Electrochemical Determination of Transmembrane Protein Na⁺/K⁺‐ATPase and Its Cytoplasmic Loop C45

Abstract

Electrochemistry of membrane proteins is complicated by the fact that the studied substances are poorly soluble or insoluble in aqueous environment. The solubilization of proteins using surfactants (detergents) affects the electrochemical analysis or even renders it impossible. In the present study, the electrochemistry of the transmembrane protein Na+/K+‐ATPase (NKA) and its water‐soluble isolated cytoplasmic loop C45 is described. The proteins were studied using adsorptive transfer cyclic voltammetry and square‐wave voltammetry on basal‐plane pyrolytic graphite electrode (PGE) as well as constant‐current chronopotentiometric stripping analysis on hanging mercury drop electrode (HMDE). The nonionic surfactant octaethylene glycol monododecyl ether (C12E8) was used for NKA solubilization. Under these conditions the oxidation currents of Tyr and Trp (peak Y: +0.55 V and peak W: +0.7 V, vs. Ag/AgCl/3 M KCl) and catalytic reduction currents (peak H: −1.8 V) of NKA and C45 loop can be observed. Using the experimental procedures suggested in this study, we were able to investigate the oxidation, reduction and adsorption of NKA and C45 at femtomole level without the necessity of labeling by electroactive markers or techniques based on protein immobilization within the lipid bilayer attached to the electrode surface.

Na⁺/K⁺-ATPase inhibition by cisplatin and consequences for cisplatin nephrotoxicity

“…[19][20][21] ). For this purpose, hanging mercury drop electrode (HMDE) was first dipped into a 10-μL aliquot of the studied sample.…”

Protein structural transition at negatively charged electrode surfaces. Effects of temperature and current density

“…By means of peak H, nanomolar concentrations of practically any protein can be analyzed at low current densities [6]. This peak showed its usefulness also in the analysis of membrane proteins [9] and protein glycation [10]. At high current densities peak H is sensitive to changes in protein structures [6,11], capable to recognize a single aa exchange in mutants as compared to parent wild type proteins [12].…”

Electrochemical sensing of concanavalin A and ovalbumin interaction in solution

“…For almost 15 years we used constant current chronopotentiometric stripping (CPS) analysis and Hg-containing electrodes, which allowed observation of the well-developed electrocatalytic peak of peptides and proteins. This peak, termed peak H, was due to the catalytic hydrogen evolution, displaying sensitivity to local and global changes in protein structure [4][5][6][7][8] even for poorly soluble membrane proteins [9,10]. This peak can be useful in protein analysis for monitoring protein aggregation [7], denaturation [4,5], glycation [11] and for analysis of protein redox states [4].…”