Microanalysis of hyaluronan oligosaccharides by polyacrylamide gel electrophoresis and its application to assay of hyaluronidase activity.

We established a rapid, sensitive polyacrylamide gel electrophoresis (PAGE) method for the analysis of hyaluronan (HA) oligosaccharides. Using mini-slab gels, but not large-slab gels so far reported, HA oligosaccharides of 5 to more than 50 repeating disaccharide units could be separated into discrete ladder-like bands in a short electrophoresis time of 45 min. Using a combined Alcian blue and silver staining protocol, the detection limit was less than 1 ng per band for 11 repeating disaccharide units, indicating 50 times higher sensitivity than that of an earlier-described sensitivity-enhanced PAGE method. Our PAGE method was applicable to the assay of hyaluronidase activity. When a total of multiple band intensities for 18-24 repeating disaccharide units was used as a measure of activity, as little as 3 x 10(-4) NFU of bovine testicular hyaluronidase was detectable on a 1-h incubation. This sensitivity permitted rapid measurements of human and rabbit serum hyaluronidases, the latter of which having never been detected even by a sensitive enzyme-linked immunosorbent assay (ELISA). Since this PAGE assay does not require specialized reagents and instruments and since it provides information on both the activity and the enzymatic HA degradation pattern, there may be many potential applications.