Download Sialic Acid Linkage Analysis Kit

Sialic Acid Linkage Analysis Kit
not affect the ability of Sialidases from S.pneumoniae and
C.perfringens to cleave the non-reducing terminal sialic acid
Catalog No
GK80010
Contents
No. of
Vials
1
Description
GK80020 Sialidase S™
(S.pneumoniae rec.)
1
GK80030 Sialidase C™
(C.perfringens rec.)
GK80040 Sialidase A™
(A.ureafaciens rec.)
3
WS0049 5X Incubation buffer B
1
Quantity
1 Unit
(lyophilized)
1Unit in 100 µl
of DSNT. The linkage of an internally linked sialic acid can,
however, still be determined. Sialidase from C.perfringens,
but not from S.pneumoniae will remove sialic acid from
DSNT once the galactose has been removed by a
β-galactosidase indicating a α2-6 linked sialic acid.
Galβ1— 3 GalNAcβ1−− 4 Galβ1−− 4Glc
3
GM1 Oligosaccharide
Neu5Acα2
1 Unit in 200 µl
Neu5Acα2
1 ml
Unit Definition
One unit is defined as the amount of enzyme required to
catalyze the release of 1 µmole of pNP from pNP-α-Nacetylneuraminic acid per minute at pH 5.5 and 37°C.
Storage
The enzymes should be stored at 4°C
Purity
The absence of exoglycosidase contaminants was confirmed
by extended incubations with the corresponding pNPglycosides.
No protease activity was detectable. Call Glyko for certificate
of analysis for details.
Specificity
Sialidase S (S.pneumoniae) releases α2-3 linked sialic acids.
Sialidase C (C.perfringens) releases α2-3 & 6 linked sialic
acids while Sialidase A (A.ureafaciens) releases α2-3,6 & 8
linked sialic acids.
Position Effects
The linkage specificities of the Sialidases from S.pneumoniae
and C.perfringens are valid for sialic acid residues situated at
the non-reducing terminus of oligosaccharides. For
oligosaccharides such as GM1 or DSNT (see structures above)
in which the sialic acid is linked to an internal residue (a
residue linked to two additional monosaccharides) Sialidases
from S.pneumoniae and C.perfringens are unable to remove
the sialic acid regardless of their linkage. However Sialidase
from A.ureafaciens can cleave sialic acids linked to internal
residues. Longer incubation times may be needed for complete
cleavage. The presence of an internally linked sialic acid does
Disialyllacto-N-tetraose
(DSNT)
6
Neu5Acα2— 3 Galβ1−− 3GlcNAcβ1−− 3Galβ1—4Glc
Enzyme Concentrations
Sialidase S (S.pneumoniae):
Sialidase C (C.perfringens):
Sialidase A (A.ureafaciens):
≥5 U/mg
≥10 U/ml
≥5 U/ml
All are formulated in/from 20 mM Tris HCl pH 7.5,
containing 25 mM NaCl. See GK80020 Certificate of
Analysis for lot specific instructions on reconstituting
Sialidase S.
Incubation Buffer:
1 vial of WS0049 5X Incubation buffer B containing 250 mM
sodium phosphate pH 6.0 is provided with each enzyme.
Sample Protocol
For the determination of the linkage position of Nacetylneuraminic acid in a complex oligosaccharide.
To
determine
the
type
of
linkages
between
N-acetylneuraminic acid and galactose in a triantennary
complex oligosaccharide (see structure below) the following
protocol was performed. Note that with the FACE
(Fluorophore Assisted Carbohydrate Electrophoresis), removal
of a sialic acid results in a decrease in mobility.
1.
2.
3.
Prepare a 50 µM (50 pm/µl) fluorophore labeled
oligosaccharide.
Set up four tubes labeled with each of the sialidases
and the fourth as a control.
Add the following to each tube:
4 µl 5X Incubation buffer B
2 µl 50µM oligosaccharide
4.
5.
12 µl water
Add 2 µl of the appropriate sialidase to the tubes and
water to the control tube.
Incubate at 37°C for 1 hour.
Cleavage
Sialidase S (S.pneumoniae) SP
Sialidase C (C.perfringens) CP
Sialidase A (A.ureafaciens) AU
Interpretation of Gel Results
The above gel shows that the digestion with a linkage specific
sialidase from S.pneumoniae results in decreased mobility of
the oligosaccharide due to the loss of α2-3 sialic acid. The
α2-6 linked sialic acid remains intact on the oligosaccharide.
Digestion with sialidase from C.perfringens and A.ureafaciens
results in a greater shift on the gel indicating the presence of
both α2-3 and α2-6 linked sialic acids. The equivalence of the
bands in lanes 4 and 5 indicate that no α2-8 linkages are
present on the oligosaccharide.
Refer to individual enzyme certificates of analysis for
additional information on enzymes in this kit.
FACE analysis of N-acetylneuraminic acid linkage
This product is intended for in vitro research only
Rev May2404
G4
1
2
3
4
5
Lane 1 Standard Ladder of glucose polymers: maltotetraose
is designated as G4
Lane 2 Complex Oligosaccahride (Trisialylated,
triantennary)
Lane 3 Digested with Sialidase S (S.pneumoniae rec.)
Lane 4 Digested with Sialidase C (C.perfringens rec.)
Lane 5 Digested with Sialidase A (A.ureafaciens rec.)