Abstract
In order to investigate the ability of the Vitreoscilla hemoglobin (VHb) to act as a peroxidase, the protein was overexpressed in Escerichia coli and purified using a 6xHis-tag. The peroxidase activity of VHb was studied using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferrocene carboxylic acid (FcCOOH) dopamine and l-dopa as substrates. The effects of external agents such as pH, salt concentration/ionic strength, and the thermal stability of VHb on the catalytic activity were assessed. The optimum pH for VHb using ABTS as a substrate was estimated to be 6–7. The VHb protein proved to be stable up to 80 °C, as judged by its peroxidase activity. Furthermore, NaCl concentrations up to 100 mM did not exert any significant effect on the activity. The catalytic activity against ABTS and FcCOOH was similar to that measured for horseradish peroxidase, whereas in the case of the phenolic substrates dopamine and l-dopa the activity was several orders of magnitude lower. The Michaelis constants, \( {\mathop K\nolimits_{\text{m}}^{{\text{H}}_{2} {\text{O}}_{2} } }, \) were in good agreement with the data for human and bovine hemoglobin. No activity could be detected for the negative controls lacking VHb. These results demonstrate that VHb exhibits peroxidase activity, a finding in line with the hypothesis that VHb has cellular functions beyond the role as an oxygen carrier.
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Abbreviations
- ABTS :
-
2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
- FcCOOH:
-
Ferrocene carboxylic acid
- Hb:
-
Hemoglobin
- IMAC:
-
Immobilized metal ion affinity chromatography
- IPTG:
-
Isopropyl-β-d-thiogalactopyranoside
- Mb:
-
Myoglobin
- PCR:
-
Polymerase chain reaction
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- Tris–HCl:
-
Tris(hydroxymethyl)aminomethane buffer adjusted with hydrogen chloride
- VHb:
-
Vitreoscilla hemoglobin
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Acknowledgements
This work was supported by grants from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas) and the Swedish Research Council (VR).
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Malin Kvist and Ekaterina S. Ryabova contributed equally to this work.
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Kvist, M., Ryabova, E.S., Nordlander, E. et al. An investigation of the peroxidase activity of Vitreoscilla hemoglobin. J Biol Inorg Chem 12, 324–334 (2007). https://doi.org/10.1007/s00775-006-0190-x
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DOI: https://doi.org/10.1007/s00775-006-0190-x