Partial Purification and Kinetics of Indoleacetic Acid Oxidase from Tobacco Roots

Abstract

Extracts from roots of Nicotiana tabacum L var. Bottom Special contain oxidative enzymes capable of rapid degradation of indoleacetic acid (IAA) in the presence of Mn(2+) and 2, 4-dichlorophenol. Purification of IAA oxidase was attempted by means of ammonium sulfate fractionation and elution through a column of SE-Sephadex. Two distinct fractions, both causing rapid oxidation of IAA in the absence of H(2)O(2), were obtained. One fraction exhibited high peroxidase activity when guaiacol was used as the electron donor; the other did not oxidase guaiacol. Both enzyme fractions caused similar changes in the UV spectrum of IAA; absorption at 280 mmu was reduced, while major absorption peaks appeared at 254 and 247 mmu. The kinetics of IAA oxidation by both fractions were followed by measuring the increase in absorption at 247 mmu. The peroxidase-containing fraction showed no lag or a slight lag which could be eliminated by addition of H(2)O(2) (3 mumoles/ml). The peroxidase-free fraction showed a longer lag, but addition of similar amounts of H(2)O(2) inhibited the rate of IAA oxidation and did not remove the lag. With purified preparations, IAA oxidation was stimulated only at low concentrations of H(2)O(2) (0.03 mumole/ml). A comparison of K(m) values for IAA oxidation by the peroxidase-containing and peroxidase-free fractions suggests that tobacco roots contain an IAA oxidase which may have higher affinity for IAA and may be more specific than the general peroxidase system previously described from other plant sources. A similar oxidase is present in commercial preparations of horseradish peroxidase. It is suggested that oxidation of IAA by horseradish peroxidase may be due to a more specific component.