JBC, Vol. 252, Issue 2, 420-426, Jan, 1977
Isolation and physical characterization of hyaluronic acid prepared from bovine nasal septum by cetylpyridinium chloride precipitation
R. L. Cleland and A. P. Sherblom
Raw extract in 2 m CaCl2 of bovine nasal septum cartilage was eluted from 4
per cent agarose gel to give a "void volume" Fraction v-4, which was
indistinguishable in composition and behavior on viscometric and
sedimentation analysis from the densest fraction obtained by associative
centrifugation in a cesium chloride density gradient. The sulfated
proteoglycan was precipitated (Fraction A) by cetylpyridinium chloride from
acidic solutions of Fraction v-4 or of dialyzed raw ectract. Neutralization
under conditions of low ionic strength precipitated a further small
fraction (B), which contained from 0.5 to 1 per cent of the uronic acid in
the original extract. Analysis by associative and dissociative density
gradient centrifugation demonstrated that Fraction B resembled in effective
density known samples of hyaluronic acid from other sources. Gel
chromatography of proteolytic digests of Fractions A and B on 6 per cent
agarose indicated that cetylpyridinium chloride precipitation essentially
separated sulfated proteoglycan (A) from hyaluronic acid (B). A
viscosity-average molecular weight of about 5 x 10(5) was estimated for a
sample of Fraction B purified in a dissociative (4 M guanidine
hydrochloride + CsCl) density gradient. Sedimentation velocity data were
consistent with this result. Analysis of hexosamines showed that the sample
contained 96 per cent glucosamine, confirming the identification of
hyaluronic acid. The proteoglycan fraction (A) resembled "subunits" in its
sedimentation behavior.
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