Variability in heat-induced fragmentation of a protein in the presence of dodecyl sulfate: the role of an intramolecular sulfhydryl/disulfide exchange.
alpha-Amylase from Aspergillus oryzae was investigated to better understand how disulfide-bonded proteins behave during denaturation with dodecyl sulfate. It was previously reported that the alpha-amylase, when denatured with dodecyl sulfate, forms two species, D1 and D2. In D1, the disulfide bonds remain intact, while in D2, there is a restricted single sulfhydryl/disulfide (SH/SS) exchange reaction. This phenomenon was re-examined as follows: electrophoretic analysis of fragments created with and without modification of a free SH group or disulfide-reduced SH groups; N-terminal sequence analysis of the fragments; reactivity of a free SH group with Ellman reagent. Data from the former two analyses showed that the variability in the electrophoretic patterns results from cleavage at Asp163-Cys164 or Asp282-Cys283, provided that these Cys residues are reduced. Different reactivities of a SH group in D1 and D2 and the appearance of a polypeptide with a nonnative SS pairing in D2 fragments confirmed that the different electrophoretic patterns result from an intramolecular SH/SS exchange. This rearrangement accounts for the variety of electrophoretic patterns observed with D2 amylase and is a result of the creation of a specific free Cys283. Specifically, the variety appears to be due to the breaking of either Cys150-Cys164 or Cys240-Cys283, which in turn leads to the labilization of Asp163-Cys164 or Asp282-Cys283, respectively.
Kubo
K
K
Faculty of Pharmaceutical Sciences, Kinki University, Osaka.
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001236:Aspergillus oryzae / Q000201:enzymology; D003545:Cysteine; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007700:Kinetics; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate; D013438:Sulfhydryl Compounds; D013816:Thermodynamics; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification
D004220:Disulfides; D010446:Peptide Fragments; D011506:Proteins; D013438:Sulfhydryl Compounds; D001224:Aspartic Acid; D012967:Sodium Dodecyl Sulfate; D004228:Dithionitrobenzoic Acid; D000516:alpha-Amylases; D003545:Cysteine