360
N.K.KARAMANOS
Chondroitinase B degrades DS chains only at the sites ofIdoA, whereas chondroitinase AC only at the sites of GlcA (Fig. 24.4B). Using these two enzymes separately, CE analysis of the digests may provide useful information on the presence
of IdoA-repeats and the distribution of GlcA in the DS chain (Karamanos et al.
1995a, Karamanos and Hjerpe 1996).
5.2.3
Sequence Analysis of OS
Chondroitinases AC and B degraded DS chain at GlcA and IdoA sites, respectively resulting in longer fragments that can be analyzed by ion-pair HPLC (Karamanos et al. 1995b). Complete separation of the variously sized non-sulfated Lldisaccharides with sizes varying from Ll-(di-nonSh to Ll-(di-nonS)s and variously sized sulfated Ll-saccharides with sizes ranging from Ll-(di-monoS)\ to
Ll-(di-monoS)4 is obtained by one single HPLC run.
The profiles obtained following the separate action of these chondro- and
dermato- lyases on the DS polymer may provide useful information on the size
and degree of sulfation. For further information on the strategy to sequence
uronic-acid containing GAGs see the survey by Karamanos and Hjerpe 1996.
With the proposed strategy the structure of DS-1S fraction from pig skin has
been elucidated (Karamanos et al. 1995b). Apart from HPLC analysis the electrophoretic method described by Cheng et al. 1994 may also be used to obtain information on the primary sequence of DS.
5.3.3
Determination of GAG Reducing Terminal Monosaccharides
and Sulfated and/or Phosphorylated Linkage-Region Oligosaccharide
Following separate digestions with chondro- and dermato-Iyases and HPLC analysis the separated Ll-saccharides can be further analyzed to determine the type
of their disaccharide units. This can be easily performed by a further digestion of
the isolated Ll-saccharides with chondroitinase ABC and analysis of the obtained
Ll-disaccharides with HPLC and/or CE by the methods described above.
Linkage-region oligosaccharides of GAGs to protein core can be analyzed
using the same ion-pair HPLC set-up. Due to the presence of only one linkage oligosaccharide per chain the available amount is limited and therefore it is necessary to increase sensitivity. Utilization of [3Hl-NaBH4 in a proportion of 25 % of
the total NaBH4 used for alkaline borohydride treatment will convert the liberated reducing terminal monosaccharide to alditole, e.g., xylose to [3Hl-xylitole,
making possible the detection and determination of the linkage-region fragment.
Sulfated and/or phosphorylated linkage-region oligo saccharides are completely
separated and determined by HPLC analysis (Gioldassi and Karamanos 1999). To
determine the type of monosaccharide present in the reducing terminal, the
reduced oligosaccharide fragment may further be hydrolyzed with S M trifluoroacetic acid at 100°C for 6h and the obtained alditoles can be resolved by
HPLC analysis as per-O-benzoylated derivatives (Karamanos 1992).
N.K.KARAMANOS
Chondroitinase B degrades DS chains only at the sites ofIdoA, whereas chondroitinase AC only at the sites of GlcA (Fig. 24.4B). Using these two enzymes separately, CE analysis of the digests may provide useful information on the presence
of IdoA-repeats and the distribution of GlcA in the DS chain (Karamanos et al.
1995a, Karamanos and Hjerpe 1996).
5.2.3
Sequence Analysis of OS
Chondroitinases AC and B degraded DS chain at GlcA and IdoA sites, respectively resulting in longer fragments that can be analyzed by ion-pair HPLC (Karamanos et al. 1995b). Complete separation of the variously sized non-sulfated Lldisaccharides with sizes varying from Ll-(di-nonSh to Ll-(di-nonS)s and variously sized sulfated Ll-saccharides with sizes ranging from Ll-(di-monoS)\ to
Ll-(di-monoS)4 is obtained by one single HPLC run.
The profiles obtained following the separate action of these chondro- and
dermato- lyases on the DS polymer may provide useful information on the size
and degree of sulfation. For further information on the strategy to sequence
uronic-acid containing GAGs see the survey by Karamanos and Hjerpe 1996.
With the proposed strategy the structure of DS-1S fraction from pig skin has
been elucidated (Karamanos et al. 1995b). Apart from HPLC analysis the electrophoretic method described by Cheng et al. 1994 may also be used to obtain information on the primary sequence of DS.
5.3.3
Determination of GAG Reducing Terminal Monosaccharides
and Sulfated and/or Phosphorylated Linkage-Region Oligosaccharide
Following separate digestions with chondro- and dermato-Iyases and HPLC analysis the separated Ll-saccharides can be further analyzed to determine the type
of their disaccharide units. This can be easily performed by a further digestion of
the isolated Ll-saccharides with chondroitinase ABC and analysis of the obtained
Ll-disaccharides with HPLC and/or CE by the methods described above.
Linkage-region oligosaccharides of GAGs to protein core can be analyzed
using the same ion-pair HPLC set-up. Due to the presence of only one linkage oligosaccharide per chain the available amount is limited and therefore it is necessary to increase sensitivity. Utilization of [3Hl-NaBH4 in a proportion of 25 % of
the total NaBH4 used for alkaline borohydride treatment will convert the liberated reducing terminal monosaccharide to alditole, e.g., xylose to [3Hl-xylitole,
making possible the detection and determination of the linkage-region fragment.
Sulfated and/or phosphorylated linkage-region oligo saccharides are completely
separated and determined by HPLC analysis (Gioldassi and Karamanos 1999). To
determine the type of monosaccharide present in the reducing terminal, the
reduced oligosaccharide fragment may further be hydrolyzed with S M trifluoroacetic acid at 100°C for 6h and the obtained alditoles can be resolved by
HPLC analysis as per-O-benzoylated derivatives (Karamanos 1992).