A fluorescent dual labeling technique for the quantitative measurement of reduced and oxidized protein thiols in tissue samples

Abstract

Oxidative stress can result in the reversible oxidation of protein thiols. Because the activity of numerous proteins is sensitive to thiol oxidation, this has the potential to affect many cellular functions. We describe a highly sensitive, quantitative labeling technique that measures global and specific protein thiol oxidative state in skeletal muscle tissue. The technique involves labeling the reduced and oxidized protein thiols with different fluorescent dyes. The resulting sample is assayed using a 96-well plate fluorimeter, or individual protein bands are separated using SDS-PAGE. We show that artifactual oxidation during sample preparation and analysis has the potential to confound results, and techniques to prevent this are described. We tested the technique by analyzing the muscles of mdx and c57 mice and found that the muscles of mdx mice were significantly (p<0.05) more oxidized (13.1±1.5% oxidized thiols) than those of c57 mice (8.9±0.7% oxidized thiols). This technique provides an effective means to measure the extent to which oxidative stress affects the oxidation of protein thiols in biological tissues.