360
J. T . D I N G L E A N D M. W E B B
from the tissue and the tissue protein. The principal consideration
that determines the method adopted for extraction and isolation of
these materials has been the need to compromise between the conflicting requirements for purification on the one hand and the avoidance
of degradative artifacts on the other. At present it would appear that
mucopolysaccharides are best extracted from tissues after a preliminary digestion with proteolytic enzymes—a procedure that causes
a loss in viscosity of isolated mucoproteins (Muir, 1956), but does not
eliminate protein or peptide components completely (e.g. Partridge
and Davis, 1958). Both trypsin and pepsin (Grossfeld, Meyer, Godman
and Linker, 1957) and also papain (Schiller, Slover and Dorfman, 1961)
have been used for the preliminary digestion. An initial extraction of
the tissue with 0-5N-NaOH before treatment with proteolytic enzymes
has been used by Schiller, Mathews, Jefferson, Ludowieg and Dorfman
(1954) and Bollet (1958) among others. Whereas aqueous alkali gives
a more complete extraction of acid mucopolysaccharides than do
neutral solutions, the high p H leads to degradative changes. Although
depolymerization of the mucopolysaccharides may be unimportant
in analytical work, initial alkali extraction is to be avoided in studies
of the macromolecular structure and physical properties of these
substances (e.g. Shatton and Schubert, 1954; Hallen, 1959). Tissue
extracts prepared without the use of proteolytic enzymes usually contain the mucopolysaccharides in combination with protein as mucoproteins, which may be isolated and purified by physical methods.
Thus chondromucoprotein has been obtained from extracts of bovine
nasal septa (Gerber, Franklin and Schubert, 1960) and from human
costal cartilage (Johnson and Schubert, 1960) by ultra-centrifugation.
F. P U R I F I C A T I O N A N D I D E N T I F I C A T I O N OF
M U C O P O L Y S A C C H A R I D E S
1. Precipitation Methods
A number of procedures are available for the purification of the
crude mucopolysaccharide fraction. The classical methods of Meyer,
Davidson, Linker and Hoffman (1956) involve the ethanolic precipitation of the mucopolysaccharides in the presence of calcium ions.
Barium has been used instead of calcium by Jorpes and Gardell (1948)
and by Smith and Gallop (1953). This method requires relatively large
quantities of material and has little application in Tissue Culture.
Progress in the development of micro-methods has been made by
Gardell (1961) and Scott (1955, 1960). Precipitation of mucopolysaccharides by cetylpyridinium chloride, and the differential solubility
of these complexes in salt solutions (Scott, 1955; Slack, 1958; Schiller
J. T . D I N G L E A N D M. W E B B
from the tissue and the tissue protein. The principal consideration
that determines the method adopted for extraction and isolation of
these materials has been the need to compromise between the conflicting requirements for purification on the one hand and the avoidance
of degradative artifacts on the other. At present it would appear that
mucopolysaccharides are best extracted from tissues after a preliminary digestion with proteolytic enzymes—a procedure that causes
a loss in viscosity of isolated mucoproteins (Muir, 1956), but does not
eliminate protein or peptide components completely (e.g. Partridge
and Davis, 1958). Both trypsin and pepsin (Grossfeld, Meyer, Godman
and Linker, 1957) and also papain (Schiller, Slover and Dorfman, 1961)
have been used for the preliminary digestion. An initial extraction of
the tissue with 0-5N-NaOH before treatment with proteolytic enzymes
has been used by Schiller, Mathews, Jefferson, Ludowieg and Dorfman
(1954) and Bollet (1958) among others. Whereas aqueous alkali gives
a more complete extraction of acid mucopolysaccharides than do
neutral solutions, the high p H leads to degradative changes. Although
depolymerization of the mucopolysaccharides may be unimportant
in analytical work, initial alkali extraction is to be avoided in studies
of the macromolecular structure and physical properties of these
substances (e.g. Shatton and Schubert, 1954; Hallen, 1959). Tissue
extracts prepared without the use of proteolytic enzymes usually contain the mucopolysaccharides in combination with protein as mucoproteins, which may be isolated and purified by physical methods.
Thus chondromucoprotein has been obtained from extracts of bovine
nasal septa (Gerber, Franklin and Schubert, 1960) and from human
costal cartilage (Johnson and Schubert, 1960) by ultra-centrifugation.
F. P U R I F I C A T I O N A N D I D E N T I F I C A T I O N OF
M U C O P O L Y S A C C H A R I D E S
1. Precipitation Methods
A number of procedures are available for the purification of the
crude mucopolysaccharide fraction. The classical methods of Meyer,
Davidson, Linker and Hoffman (1956) involve the ethanolic precipitation of the mucopolysaccharides in the presence of calcium ions.
Barium has been used instead of calcium by Jorpes and Gardell (1948)
and by Smith and Gallop (1953). This method requires relatively large
quantities of material and has little application in Tissue Culture.
Progress in the development of micro-methods has been made by
Gardell (1961) and Scott (1955, 1960). Precipitation of mucopolysaccharides by cetylpyridinium chloride, and the differential solubility
of these complexes in salt solutions (Scott, 1955; Slack, 1958; Schiller