Chemistry Reference
In-Depth Information
To confi rm the suggested structure, partial degradation is performed. The
elution positions of the sample oligosaccharide treated serially with
α
- or
β
- galac-
tosidase,
- mannosidase, neuraminidase
and weak acid lysis are then analyzed using HPLC. Using the database, we can
predict the structure of the treated products from the estimated structure. After
treatment we confi rm that the elution positions of the treated sample coincides
with those of the predicted product. After several steps of treatment, we can be
certain of the oligosaccharide structure. Of help, several enzymes have substrate
specifi city for distinct linkages and so that we can distinguish between isomers
(for example
β
-
N
- acetylhexosaminidase,
α
- fucosidase,
α
1,3/4 - galactosylation). An example of the
analysis of isomers is shown in Figure 5.4. Four monosialyl- biantennary oligosac-
charide isomers, 1A1 - 200.4, 1A2 - 200.4, 1A3 - 200.4 and 1A4 - 200.4, are separated on
ODS and amide columns. The elution positions of 1A3- 200.4 and 1A4 - 200.4 are
close to each other; these could be further differentiated if necessary. They each
α
2,3/6 - sialylation and
β
Figure 5.4
Estimation of glycan structure from
its elution position. The
x
- axis represents the
elution position on the ODS column, and the
y
-axis, the position on the amide column.
Broken arrows represent
isomers of monosialyl-biantennary glycans
have different elution positions and estimated
structures. If there are two or more candidates
with similar elution positions, partial degrada-
tion is effective. As an example, the elution
positions of 1A3-200.4 and 1A4- 200.4 are very
near to each other, but the elution positions of
the products obtained after treatment with
- galactosidase treat-
ment, and solid arrows, treatment with
β
β
-
N
-
acetylhexosaminidase, which releases
β
- GlcNAc
and
-GalNAc. Open circles represent the prod-
ucts of
β
β
- galactosidase treatment and the
closed circles, the products after further treat-
ment with
β
galactosidase and
β
-
N
- acetylhexosaminidase,
1A2 - 100.4
and
1A2 - 100.3,
can
be
easily
β
-
N
- acetylhexosaminidase. The four
distinguished.
