A direct and continuous assay for the determination of thioredoxin reductase activity in cell lysates

Anal Biochem. 2013 Dec 1;443(1):34-40. doi: 10.1016/j.ab.2013.08.013. Epub 2013 Aug 21.

Abstract

Thioredoxin reductase (TR) is an oxidoreductase responsible for maintaining thioredoxin in the reduced state, thereby contributing to proper cellular redox homeostasis. The C-terminal active site of mammalian TR contains the rare amino acid selenocysteine, which is essential to its activity. Alterations in TR activity due to changes in cellular redox homeostasis are found in clinical conditions such as cancer, viral infection, and various inflammatory processes; therefore, quantification of thioredoxin activity can be a valuable indicator of clinical conditions. Here we describe a new direct assay, termed the SC-TR assay, to determine the activity of TR based on the reduction of selenocystine, a diselenide-bridged amino acid. Rather than being an end-point assay as in older methods, the SC-TR assay directly monitors the continuous consumption of NADPH at 340 nm by TR as it reduces selenocystine. The SC-TR assay can be used in a cuvette using traditional spectrophotometry or as a 96-well plate-based format using a plate reader. In addition, the SC-TR assay is compatible with the use of nonionic detergents, making it more versatile than other methods using cell lysates.

Keywords: Assay; Redox homeostasis; Thioredoxin reductase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cystine / analogs & derivatives*
  • Cystine / chemistry
  • Enzyme Assays
  • Epithelial Cells / chemistry
  • Epithelial Cells / enzymology*
  • Mice
  • NADP / chemistry*
  • Organoselenium Compounds / chemistry*
  • Oxidation-Reduction
  • Respiratory Mucosa / chemistry
  • Respiratory Mucosa / enzymology
  • Selenocysteine / chemistry*
  • Sensitivity and Specificity
  • Spectrophotometry
  • Thioredoxin-Disulfide Reductase / analysis*

Substances

  • Organoselenium Compounds
  • Selenocysteine
  • selenocystine
  • Cystine
  • NADP
  • Thioredoxin-Disulfide Reductase