Molecular weight determination of hyaluronic acid and its separation from mouse skin extract by high-performance gel permeation chromatography using a precision differential refractometer
✍ Scribed by Noriko Motohashi; Itsuhiko Mori
- Book ID
- 104146800
- Publisher
- Elsevier Science
- Year
- 1984
- Tongue
- English
- Weight
- 320 KB
- Volume
- 299
- Category
- Article
- ISSN
- 1873-3778
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✦ Synopsis
Hyaluronic acid, a glycosaminoglycan, is ubiquitous in soft connective tissues such as the umbilical cord, synovial fluid and vitreous humour, and is also found in skin and cartilage. Its primary structure consists of repeating subunits of /l-1,Clinked dissaccharides of glucuronic acid /.I-1,3-N-acetylglucosamine. A wide distribution of molecular weight has of the order of 104-10' been found. In biological fluids, hyaluronic acid is strikingly viscoelastici but, in ageing or pathological fluid, loss of fluid viscosity and degradation of the hyaluronic acid occurz.3. Chromatography of hyaluronic acids has been studied using soft gels such as agarose ge14s5 and ion-echange ge16. Their molecular weights have been determined by gel chromatography5 and viscosity, ultracentrifuge and light-scattering measurements7T8. However, all of these methods require relative large amounts of material and long analysis times. For proteins, high-resolution separations and molecular weight determinations have become possible because of the development of pre-packed high-performance gel permeation chromatographic colums such as TSK-GEL SW type made from silica gel chemically bonded with hydrophilic compounds9-l3 and TSK-GEL PW type made from cross-linked hydrophilic polymers l 2-1 5. However, high-performance gel permeation chromatography of hyaluronic acid, a macromolecular glycosaminoglycan, has not yet been studied.
We have tried to achieve the separation of hyaluronic acid from other glycosaminoglycans and the determination of its molecular weight in a single run using small amounts of sample. This paper describes a rapid chromatographic procedure using the hydrophilic organic polymer gel column marketed by Showa Denko (Tokyo, Japan) as the Shodex OHpak B-800 series. As little as about 5 pg of hyaluronic acid can be detected on this support. This technique should serve as a powerful tool for examining the distribution of hyaluronic acid in connective tissues. EXPERIMENTAL Hyaluronic acid, grade I, from human umbilical cord was purchased from Sigma (St. Louis, MO, U.S.A.) and was used after purification through a Sephacryl S-500 column (45 x 2.5 cm I.D.) with 0.05 M phosphate buffer (pH 7.0) containing