Elucidation of the disulfide bond positions of the β-subunit of human follicle-stimulating hormone

From a proteolytic digest of human follicle-stimulating hormone (FSH), three of the five disulfide bonds in the α-subunit of human FSH, namely, those between half-cystine residues at positions 7–10, 28–87 and 82–84, and three of the six disulfide bonds in the β-subunit of human FSH, namely, those between half-cystine residues at positions 3–28, 17–51 and 32–104, have been previously identified. The remaining fractions of the proteolytic digest containing the rest of the cystine peptides of human FSH were pooled, and fractionated by gel filtration on Sephadex G-50 (superfine) and by high-pressure liquid chromatography. One high molecular weight fragment containing half-cystine residues was recovered. The fragment was resistant to a sequential digestion with specific proteolytic enzymes, such as thermolysin and l -(1-tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin. This fragment, on the basis of amino acid analysis, N- and C-terminal and limited amino acid sequence analysis, was identified as a single peptide containing the remaining six half-cystine residues or three disulfide bonds of the human FSH β-subunit. Hence, the fragment was digested with a mixture of proteases such as pronase, papain, subtilisin, chymotrypsin, thermolysin and TPCK-treated trypsin, in order to achieve a random cleavage of the peptide. From this enzyme digest, two cystine-containing peptides were isolated by gel filtration on Sephadex G-50, and high-voltage paper electrophoresis at pH 2 and pH 3.5. The peptides were identified by amino acid, N- and C-terminal and sequence analyses as containing disulfide bonds between βCys-66-βCys-94 and βCys-20-βCys-82. The single remaining bond was assigned between βCys-84-βCys-87 by elimination, thus completing the identification of all six disulfide bonds of the β-subunit of human FSH.