Dipolar coupling-based electron paramagnetic resonance method for protease enzymatic characterization and inhibitor screening

Abstract

Herein, we report an EPR-based method for protease enzymatic characterization and inhibitor screening. This method utilizes dual paramagnetically-labeled probes consisting of a nitroxide spin probe and a Gd3+ ion flanking...

Highly Sensitive “Off/On” EPR Probes to Monitor Enzymatic Activity

“…We deemed of interest to investigate alternative routes taking advantage of the fact that nitroxide radicals are “quenched” when they are immobilized on nanostructures like proteins, [7] vesicles, micelles or liposomes [8,9] as a consequence of dipole‐dipole interactions and increasing spin exchange between the closely packed radical molecules [10] . In alternative, Lu Yu and coworkers [11] recently observed a significant quenching of the EPR signal generated by the dipolar interaction with a paramagnetic center (Gd‐complex) linked through a short protease‐specific peptide to the nitroxide radical. Direct detection of caspase‐3 activity was possible by analyzing the EPR signal increase upon specific peptide cleavage.…”

EPR-basedin situenzymatic activity detection of endogenous caspase-3 in apoptosis cell lysates

“…[17][18][19][20] Also, EPR spectroscopy has advantages such as the absence of endogenous background signals and simple experimental procedure, offering a highly promising route for the detection and characterization of apoptosis-related proteases under ambient conditions. 21 In this work, we report an EPR-based enzymatic assay for reliable and rapid detection of caspase-3 activity in cell apoptosis. A caspase-3-specific peptide segment (DEVD) was synthesized, biotinylated and labeled with an EPR-detectable nitroxide radical (MTSL).…”

“…Previously, we reported on an EPR assay for protease enzymatic characterization and inhibitor screening based on the evaluation of dipolar coupling interaction between two paramagnetic centers. 21 However, a weak EPR signal could be detected for the probe itself even in the absence of protease, which hindered accurate estimation of the LOD and the application of the previous assay for samples with low concentrations of caspase-3. 21 The EPR assay reported in this study is a significant extension and improvement of our previous report because the distinct ''off/on'' transition of the EPR signal gives rise to remarkably enhanced sensitivity and precision in quantitative studies by avoiding background interference.…”

“…21 However, a weak EPR signal could be detected for the probe itself even in the absence of protease, which hindered accurate estimation of the LOD and the application of the previous assay for samples with low concentrations of caspase-3. 21 The EPR assay reported in this study is a significant extension and improvement of our previous report because the distinct ''off/on'' transition of the EPR signal gives rise to remarkably enhanced sensitivity and precision in quantitative studies by avoiding background interference. Moreover, this assay can be readily applied to the detection and quantitative studies of other proteases by adopting appropriate recognition sequences in the probe instead of DEVD.…”

“…17–20 Also, EPR spectroscopy has advantages such as the absence of endogenous background signals and simple experimental procedure, offering a highly promising route for the detection and characterization of apoptosis-related proteases under ambient conditions. 21…”