caa a22 4500 397494378 CHVBK 20180308164517.0 cr unu---uuuuu 161202e199512 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a124995 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a124995 Kubo Kanenobu Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashiosaka, Osaka 577 Variability in Heat-Induced Fragmentation of a Protein in the Presence of Dodecyl Sulfate: The Role of an Intramolecular Sulfhydryl/Disulfide Exchange [Elektronische Daten] [Kanenobu Kubo] α-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 α-amylase, when denatured with dodecyl sulfate, forms two species, D1 and D2. In Dl, 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 Dl 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. © Oxford University Press Regular Papers nationallicence denaturation nationallicence fragmentation nationallicence protein nationallicence SDS-PAGE nationallicence sulfhydryl/disulfide exchange nationallicence The Journal of Biochemistry Oxford University Press 118/6(1995-12), 1112-1117 0021-924X 118:6<1112 1995 118 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a124995 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a124995 text/html Onlinezugriff via DOI NATIONALLICENCE 100 1- Kubo Kanenobu Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashiosaka, Osaka 577 NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 118/6(1995-12), 1112-1117 0021-924X 118:6<1112 1995 118 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford