Assignment of disulfide bonds in the beta subunit of human chorionic gonadotropin.
The assignment of five disulfide bonds in the alpha subunit of human chorionic gonadotropin (hCG) using partial reduction and S-[14C]carboxymethylation has been reported earlier (Mise, T., and Bahl, O. P. (1980) J. Biol. Chem. 255, 8516-8522). Employing a similar approach, we have determined the locations of six disulfide bonds in hCG-beta. Two partially reduced and S-[14C]carboxymethylated hCG-beta derivatives, DS1.4-hCG-beta and DS3.4-hCG-beta in which on the average 1.4 and 3.4 disulfide bonds were modified, respectively, were prepared. The 14C-labeled derivatives were then completely reduced and S-carboxymethylated with nonradioactive iodoacetic acid and subjected to hydrolysis with trypsin. The radioactive peptides were purified by gel filtration and high voltage paper electrophoresis. The tryptic peptides containing two or more S-[14C]carboxymethylcysteines were further degraded using various proteolytic enzymes such as thermolysin, carboxypeptidase A and Y, cathespin C, and subtilisin to obtain individual S-[14C]carboxymethylcysteine-containing peptides. From the specific radioactivities of S-[14C]carboxymethylcysteines in DS3.4-hCG-beta, four out of six disulfide bonds, 9-90, 26-110, 34-88, and 93-100 were assigned. Similar data from DS1.4-hCG-beta gave the locations of the other two disulfide bonds, 23-72 and 38-57, while confirming the locations of four disulfide bonds derived from the radioactivity distribution in DS3.4-hCG-beta. Thus, all six disulfide bonds in hCG-beta have been located. The results of controlled reduction and S-[14C]alkylation also indicate that disulfide bond 93-100 is the most reactive, followed by disulfide bond 26-110, and that the least reactive among all is 34-88.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D006063:Chorionic Gonadotropin; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D007461:Iodoacetates; D019807:Iodoacetic Acid; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D014108:Tosylphenylalanyl Chloromethyl Ketone
D000596:Amino Acids; D006063:Chorionic Gonadotropin; D004220:Disulfides; D007461:Iodoacetates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014108:Tosylphenylalanyl Chloromethyl Ketone; D019807:Iodoacetic Acid