % -LRB- v/v -RRB- and again recovered by centrifugation . Following dissolution in distilled water -LRB- 5 ml -RRB- , the preparation was dialysed against distilled water , centrifuged -LRB- 100,000 g , 1 h -RRB- and the supernatant containing purified polydisperse K5 polysaccharide -LRB- 20 mg/l -RRB- was lyophilized . Lyophilized 15N-enriched K5 polysaccharide -LRB- ~ 30 mg -RRB- was dissolved in 5 ml of digest buffer -LRB- 100 mM Tris -- HCl , 100 mM Na-acetate , pH 8.5 -RRB- and incubated at 37 °C for 16 h with 180 U of K5-lyase enzyme -LRB- kindly supplied by Professor Ian S. Roberts -RRB- . The reaction was stopped by incubation in a boiling water bath for 5 min and the precipitated enzyme was removed by centrifugation -LRB- 13,000 g , 2 min -RRB- . Differing lengths of oligosaccharides from the K5-polysaccharide digest were separated using anion exchange FPLC on an ÄKTA system -LRB- GE Healthcare -RRB- . Briefly , digest supernatant was loaded onto a Sepharose-Q HR column -LRB- GE Healthcare -RRB- , equilibrated in Milli-Q H2O at flow rate of 10 ml/min , and eluted with a gradient of 0 -- 100 mM NaCl over 90 min . The eluent was monitored at both 214 and 232 nm . Following lyophilization , each oligosaccharide was desalted using a Bio-Gel P2 column -LRB- BioRad -RRB- running in 50 mM HN4Ac , and then repeatedly lyophilized to remove residual NH4Ac by sublimation . Purity was confirmed by analytical ion-exchange chromatography using a Mono-Q HR 5/5 -LRB- GE Healthcare -RRB- , NMR and mass spectrometry . Sample preparation Samples of oligosaccharides for NMR spectroscopy were prepared from lyophilized material reconstituted in 5 % -LRB- v/v -RRB- D2O , 0.02 % -LRB- w/v -RRB- NaN3 , 0.3 mM DSS , pH 6.0 ; pH 6.0 was chosen to allow amide HN protons to be observed . The final concentration of all samples was calculated to be approximately 5 mM . NMR spectroscopy The T1-relaxation and nOe experiments were performed at 24.5 °C , using standard pulse sequences -LSB- 40 -RSB- . All experiments were repeated at three different magnetic-field strengths : 14.1 , 18.1 and 21.1 T . The T1-relaxation delays were : 4 , 12 , 16 , 32 , 48 , 80 , 120 , 160 , 240 , 300 , 360 and 480 ms , performed in a random order to avoid systematic errors . 1024 complex data points were acquired in the direct -LRB- 1H -RRB- dimension and 18 complex points in the indirect -LRB- 15N -RRB- dimension , using a spectral window of 10 ppm in the direct dimension and 2.2 ppm in the indirect dimension . In addition , a second spectrum was collected for three -LRB- for all experiments -RRB- , which had a narrower spectral width of 0.35 ppm in the indirect dimension . The data was processed using the NMRPipe processing software -LSB- 41 -RSB- . The data was typically zero-filled in the acquisition dimension to 16,384 complex points and 1,024 complex points in the indirect dimension . The acquisition dimension was apodized using an exponential window function -LRB- typically with 15 Hz line-broadening -RRB- and the indirect dimension was apodized using a Lorentzian to Gaussian apodization function , in which the line broadening and narrowing parameters were adjusted as appropriate for the spectrum . The various peaks were picked using the peak-picking algorithm within the NMRPipe software and the peaks heights were used in all cases . The nonlinLS line shape fitting algorithm within the NMRPipe software was used on a subset of the peaks to validate this approach , and the agreement between the two approaches was found to be excellent -LRB- with the zero-filling used here -RRB- . The J-couplings were measured by finding the difference between peak frequencies , determined also using the nonlinLS algorithm . The assignment of the NH resonances follow the strategy employed by Blundell et al. -LSB- 38 , 39 -RSB- , applied to the oligosaccharides of hyaluronan . The full assignment of these oligosaccharides will be published elsewhere , the assignment of the resonances of interest , however , is given here . The assignment of the tetrasaccharide is straightforward as there are only three cross-peaks , two of which are weaker than the third . The weaker peaks have intensities equal to 60 and 40 % of the intensity of the third peak . Based on their intensities , the weaker peaks were assigned to the anomeric protons -LRB- α and β -RRB- , which are further assigned based on their intensities , since the α-anomer is dominant with preserved ratio in both free GlcNAc -LSB- 32 -RSB- and in the terminal GlcNAc of hyaluronan -LSB- 39 -RSB- -LRB- this assignment is further verified by a NOESY cross-peak between the NH proton and the previously assigned -LSB- 37 -RSB- anomeric proton at position 1 of α/β-GlcNAc -RRB- . The remaining cross-peaks can thus be assigned to the remaining NH atoms in the ω-residue of the tetrasaccharide . In the hexasaccharide all of the cross-peaks present in the tetrasaccharide are observed together with an additional cross-peak . This additional peak is assigned as the more central γ-residue , as it does not experience the same `` end effects '' -LRB- see also references to end effects in Blundell et al. -LSB- 38 , 39 -RSB- -RRB- . Finally , the octasaccharide was assigned based on the known assignment of the shorter fragments and the trend towards polymer properties . The cross-peaks for the NHs at α - , β - and ω-positions were nearly identical to those in the hexasaccharide , as these experience the same end-effects and could thus be readily assigned . The position of the NH cross-peaks of the γ - and ψ-residues in the octasaccharide are close to each other . However , one of these resonances has 1H and 15N chemical shifts that are almost identical to γ-residue of the hexasaccharide and is thus assigned as the γ-amide in the octasaccharide . The remaining cross-peak is therefore due to the ψ-amide . These assignments were further verified using the HSQC spectrum of a decasaccharide . In this spectrum , the intensity of the cross-peak corresponding to the ψ-amide increases -LRB- not shown -RRB- , due to convergence to polymer properties and overlap , whereas the intensity of the remaining resonances are unchanged . This tendency towards polymer behaviour was additionally used to verify the assignment of the NH cross-peak in the ψ-residue of the octasaccharide , which experiences the least amount of end-effects . Model-free NMR relaxation analysis Computer programs were written in the C-programming language to calculate the relaxation times for an isolated N -- H vector , according to the equations presented in the theory section . The internal correlation time at each vector was fixed at 30 ps -LRB- as described in the text -RRB- , with an associated variable S2 at each position and for each molecule a single variable for the isotopic model . For the anisotropic model , the rotational correlation time and angles between the long axis and each N -- H vector -LRB- α -RRB- had to be additionally considered . Predictions were compared with experimental data using the χ2 function and optimized using a Monte Carlo algorithm -LSB- 19 -RSB- . This process was carried out ten times for each oligosaccharide and the mean and standard deviation values for each fitted parameter were reported . The N -- H bond length and the chemical shift anisotropy of the amide nitrogen were calculated by ab initio DFT calculations on N-acetyl-d-glucosamine -LSB- 32 -RSB- -LRB- while the calculations were performed previously these values have not been reported before -RRB- . Briefly , the computation of the chemical shift anisotropy was performed using the GIAO method in the Gaussian software at the B3LYP/IGLOO-III level of theory , which resulted in a value of − 135 ppm , in close agreement with previously-reported values that used similar methods -LSB- 42 , 43 -RSB- . Similarly , the N -- H bond length was taken from quantum mechanical geometry optimization at B3LYP/631G -LRB- d , p -RRB- level of theory . The N -- H distance was found to range from 1.008 to 1.020 Å , depending on solvation model used -LRB- 1.008 , 1.020 and 1.015 Å for calculations performed in vacuo , with implicit solvent and with explicit solvent , respectively -RRB- , which is in agreement with similar calculations on proteins -LSB- 42 , 44 -RSB- . Due to this small variability of the N -- H distance , the relaxation calculations were performed using a bond length of both 1.01 and 1.02 Å -LRB- the results of the latter calculation are included in supplementary Table 2 -RRB- . Molecular dynamics simulations Molecular dynamics -LRB- MD -RRB- simulations were performed using CHARMm and a force-field suitable for glycosaminoglycans , as described previously -LSB- 45 -RSB- using an integration time-step of 2 fs . Briefly , oligosaccharides were built in a low-energy conformation consistent with preliminary NOESY measurements -LRB- unpublished results -RRB- and placed into rhomboidal dodecahedral water-boxes . The boxes had dimensions of 4.0 and 4.5 nm , containing 1504 and 2141 TIP3P water molecules for the tetrasaccharide and hexsaccharide , respectively . Long-range electrostatic interactions were treated using the particle-mesh Ewald approach and truncated to zero between 0.8 and 1.2 nm . Simulations -LRB- using the CPT ensemble -RRB- were carried out at 298 K on both α and β-anomers of the tetrasaccharide and on the β-anomer of the hexasaccharide , for a total of 50 ns in each case . Coordinates were stored every 5 ps for later analysis . The S2 values were calculated as the autocorrelation of N -- H vectors extracted from the MD simulation . The N -- H vectors were extracted by overlaying all frames from the MD simulation on the same set of atoms . These atoms were the linkage atoms adjacent to each amide -LRB- O4 of the same residue GlcNAci to C1 of previous residue GlcAi − 1 -LRB- d-glucuronic acid -RRB- and O4 of next residue GlcNAci +1 to C4 of that residue GlcNAci +1 -RRB- . For the GlcNAc residue at the reducing terminus the corresponding anomeric atoms -LRB- O1 and its attached proton -RRB- were used instead -LRB- as there is no i + 1 residue -RRB- . Values for cos -LRB- α -RRB- were calculated from the simulation by alignment -LRB- rotation and translation -RRB- of each coordinate frame such that the radius of gyration tensor was diagonal . Each N -- H vector was normalized and then projected along the unit vector coincident with the long axis of the aligned molecule , which resulted in estimates for cos -LRB- α -RRB- . These values were arithmetically averaged over the whole simulation . Diffusion ordered spectroscopy -LRB- DOSY -RRB- experiments Diffusion measurements were carried out without spinning on a 400 MHz Varian Inova instrument , using a 5 mm diameter indirect detection probe equipped with a z-gradient coil , allowing gradient pulses up to 30 G cm − 1 . No sample temperature control was used ; experiments were carried out at the probe quiescent temperature in a room air-conditioned to about 20 °C . Nominal gradient strengths were calibrated according to manufacturer 's recommendations . The Oneshot pulse sequence -LSB- 46 -RSB- was used with a diffusion delay Δ of 0.2 s and a net diffusion-encoding pulse width δ of 2 ms -LRB- i.e. , each bipolar pulse-pair consisted of two 1 ms gradient pulses -RRB- with 10 gradient strengths , ranging from 3.0 to 27.3 G cm − 1 , chosen to give equal steps in gradient squared . A spectral width of 4,000 Hz and 16,384 complex data points were acquired for each gradient strength . Correction for instrumental imperfections by reference deconvolution -LSB- 25 -RSB- , using the TSP peak as reference , were performed on the DOSY data using the standard instrument software -LRB- Vnmr6 .1 C -RRB- . The diffusion coefficients were fitted to Eq . 10 using four coefficients in the power series , where the coefficients for correction of the non-uniform field gradients -LRB- 0.928 , − 9.78 × 10 − 3 , − 3.83 × 10 − 4 and 2.51 × 10 − 5 -RRB- were obtained as previously described -LSB- 27 -RSB- . Electronic supplementary material Below is the link to the electronic supplementary material . Supporting Table 1 Experimental and calculated nOe ratios -LRB- η -RRB- , and T1 relaxation data for isotopic models of diffusion . -LRB- PDF 24.5 kb -RRB- Supporting Table 2 Calculated model-free parameters for the tetra - , hexa - , and octa sacchrides -LRB- 1 , 2 , and 3 respectively -RRB- of the NA-domain of heparan sulfate , using a constant internal correlation time of 30 ps , fitted to isotopic and anisotropic models of rotational diffusion -LRB- PDF 23.5 kb -RRB-

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

carbohydrate polysaccharide glycosaminoglycan heparin proteoglycan dynamics

n-acetyl heparosan
nuclear magnetic resonance (nmr)