Abstract
Publisher Summary This chapter discusses the use of chemical modification reactions that adapt the method for the determination of protein S-thiolation, protein S-nitrosylation, and even irreversible damage (oxidation) to reactive sulfhydryls on a specific protein. Oxidative modifications of protein sulfhydryls can increase the negative charge on a protein—that is, either S-glutathiolation or sulfhydryl oxidation to a cysteic acid. Other oxidative modifications such as S-cysteylation or Snitrosylation produce charge-silent modifications. This method produces stable isoforms where S-glutathiolated and cysteic acid containing protein species have a net negative charge. S-nitrosylated proteins may also be analyzed by electrofocusing with little concern for decomposition. The electrofocusing method can be extended to the study of protein sulfhydryl modification in complex mixtures of proteins. The method can also be extended to proteins that are not completely water-soluble by including detergents in the electrophoresis gel. However, it must be emphasized that protein denaturants expose sulfhydryls normally buried within the protein structure. With no added detergent, the most reactive and accessible protein sulfhydryls are easily protected from artifactual modification during processing by the alkylating agents used to stop the reaction.
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