Fig. 8

Thin-layer chromatographic separation of the oligosaccharides released from rat gastric mucin by β-elimination reaction. Those remaining with mucin binding protein (MBP) following partition into Triton X-114 are as described in the “Materials and Methods.” The material from incubation of MBP with oligosaccharides that partitioned into Triton X-114 was hydrolyzed with nitrous acid (HONO) and the sample for thin layer chromatography was recovered from aqueous phase of the second partition with the detergent. The water soluble phase was applied to thin layer plate and developed in solvent system consisting of n-butanol/acetic acid/water (5:3:2, v/v/v). After 6 hr of ascending chromatography, the plate was dried and sprayed with orcinol (28). Lanes 1 and 2 represent oligosaccharides recovered in the water phase of the first partition of MBP incubates with Triton X-114. Lanes 3 and 4 contain the water phase of the HONO hydrolyzed Triton X-114 extract of MBP. Lane 5 represents the oligosaccharides mixture released from rat gastric mucin by β-elimination reaction (28). The HONO hydrolysis of the Triton X-114 extract of MBP incubated with mucin oligosaccharides was instituted to remove the sample from the Triton X-114 that interfered in the thin layer chromatography. The analyses reveal that the oligosaccharides interacting with MBP are mainly in the fraction contained in region of octa- to decasaccharides or larger oligomers. As apparent from the lanes 1 and 2, the smaller oligosaccharides are not retained with MBP samples and are not binding, even if introduced as separate fraction (unpublished results).