Correlation of NADH/NAD⁺ electrochemical potential and enzymatic activity for investigating protein folding and...

The biological activity of a protein is determined by its native three-dimensional structure. Various stress factors induce structural changes in the proteins that leads to altered protein homeostasis with the accumulation of misfolded and aggregated protein conformers. Standard methods such as spectrophotometry, luminometry and fluorimetry are conventionally used to study protein activity. Here, an electrochemical method has been developed to measure the change in anodic peak current (I_p,a) of NADH generated during the oxidation reaction. The method showed a linear range of detection from 62.5 μM to 1.0 mM (R² = 0.999) for NADH with calculated detection limit of 16.02 μM and sensitivity of 1.75 × 10³ μA mM^-1 cm^-2. The method was further employed to investigate the enzymatic activity and folding kinetics of malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G6PDH). Also, the effect of aluminium ion (Al³⁺) on the activity and folding kinetics was investigated electrochemically. The Al³⁺ induced structural alterations in MDH and G6PDH were assessed using circular dichroism (CD), Thioflavin-T (ThT) and nuclear magnetic resonance (NMR). The developed label-free electrochemical method provides an alternative method for investigating protein activity and folding kinetics.