Rydén L, Norder H
J Chromatogr. 1981 Oct 23;215:341-50. doi: 10.1016/s0021-9673(00)81413-2.
The use of activated thiopropyl-Sepharose for simple and rapid isolation of thiol peptides from large proteins was investigated using ceruloplasmin (a copper protein of molecular weight 134,000 containing three cysteines and six disulphides) as a test case. Optimal results for the immobilization of the protein to the activated gel were obtained at pH 4.0 in the presence of 8 M urea and 0.05 M ethylenediaminetetraacetic acid. In these conditions 96% of the protein thiol groups were attached to the adsorbent. The immobilized protein was digested with either pepsin or trypsin. The liberated non-thiol peptides were eluted from the gel together with the protease after the digestion. After washing, the covalently attached thiol peptides were eluted in reducing buffer, desalted on the hydrophobic gel Sephadex LH-20 and carboxymethylated. The peptides were purified in a two-step procedure involving gel filtration on Sephadex G-25 and either column electrophoresis or ion-exchange chromatography. The two sets of peptides were derived from four different regions in the protein. They were 12-39 residues in length and together accounted for 152 residues. It is shown that the peptide chain was susceptible to proteolytic attack also close to the point of attachment (two residues away). One peptide with two thiol groups proved to be derived from an area containing one disulphide bridge in addition to cysteine. This bridge could be identified in a separate experiment where a second enzyme was used to release the disulphide peptide after the first digestion and washings.
