Carbohydrate Analysis
247
and
glycolipids.
As
these
glycans
are
not directly genetically coded, but their
organization depends on the sequential
action of enzymes, all such carbohydrates
are commonly found to be somewhat
heterogeneous. This may be a micro-
heterogeneity as evident in glycoprotein
glycans in which most of the structure
remains constant, but a variety of struc-
tures exist due to missing residues at the
molecule’s periphery. Alternatively, there
may be sequence and linkage heterogene-
ity, as found in heteropolysaccharides, or
molecular weight heterogeneity, as is com-
mon in homopolysaccharides.
The principal classes of carbohydrates
are listed in Table 1, together with the most
commonly encountered analytical meth-
ods. Since glycoproteins form the class of
carbohydrates containing material that is
of major importance to cell and molecu-
lar biologists, this article concentrates on
methods for their analysis. The analytical
problems surrounding the carbohydrates
are clearly orders of magnitude more difF-
cult than that of protein analysis; indeed it
is apparent that for many polysaccharides
no two molecules are likely to be identical
and the concept of ‘‘structure’’ must be in-
vestigated and interpreted differently from
that for proteins with more emphasis on
the statistical relevance. There is a further
level of complexity if the conformation of
the carbohydrates in solution is sought as,
in contrast to protein structures, these are
generally not Fxed but fluctuate widely and
may change irreversibly during isolation
or with apparently minor changes in the
temperature, solvent, pH, ionic strength,
concentration, or co-solutes.
Tab. 1
The important analytical methods for carbohydrates.
Carbohydrate
Usual analytical method(s)
Monosaccharides and
disaccharides
In syrups
ICUMSA empirical methods
In food
Enzymatic methods
In glycoconjugates and
polysaccharides
Colorimetric assays
Hydrolyzed
carbohydrate
HPLC, HPAEC-PAD, GC, GC-MS
Oligosaccharides
HPLC, HPAEC-PAD, GC-MS
Neutral
polysaccharides
Separation and extraction techniques, component
analysis, methylation analysis, partial acid and
enzymatic hydrolysis, mass spectrometry, and
13
C
and
1
HNMRspectroscopy
Proteoglycans
Separation and extraction techniques, component
analysis,
β
-elimination, and enzymatic hydrolysis
followed by HPAEC-PAD, and
13
Cand
1
HNMR
spectroscopy
Glycoproteins and
glycolipids
Cleavage from protein/lipid, lectin binding,
component analysis, methylation analysis,
enzymatic hydrolysis, mass-spectrometric
techniques (fast atom bombardment, laser
desorption, and electrospray),
13
Cand
1
HNMR
,
and two-dimensional NMR.
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