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.