ИСТИНА

Electrochemistry of proteinogenic amino acids is generally limited to sulfur-containing methionine (Met), cysteine (Cys), and cystine (Cys-Cys), and aromatic tryptophan (Trp), histidine (His), and tyrosine (Tyr) which can be oxidized on solid electrodes. In anodic voltammograms, Cys, Tyr, and Trp demonstrate well-defined peaks (at 0.5–0.8 V), while His, Met, and Cys-Cys exhibite fuzzy waves (at high positive potentials of 1–1.5 V, vs. Ag/AgCl, neutral pH). Electrochemical oxidation reactions of amino acids on solid electrodes are irreversible and pH dependent [1]. For many years a lot of various modifiers of electrode surfaces have been tested to improve electrochemical activity of amino acids, but the list of oxidizable amino acids has not been significantly extended. However, the stereotype about six “electroactive” (“easily oxidizable”) amino acids is outdated now. Recently, the specific electrochemical oxidation, both direct and electrocatalytic, of nearly all protein amino acids (except for glutamic acid, Glu) was shown by our group [2]. Particularly, the oxidation signals of amino acids were demonstrated at potentials below 1 V by cyclic voltammetry (CV) and amperometric flow injection analysis (FIA) on carbon screen printed electrodes (SPE) and SPE modified with Prussian blue (PB, Fe4[Fe(CN)6]3) in phosphate buffer, pH 6.0. For 20 amino acids out of 21 tested, the electrogenerated Berlin Green (Fe[Fe(CN)6], fully oxidized form of PB) has been reduced back to PB, forming a catalytic cycle thus resulted in an increase of oxidation currents [2]. Moreover, protein molecules were shown to be oxidized via “non-electroactive” amino acid residues on unmodified and PB modified carbon SPE by amperometric FIA [2, 3]. Modification of carbon SPE with PB led to significant increase of sensitivity of protein and peptide detection. Interestingly, the pronounced catalytic effect of PB was found for amyloid-beta peptides (Aβ) lacking the Tyr-10 residues, while practically no effect of PB was observed for Aβ mutants contained Tyr-10 [3]. In the present work, we continue to study the electrooxidation reactions of proteinogenic amino acids on carbon SPE modified with transition metal hexacyanoferrates (MeHCF, where Me is varied as Fe(II), Cu(II), Co(II), or Ni(II)). The dependencies of the analytical signal on pH (5.0–9.0) and analyte concentration were investigated. Surprisingly, it has been found by CV and amperometric FIA that PB (FeHCF) is not the best electrocatalyst of amino acid oxidation due to its instability in alkaline solutions. Only for Cys and Cys-Cys, the highest values of anodic currents were observed with SPE/PB in acidic media. Interestingly, Cys was found to be a unique amino acid for which the optimal pH values of electrooxidation reactions on blank electrodes (pH ≥ 7.0) and on PB modified electrodes (pH ≤ 5.0) were significantly different. PB was shown to be the best electrocatalyst for Cys oxidation (pH 5.0). Cys-Cys was poorly soluble in neutral and alkaline solutions. Among the metal hexacyanoferrates tested, CuHCF was shown to be stable only in strong acidic media (KCl + HCl, pH ~ 1–3) and suitable only for Cys electrocatalytic oxidation. In most cases, CoHCF surpassed NiHCF and PB catalysts in their ability to catalyze the oxidation reactions of amino acids, especially in alkaline solutuins. It was found that the optimal pH value for these reactions is 8.0–8.5. Except for six “electroactive” amino acids, the responses of other proteinogenic amino acids were not obtained on blank SPE by CV. Using arginine (Arg) as an example, a representative voltammogram of the oxidation of a “non-electroactive” amino acid on SPE/CoHCF is shown in Fig. 1. The expansion of the list of oxidazible protein amino acids opens new horizons for electrochemistry and analytical chemistry of proteins and peptides. Short chain bioactive peptides [4] are promising objects for future research using electrochemical methods. Acknowledgements This work was financially supported by the Russian Science Foundation, grant 24-13-00049, https://rscf.ru/project/24-13-00049/. References 1. E.V. Suprun, Electrochem. Commun., 125, 106983 (2021) 2. E.V. Suprun, E.V. Karpova, S.P. Radko, A.A. Karyakin, Electrochim. Acta, 331, 135289 (2020) 3. E.V. Suprun, E.V. Daboss, V.M. Pleshakov, D.V. Vokhmyanina, S.P. Radko, A.A. Karyakin, S.A. Kozin, A.A. Makarov, V.A. Mitkevich. Electrochim. Acta, 406, 139829 (2022) 4. A. Chawathe, V. Ahire, K. Luthra, B. Patil, K. Garkhal, N. Sharma, Anal. Biochem., 696, 115699 (2025)