Affinity labeling of human serum prealbumin with N-bromoacetyl-L-thyroxine.
Affinity labeling of human serum prealbumin with N-bromoacetyl-L-thyroxine (BrAcT4) was used to investigate the binding domain for L-thyroxine (T4) on prealbumin. Fluorescence titration with 8-anilinonaphthalene-1-sulfonate revealed a strong and a weak binding site for BrAcT4 (K1 = 1 X 10(8) M-1; K2 = 1 X 10(6) M-1). The reaction of BrAcT4 with prealbumin to form a covalent bond was inhibited in the presence of T4 and binding of T4 to prealbumin was nearly abolished after affinity labeling with BrAcT4. Affinity labeling with 2 mol of BrAcT4/mol of prealbumin resulted in covalent binding of 1 mol of ligand. Acid hydrolysis of affinity-labeled prealbumin gave Nepsilon-carboxymethyllysine and iminodiacetic acid, the latter being derived from the NH2-terminal glycine. A combination of analytical procedures, including tryptic digestion after maleylation, cyanogen bromide cleavage, digestion with yeast protease C, and sequential Edman degradations, revealed that the Nepsilon-carboxymethyllysine was derived from lysine-9 and lysine-15 and that the affinity label had distributed itself among glycine-1, lysine-9, and lysine-15 in a ratio of 29:63:8.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011228:Prealbumin; D011485:Protein Binding; D012709:Serum Albumin; D013050:Spectrometry, Fluorescence; D013974:Thyroxine / Q000031:analogs & derivatives*; D014357:Trypsin
D000345:Affinity Labels; D000596:Amino Acids; D010446:Peptide Fragments; D011228:Prealbumin; D012709:Serum Albumin; C015042:N-bromoacetylthyroxine; D014357:Trypsin; D013974:Thyroxine