Investigation of the conformation changes of myoglobin by an electrochemical method and a biosensing application based on controlled protein unfolding

Abstract

The conformational changes of myoglobin (Mb) during urea-induced protein unfolding were investigated using an electrochemical method. Using several different concentrations of urea, Mb adsorbed onto a montmorillonite clay modified glassy carbon electrode (GCE) was denatured. It was determined from the relative differences in the percentage of Mb unfolding that urea-induced Mb unfolding is a one-step, two-state transition process. The results obtained using electrochemical analysis were in agreement with those obtained by UV-vis spectroscopy and fluorescence spectroscopy, confirming our observations. Thermodynamic parameters during the conformational changes were also calculated to further characterize the unfolding process of Mb. Furthermore, two typical denaturants, urea and acid, were synergistically utilized to maintain GCE incorporated Mb in its most unfolded state, while simultaneously maintaining the presence of heme groups. Under optimal conditions, the unfolded Mb/clay/GCE exhibited accelerated direct electron transfer relative to the native Mb/clay/GCE. Additionally, the sensitivity for the detection of H₂O₂ was increased nearly 10-fold, and the limit of detection (LOD) for H₂O₂ was reduced to 0.3 μM for the unfolded Mb/clay/GCE relative to the native Mb/clay/GCE. The present work introduces a simple and effective way to study the unfolding of metalloproteins and holds great promise for the design of novel sensitive biosensors.