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N. K. KARAMANOS
eliminated from the extraction solution since nucleic acids are also extracted and
may interfere with subsequent steps. Protease activity is another crucial parameter that should be taken into consideration during extraction and further purification steps. A cocktail of protease inhibitors should be used for this purpose. An
effective mixture which is widely used contains: 0.1 M 6-amino-hexanoic acid,
5 mM benzamidine hydrochloride, 0.2 to 1 mM phenylmethansulfonyl fluoride
and 10 mM EDTA for cathepsin D-like activity, trypsin-like activity, serinedependent proteases and metalloproteases, respectively. They are added just
before use to the extraction buffer consisting of 4 M GdnHCI-0.1 M AcONa, pH
5.8, and the suitable detergent. It should be noted that 80-100 % of PGs is usually
extracted and solubilized. When low yield is obtained, such as in some exceptional cases, for instance in invertebrate tissues, SDS (2 %) in the presence of
other detergent may help to improve extractability (Vynios and Tsiganos 1990).
Treatment of tissue with collagenase and/or elastase may also increase extractability disrupting the PG-collagen network.
Extraction should be performed at 4°C overnight with gentle stirring and
using 10 vol. per g of wet weight tissue or 1-2 ml per 35-mm dish for cell cultures. Following low-speed centrifugation the residue can be re-extracted using
half the volume of the extractant for 3-4 h and the extracts can be combined.
Extractability yield can be estimated by degrading the PGs present in the residue
and the extract with papain and measuring the GAG content. This can be done by
applying the digest to a micro column packed with an anion-exchange resin, such
as DEAE-Sephacel (Pharmacia). Following washing with 0.1 M NaCI, GAGs are
eluted with 1.2 M LiCl.
4.3
Fractionation and Purification of PGs
A schematic strategy followed to isolate and characterize PG is given in Fig. 24.7.
Both the culture medium and the cell extract will contain labelled macromolecules and unincorporated precursors. To remove unincorporated precursors and
quantitate the incorporated radioactivity in macromolecules as well as to
exchange the extraction solvent with a solvent which will be suitable for subsequent anion-exchangers used in later steps, a molecular sieve step is necessary.
Sephadex G-25 (PD-lO prepacked columns, BioRad) is equilibrated with chaotropic and non-ionic solvent, such as 8 M urea or 10 M formamide. Column is
equilibrated with one column vol. of elution buffer: 10 M formamide-0.05 M
sodium acetate, pH 6, containing 0.30 M NaCI, 0.5 % (w/v) CHAPS and protease
inhibitors. Formamide is preferably used since it is more stable than urea, which
decomposes with time. The sample is then applied to the column and following
washing with 1.5 ml, the macromolecular fraction is recovered by applying a total
of 3.5 ml of eluting solution (elution between 1.5 and 5.0 ml after sample application). This procedure ensures that macromolecules are free from unincorporated
radioactivity). The total included substances are then eluted with 7.0 ml and this
results in column re-equilibration. Instead of the last step, the column, which
contains the majority of unincorporated radioactivity, can be discarded in radioactive waste, a step that effectively minimizes contamination of the laboratory
N. K. KARAMANOS
eliminated from the extraction solution since nucleic acids are also extracted and
may interfere with subsequent steps. Protease activity is another crucial parameter that should be taken into consideration during extraction and further purification steps. A cocktail of protease inhibitors should be used for this purpose. An
effective mixture which is widely used contains: 0.1 M 6-amino-hexanoic acid,
5 mM benzamidine hydrochloride, 0.2 to 1 mM phenylmethansulfonyl fluoride
and 10 mM EDTA for cathepsin D-like activity, trypsin-like activity, serinedependent proteases and metalloproteases, respectively. They are added just
before use to the extraction buffer consisting of 4 M GdnHCI-0.1 M AcONa, pH
5.8, and the suitable detergent. It should be noted that 80-100 % of PGs is usually
extracted and solubilized. When low yield is obtained, such as in some exceptional cases, for instance in invertebrate tissues, SDS (2 %) in the presence of
other detergent may help to improve extractability (Vynios and Tsiganos 1990).
Treatment of tissue with collagenase and/or elastase may also increase extractability disrupting the PG-collagen network.
Extraction should be performed at 4°C overnight with gentle stirring and
using 10 vol. per g of wet weight tissue or 1-2 ml per 35-mm dish for cell cultures. Following low-speed centrifugation the residue can be re-extracted using
half the volume of the extractant for 3-4 h and the extracts can be combined.
Extractability yield can be estimated by degrading the PGs present in the residue
and the extract with papain and measuring the GAG content. This can be done by
applying the digest to a micro column packed with an anion-exchange resin, such
as DEAE-Sephacel (Pharmacia). Following washing with 0.1 M NaCI, GAGs are
eluted with 1.2 M LiCl.
4.3
Fractionation and Purification of PGs
A schematic strategy followed to isolate and characterize PG is given in Fig. 24.7.
Both the culture medium and the cell extract will contain labelled macromolecules and unincorporated precursors. To remove unincorporated precursors and
quantitate the incorporated radioactivity in macromolecules as well as to
exchange the extraction solvent with a solvent which will be suitable for subsequent anion-exchangers used in later steps, a molecular sieve step is necessary.
Sephadex G-25 (PD-lO prepacked columns, BioRad) is equilibrated with chaotropic and non-ionic solvent, such as 8 M urea or 10 M formamide. Column is
equilibrated with one column vol. of elution buffer: 10 M formamide-0.05 M
sodium acetate, pH 6, containing 0.30 M NaCI, 0.5 % (w/v) CHAPS and protease
inhibitors. Formamide is preferably used since it is more stable than urea, which
decomposes with time. The sample is then applied to the column and following
washing with 1.5 ml, the macromolecular fraction is recovered by applying a total
of 3.5 ml of eluting solution (elution between 1.5 and 5.0 ml after sample application). This procedure ensures that macromolecules are free from unincorporated
radioactivity). The total included substances are then eluted with 7.0 ml and this
results in column re-equilibration. Instead of the last step, the column, which
contains the majority of unincorporated radioactivity, can be discarded in radioactive waste, a step that effectively minimizes contamination of the laboratory