Microbial Redox Regulator-Enabled Pulldown for RapidAnalysis of Plasma Low-Molecular-Weight Biothiols

Although low-molecular-weight (LMW) biothiols function as a disease indicator in plasma, rapidly and effectively analyzing them remains challenging in the extracellular oxidative environment due to technical difficulties. Herein, a microbial redox sensor protein-enabled method for rapid and cost-effective analyses of free and total LMW biothiols is described. We newly designed an affinity pull-down analysis of plasma LMW biothiols using a Bacillus subtilis-derived organic hydroperoxide resistance regulatory (OhrRBS) protein and its operator dsDNA. LMW biothiols triggered the rapid dissociation of FLAG-tagged OhrRBS (FLAG-OhrRBS) from FAM-labeled operator dsDNA (FAM-dsDNA) via S-thiolation of FLAG-OhrRBS in the presence of organic hydroperoxide, leading to a strong increase of fluorescence intensity in bead-containing solution supernatant. This method was easily extended by using a reducing agent to detect free and total LMW biothiols simultaneously in mouse plasma. Free plasma LMW biothiol was similar in ...