1 1 . M U C O P O L Y S A C C H A R I D E S IN T I S S U E C U L T U R E
361
et al., 1961) seems potentially of considerable value in the analysis
and characterization of fractions obtained from cultured material.
Thus Korn (1959) obtained quantitative precipitation of heparin even
at concentrations as low as 25 /xg/ml with cetyltrimethyl-ammonium
bromide, provided celite was added as an inert adsorbent. The major
advantage of this method is that the initial precipitation from dilute
salt solution gives considerable purification, since most of the protein
remains in solution. It should be remembered, however, that these
quaternary ammonium compounds also precipitate nucleic acids
(Jones, Marsh and Rizvi, 1957). As cetylpyridinium chloride in these
fractions may interfere with the estimation of amino sugar, the mucopolysaccharide concentration is determined preferably by uronic
acid analysis. Procedures that utilize the acidic properties of mucopolysaccharides in the formation of insoluble complex salts (e.g.
Ehrenprieis and Fishman, 1960) serve to eliminate neutral polysaccharides. Although, as in other methods, glycogen may be removed
by treatment with amylase or diastase, this does not apply to cellulose,
which Hall, Happey, Lloyd and Saxl (1960) have shown to be a normal
though minor component of mammalian connective tissue.
Further modifications of these methods were described by Antonopoulos, Borelius, Gardell, Hamstrom and Scott (1961) and applied to
papain-digests of 200-400 fxg (dry weight) of (connective) tissue by
Antonopoulos, Gardell, Szirmai and De Tyssonsk (1962). The method
described by these authors not only gave a quantitative recovery of
mucopolysaccharides from both pure preparations and biological
materials but was applicable to serial sections of connective tissue and
was capable, therefore, of histological control.
Although the chondroitin sulphate isomers are not separated by this
method, Roden and Dorfman (1958) described the isolation of chondroitin sulphate-B of high purity from the mixture of chondroitin
sulphuric acids obtained from rat skin, by digestion with testicular
hyaluronidase followed by the precipitation of the resistant chondroitin sulphate-B with cetylpyridinium bromide.
2. Chromatography
Schiller et al. (1961) found chromatography on the anion exchange
resin Dowex-1-chloride to be a useful supplement to the cetylpyridinium chloride method of fractionation. From the Dowex column the
fractions were eluted with sodium chloride solutions of increasing
molarity.
Mixtures of hyaluronic acid, chondroitin sulphate-A and/or heparin
can be separated on columns of celite and calcium phosphate (Bowness,
1960) and on cellulose by elution with increasing concentrations of
N *
361
et al., 1961) seems potentially of considerable value in the analysis
and characterization of fractions obtained from cultured material.
Thus Korn (1959) obtained quantitative precipitation of heparin even
at concentrations as low as 25 /xg/ml with cetyltrimethyl-ammonium
bromide, provided celite was added as an inert adsorbent. The major
advantage of this method is that the initial precipitation from dilute
salt solution gives considerable purification, since most of the protein
remains in solution. It should be remembered, however, that these
quaternary ammonium compounds also precipitate nucleic acids
(Jones, Marsh and Rizvi, 1957). As cetylpyridinium chloride in these
fractions may interfere with the estimation of amino sugar, the mucopolysaccharide concentration is determined preferably by uronic
acid analysis. Procedures that utilize the acidic properties of mucopolysaccharides in the formation of insoluble complex salts (e.g.
Ehrenprieis and Fishman, 1960) serve to eliminate neutral polysaccharides. Although, as in other methods, glycogen may be removed
by treatment with amylase or diastase, this does not apply to cellulose,
which Hall, Happey, Lloyd and Saxl (1960) have shown to be a normal
though minor component of mammalian connective tissue.
Further modifications of these methods were described by Antonopoulos, Borelius, Gardell, Hamstrom and Scott (1961) and applied to
papain-digests of 200-400 fxg (dry weight) of (connective) tissue by
Antonopoulos, Gardell, Szirmai and De Tyssonsk (1962). The method
described by these authors not only gave a quantitative recovery of
mucopolysaccharides from both pure preparations and biological
materials but was applicable to serial sections of connective tissue and
was capable, therefore, of histological control.
Although the chondroitin sulphate isomers are not separated by this
method, Roden and Dorfman (1958) described the isolation of chondroitin sulphate-B of high purity from the mixture of chondroitin
sulphuric acids obtained from rat skin, by digestion with testicular
hyaluronidase followed by the precipitation of the resistant chondroitin sulphate-B with cetylpyridinium bromide.
2. Chromatography
Schiller et al. (1961) found chromatography on the anion exchange
resin Dowex-1-chloride to be a useful supplement to the cetylpyridinium chloride method of fractionation. From the Dowex column the
fractions were eluted with sodium chloride solutions of increasing
molarity.
Mixtures of hyaluronic acid, chondroitin sulphate-A and/or heparin
can be separated on columns of celite and calcium phosphate (Bowness,
1960) and on cellulose by elution with increasing concentrations of
N *