354
N.K.KARAMANOS
minimize nonspecific adsorption and simultaneously permit binding of PGs to
the active groups of the resin. Small volume of resins (-1 ml), due to their high
binding capacity, is often sufficient for -3 mg of PGs. Proteoglycans are then
eluted with a solvent that contains a sufficient salt concentration. 4 M GdnHCI or
a 10 M formamide that contains -1.0-1.2 M NaCI are commonly used. Utilization of detergents and salts in the eluants reduces nonspecific adsorption of contaminating macromolecules and results in high column recoveries.
In samples containing nucleic acids, e.g., cell extract obtained with guanidine
extraction, they co elute with the PG-containing fraction in anion-exchange chromatography. In this case further purification steps, such as gel-permeation chromatography (GPC) and enzymic digestion with DNAse are often useful in removing nucleic acids.
4.4
Techniques to Separate PG Population
Due to the type of the chains present in various PG populations and the degree
of substitution of PGs with GAG chains, PGs may differ in their charge density,
average buoyant densities, average hydrodynamic sizes, and the hydrophobicities
of their core proteins. Each of these properties can be used to separate and identify different PG classes.
4.4.1
Separation According to Charge Density
The batch ion-exchange step results in a reasonably high level of purification of
PGs from other types of macromolecules. However, a second ion-exchange step
with a continuous salt gradient elution often results in better purity and can separate different PG populations. A commonly used starting eluant is 10 M
formamide-O.OS M sodium acetate, pH 6, containing 0.3 M NaCI and 0.5 % (w/v)
CHAPS. The binary gradient system is composed of equal volumes of the starting
buffer solution and of the same solution containing -1.0-1.2 M NaCl.
PGs with different charge densities can be separated, at least partially, into
separate peaks, as often occurs when both HSPGs and CSIDSPGs are present
(Fig. 24.8A). Single peaks represent homogeneously sized PGs. Further purification of these PGs may be achieved by size exclusion chromatography and buoyant
density gradient centrifugation.
Various types of ion-exchangers are available in high-performance liquid
chromatography (HPLC) and membrane cartridge forms. They offer a variety of
advantages, such as short analysis time, high capacity and recoveries and better
separation profiles.
4.4.2
Separation According to Hydrodynamic Volume
GPC is widely used to separate and characterize macromolecular properties of
PGs, such as average Mr and binding with HA. Furthermore, the presence of
selectively degraded PGs can be assessed. The choice of support matrix and elu-
N.K.KARAMANOS
minimize nonspecific adsorption and simultaneously permit binding of PGs to
the active groups of the resin. Small volume of resins (-1 ml), due to their high
binding capacity, is often sufficient for -3 mg of PGs. Proteoglycans are then
eluted with a solvent that contains a sufficient salt concentration. 4 M GdnHCI or
a 10 M formamide that contains -1.0-1.2 M NaCI are commonly used. Utilization of detergents and salts in the eluants reduces nonspecific adsorption of contaminating macromolecules and results in high column recoveries.
In samples containing nucleic acids, e.g., cell extract obtained with guanidine
extraction, they co elute with the PG-containing fraction in anion-exchange chromatography. In this case further purification steps, such as gel-permeation chromatography (GPC) and enzymic digestion with DNAse are often useful in removing nucleic acids.
4.4
Techniques to Separate PG Population
Due to the type of the chains present in various PG populations and the degree
of substitution of PGs with GAG chains, PGs may differ in their charge density,
average buoyant densities, average hydrodynamic sizes, and the hydrophobicities
of their core proteins. Each of these properties can be used to separate and identify different PG classes.
4.4.1
Separation According to Charge Density
The batch ion-exchange step results in a reasonably high level of purification of
PGs from other types of macromolecules. However, a second ion-exchange step
with a continuous salt gradient elution often results in better purity and can separate different PG populations. A commonly used starting eluant is 10 M
formamide-O.OS M sodium acetate, pH 6, containing 0.3 M NaCI and 0.5 % (w/v)
CHAPS. The binary gradient system is composed of equal volumes of the starting
buffer solution and of the same solution containing -1.0-1.2 M NaCl.
PGs with different charge densities can be separated, at least partially, into
separate peaks, as often occurs when both HSPGs and CSIDSPGs are present
(Fig. 24.8A). Single peaks represent homogeneously sized PGs. Further purification of these PGs may be achieved by size exclusion chromatography and buoyant
density gradient centrifugation.
Various types of ion-exchangers are available in high-performance liquid
chromatography (HPLC) and membrane cartridge forms. They offer a variety of
advantages, such as short analysis time, high capacity and recoveries and better
separation profiles.
4.4.2
Separation According to Hydrodynamic Volume
GPC is widely used to separate and characterize macromolecular properties of
PGs, such as average Mr and binding with HA. Furthermore, the presence of
selectively degraded PGs can be assessed. The choice of support matrix and elu-