Assignment of disulfide bonds in the alpha subunit of human chorionic gonadotropin.
The alpha subunit of human chorionic gonadotropin (hCG) was partially reduced and S-alkylated with [2-14C]iodoacetic acid. The resulting derivative in which on the average 1.6 residues of cystine were modified was completely reduced and S-alkylated. The S-[14C]-carboxymethylated hCG-alpha was then subjected to hydrolysis with trypsin and the hydrolysate was fractionated by gel filtration. The radioactive fractions were further purified by high voltage paper electrophoresis at pH 4.7 to yield tryptic peptides, alpha T-1, alpha T-8, and alpha T-11a, containing 5, 2, and 3 S-carboxymethyl cysteinyl residues, respectively. These peptides were further fragmented by a variety of cleavage reagents such as cyanogen bromide, chymotrypsin, Staphylococcus aureus protease, subtilisin, and cathepsin C to isolate individual S-[14C]carboxymethylcysteine-containing peptides. After ensuring their purity, the specific radioactivity of each S-[14C]carboxymethylcysteine was determined following its isolation from the acid hydrolysate of the peptide by high voltage paper electrophoresis at pH 4.7. The 2 S-[14C]carboxymethylcysteine residues with identical specific radioactivity yielded the precise location of the disulfide bridge in the polypeptide chain. Thus, all five disulfide bonds in hCG-alpha were assigned and are located at positions 7 and 31, 10 and 32, 28 and 60, 59 and 87, and 82 and 84.
D000596:Amino Acids / Q000032:analysis; D006063:Chorionic Gonadotropin; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D014357:Trypsin
D000596:Amino Acids; D006063:Chorionic Gonadotropin; D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014357:Trypsin; D003488:Cyanogen Bromide