Glycobiology vol. 19 no. 1 pp.
52–67, 2009
doi:10.1093/glycob/cwn103
Advanced Access publication on October 2, 2008
Glycosaminoglycan origin and structure revealed by multivariate analysis of NMR
and CD spectra
Timothy R Rudd2 , Mark A Skidmore2 , Scott E Guimond2 ,
Cesare Cosentino3 , Giangiacomo Torri3 , David G Fernig2 ,
Robert M Lauder4 , Marco Guerrini3 , and Edwin A Yates1,2
2 School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB,
UK; 3 Institute of Chemical and Biochemical Research “G Ronzoni”, Via G
Colombo 81, Milan 20133, Italy; and 4 School of Health and Medicine,
Division of Biomedical and Life Sciences, Lancaster University, Bailrigg,
Lancaster, UK
Received on July 11, 2008; revised on September 29, 2008; accepted on
September 29, 2008
Principal component analysis (PCA) is a method of sim-
plifying complex datasets to generate a lower number of
parameters, while retaining the essential differences and al-
lowing objective comparison of large numbers of datasets.
Glycosaminoglycans (GAGs) are a class of linear sulfated
carbohydrates with diverse sequences and consequent com-
plex conformation and structure. Here, PCA is applied to
three problems in GAG research: (i) distinguishing origins
of heparin preparations, (ii) structural analysis of heparin
derivatives, and (iii) classification of chondroitin sulfates
(CS). The results revealed the following. (i) PCA of heparin
13
C NMR spectra allowed their origins to be distinguished
and structural differences were identified. (ii) Analysis of the
information-rich 1 H and 13 C NMR spectra of a series of sys-
tematically modified heparin derivatives uncovered under-
lying properties. These included the presence of interactions
between residues, providing evidence that a degree of degen-
eracy exists in linkage geometry and that a different degree
of variability exists for the two types of glycosidic linkage.
The relative sensitivity of each position (C or H nucleus) in
the disaccharide repeating unit to changes in O-, N-sulfation
and N-acetylation was also revealed. (iii) Analysis of the
1
H NMR and CD spectra of a series of CS samples from
different origins allowed their structural classification and
highlighted the power of employing complementary spec-
troscopic methods in concert with PCA.
Keywords: CD/chondroitin sulfate/heparin/NMR/PCA
Introduction
Glycosaminoglycans (GAGs) are a family of biologically im-
portant anionic carbohydrates, consisting of repeating disac-
charide units of a uronic acid and an aminosugar. GAGs are
found in connective tissue, e.g., hyaluronic acid, or conjugated
to proteins as proteoglycans (PGs). PGs are associated with
1 To whom correspondence should be addressed: Tel: +44-151-795-4429;
e-mail: eayates@liv.ac.uk

c The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org
either the extracellular matrix (ECM), for instance, in perlecan,
or with the cell surface, for example, in syndecan or glypican,
and are involved in many aspects of healthy and disease-related
mammalian physiology (Bishop et al. 2007). In this paper, two
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members of the GAG family were studied, heparin and the chon-
droitin/dermatan sulfates (CS/DS). Heparin consists of a general
disaccharide repeating unit of 1,4-linked α-L-iduronic and α-D-
glucosamine, in which the predominant substitution pattern is
2-O-sulfation of the iduronate residues and N- and 6-O-sulfation
of the glucosamine residues, although a considerable number of
other substitutions including N-acetylation and 3-O-sulfation in
glucosamine and the presence of both nonsulfated iduronate and
β-D-glucuronate in place of iduronate lend the sequence consid-
erable heterogeneity (Esko and Lindahl et al. 2001). Chondroitin
sulfates (CS-A and CS-C) and dermatan sulfate (DS; also known
as chondroitin sulfate B, CS-B) possess the same underlying
structure of repeating disaccharide units of D-glucuronic acid-
β-(1→3) N-acetyl-D-galactosamine-β-(1→4). CS can be sul-
fated at positions 4 (to form CS-A) or 6 (to form CS-C) of the
galactosamine residues, while in DS (CS-B), the D-glucuronic
acid is epimerized to L-iduronic acid (hence, is linked to
-α-(1→4)) and can be 2-O-sulfated. CS sulfation varies (Lauder
et al. 2000) with tissue type (Bayliss et al. 1999), location, and
age (Bayliss et al. 1999; Lauder et al. 2001).
These, and other GAGs, exhibit varying degrees of sequence
heterogeneity as a function of their origin and as a result of the
treatments to which they have been subjected during their prepa-
ration. It has proved valuable to study these polysaccharides by
a number of complementary spectroscopic techniques including
NMR, FTIR, and CD (Guerrini et al. 2001; Rudd et al. 2008) to
obtain detailed structural information related to such parameters
as substitution pattern and conformation, to undertake quality
control, and to determine their origin. These are all challenging
issues both technically and in terms of the amount of data that
must be accumulated, compared, and analyzed. A widely used
approach to tackle the problem of handling such large sets of
data, in this case a series of NMR or CD spectra, is to first reduce
their complexity to those basic elements which best encapsulate
the crucial differences between them by employing principal
component analysis (PCA).
PCA is a simple, nonparametric method (it does not depend on
adjusting any values, i.e., parameters, determined through previ-
ous experience) for the reduction of a complex dataset to one of
lower dimension, usually with the aim of revealing simplified
relationships (Ruiz-Calero et al. 2000; Ruiz-Calero, Saurina,
Galceran et al. 2002; Ruiz-Calero, Saurina, Hernandez-Cassou
et al. 2002; Duarte et al. 2004; Almeida et al. 2006). PCA per-
forms the optimum coordinate rotation to align the axes so that
the variance within the data is maximized. When the observed
data have a high signal-to-noise ratio, the principal components
(PCs) with higher variance and of lower order, e.g., first and
52

second principal components (principal components are also
known as factors, eigenvalues, singular vectors or loadings, de-
pending on context), correspond to those which contain differ-
ences in the data and are retained, while those of lower variance
(and of corresponding higher orders) containing noise are dis-
carded. The usual aim of applying PCA is, therefore, to reduce
the dimensionality of the dataset while retaining those charac-
teristics which contribute most to the variance or, put another
way, to find the most effective way to compare data containing
both meaningful signals and noise. A detailed description of the
linear algebra underlying PCA and related techniques is beyond
the scope of this article, although a brief overview is given in
Materials and Methods. For more detailed information, read-
ers are referred to the literature (Jolliffe 2002). Examination of
the individual components does not only reveal simplified rela-
tionships, but also unexpected ones. Another property of PCA is
that it allows the objective comparison of many complex spectra,
which is useful, especially when these are difficult to compare by
visual inspection. Such spectra can then be clustered, or a den-
drogram constructed, to represent their relatedness to each other,
and this can assist recognition or, equally, be used to identify any
differences (which might be subtle) between them. The identity
of essential differences between such groupings can then also
be extracted. In the present article, PCA has been employed first
for the differentiation and structural analysis of heparin samples
obtained from different sources. It was then used to analyze 1 H
and 13 C NMR chemical shift data of systematically modified
heparin polysaccharides to reveal structural subtleties and, fi-
nally, for the analysis of a series of the 1 H NMR and CD spectra
of CS samples for the purposes of classifying them in terms of
their underlying structures.
Results and discussion
A. Differentiation of the 13 C NMR spectra of heparin samples
of distinct origins
It is often desirable to compare samples of commercial pharma-
ceutical grade heparin, or their precursor crude heparin, which
may have been obtained from a variety of sources, for reasons of
quality control. One common requirement is to be able to differ-
entiate rapidly between heparin samples from distinct sources,
for example, porcine (a major source of commercial heparin) or
bovine (now more rarely encountered since the advent of bovine
spongiform encephalopathies; BSE). This has been done previ-
ously using 13 C NMR spectroscopy (Casu et al. 1996) to distin-
guish heparin samples from bovine or porcine origin, essentially
by visual inspection, and the selection of obvious spectral fea-
tures, but here, the ability of PCA to differentiate between three
heparin samples of porcine and four of bovine origin (Table I)
(spectra shown in supplementary data) was tested. The compo-
nent plot revealed that the samples fell into two defined clusters
(Figure 1A and B), which were almost orthogonal. Plotting com-
ponents as a function of sample revealed that component 1 was
strongly correlated with bovine and component 2 with porcine
origin; this strong correlation can be seen in Figure 1C, in which
the contribution of each compound to component 1 is plotted.
Figure 1C also clearly demonstrates that only one component
is required to differentiate between bovine and porcine hep-
arin. PCA with mean centering, which provides slightly better
differentiation, is also included in the supplementary data.
Glycosaminoglycan origin and structure
Table I. Origins of the heparin
samples 1–7
Sample Origin
1 Bovine
2 Porcine
3 Bovine
4 Bovine
5 Porcine
6 Porcine
7 Bovine
Furthermore, plotting component factor regression scores
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(FRS) for components 1 or 2 indicated those spectral details
which differentiated the two groups (Figure 2). The spectrum of
bovine heparin contained more clearly distinct signals; at least
23 positive peaks were found through initial peak picking (see
Table II). One of the peaks found in component 1 FRS (strongly
correlated with bovine origin) had been shown previously to be
a key marker (by visual inspection) for the differentiation of
bovine and porcine heparin spectra (Casu et al. 1996). The peak
at 62.04 ppm was assigned to non-6-O-sulfated glucosamine
residues and was significantly stronger in bovine mucosal hep-
arin (Figure 2A). Conversely, the sample of porcine origin had
a slightly higher level of 6-O-sulfated glucosamine, with a peak
at 68.6 ppm (Figure 2A). The two structures were also differen-
tiated by their respective signal around 73 ppm (Figure 2A and
tentative assignments in Table II) arising from A5 (Yates et al.
1996). Peaks of interest are highlighted in Figure 2.
This simple example illustrates the ability of PCA to catego-
rize datasets and extract key differentiating variables from the
GAG NMR spectra. These results suggested that PCA may also
be applied to the analysis of more complex datasets such as
those arising from a series of systematically modified heparin
derivatives.
B. Analysis of the 1 H and 13 C NMR spectra of a series of
systematically modified heparin derivatives to reveal
correlations with substitution pattern and underlying
structural features
The 1 H and 13 C NMR chemical shift data and assignments
for 12 modified heparin polysaccharides were reported previ-
ously (Yates et al. 1996). The basic disaccharide repeating unit
of bovine lung heparin, -4)-α-L-idoA-2S-(1-4)-α-D-GlcNS,6S-
(1-, was systematically modified exhaustively at each position to
provide combinations of essentially homogeneous polysaccha-
rides (8–19), repeating structures of which are shown in Table
III. The 1 H and 13 C spectra were recorded and assigned (by
one- and two-dimensional homo- and heteronuclear NMR) to
provide datasets consisting of chemical shift values, correlating
with particular (and known) substitution patterns. One challenge
in analyzing such data was to determine whether other subtle
structural relationships existed which might indicate, for ex-
ample, that conformational changes had occurred at positions
remote from the sites of substitution. These may have arisen,
for instance, from changes in hydrogen-bond formation and
could involve changes in glycosidic linkage geometry, altered
ring conformation, or both. These possibilities were investigated
through analysis of the effects on chemical shift values at these
positions.
53
T R Rudd et al.

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Fig. 1. (A) A plot of components 1 and 2 and cluster analysis, for PCA of the 13 C NMR spectra of heparin samples showing their clear differentiation into two
groups. Note that samples 1 and 4 are superimposed. (B) Dendrogram for compounds 1–7 showing the clear differentiation of those derived from bovine (1, 3, 4,
and 7, while closely related samples 1 and 4 can be differentiated by the cluster analysis) and porcine (2, 5, and 6) sources (B). (C) A bar plot of component 1.

Table II. Peak picking of component 1 and 2 FRS with selected assignments

Component 1 FRS Component 2 FRS

Positive (ppm) Negative (ppm) Positive (ppm) Negative (ppm)

60.13 A2 55.86 A2 55.86 A2 60.42 A2

60.42 A2 71.30 58.79 62.03 A6(OH)
62.03 A6(0H) 75.63 60.05 A2 70.04
65.51 78.34 A4 63.14 70.52
70.04 97.58 68.56 A6(S)/I3(NH2/NAc) 73.23 A3/A5/I2OH/I3/I4/I4
70.54 104.18 71.33 76.99
71.10 75.62 79.61 A4
71.45 78.30 A4 79.87 A4
71.78 97.58 99.37
73.23 A3/A5/I2OH/I3/I4/I4 98.86 A1
75.05 104.18
76.96
77.98 I4
78.11 A4
78.81 A4
79.60 A4
79.87 A4
81.93 A4
99.36 A4
99.77
101.45 I1(2S)
102.91 I2(2S)
104.39 I1(2OH)

54
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Fig. 2. (A) Component 1 and 2 factor regression score (FRS) plots, which reproduced features of the bovine spectra and the differences between the porcine and
bovine spectra, respectively. (B) 13 C NMR spectra (as examples) of 2 (of porcine origin) and 7 (of bovine origin). Peaks picked from the FRS plots were tabulated
and tentative assignments made (Table II).

The chemical shift positions of 12 proton and 11 carbon nu- i. Analysis of 1 H NMR Chemical Shift Data. The scree plot
clei (those of the carboxylic acids and N-acetyl groups were revealed three distinct components (Figure 3A). Hierarchical
not included in the original study) comprised the variables for cluster analysis (Figure 3C) of the 1 H NMR chemical shift data
each of the 12 compound cases. Three pieces of information component plot (Figure 3B) indicated that, over the series of
were generated for the 1 H and 13 C data of the 12 compounds: modifications, the highest variation in 1 H chemical shift val-
the scree plot (showing how many components could be found ues occurred at A2. The signal from iduronate most closely
for each dataset), the component value for each chemical shift correlated with A2 was I3. This is consistent with a proposed
position (indicating how significantly a given position, i.e., sin- hydrogen bond between A2-NS and I3-OH, which disappeared
gle chemical shift value, contributed to that component), and following the removal of NS (or substitution with N-acetyl) and
the component factor regression score (FRS, the relative ex- resulted in substantial changes in the chemical shift value of I3
tent to which each modified derivative contributed to a given (Casu et al. 1996; Yates et al. 2000). The majority of iduronate
component). and glucosamine positions lay in the same clusters, I1, I5, I2,
I3, and A4, A5, A6, and A6 , respectively. Interestingly, A3 was
closely linked to the iduronate positions, indicating that the sub-
stitution and conformation of the iduronate residues may directly
affect this position, presumably also through hydrogen bonding.
Table III. The systematically modified heparin samples (8–19)
Of the four glycosidic linkage-related positions, variations in the
Position of substitution chemical shift positions of A1 and I4 were closely correlated,
while more independent variation was evident between I1 and
Compound Notation I2 A6 A2 A4. This is again consistent with the existence of hydrogen
bonding between glucosamine and iduronate, but may indicate
8 I2OH A6OH NH2 H H H
9 I2OH A6OH NS H H SO3 − considerably weaker interactions between iduronate and glu-
10 I2OH A6S NS H SO3 − SO3 − cosamine than between glucosamine and iduronate residues in
11 I2S A6S NH2 SO3 − SO3 − H many derivatives. The torsion angles in the glycosidic linkage
12 I2S A6S NAc SO3 − SO3 − COCH3 I1-Og -A4 are, therefore, presumably less restrained generally
13 I2S A6OH NS SO3 − H SO3 −
14 I2OH A6S NH2 H SO3 − H
than those in A1-Og -I4 (see also section (iii)). I1, I5, and I2 are
15 I2OH A6S NAc H SO3 − COCH3 the positions at which changes due to structural modifications
16 I2OH A6OH NAc H H COCH3 with the highest similarity in chemical shift positions, with I1
17 I2S A6OH NH2 SO3 − H H chemical shift values being correlated with those at I5 and I2
18 I2S A6OH NAc SO3 − H COCH3 which, in the case of I5 do, or I2 can, carry negatively charged
19 I2S A6S NS SO3 − SO3 − SO3 −
groups (a carboxylic acid and a sulfate group, respectively).
55
T R Rudd et al.
Fig. 3. (A) Scree plot of components. (B) Component plot. (C) Hierarchical cluster analysis (clusters defined by at least 80% similarity) produced by PCA of the
1 H chemical shift data for the chemically modified heparin derivatives 8–19 showing correlations between changes in chemical shift positions within the repeating
disaccharide units.
This analysis indicated that there were positions (e.g., A3, A1,
I4, and A4) whose 1 H chemical shift values (inferring also their
environments) were altered, even though they were remote from
the positions at which the substitutions were made. This demon-
strated that subtle structural changes had occurred in the heparin
molecule as substitution patterns were modified.
When the contribution made to each component by each po-
sition was plotted (Figure 4), a number of strong correlations
were revealed: component 1 with A3, A4, I1, I2, I3, and I5;
component 2 with A4, A5, A6, and A6 ; component 3 corre-
lated with A1, I3, and I4 and had negative correlations with A2,
A3, and A4; and component 4 correlated with A3, A4, A6 , I2,
I4, and I5 (supplementary data). When compared with the FRS
for each component, it was apparent that certain modifications
correlated with specific chemical shifts. The significant finding
was that those chemical shifts did not all arise from the obvi-
ous positions of substitution (that is, from those chemical shift
changes that would be expected following the selective removal
of the electronegative sulfate group at I2, A2, or A6, in the case
of 2-O-sulfation in iduronate, N-sulfation, or 6-O-sulfate, or
the addition of N-acetyl groups in glucosamine, respectively).
This indicated structural subtleties and interdependences that
were difficult, or impossible, to delineate by the simple visual
inspection of the spectra.
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For example, the component 1 FRS clearly illustrated
that component 1 differentiated between O-sulfation at I2
(Figure 5A) and was highly correlated with I1, I2, and I3,
(Figure 4A) which was to be expected. However, the correla-
tions with A3, A4, and I5 were less obvious and the correlation
with A3 is consistent with interactions between the hydroxyl
group at A3 and the iduronate 2-O-sulfate, resulting in altered
glycosidic bond geometry involving A4. The correlation with I5
indicated a conformational change when the iduronate residue
was 2-O-sulfated. There was also a weak correlation with A6 .
The component 2 FRS indicated that component 2 dif-
ferentiated between 6-O-sulfation and de-O-sulfation at A6
(Figure 5B). Component 2 was highly correlated with A5, A6,
and A6 (Figure 4B). A4 was also highly correlated with the
de-O-sulfation at A6, indicating probable changes in glycosidic
linkage geometry. There were weaker correlations with A1, A2,
A3, I1, 12, I4, and I5 indicating widespread conformational
effects on the iduronate conformation and glycosidic linkage
geometry. These were investigated further (see section (iii)).
Component 3 FRS differentiated between free-amine/
N-sulfate and N-acetylation at A2 (Figure 5C). Component 3
was correlated positively with A1, I3, and I4, whereas the corre-
lation with A2, A3, and A4 was negative (Figure 4C), suggesting
that the immediate environment in glucosamine was unchanged

Fig. 4. (A) The first, (B) second, and (C) third principal components from the 1 H chemical shift data plotted against proton identity in the disaccharide repeating
units for chemically modified heparin derivatives (8–19). These plots reveal the relative extent to which components 1, 2, and 3 were influenced by the substitutions
made in 8–19 at each position of the repeating disaccharide unit.
by N-acetylation and this is consistent with a stable conforma-
tion in the glucosamine residue. The correlation with A1 and I4
indicates an effect on the glycosidic linkage between A1 and I4,
whereas, the correlations at I3 and I4 represent an interaction
between adjacent residues via the hydroxyl group at I3 with the
amine or N-sulfate at A2, which was obviously distinct when
A2 was N-acetylated. The component 4 FRS differentiated be-
tween N-sulfation at A2 and either free amine or N-acetylation
(see supplementary data).
The relationship between each component and the derivatives
was also studied, to reveal which type of substitution correlated
with each component. Component 1 was strongly related to
those compounds containing O-sulfation at I2, component 2 to
O-sulfation at A6, and component 3 to de-N-acetylation at A2.
Plotting each component against each other (Figure 6) revealed
those positions which correlated specifically with single chem-
ical modifications. Obviously the modifications at I2, A2, and
A6 correlated directly with those positions, but from these plots,
it was also possible to deduce which positions were insensitive
to the chemical modification or, conversely, which seemingly
unrelated positions were affected. If a particular position lay
on either of the axes, this indicated that the position was only
sensitive to that modification. On the other hand, if a position
lay between the axes, they were sensitive to both modifications.
Glycosaminoglycan origin and structure
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I4 was sensitive to modifications only in the glucosamine
residue, either at A2 or A6 (Figure 6B and C), and insensitive
to modifications at I2 (Figure 6A and B) (see also section (iii)).
A3 was sensitive to O-sulfation of I2, which was consistent with
the proposed hydrogen bond between the hydroxyl group at A3
and the O-sulfate at I2 (Figure 6A and B). A6, A6 , and A5 were
sensitive only to O-sulfation at A6 (Figure 6A and C).
ii. Analysis of 13 C Chemical Shift Data. The scree plot generated
by analysis of the 13 C NMR chemical shift values for chemically
modified heparin derivatives 8–19 revealed that there were three
clearly distinct components (Figure 7C).
Variations in the 13 C chemical shift positions for A6 and I2
were observed for all modifications, whereas modifications at
A2 (free amine, N-sulfate or N-acetyl) induced variations at A1,
I3, I4, and I5, in particular, and I1 to a lesser extent (Figure 7).
The component 1 FRS differentiated between O-sulfation and
hydroxyl at I2 (Figure 8A). Desulfation at I2 affected the whole
of the iduronate residue, with a substantial effect in the 13 C spec-
tra at I2; the changes of the other iduronate chemical shifts prob-
ably arose from a change in conformation of the iduronate ring.
There was little change to the glucosamine residue (Figure 9A),
indicating that both conformational stability and interactions
between the two residues probably remained largely unaffected.
57
T R Rudd et al.
Fig. 5. Component FRS plots for (A) component 1, (B) component 2, and (C) component 3 from PCA of 1 H chemical shift data for each chemically modified
heparin derivative 8–19 revealing how the specific chemical modification was correlated with each component.
This agrees with findings in the literature (Hricovini and Torri
1995) in which the glucosamine residue has been observed pre-
dominantly in the 4 C1 conformation.
The component 2 factor FRS differentiated between N-
sulfation at A2 or free amine/N-acetylation (Figure 8B). Com-
ponent 2 was highly correlated with A1, A2, I2, I3, I4, and I5.
The correlation at A1 and A2 was expected, as the substitution
occurred at A2, with correlations at A1 and A4 indicating prob-
able changes in glycosidic linkage geometry. The correlation
of almost all positions in the iduronate residue was more un-
expected, suggesting a conformational change in the ring. The
correlation with I3 indicated a change in the interaction between
I3 and A2 (e.g., in hydrogen bonding). This was corroborated
by the correlation at A1 and I4, consistent with the glycosidic
linkage undergoing geometric rearrangement due to changes in
the interactions between A2 and I3 (Figure 9B)
The component 3 FRS differentiated between O-sulfation and
free hydroxyl at A6 (Figure 8C). Component 3 was correlated
with A4, A5, and A6, with 6-de-O-sulfation only affecting A4
and A5 (Figure 9C). The slightly different information obtained
from the 1 H and 13 C NMR analyses reflected the distinct loca-
tions of 1 H and 13 C nuclei and the influences to which each was
sensitive. The least sensitive position to chemical modification
apparent from analysis of the 13 C chemical shift data was A3
(Figure 9A–D and supplementary data) while I1 was only sen-
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sitive to modification at I2 (i.e., component 1) (Figure 9A and
D) (see also section (iii)). For most modifications, the positions
were orthogonal (supplementary data), except for I4, I3, and I5,
which were sensitive to modifications at I2 and A2 (Figure 9A–
C). N-Sulfation of A2 affected not only the adjacent linkage
position I4 and the I3 hydroxyl group, but also I5 (Figure 9B).
There was also a weak correlation with I2, possibly suggesting
an interaction between de-O-sulfated I2 and the A2-N-sulfated
positions (Figure 9B and supplementary data).
iii. Analysis of the Linkage Position: 13 C Chemical Shift Data.
The possibility of attributing the relative weights of a partic-
ular component to chemical shift changes at each position in
the repeating disaccharide (e.g., Figures 8A and 9A), allowed
selected areas of the molecule (e.g., ring components, those
involving glycosidic linkages, etc.) to be examined. If the as-
sumption is made that similar chemical shift values equated to
similar environments and hence, similar geometries, the anal-
ysis of 13 C chemical shift data (Figure 9) would reveal some
unexpected findings. Figures 7A and 10A demonstrate that the
considerable variation in 13 C NMR shifts existed at the positions
of the linkage (A1, I4, A4, and I1), but one interesting question
in these derivatives is the extent to which the backbone geometry
(represented by 13 C chemical shift values of the linkage posi-
tions) differed for 8–19. This allowed (within the framework
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Fig. 6. Component plots for the 1 H chemical shift data. Component 1 represents 2-O-sulfation/de-O-sulfation, component 2 6-O-sulfation/de-O-sulfation, and
component 3 N-acetylation. Points (representing the chemical shift values at each position, i.e., 1 H nucleus) lying on either the horizontal or vertical axis essentially
contributed to one or other component, those lying away from the axes contributed to both components.

of the assumption made above) the issue of whether a level of
degeneracy existed in relation to the glycosidic geometry to be
addressed.
Figure 10B, in which the chemical shifts arising from the
positions involved in the glycosidic linkages (I4, I1, A4, and
A1), showed distinct groupings comprising four loosely defined
groups: one contained 8, 9, 10, 14, 15, 16, and 19 and another, 12,
17, and 18, while both 11 and 13 lay in more distinct positions.
All except one of the former group contained I2OH residues and
all three of the second group contained I2S residues. This implies
that the linkage geometry (again, inferred through changes in
chemical shifts) was little affected when the iduronate residue
lacked O-sulfates. When the I2 position was O-sulfated (in com-
pounds 11, 12, 13, 17, 18, and 19), the chemical shift values
(hence, glycosidic geometries) were more varied: compounds
17 and 18 (I2S A6OH NH2 and I2S A6OH NAc ) were very similar,
while 11 (I2S A6S NH2 ) and 13 (I2S A6OH NS ) were the most dis-
tinct. There was almost complete identity apparent between a
number of pairs: 9 and 10 (I2OH A6OH NS and I2OH A6S NS ), 15 and
16 (I2OH A6S NAc and I2OH A6OH NAc ), and 17 and 18 (I2S A6OH NH2
and I2S A6OH NAc ). For each of these pairs, the structural differ-
ence was the position of substitution in the glucosamine residue.
To establish the extent to which this approach had succeeded
in distinguishing distinct glycosidic geometries from analysis
of the chemical shifts of these four positions alone, NOE exper-
iments were conducted under identical conditions for some of
these pairs (Table IV). Compounds 9 and 10 were selected as
two examples with almost identical chemical shift values and
16 as an example clearly distinct from these two.
The interacting protons and the dimension of their NOE val-
ues were strikingly similar for 9 and 10 across the A–I glycosidic
Table IV. Percent NOEs between A1 and other positions and between I1 and
other positions across the glycosidic linkages for three derivatives 9, 10, and
16. The presence of an NOE between protons indicates close proximity and the
magnitude of the NOE is sensitive to the conformation
Interacting protons (% NOE)
Compound A1 I1
9 I3 (35), I4 (20), A2 (41) A6/I2 (81), A4 (44)
10 I3 (30), I4 (17), A2 (37) A6/A6 (22), I2 (2), A4 (25)
16 I3 (52a ), I4 (17), A2 (49) A6 (53a ), I2 (14), A4/I2 (41)
a Indicates overlapping signals.

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Fig. 7. (A) Plot of the first three principal components, (B) hierarchical cluster analysis, and (C) scree plot for PCA of the 13 C chemical shift data of 8–19.

linkage between A1 and I4 and were substantially different for sources as shown in Table V. The aim was to determine whether
16. On the other hand, the wider variation was observed in the PCA could be used to group these according to their structural
NOE interactions between I1 and positions across the I–A gly- content and type by analysis of their 1 H NMR or CD spec-
cosidic linkage. This agrees with the earlier observation that tra alone and to determine which spectroscopic method would
the A–I glycosidic linkage is generally less varied than the I–A provide the best means of identification. Their classification
linkage (section (ii)). A comparison of the repeating structures
of 9 and 10 confirmed that the removal of 6-O-sulfate groups
in the presence of N-sulfates, but the absence of 2-O-sulfates,
had very little effect on the A–I linkage geometry, but more Table V. Origins and composition (by disaccharide analysis) of samples of
significant effects on I–A geometry. This implies that, in the ab- chondroitin sulfate (20–29)
sence of 2-O-sulfate groups in iduronate residues, 6-O-sulfates Sample Origin 4-Sulfated 6-Sulfated 2,6-Disulfated
do not interact with the iduronate residues across A–I glycosidic (%) (%) (%)
linkages, but rather, across I–A linkages (Table IV). This also
implies that, through specific modifications, it may be possi- 20 Bovine tracheal CS 65 35 –
ble to alter the geometry of one glycosidic linkage significantly 21 Shark cartilage CS 35 50 15
22 Pig tracheal (6 months) CS 90 10 –
more strongly than the other. 23 DS 100 0 –
24 Ca2+ :DS 100 0 –
25 Whale cartilage CS High nd –
C. Differentiation of chondroitin sulfate samples 26 Pig tracheal CS (old) 80 20 –
by a combination of 1 H and CD spectroscopy 27 Bovine tracheal CS 65 35 –
28 Pig tracheal (6 months) CS 90 10 –
The 1 H NMR and circular dichroism (CD) spectra were recorded 29 Pig tracheal CS (old) 80 20 –
for 10 samples of chondroitin sulfate from different biological
60

Fig. 8. Component FRS plots, (A) component 1, (B) component 2, and (C) component 3 from PCA of 13 C chemical shift data for each chemically modified
heparin derivative 8–19 revealing how the specific chemical modification was correlated with each component.
essentially depends on their disaccharide content compris-
ing characteristic proportions of 4- and 6-O-sulfation and the
presence of iduronate or glucuronate residues. The disaccharide
compositions of the samples were also determined to allow
comparisons to be made and are shown in Table V. The 1 H
NMR spectra for the 10 samples are shown in supplementary
data.
PCA of the 1 H NMR spectra (Figure 11) produced two pri-
mary components, which allowed differentiation of CS (com-
ponent 1 (20, 21, 22, 25, 26, 27, 28, 29)) and DS (component
2 (23, 24)), but the addition of four other components covered
99% of the total variance. Inclusion of these components al-
lowed differentiation between bovine tracheal (27), Ca2+ : DS
(24), shark cartilage (2,6 disulfated) (21), and bovine tracheal CS
(20). From the inspection of component FRS plots (Figure 11D),
it was clear that major structural differences resided in the N-
acetyl spectral region around 2 ppm. As the N-acetyl region has
clear, definable peaks, PCA was also performed on that spectral
region alone. The component plot was very similar to that for
the full spectra (supplementary data), with the two DS samples
(23 and 24) being the most distinct. Again, 27, 22, and 21 were
clearly divided in the three component plots (Figure 11C). As
an example, Figure 12A shows the 1 H spectra for 21, 23, and 28,
with the relevant component FRS values also plotted; these are
the components that correlated with the key spectral features of
Glycosaminoglycan origin and structure
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samples 21, 23, and 28. This illustrated how PCA can be used
to select meaningful features from the NMR spectra, but also
showed that the variation within the N-acetyl region alone was
sufficient for their differentiation into CS and DS. As for the
full spectra, the N-acetyl regions clearly differentiated CS from
DS (23 and 24) and also 27, illustrating that the main difference
in these samples was the position of the peak due to N-acetyl
(Figure 12B). In the analysis of the CD and 1 H NMR spectra of
CS, six components were required to represent fully the underly-
ing features of the different CS/DS samples while differentiation
between CS and DS only required component 1. The structural
differences present in the 10 samples gave rise to several distinct
spectra, and hence 6 principal components were found. Circular
dichroism (CD) is known to be sensitive to uronic acid confor-
mation (and configuration) and was, therefore, also employed.
PCA on the CD spectra produced two components (Figure 13B),
which when plotted (Figure 13C and D) confirmed compounds
23 and 24 (DS and Ca2+ :DS, respectively) as distinct, but also
clearly differentiated compound 21 (shark cartilage), which in
the 1 H NMR analysis needed five components. Furthermore,
by utilizing only two components, it was possible to differenti-
ate between samples CS containing low and high 4-O-sulfation
(Figure 13) 22, 25, and 28 having higher 4-O-sulfation than 20,
27, and 26. This differentiation was confirmed by the disac-
charide analysis shown in Table V. Thus, the combined use of
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Fig. 9. (A) The first, (B) second, and (C) third principal components and (D) component 1 plotted against component 2 from the 13 C chemical shift data plotted
against proton identity in the disaccharide repeating unit for chemically modified heparin derivatives (8–19). These plots show the relative extent to which
components 1, 2, and 3 were influenced by the substitutions made in 8–19 at each position of the repeating disaccharide unit.

complementary spectroscopic techniques and PCA was able to
detect distinct structural differences but CD, somewhat surpris-
ingly given the rather broad features of the CD spectra, was the
more effective of the two. Analysis of these spectra using prior
mean centering, which provided slightly cleaner differentiation,
is shown in supplementary data.
Material and methods
Materials
Chondroitin sulfates were isolated as previously described
(Lauder et al. 2001, 2000). Briefly, diced tissue was digested
by papain (1 U/100 mg tissue) in 0.1 M sodium acetate, 2.4 mM
EDTA, pH 6.8, with 10 mM cysteine HCl added just prior to
digestion, for 24 h at 65◦ C. Following digestion, the GAGs
were precipitated from the soluble fraction by the addition of
4 volumes of ethanol and cooling to 4◦ C overnight. The precip-
itate was resuspended in a minimum volume of 50 mM sodium
acetate and the CS precipitated by the dropwise addition of
2 volumes of ethanol under constant agitation. The solution was
cooled to 4◦ C and allowed to stand overnight before the re-
covery of the precipitate, which was dialyzed overnight against
62
D2 O and lyophilized. The CS chains were released from the
attached amino acids by β-elimination with 0.05 M NaOH con-
taining 1 M sodium borohydride at 45◦ C for 48 h after which
they were dialyzed exhaustively against D2 O then lyophilized.
Dermatan sulfate was isolated from porcine intestinal mucosa as
previously described (Lauder et al. 2000). The Ca2+ form was
generated using cation exchange resin and dialyzing the column
eluate against calcium carbonate before final extensive dialysis
against distilled water.
SRCD
The SRCD spectra were recorded on the CD-12 beamline at
Daresbury Laboratory, a purpose built SRCD facility, using a
quartz sample cell with 0.02 cm path length, 1 nm resolution,
between 260 nm and 175 nm. The CD spectra were recorded
at 10 mg/mL and are relative to (+)-10-camphorsulfonic acid
(1.0 mg/mL).
NMR
The 1 H and 13 C NMR spectra were recorded as described previ-
ously (Yates et al. 1996) with delays of 8 s and 1.5 s for the 1 H and
13
C spectra, respectively. Briefly, the 1 H (COSY and HMQC)
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Fig. 10. (A) A plot of components 1 and 2 (where a correspondence between components 1 and 2 with N-sulfation and I2 O-sulfation, respectively, is revealed in
(C) and (D)) for the PCA analysis performed for the linkage regions (i.e., on 13 C chemical shift positions for I1, A4, I4, and A1) for 8–19. I1 is almost entirely
dependent on component 2, A4 similarly on component 1, whereas I4 and A1 depend on both. (B) The component 1 and 2 FRS plotted against each other for 8–19
showing that the linkage positions are to some extent all influenced by either component 1, 2, or both. (C) The component 1 FRS from the PCA analysis performed
on positions A1, A4, I1, and I4 for compounds 8–19. All those with positive values bear an N-sulfate except 17 and 18 D. The component 2 FRS from the PCA
analysis performed on linkage positions for 8–19. All those with positive values except 19 bear O-sulfate groups at I2.

spectra were obtained at 500 MHz with a Bruker AMX500 spec-
trometer equipped with a 5 mm 1 H/X inverse probe and the 13 C
spectra were obtained at 100 MHz with a 10 mm probe using
a Bruker AM400 instrument. All spectra were recorded in D2 O
at 40◦ C and chemical shift values (ppm) are expressed relative
to external (nondeuterated) trimethylsilyl propionate (TSP) in
D2 O as standard. The NOESY spectra were measured using the
Bruker library sequence (NOESYPHPR) with mixing times of
200 and 400 ms with presaturation of the residual HOD signal
and a delay of 4 s between experiments. The 2D matrix size
of 2000 × 320 was zero filled to 4000 × 2000 by application
of a squared cosine function before Fourier transformation. The
percentage NOE enhancement was measured by integration of a
projection of the cross peak and is expressed relative to a signal
given a nominal value of 1 proton.
Multivariate analysis
Consider a dataset xij (cases i and variables j (i = 1, . . . , n;
j = 1, . . . ,k)); factor analysis allows xij to be modeled as a com-
bination of r factors, ξ. xij = λj1 ξi1 + λj2 ξi2 + · · · +λjr ξir + σij .
The λ terms correspond to the factor loadings to be estimated,
and σij is the measurement error in xij , or the part of xij that is not
accounted for by the r underlying factors. In matrix notation,
this representation can be expanded to X =
  +, where X
(n by the k-matrix) is the observed cases,
(n by the r-matrix)
is the scores on the underlying factors,   (r by the k-matrix) is
the transpose of the factor loadings, and  (n by the k-matrix)
is the measurement errors.
Definitions
Factor/components: Factors/components represent the underly-
ing dimensions that summarise or account for the original set of
observed data.
Loadings/component factor regression scores: Correlation be-
tween the original variable and the factors, the squared factors
loadings indicate what percentage of the variance in the original
variable is explained by a factor.
Score/component values: Composite measure created for each
observation on each factor extracted in the factor analysis.
Multivariate analysis was performed using SPSS 15.0 (SPSS
UK Ltd, Woking, UK), without prior mean centering, unless
63
T R Rudd et al.
Fig. 11. (A) Scree plot from PCA of the 1 H NMR spectra of CS/DS samples, identifying the number of principal components. (B) Cluster plot indicating the
degree of apparent relatedness by analysis of their 1 H NMR spectra, (C) plot of components 1, 2, and 3, and (D) plot of component 1, 2, and 3 FRSs for the analysis
of the 1 H spectra of CS samples 20–29 by PCA.
stated otherwise. Factor analysis was performed, with factor/
components extracted by PCA with a correlation matrix; the
components were then rotated using the varimax method. Clus-
ter analysis was performed using Minitab (Minitab Ltd, Coven-
try, UK) with single linkage. Euclidean distances were measured
and clusters were determined by 80% similarity.
Discussion
Heparin samples of different origins can be distinguished by
PCA of their 13 C NMR spectra and the structural differences
between them were identified. PCA can reveal a wealth of de-
tails concerning complex datasets that would otherwise be dif-
ficult to identify. Analysis of a series of chemically modified
heparin derivatives by NMR revealed that the chemical shift
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changes that most accurately indicate structural changes may
not always occur at the positions of substitution (this much
was evident from the inspection of data carried out in Yates
et al. 1996, 2000) but PCA also identified those positions which
were most strongly correlated with particular changes, as well
as the location of interactions between residues. Furthermore,
changes in the chemical shift values at the glycosidic linkage
positions were identified by PCA and, making the assumption
that similar chemical shift values indicated similar geometries,
allowed their analysis. The close similarity observed in sev-
eral derivatives indicated a possible level of degeneracy in the
geometry of the backbone region (again, assuming that geom-
etry was closely related to chemical shift values at linkage po-
sitions). Some caution must be exercised, however, because
conformational changes in the flexible iduronate residue may

Fig. 12. (A) Component 1, 3, and 6 FRS values derived by PCA of the N-acetyl (CH3 ) region (2.00–2.16 ppm) of the 1 H NMR spectra of CS/DS samples and the
1 H spectra of 21 (shark cartilage), 23 (DS), and 28 (CS). (B) Plot of components 1, 2, and 3 and (C) plot of components 1, 3, and 6 of the N-acetyl (CH ) region
(2.00–2.16 ppm) of the 1 H NMR spectra of CS/DS samples.
also affect the environment of I4 and I1 and could compen-
sate for changes in glycosidic linkage geometry. Interestingly,
the structures that PCA revealed as being the most distinct
were not predictable from a consideration of their substitu-
tion patterns alone and certain structural changes had much
more dramatic effects than others. An example was the dif-
ference between 11 (a repeating structure of I2S A6S NH2 ) and
13 (I2S A6OH NS ). The difference in substitution pattern in
this pair effectively involved the transfer of a sulfate group
from A6 (in 11) to A2 (in 13) and this resulted in the most
different pair in terms of distinct linkage regions of com-
pounds among the heparin derivatives studied (Figure 10B).
On the other hand, the pairs of compounds with almost identical
chemical shift values for the positions involved in the glyco-
sidic linkages (A1, I4, A4, and I1), such as 9 (I2OH A6OH NS ) and
10 (I2OH A6S NS ) or 15 (I2OH A6S NAc ) and 16 (I2OH A6OH NAc ),
suggested that with the I2 position lacking an O-sulfate group,
modifications in glucosamine such as 6-hydroxyl to 6-O-sulfate
at A6 in 9 and 10, or 6-hydroxyl to 6-O-sulfate in 15 and 16 had
little effect on the glycosidic linkages.
Glycosaminoglycan origin and structure
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3
To test the extent to which the glycosidic linkages of such
polysaccharides, with apparently almost identical geometries
(according to PCA of the 13 C chemical shifts of the linkage
positions) varied, a number of NOE experiments were under-
taken. For these polysaccharides, in which the iduronate lacked
a 2-O-sulfate group (9 (I2OH A6OH NS ), 10 (I2OH A6S NS )), the gly-
cosidic linkage geometry between glucosamine and iduronate
was essentially maintained, while that between iduronate and
glucosamine residues showed more variation. Interestingly, this
also indicated that these particular changes in substitution pat-
tern had little effect on one of the linkages, but more on the
other. Similar measurements on another derivative (16 (I2OH
A6OH NAc )) with a distinct location on the PCA plot (Figure 10B),
were confirmed as possessing distinct geometries at both A–I
and I–A linkages. PCA of these data was, therefore, apparently
not particularly sensitive to the changes across the I–A linkage.
The finding that some changes in substitution pattern can
apparently alter one, but not the other, glycosidic linkage (sug-
gesting that a level of degeneracy exists) has implications for
the nature of HS and heparin chains. For example, modifications
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Fig. 13. (A) CD spectra for 20–29. (B) Scree plot indicating two principal components. (C) Plot of component 1 against component 2 indicating differentiation into
three groups: one containing 23 and 24 (both DS samples), a main group comprising 22, 25, 26, 27, 28, and 29 (CS samples), with 21 (shark cartilage) distinct.
(D) Cluster analysis showing the degree of relatedness between the members of these groups.

such as 6-O-sulfation in stretches of I2OH –ANS will result in a
specific geometric modification in one glycosidic linkage and
not the other. In the case of a hydroxyl group at I2 in iduronate,
the A–I linkage geometry appears indifferent to the presence of
sulfate or hydroxyl at A6. On the other hand, in the presence
of 2-O-sulfate at iduronate, the A–I linkage becomes sensitive
to the sulfation status of position A6. Likewise, when N-acetyl
is substituted with N-sulfate in the presence of both 2-O and
6-O-sulfation, comparing 12 and 19 in Figure 10, more substan-
tial geometric changes occur. This has also been confirmed by
NOE experiments (Rudd et al. 2007) in which the magnitude
of the NOEs measured between A1 and I3 or I4 for heparin
was significantly altered in the N-acetylated derivative. The ge-
ometric consequences of substitution patterns including 2-O-
sulfates, with or without 6-O-sulfation, for example, comparing
compounds 19 and 13 in Figure 10, are more significant and
also affect both glycosidic linkages. These results show that,
for particular substitution patterns, certain groups interact with
adjacent residues changing one, or both, glycosidic geometries
and that 2-O-sulfation renders the A–I linkage more sensitive to
the substitution pattern of glucosamine. The ability to influence
the location of these combinations of sulfation and acetyla-
tion, rather than just a single substitution, therefore, provides a
66
method of fine control over the conformational consequences.
The subtle nature of the geometric influence of particular struc-
tural modifications and the first indications and nature of the
degeneracy of this system have been identified. This is the first
step in relating the substitution pattern to conformational out-
comes in these polysaccharides, which will have implications
for their activities.
For the analysis of chondroitin sulfate, PCA allowed classifi-
cation into groups that corresponded to their identification as CS
or DS. By utilizing only the N-acetyl region of the CS/DS spec-
tra, PCA was able to differentiate between the samples to the
same level as using the full spectra; however, limitations in the
ability of 1 H NMR to differentiate CS samples were exposed.
Surprisingly, the CD spectra were better able to differentiate
the samples than 1 H NMR, despite the generally broad features
of the former and apparent information-rich content of the lat-
ter. Indeed, analysis of the CD spectra (Figure 13D) grouped
the CD spectra into clusters: 20, 27, and 26, corresponding to
those with lower ratios of 4- to 6-O-sulfates; 22, 25, 28, and 29,
which contained those with higher ratios of 4- to 6-O-sulfation,
and others, comprising 21, 23, and 24, containing DS samples.
The apparent correlation of the CD spectra with sulfation pattern
suggests that these substitutions may also affect the environment

of the uronic acid to an extent that can be detected by CD. The
ability of CD to differentiate these samples is presumably a con-
sequence of its sensitivity to the conformation or configuration
of the carboxylic acid chromophore of the uronic acid (Rudd et
al. 2007), one of the key differentiating features of CS (Linhardt
et al. 1994).
The potential for the application of PCA to the NMR spectra
of heparin samples for quality control and monitoring the batch-
to-batch variation as well as for the purposes of distinguishing
the origins of heparin samples is clear. The approach can also
comfortably accommodate the sample-to-sample spectral vari-
ation. The component FRS plots determined those characteris-
tic peaks which were present or absent from a spectrum, and
this could serve as an alternative means of structural analysis,
forming the basis of their differentiation in industrial applica-
tions with automated spectral analysis. PCA also has consider-
able potential for identifying structural features from complex
datasets. It is interesting that indications of geometric similar-
ity or change for the series of systematically modified heparin
derivatives could be found by analysis of chemical shift values
at the anomeric positions alone. This highlights the depth of
information that the NMR spectra of heparin derivatives con-
tain. Further investigation indicated that a level of degeneracy
exists in the geometry of the glycosidic linkages. In contrast,
the SRCD spectra were found to be a more sensitive way than
NMR of differentiating between CS samples of varied origins.
PCA combined with spectroscopic approaches is a powerful
combination that can reveal a wealth of structural information
in the GAGs that otherwise would remain obscured.
Supplementary Data
Supplementary data for this article is available online at
http://glycob.oxfordjournals.org/.
Funding
The Biotechnology and Biological Sciences Research Council;
Wellcome Trust; Medical Research Council; and North West
Cancer Research Fund (to D.G.F.).
Acknowledgements
The authors thank David Clarke and Alan Brown of Daresbury
Laboratory for their assistance in collecting the CD spectra and
the STFC for provision of facilities on the CD beamline 12.1,
Daresbury Laboratory, Warrington, UK.
Conflict of interest statement
None declared.
Abbreviations
BSE, bovine spongiform encephalopathy; CS, chondroitin sul-
fate; DS, dermatan sulfate; FRS, factor regression score; GAG,
glycosaminoglycan; HS, heparan sulfate; NOE, nuclear over-
hauser effect; PC, principal component; PCA, principal compo-
Glycosaminoglycan origin and structure
nent analysis; PG, proteogylcan. Throughout the text An or In
refer to position n (either C atom or H atom depending on the
context) of the amino sugar (glucosamine) or iduronate residue,
respectively.
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