Hyaluronan (HA) substrate gel electrophoresis of chondrocyte cell layers extracted with Triton X-100. (a) Specimens of fibroblast cell layers extracted with Triton X-100 (3 mg as total protein) were applied to an anti-PH-20-antibody Sepharose gel and eluted with either the ImmunoPure Gentle Ag/Ab buffer (lane 1) or the glycine–HCl buffer (lane 3) were subjected to HA substrate gel electrophoresis. A band of activity with an unexpected molecular weight (>200 kDa) was observed in both specimens. (b) When HA oligosaccharides were added to specimens eluted with either the 'gentle' buffer or the glycine–HCl buffer, the band of activity appeared at the expected molecular weight: 'gentle' buffer-eluted specimens without (lane 1) and with (lane 2) HA oligosaccharides; glycine–HCl buffer-eluted specimens without (lane 3) and with (lane 4) HA oligosaccharides; lane 5, commercial preparation of testicular hyaluronidase. Similar results were obtained with specimens from fibroblast and synoviocyte cell layers. (c) No hyaluronidase activity was detected in the HA oligosaccharide preparation (lane 1) or in the 'gentle' buffer preparation (lane 2); lane 3, commercial preparation of testicular hyaluronidase.