Hyaluronan is a polyanion with a constant charge to mass ratio, regardless of molecular weight. In order to obtain a molecular weight-dependent separation of hyaluronan using electrophoresis, a gel matrix is commonly employed as a sieving and separation-stabilizing medium. Under appropriate conditions, hyaluronan molecules may thus be separated according to molecular weight, with the smallest species having the greatest mobility. The size range over which separation can be achieved depends on the size of the gel pores relative to the size of the hyaluronan molecules. The porosity of the gel matrix, in turn, depends on the concentration of the gel-forming polymer and its degree of crosslinking. Gels with a small average pore size, such as crosslinked polyacrylamide gels are suitable for the separation of low molecular weight oligosaccharides and fragments of hyaluronan. Gels with a large average pore size such as agarose gels are used for the separation of high molecular weight hyaluronan.

A. Polyacrylamide Gel Electrophoresis (PAGE) of Hyaluronan Oligosaccharides and Fragments

Early electrophoretic analyses of hyaluronan and other glycosaminoglycans were designed to separate polysaccharides primarily according to charge density (33-35). Size separation had not been established by analysis of well-characterized samples differing solely in molecular weight. In 1984, three separate groups (36-38) reported high-resolution separation of glycosamino-glycan oligosaccharides according to size. In each case, a polyacrylamide gel was used to separate oligosaccharides into discrete bands, with adjacent bands in the final pattern differing in size by one disaccharide repeat. The gel composition varied from 10 to 25% acrylamide and the continuous buffer systems used were 50-100 mM Tris-borate, pH 8.3, with 1-2.4mM EDTA or 100 mM Tris-glycine, pH 8.9, with 1.25 mM EDTA. If the glycosaminoglycan fragments were radiolabeled, the separation patterns were visualized by fluorography of the gel after incorporation of a fluor and subsequent drying of the gel. Separation patterns for non-labeled oligosaccharides were visualized by staining with alcian blue, which effectively precipitated the fragments in the gel. The separation of up to 30-40 different hyaluronan oligosaccharides into distinguishable bands could be observed (37). Turner and Cowman (39) showed that the bands could be identified by co-electrophoresis of purified hyaluronan oligosaccharides. They also showed that short oligosaccharides (less than about eight disaccharides in length) were not visualized using alcian blue in water and oligosaccharides shorter than about 12 disaccharides were underrepresented in the stained pattern for digests as a result of the difficulty in immobilizing the separated fragments in the gel by dye binding. A number of useful modifications were subsequently made to the electrophoretic methods. Min and Cowman (40) developed an improved procedure using long thin gels and a two-step staining process: precipitation of fragments in the gel with alcian blue followed by silver staining.

The sensitivity of the improved alcian blue/silver stain procedure was approximately 50 ng per band, or 2-5 mg for a complex mixture. Multiple loadings of samples on the gel after different delay times, a technique employed by Hampson and Gallagher (37) for the separation of dermatan sulfate oligosaccharides, was used to separate HA fragments containing up to 250 disaccharides into discrete bands. Lyon and Gallagher (41) found even more sensitive detection (as low as 1-2 ng per band for sulfated glycosaminoglycan fragments) using azure A and an ammoniacal silver stain, but data for hyaluronan were not detailed. Separation techniques using gradient gels (12-25% or 20-30% acrylamide) and discontinuous buffer systems were developed primarily for sulfated glycosaminoglycans with significant charge density heterogeneity (42,43), but were also reported to work for hyaluronan. Electrotransfer to positively charged nylon membranes allowed better fluorography of labeled fragments, and/or isolation of separated fragments (43,44). The trend toward mini-gel systems was recently exploited by Ikegami-Kawai and Takahashi (45) in the development of a rapid method for hyaluronan fragment analysis, using a 15% polyacrylamide gel with a Tris-borate-EDTA continuous buffer system, and staining using alcian blue/silver. Excellent separation was achieved in a 45 min run for fragments containing up to approximately 50 disaccharides. As previously observed, short fragments are poorly retained in the gel during staining. Samples containing species shorter than about 11 disaccharides should be analyzed with this restriction in mind.

Some of the applications of PAGE to the molecular weight characterization of hyaluronan samples include [1] calibration of gel permeation chromatography columns by direct analysis of the MWD in each fraction (46); [2] determination of the weight-average and number-average molecular weights of isolated hyaluronan subfractions for the purpose of comparison with light-scattering data showing self-association of hyaluronan fragments (Fig. 5) (11); [3] assays of extremely high sensitivity for hyaluronidase activity (45); and [4] characterization of oligosaccharides with both odd and even numbers of sugars prepared by chemoenzymatic synthesis of hyaluronan fragments (47).

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