DNA-based maleimide compound enables titration of reactive cysteinyl thiols in proteins

Thiols in protein are often subjected to a variety of oxidative modifications, which play a key role in many cellular processes and are referred to as redox regulation. Therefore, detection of thiol redox states in a specific protein is essential for a deeper understanding of the redox regulation at the molecular level.

Although conventional maleimide compounds can distinguish the redox states of thiols in a given protein, it is difficult to accurately determine the precise number of redox-related thiols due to limitations related to their chemical characteristics.

Here, Satoshi Hara and his colleagues at the Chemical Resource Laboratory of Tokyo Institute of Technology describe the development of a DNA-based maleimide compound (DNA-Mal) and its versatility as a new reagent for investigating redox reagents.

The DNA-Mal was synthesized by conjugating a single stranded DNA to the maleimide group at the 5'-terminus via its aminohexylation.

DNA-Mal with 24 bases can be effectively used to label protein thiols and lead to SDS-PAGE electrophoretic mobility shifts equivalent to ~9 kDa per incorporated DNA-Mal molecule, enabling accurate determination of the number of protein thiols.

The versatility of DNA molecules opens up the possibility of using DNA-Mal for a broader range of cysteine containing proteins.

Graphical representation of DNA-Mal. DNA conjugated maleimide group binds to thiols in the protein of interest. The number of bound maleimide is countable from SDS-PAGE migration distance and indicates their redox states.

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