Amino acid sequences containing cysteine or half-cystine residues in beta-glucuronidase

Amino acid analysis of oxidized or reduced and carboxymethylated beta-glucuronidase have shown the presence of 24 cysteic acid or S-carboxymethylcysteine residues respectively per mole of the tetrameric enzyme. Titration of sulfhydryl groups gave eight cysteine residues, and by difference 16 half-cystine residues per mole. Six peptides containing radiolabelled cysteine residues were isolated from pepsin and chymotrypsin digest of reduced and S-carboxymethylated beta-glucuronidase by ion-exchange chromatography or gel filtration, followed by paper ionophoresis and paper chromatography. The peptides were analysed for amino acids and sequenced by the dansyl-Edman procedure. Peptides containing cysteic acid were selectively recovered from thermolysin digests of performic acid-oxidized glucuronidase. The amino acid sequences confirmed that there were only six different peptide sequences containing either cysteine or half-cystine residues in the tetrameric enzyme, supporting the presence of four identical subunits. These sequences wer: (A)-Val-Asx-Val-Ile-Cys-Val-Asx-Ser-Tyr- (B)-Gly-Asx-Leu-Cys-Ser-Gly- (C)-Phe-Val-Val-Ile-Asx-Glx-Cys-Pro-Gly-Val-Gly- (D)-Val-Val-Cys-Leu- (E)-Gln-Ser-Gly-Cys-Leu-Val-Lys-Gly-Tyr- (F)-Cys-Asp-Arg-Tyr-Gly-Ile-Val-Val-.