1 1 . M U C O P O L Y S A C C H A R I D E S IN TISSUE C U L T U R E
363
subsequently with hydrochloric acid. If borate ions are incorporated
into the reaction, less colour is given by glucosamine than by galactosamine, a fact that has been utilized by Tracey (1955) for the determination of mixtures of these amino sugars. Parekh and Glick (1962) have
described a modification of the colorimetric determination which
gives 100°/o recovery with 0-01-0-1 /xg hexosamine. Mixtures of the
amino sugars can be separated by chromatography on paper as
described by Fisher and Nebel (1955), or quantitatively on columns of
Zeo-Karb 225, or Dowex-50 (Drake and Gardell, 1952; Rondle and
Morgan, 1955; Gardell, 1953; Crumpton, 1957; Pogell and Koenig,
1959). Recently, Gabriel and Igals (1960) have reported the separation
of monosaccharides on glass-fibre sheets, and Partridge and Elsden
(1961b) have described a rapid method for separation of glucosamine
and galactosamine. These amino sugars may be identified also by
oxidation with ninhydrin to the corresponding pentoses which are
separated readily by paper chromatography (Stoffyn and Jeanloz,
1954).
The uronic acid component of the mucopolysaccharide may be
determined by measurement of the carbon dioxide produced on treatment with strong acids (Tracey, 1948), or by the colorimetric method
of Dische (1947, 1955). Iduronic acid, which occurs in chondroitin
sulphate-B, is more stable to hydrolysis than glucuronic acid and can
be identified by paper chromatography (Hoffman et al., 1956; Berenson
et al., 1958). Iduronic acid and glucuronic acid also differ in their
behaviour in the colorimetric procedure of Dische (1955).
In addition to the conventional methods, acetyl groups in the
1-10 /xmole (about 40-400 fig) range have been determined by hydrolysis in 2 N - H C 1 in dry methanol, and the methyl acetate formed determined colorimetrically as the Fe
8 +
-hydroxamic acid complex (Ludowieg
and Dorfman, 1960).
Sulphate is difficult to determine accurately in small amounts;
probably the best procedure at present available is Spencer's (1960)
modification of the barium chloranilate method. This is less sensitive
than the benzidene assay, which is recommended for S 0 4
a _ concentrations less than 25 ju,g/ml (Spencer, 1960), but is more rapid and can
be used in the presence of P 0 4
3 ~ ions. Slack (1958) found precipitation
with 4-chloro-4'-aminodiphenyl (Belcher and Wilson, 1956) also
suitable for the determination of microgram amounts of S 0 4
a _ liberated
from mucopolysaccharides on hydrolysis with 2 5 % formic acid. As
3 5
S 0 4
2 _ is now readily available, isotopic dilution offers a convenient
method for the determination of small amounts of sulphate.
All of the above mentioned analyses require the prior hydrolysis of
the mucopolysaccharides. Not only is glucuronic acid particularly
363
subsequently with hydrochloric acid. If borate ions are incorporated
into the reaction, less colour is given by glucosamine than by galactosamine, a fact that has been utilized by Tracey (1955) for the determination of mixtures of these amino sugars. Parekh and Glick (1962) have
described a modification of the colorimetric determination which
gives 100°/o recovery with 0-01-0-1 /xg hexosamine. Mixtures of the
amino sugars can be separated by chromatography on paper as
described by Fisher and Nebel (1955), or quantitatively on columns of
Zeo-Karb 225, or Dowex-50 (Drake and Gardell, 1952; Rondle and
Morgan, 1955; Gardell, 1953; Crumpton, 1957; Pogell and Koenig,
1959). Recently, Gabriel and Igals (1960) have reported the separation
of monosaccharides on glass-fibre sheets, and Partridge and Elsden
(1961b) have described a rapid method for separation of glucosamine
and galactosamine. These amino sugars may be identified also by
oxidation with ninhydrin to the corresponding pentoses which are
separated readily by paper chromatography (Stoffyn and Jeanloz,
1954).
The uronic acid component of the mucopolysaccharide may be
determined by measurement of the carbon dioxide produced on treatment with strong acids (Tracey, 1948), or by the colorimetric method
of Dische (1947, 1955). Iduronic acid, which occurs in chondroitin
sulphate-B, is more stable to hydrolysis than glucuronic acid and can
be identified by paper chromatography (Hoffman et al., 1956; Berenson
et al., 1958). Iduronic acid and glucuronic acid also differ in their
behaviour in the colorimetric procedure of Dische (1955).
In addition to the conventional methods, acetyl groups in the
1-10 /xmole (about 40-400 fig) range have been determined by hydrolysis in 2 N - H C 1 in dry methanol, and the methyl acetate formed determined colorimetrically as the Fe
8 +
-hydroxamic acid complex (Ludowieg
and Dorfman, 1960).
Sulphate is difficult to determine accurately in small amounts;
probably the best procedure at present available is Spencer's (1960)
modification of the barium chloranilate method. This is less sensitive
than the benzidene assay, which is recommended for S 0 4
a _ concentrations less than 25 ju,g/ml (Spencer, 1960), but is more rapid and can
be used in the presence of P 0 4
3 ~ ions. Slack (1958) found precipitation
with 4-chloro-4'-aminodiphenyl (Belcher and Wilson, 1956) also
suitable for the determination of microgram amounts of S 0 4
a _ liberated
from mucopolysaccharides on hydrolysis with 2 5 % formic acid. As
3 5
S 0 4
2 _ is now readily available, isotopic dilution offers a convenient
method for the determination of small amounts of sulphate.
All of the above mentioned analyses require the prior hydrolysis of
the mucopolysaccharides. Not only is glucuronic acid particularly