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 Asp(163)-Cys(164) Or Asp(282)-Cys(283) 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 Cys(283), Specifically, the variety appears to be due to the breaking of either Cys(150)-Cys(164) or Cys(240)-Cys(283), which in turn leads to the labilization of Asp(163)-Cys(164) or Asp(282)-Cys(283), respectively.