Thioredoxin Redox Status Assessment During Embryonic Development: The Redox Western

Abstract

Oxidative stress can contribute significantly to the mechanisms that cause teratogenesis and embryotoxicity, but cellular antioxidant defense systems have evolved to counteract the detrimental consequences of oxidative insult. Numerous antioxidant systems exist in developing organisms but have been shown to be dynamically expressed and may only become functional during specific periods of development. Thioredoxins (Trxs) are a class of low-molecular-weight oxidoreductase antioxidants that play an important role in reactive oxygen species detoxification and transcription factor regulation, both of which are critical to normal development. In this chapter, we describe a method by which cytosolic thioredoxin-1 (Trx1) and mitochondrial thioredoxin-2 (Trx2) redox status can be measured. In brief, derivatization of reduced Trx at the active site cysteine thiols using a large maleimide derivative, targeting the redox-sensitive residues making the protein larger. Nonreducing SDS-PAGE is performed to separate reduced (labeled) and oxidized (unlabeled) Trx based on their size. Band densitometries can be measured and used in the Nernst equation to calculate redox potentials. These approaches provide a means to evaluate this unique redox couple during development and allow for the measurement of compartmentalized (mitochondrial vs. cytosolic) oxidation during chemical insult.