Conformational state of ovalbumin at acidic pH as evaluated by a novel approach utilizing intrachain sulfhydryl-mixed disulfide exchange reactions.
Ovalbumin contains four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) and one cystine disulfide (Cys73-Cys120). A highly reactive aromatic disulfide, 2,2'-dipyridyl disulfide, reacts specifically with Cys367 of ovalbumin at pH 2.2 generating a mixed disulfide protein derivative [Tatsumi, E., and Hirose, M. (1997) J. Biochem. 122, 300-308]. The mode of conformational fluctuation in ovalbumin was investigated at pH 2.2 using the mixed disulfide derivatives of the cystine-intact and cystine-reduced protein forms. In the presence of a high concentration of urea, both the mixed disulfide derivatives underwent rapid cysteine sulfhydryl/mixed disulfide exchanges, thereby releasing the quantitative amount of 2-thiopyridone. A peptide mapping analysis for disulfide-forming cysteines revealed that this release was mostly accounted for by the nucleophile attack on the Cys367-mixed disulfide by the nearest cysteine residue in the primary structure, Cys382. At the acidic pH, the exchange reaction was practically restricted to the cysteine sulfhydryl/mixed disulfide exchanges; no other exchange reaction, such as the cysteine sulfhydryl/cystine disulfide exchange reaction, was detected. In the absence of urea, the cystine-reduced form, but not the cystine-intact form, underwent significant sulfhydryl/mixed disulfide exchange reactions at a physiological temperature, as determined by the release of 2-thiopyridone. A kinetic analysis for the generation of disulfide-forming cysteines with Cys367 at 37 degreesC revealed that the rate for the intrachain exchange reaction was quite different for the five cysteine sulfhydryls. The effective concentrations of the five cysteine sulfhydryls relative to the Cys367-mixed disulfide were determined by using three related model reactions: the obtained values were 11.4, 4.6, 15.2, 5.9, and 8.9 microM for Cys11, Cys30, Cys73, Cys120, and Cys382, respectively. Implications of the effective concentrations for the conformational state of acidic ovalbumin are discussed.
Tatsumi
E
E
The Research Institute for Food Science, Kyoto University, Japan.
Yoshimatsu
D
D
Hirose
M
M
D000143:Acids; D018360:Crystallography, X-Ray; D003538:Cystamine / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010047:Ovalbumin / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000143:Acids; D004220:Disulfides; D013438:Sulfhydryl Compounds; D010047:Ovalbumin; D003545:Cysteine; D003538:Cystamine