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Purification of the plant alternative oxidase from Arum maculatum: measurement, stability and metal requirement

journal contribution
posted on 2023-06-07, 22:43 authored by Charles Affourtit, Anthony Moore
We have purified plant alternative oxidase (AOX) protein from the spadices of thermogenic Arum maculatum (cuckoo pint) to virtual homogeneity. The obtained enzyme fraction exhibits a high specific activity, consuming on average 32 µmol oxygen min-1 mg-1, which is completely stable for at least 6 months when the sample is stored at -70 °C. This exceptionally stable AOX activity is inhibited approximately 90% (I50not, vert, similar10 µM) by 8-hydroxyquinoline (8-OHQ) and also, although to a lesser extent, by other metal chelators such as o-phenanthroline, a,a'-dipyridyl and EDTA. When inhibited by 8-OHQ, AOX activity is fully restored upon addition of 1.2 mM ferric iron, but neither ferrous iron nor manganese has any effect, whilst zinc decreases activity even further. Furthermore, we have developed a spectrophotometric assay to measure AOX activity in an accurate manner, which will facilitate future steady state and transient kinetic studies. The reliability of this assay is evidenced by retained stability of AOX protein during the course of the reaction, reproducibility of the measured initial rates, an observed 2:1 duroquinol¿oxygen stoichiometry and by the fact that, in absolute terms, the measured rates of duroquinone formation and duroquinol disappearance are identical.

History

Publication status

  • Published

Journal

BBA - Biochimica et Biophysica Acta

ISSN

0006-3002

Publisher

Elsevier

Issue

2-3

Volume

1608

Page range

181-189

Pages

9.0

Department affiliated with

  • Biochemistry Publications

Notes

ALM is the PI on the grant, helped devise and conduct the expts. Describes a technique to purify the plant alternative oxidase which results in a stable protein product which is highly active and suitable for crystallisation studies.

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2012-02-06

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