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

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Abstract

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% (I50∼10 μM) by 8-hydroxyquinoline (8-OHQ) and also, although to a lesser extent, by other metal chelators such as o-phenanthroline, α,α′-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.

Keywords

Plant mitochondrion
Alternative oxidase
Protein purification
Enzyme activation
Quinol oxidase
Diiron protein

Abbreviations

AOX
alternative oxidase
BSA
bovine serum albumin
DEAE
diethylaminoethyl
deoxyBigCHAP
N,N-bis(3-gluconamidopropy)deoxycholamide
DTT
dithiothreitol
DQ
duroquinone
DQH2
duroquinol
EDT-20
N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane
MOPS
3-(N-morpholino) propanesulfonic acid
SDS
sodium dodecyl sulfate
TES
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid
Tris
tris(hydroxymethyl)aminomethane
Tween-20
polyoxyethylene-sorbitan monolaurate

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