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Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme.

Iniesta J, Esclapez-Vicente MD, Heptinstall J, Walton DJ, Peterson IR, Mikhailov VA, Cooper HJ - Enzyme Microb. Technol. (2010)

Bottom Line: Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose.Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum.The nitration sites were confirmed as Tyr23 and Tyr20.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry and Institute of Electrochemistry, University of Alicante, Alicante 03080, Spain.

ABSTRACT
In this paper we report the successful use of a non-metallic electrode material, boron-doped diamond (BDD), for the anodic electro-oxidative modification of hen egg white lysozyme (HEWL). Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose. We also compare literature methods of electrode pre-treatment to determine the most effective in electrosynthesis. Our findings show a decrease in total nitroprotein yield with decreasing nitrite concentration and an increase with increasing solution pH, confirming that, at a BDD electrode, the controlling factor remains the concentration of tyrosine phenolate anion. Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum. The products from electro-oxidation of HEWL at BDD were confirmed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometry, which revealed unique mass increases of +45 and +90 Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20.

No MeSH data available.


Related in: MedlinePlus

Cyclic voltammograms showing the effect of pre-treatment of the BDD electrode on the electrochemical oxidation of 6 mM NaNO2 in 50 mM disodium tetraborate adjusted to pH 9.0 with H3BO3: (A) cathodic pre-treatment, and (B) anodic pre-treatment. Dotted traces correspond to the background in the absence of nitrite. Inset figure shows the cyclic voltammograms for the electrochemical behaviour of a test redox couple 1 mM K3[Fe(CN)6] plus 0.1 M KCl in aqueous solution, comparing both electrochemical pre-treatments as described in Section 2: cathodic pre-treatment (solid line) and anodic pre-treatment (dotted line). Cyclovoltammograms were obtained at a scan rate of 0.050 V s−1 and the first cycle was recorded.
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fig1: Cyclic voltammograms showing the effect of pre-treatment of the BDD electrode on the electrochemical oxidation of 6 mM NaNO2 in 50 mM disodium tetraborate adjusted to pH 9.0 with H3BO3: (A) cathodic pre-treatment, and (B) anodic pre-treatment. Dotted traces correspond to the background in the absence of nitrite. Inset figure shows the cyclic voltammograms for the electrochemical behaviour of a test redox couple 1 mM K3[Fe(CN)6] plus 0.1 M KCl in aqueous solution, comparing both electrochemical pre-treatments as described in Section 2: cathodic pre-treatment (solid line) and anodic pre-treatment (dotted line). Cyclovoltammograms were obtained at a scan rate of 0.050 V s−1 and the first cycle was recorded.

Mentions: Fig. 1 shows the effect of the different pre-treatments on the electrochemical oxidation of 6 mM sodium nitrite at a BDD electrode. There are clear differences in peak potential and current density. Cathodic pre-treatment favours a higher current density of 1.7 mA cm−2 with a peak potential of +1.32 V. In contrast, cyclic voltammetry of the anodic oxidation of sodium nitrite at platinum electrodes previously showed oxidation of nitrite (in 50 mM sodium tetraborate with 10 mM sodium nitrite [pH 9.4]) at +0.85 V (vs. SCE) with a polycrystalline platinum working anode. Thus, the oxidation potential peak is shifted some +0.5 V for nitrite oxidation at a cathodically pre-treated BDD electrode (+1.32 V vs. Ag/AgCl). The peak potential shift for nitrite oxidation is ascribed to a lower resistivity of the cathodically pre-treated BDD electrode surface [20]. Anodic pre-treatment (Fig. 1B), gave a much lower current density in the positive scan direction for the BDD electrode than was seen following cathodic pre-treatment. Upon abrasion of the electrode with 1 alumina powder/water slurry, a similar voltammetric response was obtained to that from the cathodic pre-treatment. Cathodic pre-treatment is known to produce increased surface conductivity in BDD [22,23], whereas anodic pre-treatment gave effects attributed to surface functionalisation, with the formation of alcohols, ketones and mainly carboxylic groups. In this context, the BDD presents a negatively charged surface at high pH [24], providing a lower current density for the direct electro-oxidation of nitrite. Plots of oxidative peak current Ip vs. the square root of scan rate behave linearly as previously reported for the electrochemical oxidation of nitrite on a BDD electrode [25]. A small non-zero intercept indicates an influence of irreversible kinetic behaviour.


Retention of enzyme activity with a boron-doped diamond electrode in the electro-oxidative nitration of lysozyme.

Iniesta J, Esclapez-Vicente MD, Heptinstall J, Walton DJ, Peterson IR, Mikhailov VA, Cooper HJ - Enzyme Microb. Technol. (2010)

Cyclic voltammograms showing the effect of pre-treatment of the BDD electrode on the electrochemical oxidation of 6 mM NaNO2 in 50 mM disodium tetraborate adjusted to pH 9.0 with H3BO3: (A) cathodic pre-treatment, and (B) anodic pre-treatment. Dotted traces correspond to the background in the absence of nitrite. Inset figure shows the cyclic voltammograms for the electrochemical behaviour of a test redox couple 1 mM K3[Fe(CN)6] plus 0.1 M KCl in aqueous solution, comparing both electrochemical pre-treatments as described in Section 2: cathodic pre-treatment (solid line) and anodic pre-treatment (dotted line). Cyclovoltammograms were obtained at a scan rate of 0.050 V s−1 and the first cycle was recorded.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101334&req=5

fig1: Cyclic voltammograms showing the effect of pre-treatment of the BDD electrode on the electrochemical oxidation of 6 mM NaNO2 in 50 mM disodium tetraborate adjusted to pH 9.0 with H3BO3: (A) cathodic pre-treatment, and (B) anodic pre-treatment. Dotted traces correspond to the background in the absence of nitrite. Inset figure shows the cyclic voltammograms for the electrochemical behaviour of a test redox couple 1 mM K3[Fe(CN)6] plus 0.1 M KCl in aqueous solution, comparing both electrochemical pre-treatments as described in Section 2: cathodic pre-treatment (solid line) and anodic pre-treatment (dotted line). Cyclovoltammograms were obtained at a scan rate of 0.050 V s−1 and the first cycle was recorded.
Mentions: Fig. 1 shows the effect of the different pre-treatments on the electrochemical oxidation of 6 mM sodium nitrite at a BDD electrode. There are clear differences in peak potential and current density. Cathodic pre-treatment favours a higher current density of 1.7 mA cm−2 with a peak potential of +1.32 V. In contrast, cyclic voltammetry of the anodic oxidation of sodium nitrite at platinum electrodes previously showed oxidation of nitrite (in 50 mM sodium tetraborate with 10 mM sodium nitrite [pH 9.4]) at +0.85 V (vs. SCE) with a polycrystalline platinum working anode. Thus, the oxidation potential peak is shifted some +0.5 V for nitrite oxidation at a cathodically pre-treated BDD electrode (+1.32 V vs. Ag/AgCl). The peak potential shift for nitrite oxidation is ascribed to a lower resistivity of the cathodically pre-treated BDD electrode surface [20]. Anodic pre-treatment (Fig. 1B), gave a much lower current density in the positive scan direction for the BDD electrode than was seen following cathodic pre-treatment. Upon abrasion of the electrode with 1 alumina powder/water slurry, a similar voltammetric response was obtained to that from the cathodic pre-treatment. Cathodic pre-treatment is known to produce increased surface conductivity in BDD [22,23], whereas anodic pre-treatment gave effects attributed to surface functionalisation, with the formation of alcohols, ketones and mainly carboxylic groups. In this context, the BDD presents a negatively charged surface at high pH [24], providing a lower current density for the direct electro-oxidation of nitrite. Plots of oxidative peak current Ip vs. the square root of scan rate behave linearly as previously reported for the electrochemical oxidation of nitrite on a BDD electrode [25]. A small non-zero intercept indicates an influence of irreversible kinetic behaviour.

Bottom Line: Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose.Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum.The nitration sites were confirmed as Tyr23 and Tyr20.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry and Institute of Electrochemistry, University of Alicante, Alicante 03080, Spain.

ABSTRACT
In this paper we report the successful use of a non-metallic electrode material, boron-doped diamond (BDD), for the anodic electro-oxidative modification of hen egg white lysozyme (HEWL). Platinum electrodes can give rise to loss of activity of HEWL in electrosynthetic studies, whereas activity is retained on boron-doped diamond which is proposed as an effective substitute material for this purpose. We also compare literature methods of electrode pre-treatment to determine the most effective in electrosynthesis. Our findings show a decrease in total nitroprotein yield with decreasing nitrite concentration and an increase with increasing solution pH, confirming that, at a BDD electrode, the controlling factor remains the concentration of tyrosine phenolate anion. Purification of mono- and bis-nitrated HEWL and assay of enzymic activity showed better retention of activity at BDD electrode surfaces when compared to platinum. The products from electro-oxidation of HEWL at BDD were confirmed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FT-ICR) mass spectrometry, which revealed unique mass increases of +45 and +90 Da for the mono- and bis-nitrated lysozyme, respectively, corresponding to nitration at tyrosine residues. The nitration sites were confirmed as Tyr23 and Tyr20.

No MeSH data available.


Related in: MedlinePlus