356
N. K. KARAMANOS
4.4.3
Separation with Buoyant Density Gradient Centrifugation
GAGs with their highly anionic groups incorporate the cesium cations and therefore they present much higher buoyant densities in CsCl equilibrium density gradients than those of proteins. PGs with higher GAG-to-protein ratios have, therefore, higher buoyant densities. Gradients formed in the presence of chaotropic
solvents (4 M GdnHCl, 8 urea or 8-10 M formamide) are referred to as dissociative gradients and those in the absence of chaotropic solvents are referred to as
associative gradients (Hascall and Kimura 1982). When hydrophobic PGs, such as
cell surface HSPGs, have to be analyzed addition of 0.5 % CHAPS results in better
recoveries.
4.4.4
Separation According to Protein Core Hydrophobicity
Some PGs can be separated on the basis of differences of their protein cores
hydrophobic properties. Following a detergent gradient elution of octylSepharose column, decorin and the KSPG from cornea, as well as different types
of cell surface HSPGs can be separated at different detergent concentrations
(Takeuchi et al. 1992). Eluants, such as 10 M formamide-0.05 mM sodium acetate
(pH 6.0), containing 0.3 M NaCl, or 4 M GdnHCl and 8 M urea can be used. After
sample application, the binary gradient system is developed using equal volumes
of the starting eluant and of the same solution containing 1.5 % (w/v) CHAPS.
Concentrations of CHAPS in the collected fractions can be determined with a
carbazole assay. Detergents such as Triton X-IOO or other nonpolar reagents can
also be used.
5
Isolation and Characterization of Glycans
The carbohydrate constituents of PGs - GAG chains and oligo saccharides - play
key roles in regulating physical properties and biological functions of PGs. The
structure of the most common carbohydrate constituents, the GAGs, is variable
due to various factors, such as the sulfation degree, the position of sulfates in
repeating disaccharide units, the presence or not of IdoA, the type of hexosamine
present and the structure of the region that links GAGs to protein cores. These
parameters are essential to determine the type of GAG and therefore the PG functional properties.
HA is the only GAG not bound to protein cores. This is secreted extracellularly
and may be separated from PGs by ion-exchange chromatography on DEAESephacel, where HA is eluted with 0.2 M NaCl in 10 M formamide buffer. Buoyant
density centrifugation under dissociative conditions can also be used when HAPG aggregates are present. All other GAGs should be liberated from their PGs
using either digestion with papain and/or alkaline borohydride treatment. In
case of papain-resistant PGs further treatment with alkali may be used to liberate
protein-free GAG chains.
N. K. KARAMANOS
4.4.3
Separation with Buoyant Density Gradient Centrifugation
GAGs with their highly anionic groups incorporate the cesium cations and therefore they present much higher buoyant densities in CsCl equilibrium density gradients than those of proteins. PGs with higher GAG-to-protein ratios have, therefore, higher buoyant densities. Gradients formed in the presence of chaotropic
solvents (4 M GdnHCl, 8 urea or 8-10 M formamide) are referred to as dissociative gradients and those in the absence of chaotropic solvents are referred to as
associative gradients (Hascall and Kimura 1982). When hydrophobic PGs, such as
cell surface HSPGs, have to be analyzed addition of 0.5 % CHAPS results in better
recoveries.
4.4.4
Separation According to Protein Core Hydrophobicity
Some PGs can be separated on the basis of differences of their protein cores
hydrophobic properties. Following a detergent gradient elution of octylSepharose column, decorin and the KSPG from cornea, as well as different types
of cell surface HSPGs can be separated at different detergent concentrations
(Takeuchi et al. 1992). Eluants, such as 10 M formamide-0.05 mM sodium acetate
(pH 6.0), containing 0.3 M NaCl, or 4 M GdnHCl and 8 M urea can be used. After
sample application, the binary gradient system is developed using equal volumes
of the starting eluant and of the same solution containing 1.5 % (w/v) CHAPS.
Concentrations of CHAPS in the collected fractions can be determined with a
carbazole assay. Detergents such as Triton X-IOO or other nonpolar reagents can
also be used.
5
Isolation and Characterization of Glycans
The carbohydrate constituents of PGs - GAG chains and oligo saccharides - play
key roles in regulating physical properties and biological functions of PGs. The
structure of the most common carbohydrate constituents, the GAGs, is variable
due to various factors, such as the sulfation degree, the position of sulfates in
repeating disaccharide units, the presence or not of IdoA, the type of hexosamine
present and the structure of the region that links GAGs to protein cores. These
parameters are essential to determine the type of GAG and therefore the PG functional properties.
HA is the only GAG not bound to protein cores. This is secreted extracellularly
and may be separated from PGs by ion-exchange chromatography on DEAESephacel, where HA is eluted with 0.2 M NaCl in 10 M formamide buffer. Buoyant
density centrifugation under dissociative conditions can also be used when HAPG aggregates are present. All other GAGs should be liberated from their PGs
using either digestion with papain and/or alkaline borohydride treatment. In
case of papain-resistant PGs further treatment with alkali may be used to liberate
protein-free GAG chains.