Partial Purification and Kinetics of Indoleacetic Acid Oxidase from Tobacco Roots

Abstract

Extracts from roots of tobacco (var. Bottom Special) contain oxidative enzymes capable of rapid degradation of indoleacetic acid (IAA) in the presence of Mn2+ and 2,4-dichlorophenol. Purification of 1AA 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 H2O2, were obtained. One fraction exhibited high peroxidase activity when guaiacol was used as the electron donor; the other did not oxidaze guaiacol. Both enzymes caused similar changes in the UV spectrum of IAA; absorption at 280 m[mu] was reduced, while major absorption peaks appeared at 254 and 247 m[mu]. The kinetics of IAA oxidation by both fractions were followed by measuring the increase in absorption at 247 m[mu]. The peroxidase-containing fraction showed no lag or a slight lag which could be eliminated by addition of H2O2 (3 [mu] moles/ml). The peroxidase-free fraction showed a longer lag, but addition of similar amounts of H2O2 inhibited the rate of IAA oxidation and did not remove the lag. With purified preparations, IAA oxidation was stimulated only at low concentrations of H2O2 (0.03 [mu]moles/ml). Km values suggest 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. A similar oxidase is present in commercial preparations of horseradish peroxidase.