Partial reduction with dithiothreitol of disulfide bonds in human chorionic gonadotropin.
Of the eleven disulfide bonds of human chorionic gonadotropin (hCG), two were reduced with a 10-fold molar excess of dithiothreitol (DTT) relative to hormone. An S-carboxymethyl (SCM) derivative and a reoxidized product of this reduced hCG retained full biologic activity and were likely to be immunologically identical with native hCG. These two disulfide bonds appeared to be located in the alpha-subunit of the hormone. A 40-fold molar excess of DTT was required to reduce the third disulfide bond which was located in the beta-subunit. An SCM derivative ot this hexa-SH-hCG was only one fifth as biologically active as native hCG but its immunologic activity was only slightly decreased. However, its reoxidized product exhibited over 70% of the biologic activity of and it had nearly the same immunologic activity as the hormone. Reduction of a fourth disulfide bond, probably in the beta-subunit, by a 100-fold molar excess of the reagent was accompanied by considerable alterations in the hormone conformation as evidenced by electrophoresis. The resulting SCM derivatives as well as reoxidized products showed progressive decreases in biologic and immunologic activity and both reduced deca-SCM-hCG and a reoxidized product of deca-SH-hCG were virtually devoid of biologic activity. These findings suggest that the two first-reduced disulfide bonds, apparently in the alpha-subunit, are not important to the biologic activity of the hormone and that the third-reduced disulfide bond, probably in the beta-subunit, is very important in maintaining a biologically active conformation of the hormone. Further disulfide reduction appears to result in considerable alterations in the general conformation of the hormone, including disulfide interchange.
D001665:Binding Sites; D001681:Biological Assay; D006063:Chorionic Gonadotropin / Q000276:immunology; D003168:Complement Fixation Tests; D004220:Disulfides; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D011485:Protein Binding
D006063:Chorionic Gonadotropin; D004220:Disulfides; D046911:Macromolecular Substances; D004229:Dithiothreitol