 |
|
Title
|
|
Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length.
|
|
|
Authors
|
|
D.J.Mahoney,
R.T.Aplin,
A.Calabro,
V.C.Hascall,
A.J.Day.
|
|
|
Ref.
|
|
Glycobiology, 2001,
11,
1025-1033.
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts
as a structural component of extracellular matrices and mediates cell adhesion.
There have been numerous recent reports that fragments of hyaluronan have
different properties compared to the intact molecule. Though many of these
results may be genuine, it is possible that some activities are due to minor
components in the preparations used. Therefore, it is important that
well-characterized and highly purified oligosaccharides are used in cell
biological and structural studies so that erroneous results are avoided. We
present methods for the purification of hyaluronan oligomers of defined size
using size exclusion and anion-exchange chromatography following digestion of
hyaluronan with testicular hyaluronidase. These preparations were characterized
by a combination of electrospray ionization mass spectrometry, matrix-assisted
laser desorption/ionization mass spectrometry with time-of-flight analysis, and
fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging
from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to
be homogeneous with regard to length but did contain varying amounts of
chondroitin sulfate. This contaminant could have been minimized if digestion had
been performed with medical-grade hyaluronan rather than the relatively impure
starting material used here. The 18- to 34-mer preparations were mixtures of
oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer,
10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In
addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with
terminal glucuronic acid residues were identified.
|
|
|
|