Hydrophobic binding properties of bovine gallbladder mucin.
Hydrophobic binding properties of purified bovine gallbladder mucin were studied by fluorescence spectroscopy using 1-anilino-8-naphthalene sulfonate (ANS) and N-phenyl-1-naphthylamine. The purified glycoprotein contained 75.5%, dry weight, as carbohydrate, 16.3% as protein, and 3.7% as sulfate; Mr = 2.2 X 10(6) was estimated by chromatography on Sephacryl S-500. Mucin contained a large number of low-affinity binding sites for these hydrophobic ligands. The dissociation constant, KD of mucin-ANS binding was 2.7 X 10(-5); each mucin molecule had approximately 42 binding sites for ANS. These binding sites were deduced to be on the unglycosylated portion of the protein core, as Pronase digestion completely eliminated binding. Reduction of mucin with 2-mercaptoethanol increased the fluorescence yield by formation of subunits with increased binding sites for the ligand. Increasing NaCl concentration (0.125 to 2.0 M) and decreasing pH (9 to 3) progressively increased fluorescence with the charged ligand ANS, suggesting that the binding site may have acidic groups which are shielded at high ionic strength or low pH. The fluorescent yield with N-phenyl-1-naphthylamine, an uncharged ligand, was an order of magnitude higher than with ANS. Bilirubin and bromosulfophthalein inhibited mucin-induced ANS fluorescence, but bile acids did not. Gallbladder mucin contains hydrophobic binding domains in the nonglycosylated peptide core that are involved in polymer formation and binding of biliary lipids and pigment.
D015057:1-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D001665:Binding Sites; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D005029:Ethylenediamines / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005704:Gallbladder / Q000032:analysis*; D008623:Mercaptoethanol / Q000494:pharmacology; D009077:Mucins / Q000378:metabolism*; D010860:Pigments, Biological / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D000817:Anilino Naphthalenesulfonates; D001647:Bile Acids and Salts; D002241:Carbohydrates; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D009077:Mucins; D010860:Pigments, Biological; D011108:Polymers; C005798:N,N,N',N'-tetramethylethylenediamine; D012965:Sodium Chloride; D008623:Mercaptoethanol; C027132:1-anilino-8-naphthalenesulfonate; C005444:N-phenyl-1-naphthylamine; D015057:1-Naphthylamine; D011402:Pronase