Identification of the Thiol Groups in Human Ceruloplasmin

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Abstract
Human ceruloplasmin was attached to activated thiol-Sepharose via its thiol groups and was then digested with pepsin. After appropriate washings the thiol peptides were eluted by reduction and were carboxymethylated and purified by column chromatography and electrophoresis. Amino acid sequencing showed that the peptides were derived from 5 different areas in the molecule and together accounted for 92 residues, 6 of which were cysteines. Since 1 of the peptides contained 2 cysteines prior to the reductive elution of the peptides, 1 of these had been paired in a disulfide bridge with 1 of the 4 remaining thiol peptides present in the mixture. The disulfide was isolated and identified by digesting the immobilized protein with pepsin followed by trypsin. The 2nd (tryptic) digestion released the disulfide peptide. Three of the true thiol peptides obtained occur in regions of sequence that have already been reported and which account for 564 of the .apprx. 1050 residues present in the protein. Three of them also show .apprx. 40% identity with each other, whereas no relatedness is observed with the 4th. The 3 related peptides are clearly homologous to the Cu-binding areas in the small blue plant and bacterial proteins plastocyanin and azurin. Homologous regions are also evident when the peptides are compared to the 2 sequences reported for the blue oxidase, fungal laccase, 1 of which contains a disulfide bridge.