An Optical Method for the Detection of Oxidative Stress Using Protein−RNA Interaction

Abstract

The cytosolic 4Fe−4S protein aconitase can be converted under the influence of reactive oxygen species into an iron-regulatory protein (IRP1). Therefore, the IRP1 level is considered as an indirect marker of oxidative stress. An experimental approach is presented here to detect the concentration of this marker protein by surface plasmon resonance. The optical method exploits the natural binding affinity of IRP1 to an iron-responsive element (IRE) which was in vitro transcribed with a linker sequence and subsequently immobilized on a BIACORE sensor chip. The detection was found to be reproducible and sensitive in the range 20−200 nM IRP. Conditions of the binding process, such as pH and thiol concentration, were characterized. Feasibility of the method to detect and quantify IRP1 in physiological media was demonstrated.