266
Carbohydrate Analysis
anomeric confguration oF glucopyranose
and galactopyranose are easily observed,
From the
3
J
H1–H2
coupling constant, as the
H-1 and H-2 protons oF the beta anomers
are trans-diaxial (
180
) giving large cou-
plings, whereas those in alpha anomers
are gauche (
60
) with small couplings.
2.3.6
Molecular Modeling
Molecular
modeling
techniques
treat
structures as mechanical entities in which
bond lengths, angles, and torsions are
treated as springs requiring Forces to move
them From their preFerred values. Together
with a consideration oF atomic charges and
van der Waals interactions, the structure oF
the molecule is optimized, giving preFerred
conFormations. Various molecular dynam-
ics and random walk molecular mechanics
(RAMM) methods exist to ensure that the
molecule does not settle into biologically
irrelevant local potential energy minima.
The
additional
inFormation
available
From such molecular modeling is par-
ticularly useFul For carbohydrates, when
compared to proteins, as carbohydrates
generally do not Form crystal structures
(iF they Form them at all) with similar con-
Formations to their conFormation in Freely
dissolved solution. The inFormation is also
useFul For clariFying NMR structural analy-
ses where the coupling constants may not
be measurable and where there are mixed
conFormational populations present.
2.4
Linkage Analysis
2.4.1
Glycan Detection
There are several methods available For de-
tecting the presence oF glycans attached to
proteins (glycoproteins) and lipids (glycol-
ipids). One way is to oxidize all the pairs oF
adjacent hydroxyl groups (i.e.
cis
-diols) in
the carbohydrate to aldehyde groups, using
periodate, Followed by covalent coupling
to hydrazide derivatives oF biotin or the
steroid digoxigenin. The respective labeled
glycoconjugates may be detected by an en-
zyme immunoassay using streptavidin or
a digoxigenin-specifc antibody conjugated
to alkaline phosphatase. This procedure
may be applied
in situ
For the detection
oF nanogram quantities oF glycoproteins,
previously separated by sodium dodecyl
sulFate/polyacrylamide gel electrophoresis
(SDS-PAGE) and blotted onto nitrocellu-
lose membranes. More specifc oxidation
methods, such as the use oF galactose ox-
idase or prior specifc enzyme hydrolysis
(Sect. 2.4.2), may allow the detection oF
glycan subclasses.
2.4.2
Glycan Release and the Use of
Enzymes
The
carbohydrate
structures
in
glyco-
proteins are normally investigated aFter
they are released From the protein and
chromatographically separated From each
other. There are a number oF methods by
which these glycans may be released; some
utilize specifc enzymes (Table 2), whilst
others use chemical methods (±ig. 2).
2.4.3
Methylation Analysis
M
e
th
y
l
a
t
i
onan
a
l
y
s
i
si
sap
ow
e
r
Fu
lan
d
widely used method For the determina-
tion oF the glycosidic linkage positions in
the structural analysis oF glycoprotein, as
well as glycolipids, glycans, and polysac-
charides. It involves replacing all the Free
hydroxyl groups with methyl ether groups
(±ig. 8). The methylated polysaccharide
is then hydrolyzed, converted to alditols,
acetylated, and analyzed by GC or GC-MS.
The carbohydrate is initially dissolved
ind
ryDMSO
.Inthep
resenceo
Fave
ry
strong base, such as sodium hydride or
potassium
tert
-butoxide, and the absence
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