Use of proline-specific endopeptidase in the isolation of all four "native" disulfides of hen egg white lysozyme.
The disulfide peptides from the tryptic digestion of cyanogen bromide-treated hen egg white lysozyme (HEWL) were isolated by reverse phase high performance liquid chromatography (HPLC) and identified by amino acid analysis. Three peptides containing the I-VIII, II-VII, and III-V + IV-VI disulfide bonds were obtained. The two-disulfide peptide was further digested with proline-specific endopeptidase (PCE) (EC 3.4.21.26). Amino acid analysis of digest peptides separated by HPLC showed four peptides with the IV-VI disulfide bond as well as a peptide with the III-V disulfide bond. The IV-VI peptides were produced by hydrolysis of several alanine-X bonds as well as the prolyl-cystine bond. Our studies show that alanyl peptide bonds to lysyl, seryl, and leucyl residues are susceptible to hydrolysis by PCE preparations, thus substantially extending its known specificity range. The two-disulfide peptide was also digested sequentially with thermolysin and PCE; the resulting IV-VI and III-V peptides were identified by HPLC and amino acid analysis. PCE showed substantial activity at pH 5.3 as well as at pH 8.3. The lower pH is useful in studies of proteins or peptides where base-catalyzed reactions must be limited.
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis*; D004529:Egg White; D010450:Endopeptidases / Q000378:metabolism*; D009113:Muramidase; D010446:Peptide Fragments / Q000032:analysis; D000085822:Prolyl Oligopeptidases; D011487:Protein Conformation; D012697:Serine Endopeptidases; D014357:Trypsin
D004220:Disulfides; D010446:Peptide Fragments; D009113:Muramidase; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D014357:Trypsin; D003488:Cyanogen Bromide