Chemistry Reference
In-Depth Information
proteins. For reproducibility, it is strongly recommended with both these enzymes
that the substrates are denatured before analysis (please see Info Box for further
information).
To remove
N
-glycans, the established chemical procedure uses hydrazine [2] .
This method removes the risk of the glycan remaining uncleaved due to the steric
hindrance from the protein region or the loss of enzyme activity. However, this
anhydrous reaction requires careful handling and equipment. In this chemical
method,
N
-glycolylneuraminic acid can also be converted to
N
- acetylneuraminic
acid.
As yet, there is no single enzyme for the removal of all types of
O
- glycans. Alkali
degradation and hydrazine degradation are used to release
O
- glycans [3, 4] .
However, these procedures can easily result in the formation of fragmented prod-
ucts. Endoglycoceramidase, which releases glycans from glycosphingolipids (intro-
duced by M. Ito and T. Yamagata in 1986), can be used to analyze the structure
of glycans in sphingolipids.
Info Box
N
- Glycosidase, formally peptide -
N
4
- (
N
- acetyl -
- glucosaminyl) asparagine ami-
dase (EC 3.5.1.52), is named as glycopeptidase, glycoamidase, glycanase and
peptide -
N
-glycosidase. This enzyme was originally isolated from almonds by
N. Takahashi in 1977. In 1984, T. H. Plummer
et al.
isolated another glycopep-
tidase from
Flavobacterium
spp. These enzymes cleave the N-C bond in
the Asn branch, which binds the
N
-glycan, liberating a glycan that includes the
- NH
2
, and changing Asn to Asp (Figure 5.2). There are differences in the
optimal pH of the two enzymes. The enzyme from almonds reacts under weak
acidic conditions, so the amine of the reducing end of the glycan is rapidly
removed. On the other hand, the enzyme from
Flavobacterium
reacts at pH 7-8
and the amine on the reducing end is not removed. The complexity in the
reaction mechanisms and competition between suppliers has led to the use of
several names for these enzymes. In addition, 1 U of the commercial enzyme
from
Flavobacterium
corresponds to 1 mU of the enzyme from almonds. There
are also differences in the active ingredient between manufacturers, so it is
important to take particular care when using this enzyme.
β
5.3
Glycan Purifi cation
Glycans can be purifi ed, as for other biomaterials, on gel- fi ltration, ion- exchange
or reversed-phase columns. In addition, cellulose and activated carbons, in particu-
lar, are known to interact with carbohydrates [5]. A sophisticated method for glycan
isolation involves the use of reactive materials including oxime or hydrazide func-
tional groups to bind to aldehyde on the reducing end of the carbohydrates (Figure
5.3) [6]. Sialic acids, which often form the non-reducing ends of glycans, are
