Enzymatic Formation of Indole-3-Carboxaldehyde from Indole-3-Acetic Acid.

Abstract

It was first suggested by Tang and Bonner (14) that indole-3-carboxaldehyde is the product of the oxidative degradation of indole-3-acetic acid (IAA) by crude enzyme preparations from etiolated pea seedlings. Using the more active bean root enzyme, Wagenknecht and Burris (15) attempted without success to obtain the 2,4-dinitropheny1hydrazone of the aldehyde. On the basis of chromatographic data, Racusen (9) reports that the pea enzyme forms small amounts of the aldehyde. More recently, Wagen-knecht (personal communication) has made similar observations with the bean root svstem. Ray and Thimann (10) and Ray (11) have recently reported that the enzyme from Omphalia flavida is unable to form the aldehyde. The recent work of MAaclachlan and Waygood (6) likewise has left unsettled the nature of the end product. MIanningf and Galston (7) have reported the formation in the pea system of two products having high Rf values and giving qualitative color tests which indicate a similarity to our products # 4 and # 5 (see below). They were unable to demonstrate the formation of indole-3-carboxaldehyde, and also ruled out ortho-formamidoacetophenone, ortho-ami-noacetophenone, and 4-hydroxyquinoline as major end products, although they believe that the indole ring is ruptured. Chromatographic separation and spectrophoto-metric examination of the products of the IAA oxi-dase-peroxidase system from Lupinus albus L. (13) gave no indication that the aldehyde was formed; likewise, the results obtained when the chromato-grams were tested with 2,4-dinitrophenylhydrazine hydrochloride, modified Salkowski (3), and Ehrlich re-agent suggested the absence of the aldehyde. In view of the conflicting results, attempts were made to sup-lement the purified IAA oxidase with the auxiliary enzyme systems presumed to be present in the crude enzyme preparations employed in the early investigations. The present report is concerned with the coupling of the oxidase system to the cytochrome oxi-dase system. Under these conditions the formation of indole-3-carboxaldehyde as a major reaction product was observed. Subsequent reexamination of the reaction products from the oxidase-peroxidase system, particularly those freshly prepared from etiolated lupine hypocotyls, indicated the normal formation of the aldehvde in small amounts. Therefore, in order to obtain data on 1 Received revised manuscript December 23, 1957. 2 Work performed under the auspices of the U. S. Atomic Energy Commission. 3 Present address: Research Laboratory of the Western Pine Association, 7733 SE 13th Avenue, Portland 2, Oregon. the oxidation mechanism, indole-3-carboxylic acid and a number of substituted indole-3-acetic acid derivatives were tested both with the oxidase-peroxi-dase system alone and with the added cytochrome system. It is of interest to note that since this paper was submitted, Jones and Taylor (5) have reported the isolation of both indole-3-carboxaldehyde and in-dole-3-carboxvlic acid from cabbage extracts. MATERIALS AND METHODS Preparation and purification of the IAA oxidase from Lupinus albus L. has been previously described (13). The course of the oxidation of IAA was followed with the Warburg manometric apparatus under the conditions outlined earlier (13) except where 0.4 M tris-(hy-droxymethyl)-aminomethane buffer at pH 7.2 was uised in an attempt to approach the pH optimum of the cytochrome oxidase system and still maintain IAA oxidase activity. Under these conditions a small amount of phosphate, 4 micromoles per flask, was added for optimum IAA oxidase activity. The normal 3-ml reaction mixture was enzyme; 1 ml 0.4 M orthophosphate buffer, pH 6.3; 0.3 micro-mole MInCl,; 0.3 micromole soditim-2,4-dichlorophen-olate; (in the main compartment) 0.1 ml M KOH with a standard wick in the center well; and 30 micromoles of sodium indole-3-acetate in the side arm. When the cytochrome system was to be coupled to the oxidase, cytochrome oxidase was added to each flask in the form of 0.2 ml of a 5 % suspension of homogenized rat liver, together with cytochrome c, usually 2 x 10-5 M, and 0.3 micromoles of Al+++. Ascending chromatography on Whatman 3-mm filter paper was carried out with a solvent mixture consisting of 28 % NH40H, water, and isopropvl alcohol in proportions ranging from 1 : 1 : 6 to 1: 1: 12, depending on whether it was desired to obtain greater resolution of the more acidic or the neutral fraction of the products. The reaction products to be discussed have been designated by numbers 1 to 5 in order of increasing Rf value (13). This has been done in preference to Rf designations alone because the relative positions of the components were retained in the various solvent mixtures used. The products (13) which appeared important in this study were the apparently large # 2 (Rf 0.6) group of non-indolyl fluorescent compounds and the # 4 and # 5 (Rf 0.8 and 0.9), presumably polymeric indolyl, products which bracket the aldehyde. The # 1 (Rf 0.45) indolyl component (13) was not encountered in this study-presumably because the reaction with crude enzyme was not permitted to go to exhaustion. In some experiments, the substrate was IAA, labeled with C14 either in the methvlene (12) or in the carboxyl position (13). In order to obtain suffi-207