Carbohydrate Analysis
253
5.
Structural studies on the intact glycan.
Much analytical information may be ob-
tained by the use of NMR techniques and
mass spectrometry (MS) of the intact gly-
copeptides. Although it is a powerful tool
for determining the presence, sequence,
and arrangement of carbohydrates in a
glycan chain of up to about 40 residues,
mass spectrometry alone is generally not
able to distinguish the anomeric type or
the linkage positions. NMR, on the other
hand, provides a complementary tool that
less easily determines sequences but is
able to provide detailed information on the
anomeric type of the linkages present and
the linkage positions. NMR is also often
the only experimental technique that can
provide the three-dimensional structure.
Lectin binding is a powerful, yet simple,
technique for discriminating between gly-
can types.
6.
Complete glycan analysis.
To be sure of
any conclusions drawn from spectroscopic
studies on an intact glycan, it is neces-
sary to know the complete component
composition of all such glycans. In ad-
dition, it is usual to conFrm the sequence
of carbohydrates and their anomeric link-
ages through the use of speciFc endo- and
exoglycosidases (see ┬▒ig. 2 for examples).
The linkage positions of the glycan com-
ponents are still often determined or con-
Frmed by means of methylation analysis.
1.7
Glycolipids
Glycolipids form a diverse group of bi-
ological molecules that contain glycans
covalently bound to a wide variety of
lipid molecules. The intact molecules are
generally separated by HPLC. The puri-
Fed glycolipid molecules may be struc-
turally
analyzed
by
a
combination
of
NMR and MS or by other techniques
similar to those applicable to glycopro-
tein glycans.
2
Key Methods
2.1
The Use of Enzymes in Analysis
Enzymatic methods of analysis, which use
the speciFcity of enzymes to selectively
pick out substances from mixtures, are
ideally placed for the analysis of complex
biological mixtures. There are two ways in
which enzymes are generally used:
1. Direct
determination
of
substrates
by conversion to products that may
be quantiFed.
2. Structural determination by the ability
of enzymes to speciFcally cleave car-
bohydrate moieties in di-, oligo-, and
polysaccharides and glycoconjugates.
A most important quantitative method
is
that
for
glucose.
This
is
not
only
because glucose is a common carbohy-
drate analyte but also because it is often
hydrolytically produced from other carbo-
hydrates, in their assays. Analysis is by
the completely speciFc conversion of the
glucose to 6-phosphogluconolactone with
the concomitant conversion of the coen-
zyme NADP
+
to its reduced form NADPH,
which absorbs light of 340 nm wavelength.
If care is taken to ensure the purity of the
enzyme(s), such assays are generally very
reliable methods, exhibiting good sensitiv-
ity, accuracy, linearity, and precision. All
currently accepted assays make use of end-
point determinations; quantiFcation being
determined on the basis of a change in
color or ultraviolet absorption before and
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