Carbohydrate Analysis
251
detailed
fne
structure
is
much
more
arduous.
1.6
Glycoproteins
Generally, the most important carbohy-
drates to cell and molecular biologists
are the glycoproteins, where carbohydrate
oFten conFers the specifcity inherent in
many molecular and cellular interactions.
These compounds possess diverse struc-
tures
in
which
carbohydrate
moieties,
known as glycans, are covalently attached
to proteins. A number oF diFFerent car-
bohydrates may be attached in diFFerent
ways, with specifc but varying anomeric
confgurations, linkage positions, and lo-
cations within the sequence. All these
parameters must be determined iF a com-
plete analysis oF the glycoprotein is to be
achieved.
Usually the glycans are oF relatively low
molecular weight, containing between 1
and 20 monosaccharides oF between 1 and
5 diFFerent types. They oFten involve com-
plex, highly branched structures (±ig. 2).
The same type oF monosaccharide may be
connected in diFFerent ways even within a
single glycan group. It is Frequently Found
that several diFFerent types oF glycan are
attached to one protein molecule. Since
incomplete glycan biosynthesis or subse-
quent partial hydrolysis commonly results
in glycan heterogeneity, and since such
diFFerences in structure oFten do not sig-
nifcantly influence the chromatographic
behavior oF the intact glycoprotein, a num-
ber oF diFFerent, but related, glycans may
be present and coupled to the same amino
acid residue, even in ‘‘purifed’’ glycopro-
tein preparations. The analysis oF such
structures is a serious undertaking For
which there are ground rules but no gener-
ally applicable, single analytical technique.
Methods may vary between laboratories
even For similar glycoproteins. There are,
however, stages through which any com-
plete analysis must pass.
1.
Isolation and purifcation.
This in-
volves a number oF chromatographic steps
such as Fast protein liquid chromatography
(±PLC), HPLC, gel exclusion chromatog-
raphy, and lectin aFfnity chromatography
(Sect. 2.5).
2.
Determination
oF
the
constituent
car-
bohydrates.
Monosaccharides may be
liberated From the glycoproteins by mild
acid
hydrolysis
(e.g.
2
M
HCl,
4
h,
100
C). Once liberated, and aFter any nec-
essary derivatization, they are separated
and determined in one step by either
GC or HPLC. This component analy-
sis usually indicates the type oF glycans,
hence the likely glycan–protein linkages
involved.
3.
Cleavage oF the intact glycans From the
protein moieties.
The diFFerent Families
oF glycoprotein glycans require diFFerent
methods For their cleavage (±ig. 2). As
an alternative to the use oF enzymes,
automated chemical cleavage using hy-
drazinolysis releases either O-linked or
O- and N-linked intact oligosaccharides
(±ig. 2). The glycosylation sites may be de-
termined From glycoprotein peptide map-
ping. The cleaved glycans are oFten labeled
with a radioactive tracer (e.g. by having
their reducing ends reduced with sodium
borotritide) to make it easier to Follow them
through their purifcation.
4.
Glycan isolation and purifcation.
A
combination oF size-exclusion HPLC and
HPAEC with pulsed amperometric detec-
tion (HPAEC-PAD) is commonly used For
the purifcation oF glycans. Lectin aFfnity
chromatography is a powerFul technique
used in their separation.
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