Effect of deglycosylation on the binding and immunoreactivity of human thyroxine-binding globulin.
Thyroxine-binding globulin (TBG), prepared from human serum by an improved purification method, was treated with a mixture of neuraminidase, beta-galactosidase, alpha-mannosidase, and beta-N-aectylglucosaminidase, which resulted in the removal of approximately 86% of saccharides. Purification by thyroxine-Sepharose affinity chromatography gave a homogeneous protein as shown by equilibrium sedimentation and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Amino acid and NH2-terminal sequence analysis indicated that the protein moiety was intact. Deglycosylation had no effect on the stoichiometry of the binding of L-thyroxine as shown by tryptophanyl fluorescence quenching and equilibrium dialysis at pH 8.6 and 25 degrees C. However, the affinity constant for L-thyroxine was reduced from 1.6 X 10(9) M-1 to 0.58 X 10(9) M-1. Analysis of radioimmunoassay data revealed that deglycosylation resulted in a slight decrease of the affinity constant for anti-TBG antibody from 3.9 X 10(10) M-1 to 1.8 X 10(10) M-1. These results suggest that the polypeptide moiety, rather than the heterosaccharides, contains the antigenic determinants. Removal  of the majority of the heterosaccharides of TBG has only a minor effect on its immunoreactivity and on the binding of thyroid hormone.
D000596:Amino Acids / Q000032:analysis; D000937:Antigen-Antibody Reactions; D002241:Carbohydrates / Q000032:analysis; D006026:Glycoside Hydrolases; D006027:Glycosides / Q000032:analysis*; D006801:Humans; D008970:Molecular Weight; D013050:Spectrometry, Fluorescence; D013975:Thyroxine-Binding Proteins / Q000276:immunology
D000596:Amino Acids; D002241:Carbohydrates; D006027:Glycosides; D013975:Thyroxine-Binding Proteins; D006026:Glycoside Hydrolases