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Title: Purification and preliminary characterization of a pineapple indoleacetic acid oxidase, a peroxidase 
Author: Beaudreau, Charles Arthur
Date: 1963
Publisher: [Honolulu]
Abstract: Indoleacetic acid is a normal plant product which has been established to be a plant growth regulator. Indoleacetic acid when applied to plants will cause various growth responses. One response, promotion of cell elongation, is used as the basis of a very sensitive method for the detection of indoleacetic acid (1). Initiation of cell division has also been observed after the application of indoleacetic acid as in the formation of parthenocarpic fruit, adventitious roots, activation of cambial growth. and the formation of callus tissue. Cases where the application of indoleacetic acid causes inhibition of growth have also been observed. Probably the best known of these responses are the inhibition of elongation of root length and the prevention of lateral budding due to the production of indoleacetic acid by the apical bud. The enzyme, indoleacetic acid oxidase, which catalyses the oxidative degradation of indoleacetic acid, is also found in plant tissues. The function of this enzyme might be in the regulation of hormone level within the plant. However the regulation of hormone level within the plant is a complicated matter, since indoleacetic acid oxidase, with its necessary cofactors, the inhibitor(s) of indoleacetic acid oxidase, and the enzyme responsible for the formation of indoleacetic acid, may all be found in the same tissue. Despite the amount of work that has been done with the enzyme, indoleacetic acid oxidase, relatively little is known about it. Proposals have been made that the enzyme system consists of a flavo-protein and peroxidase (2), a copper protein (3), and an oxidase-peroxidase (4). Indoleacetic acid oxidase activity free from peroxidase activity has never been observed. However, it has been shown (5), that purified preparations of horseradish peroxidase can oxidize indoleacetic acid if Mn++ ion and certain diphenols are present. Also, crystalline turnip peroxidase (6) and two crystalline wheat germ peroxidases (7) have been shown to catalyze the oxidation of indoleacetic acid. In the cases , where known peroxidase activities have been checked for indoleacetic acid oxidase activity there is only one report (8) of a failure to observe the oxidation of indoleacetic acid. McCune (9) has reported on the separation by electrophoresis of six peroxidases from extracts of corn seedlings and showed that at least the four components that moved toward the cathode all displayed indoleacetic acid oxidase activity. The first conclusive evidence of the enzymatic destruction of indoleacetic acid was reported by Larsen in 1936 (10). He worked with an "auxin inactivation substance" obtained from the juice of pressed Phasiolus seedlings. This material was thermolabile and could be purified by precipitation with 607. ethyl alcohol. It possessed the ability to inactivate indoleacetic acid as well as auxin extracted from corn. The reaction was also shown to require oxygen. Tang and Bonner in 1948 (11), working with extracts of etiolated pea seedlings, studied an indoleacetic acid oxidase system. The inactivation of the indoleacetic acid was followed by observing the decrease in biological activity in the Avena curvature test (12) and by a chemical method (13) utilizing the color produced by the indole nucleus in the presence of FeCl3 (Salkowski test). They found that one molecule of O2 was consumed for each molecule of indoleacetic acid that was inactivated and that one molecule of CO2 was released. The enzyme system was inhibited by KCN and CO. This inhibition would seem to indicate the involvement of a heme protein. In 1950 Wagenknecht and Burris (3), working with a cell-free extract of yellow wax bean, studied an indoleacetic acid oxidase system. On the basis of their experiments involving the effects of inhibitors, they concluded that the enzyme was a copper protein. Galston and Baker in 1952 (14) using pea seedling homogenates, suggested that the indoleacetic acid system as it occurs in pea seedlings consisted of two portions; a heavy metal enzyme plus a light activated flavo-protein. Galston, Bonner and Baker (2) later reported that the indoleacetic acid oxidase of pea seedlings consisted of a light-activated flavo-protein coupled, through H202, to a peroxidase. Their system was inhibited by catalase and Mn++ ions. The activity of the system was increased by the addition of H2O2. An analog of the system could be formed by combining xanthine oxidase from cream and horseradish peroxidase. Kenton in 1955 (5) reported the oxidation of indoleacetic acid by' 02 in the presence of purified horseradish peroxidase. He found that certain monophenols as well as Mn++ ions increased the rate of oxidation of the indoleacetic acid and that some of the peroxidase substrates acted as inhibitors. He also demonstrated the presence of a thermostable fraction in wax bean extracts which acted as an activator for both the indoleacetic acid oxidase system of wax bean and for purified horseradish peroxidase. A 1/1 ratio of O2 consumed to indoleacetic acid oxidized and to CO2 released was also found. He concluded from his evidence that the oxidation of indoleacetic acid by peroxidase systems was not dependent on the presence of a flavo-protein. Ray reported in 1956 (15) on the indoleacetic acid oxidase of the fungi Omphalia flavida. 1'bis enzyme was found to have a pH optimum of 3.5. Its activity was not enhanced by the addition of 2,4-dichlorophenol but was moderately stimulated by the addition of Mn++ ions. the addition of Mn++ ions also prevented the enzyme activity from declining during the course of. the reaction. The enzyme was very sensitive to the addition of cyanide. He also showed that one mole of O2 was used for the oxidation of one mole of indoleacetic acid and that one mole of CO2 was released. Later, 1960 (16) Ray reported on the relationships between peroxidase action and indoleacetic acid oxidation in the Omphali flavida enzyme. In this work he showed that the peroxidase activity had a pH optimum of 3.5 - 3.7, which was similar to the pH optimum of the oxidizing activity. He also found that the indoleacetic acid oxidizing activity and the peroxidase activity paralleled each other through a three-step purification procedure involving an eleven fold increase in activity. The activity ratios were also closely parallel during the course of a heat inactivation experiment. The conclusion drawn from these results was that both activities were caused by the same enzyme. Using Lupinus albus as a source material, Stutz in 1957 (4) reported on the purification of an indoleacetic acid oxidase-peroxidase system that had been purified until it was homogeneous by starch-block electrophoresis. the enzyme functioned both as a peroxidase and as an oxidase. When functioning as an oxidase it showed an obligate requirement for a phenolic cofactor and a stimulation by the addition of Mn++ ions. Both the oxidase and peroxidase activities of the enzyme were inhibited by the addition of heme inhibitors. However, the lack of sufficient purified material to physically characterize the enzyme limits the value of this work. Gortner and Kent (17) reported in 1953 on the presence of an indoleacetic acid oxidase system in pineapple stem tissue. They also suggested the presence of an inhibitor in the crude extracts. The crude enzyme homogenate showed a pH optimum close to pH 3.5 and required Mn++ ions for activity. They showed that the enzyme was inhibited by cyanide but not by 8-hydroxy-quinol1ne. ibis would suggest that the enzyme might be a peroxidase and that it does not require copper. A later report by Gartner and Kent (18) showed that the natural inhibitor was probably ferulic acid or an ester of ferulic acid. They also showed that the naturally-occurring coenzyme for pineapple indoleacetic acid oxidase was probably p-coumaric acid or a quiny1-p-coumarate -- both of which were found to be present in pineapple tissue. The objective of this research was to extend the work initiated by Gortner and Kent. Where they were primarily concerned with the coenzyme requirements and inhibitors of the enzyme, this study was concerned with the isolation, purification, crystallization, and study of certain properties of the purified enzyme.
Description: Typescript. Thesis (Ph. D.)--University of Hawaii, 1963. Bibliography: l. [69]-73. v, 73, [1] leaves mount. ill., diagrs., tables (part fold.)
URI: http://hdl.handle.net/10125/11379
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Keywords: Pineapple -- Analysis, Indoleacetic acid oxidase

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