Identification of the interchain disulfide bonds of dimeric human placental lactogen.

We previously isolated a naturally occurring dimer of human placental lactogen (hPL) and showed that it was linked by one or more disulfide bonds. Monomeric hPL is a 191-amino acid peptide with two disulfide bonds, one between residues 54 and 165 and the other between 182 and 189. The present studies were designed to determine the location of the disulfide bonds in dimeric hPL and to relate this to its biologic activity. Identification of individual disulfide bonds were accomplished using radioactive alkylating reagents and cyanogen bromide (CNBr) cleavage. In nondenaturing solvent, the COOH-terminal disulfide bond of hPL was selectively reduced by NaBa, while in denaturing solvent, both bonds were reduced. The COOH-terminal disulfide of dimeric hPL was also selectively reduced by NaB&, indicating that the COOH-terminal disulfide formed the interchain bond. Radioactive alkylating agents also showed that there were no free sulihydryl groups in dimeric hPL. To distinguish between the possibilities of parallel and antiparallel alignments of the chain, a map of the tryptic digest of dimeric hPL was prepared. The results indicated an antiparallel configuration was present. Therefore, dimeric hPL is formed by a symmetric pair of disulfide bonds between cysteinyl residue 182 of one chain and cysteinyl residue 189 of the other. The structure of dimeric hPL indicates that the carboxyl region of the molecule is not essential for its lactogenic activity.