THE OXIDATION OF INDOLE-B- ACETALDEHYDE AND ITS l-METHYL DERIVATIVE BY HORSERADISH PEROXIDASE Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY REN HOMER YEI-I 1967 III III III IIIIIII IIIIIII IIIIIIIIIIIII This is to certify that the thesis entitled THE OXIDATION OF INDOLE-3" ACETALDEHYDE AND ITS I-METHYL DERIVATIVE BY HORSERADISH PEROXIDASE presented by Ren Homer Yeh has been accepted towards fulfillment of the requirements for Ph.D. ' ' degree in Biochemistry vam $2311 Major professor Date j\_‘r1_ C37 0469 IIIIAISD 8- “SIM? 800K BRIBERY IIII). IIIIIIIII ABSTRACT THE OXIDATION OF INDOLE-B-ACETALDEHYDE AND ITS 1-METHYL DERIVATIVE BY HORSERADISH PEROXIDASE by Ben Homer Yeh Indole-B-acetaldehyde exerts the same physiological effect on plant growth as does indole—B-acetic acid. There- fore. it is assumed that the former compound is oxidized enzymatically within the plant tissues to indole-3-acetic acid. Since this assumption has not been completely verified this investigation was undertaken to ascertain the alternative metabolic pathways of these two plant auxins. Because peroxidase enzymes play a dual role as an "auxin oxidase” as well as an "aldehyde oxidase," horseradish peroxidase was used as a model enzyme system to study and compare the metabolism of indole-B-acetaldehyde and its 1-methy1 derivative to that of indole-B-acetic acid. In this study, a number of organic compounds including 1-methylindole-3-aceta1dehyde (not synthesized previously) were prepared by various methods. Our experimental results indicate that the peroxidase enzyme catalyzed the oxidation of indole-B-acetic acid to 3-methy1ene oxindole. However, oxidation of indole-3- acetaldehyde under the same conditions produced indole-B- Ben Homer Yeh carboxaldehyde as one of the major products instead of 3-methy1ene oxindole. The oxidation of the auxin derivatives including indole-3-acetic acid was found to be dependent on the con- ditions of the reaction. Changes in pH and the presence or absence of a cofactor altered the oxidative pathways of these compounds and gave rise to different oxidation products. For example, the presence of bisulfite ion at a pH range above neutrality stimulated the oxidation of indole-B-acet- aldehyde to h-hydroxyquinoline. The latter compound was isolated in crystalline form by use of silicic acid column chromatography. The same transformation of the 1-methy1 derivative of indole-B-acetaldehyde to 1-methyl-D-hydroxy- quinoline was not obtained, and apparently no single rule can be applied to the various patterns of peroxidase oxida- tion of indole auxins. Thin-layer chromatographic techniques were used for identifying the oxidation products resulting from the oxida- tion of indole-B-acetaldehyde and 1-methylindole-3-carbox- aldehyde by horseradish peroxidase. Manganese and 2,4-dichlorophenol had no effect on the oxidation indole-B-acetaldehyde either at alkaline or acidic pH conditions, although these two cofactors are excellent stimulators of peroxidase oxidation of indole-3-acetic acid. Indole-B-acetic acid at alkaline pH conditions inhibited strongly the formation of peroxide-peroxidase com- plexes of the horseradish peroxidase. This observation Ren Homer Yeh supports the belief that at certain pH values, oxidation of auxin by peroxidase is controlled by auxin itself. Also these studies indicate a typical case of substrate inhibi- tion of an enzyme reaction. THE OXIDATION OF INDOLE-BnACETALDEHYDE AND ITS l-METHYL DERIVATIVE BY HORSERADISH PEROXIDASE by Ben Homer Yeh A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1967 C«LI%")C>I 3-7le C 3 ACKNOWLEDGMENTS The author thanks Professor H. M. Sell for suggesting the problem and for his guidance and encouragement throughout the course of this research work. Thanks go to Professors P. Kindel, M. J. Bukovac, E. J. Benne and R. G. Hansen for their friendly criticisms and suggestions which were invaluable for the present work. The author also thanks Drs. R. W. Ritzert, K. K. Schlender and R. L. Franklin for their unselfishness in sharing research ideas: The financial support from the Department of Biochemistry of Michigan State University is very much‘appreciated. ii TABLE ACKNOWLEDGMENTS . . . . . . LIST OF TABLES . . . . . . LIST OF FIGURES . . . . . . LIST OF ABBREVIATIONS . . . INTRODUCTION . . . . . . . Literature Survey . . . Statement of Problem . . MATERIALS AND METHODS . . 0 Synthesis of Compounds. Indole-B-acetaldehyde. 1-Methylindole-3-acetaldehyde i-Methyltryptophan . 1-Methylindole-3~carboxaldehyde 1-Methyl-LI-hydroxyquinoline . . Identification of Compounds . . . 1~Methylindole-3-carboxaldehyde 1-Methyl-N-hydroxyquinoline . . Indole-B-acetaldehyde 1-Methylindole-B-acetaldehyde l-Methyltryptophan . Methods for Determination OF‘CONTENTS . , of Substrate Concentrations . . . . Enzymeeoooeoeoe Enzyme Reactions . . . Incubation Conditions for Biosynthesis of a New Oxidation Product . . . h-Hydroxyquinoline . Thin-Layer Chromatography Silicic Acid Column Chromatography . Ultraviolet and Infrared RESULTS 0 O O O O O O O O O Spectrophotometric Studie Oxidation of Indole-B-Acetic Acid, Indole-B-Acetaldehyde, and Indole- Spectra . s on the 3-Acetaldehyde-Sodium Bisulfite CompleXOeeoeeo 111 O O O O O Page ii 39 Effect of pH on the Oxidation of Indole-B- Acetic Acid, Indole-BnAcetaldehyde, and Indole-3~Acetaldehyde-Sodium Bisulfite Complex c e 0 Effect of Bisulfite or Sulfite ions on the Oxidation of Indole-BnAcetic Acid and IHdOle-Bchetaldehyde o o e c o o e e 1 Effect of Enzyme, Substrate, Hydrogen Peroxide, Bisulfite Ion, and Sulfite Ion Concentrations on the Formation of a Compound with a Spectral Absorption Of 316 and 330 mu 0 e e o e e o o e o o 0 Effect of 2 ,quichlorophenol and Manganese Ions on the Oxidation of Indole-B-Acetic Acid and Indole-B-Acetaldehyde . . . . Identification of Oxidation Products . . . Some Preliminary Studies with Thin- Layer Chromatography' e e e o o o o e 0 Isolation and Purification of a Compound with a Spectral Absorption at 316 and 330 mu by Column Chromatography . . quinoline . . . Thin-Layer Chromatographical Identification Of Compound X as Indole-B-Carboxaldehyde Nlindole-BnAcetaldehyde by Oxidation of 1-Meth Horseradish Peroxidase . . Possible Mechanism of Peroxidase Oxidation of Indole-BnAcetic Acid and Indole-B- Acetaldehyde o o c c o e o o o o o o 0 DISCUSSION 0 O O O 0 O O O O O O 0 I. O O O O SIMMARY O O O 0 O O O O O O O O O O O O O O O REFEREN CES O O O '0 I O O O O O O O O O O O 0 iv Identification of Compound X2 as h-Hydroxy- Page 46 54 60 66 71 71 75 76 81 87 9# 102 125 128 LIST OF TABLES Table Page i. R values of Indole-B-Carboxaldehyde and Igolated Indole-B-Carboxaldehyde . . . . . . . . 8A 2. R values of 1-Methylindole-3-Carboxaldehyde afid Isolated 1-Methylindo1e-3-Carboxa1dehyde . . 93 Figure 1. 7. 9. 10. 11. LIST OF FIGURES Pathways for the Metabolism of Indole-B- Acetaldehydeeoooooeeeeoone. Infrared Absorption Spectra of i-Methyl- indole-B-Carboxaldehyde and Indole-3- carboxaldehydeoeeeeococoons. Infrared Absorption Spectra of Indole-B- Acetaldehyde Sodium Bisulfite Salt, 1~Methylindole-B-Acetaldehyde Sodium Bisulfite Salt, Indole-Bchetaldehyde, and 1~Methylindole-3chetaldehyde . . . . Calibration Curves for Estimating Indole- 3-Acetic Acid and Indole-B-Acetaldehyde by the Xanthydrol Reaction . . . . . . . . Changes in Ultraviolet Absorption Spectrum during Enzymatic Oxidation of Indole-B- ACGtiCACIdooeeeoeooeeoeoo Changes in Ultraviolet Absorption Spectrum during Enzymatic Oxidation of Indole-B- AcetaldehYdeoeoooocoeoecoco Changes in Ultraviolet Absorption Spectrum during Enzymatic Oxidation of Indole-B- Acetaldehyde Sodium Bisulfite Addition PrOdUCteoooeoeeoooooeooo Effect of pH on the Oxidation of Indole-3- Acetic Acid, Indole-B-Acetaldehyde and Indole-BnAcetaldehyde Sodium Bisulfite AdditionPrOdUCtoo o o e e o co co co Effect of Sodium Bisulfite on the Oxida- tion of Indole-B-Acetaldehyde at Different pHValueS............o... Effect of Sodium Bisul.fite on the Oxida- tion of Indole-BnAcetaldehyde. . . . . . . Effect of Sodium Bisulfite on the Oxida- tion of Indole-B-Acetic Acid . . . . . . . vi Page 25 28 33 40 43 47 49 52 55 55 Figure Page 12. Spectroscopic Changes During the Oxidation of Indole-B-Acetaldehyde and Indole-3- Acetic Acid in the Presence of Sodium B18u1f1teeoooooeedoeceeeooeo 58 13. Effect of Sodium Sulfite Concentration on the Oxidation of Indole-3-Acetaldehyde . . . . 61 1%. Effect of Indole-B-Acetaldehyde Concentra- tion on the Formation of a Compound with Spectral Absorption at 316 and 330 mu . . . . . 64 15. Effect of Enzyme Concentration on the Formation of a Compound with Spectral Absorption at 316 and 330 mu . . . . . . . . . 64 16. Effect of Hydrogen Peroxide Concentration on the Oxidation of Indole-Bchetaldehyde Sodium Bisulfite Addition Product . . . . . . . 67 17. Effect of Horseradish Peroxidase on the Oxidation of Indole-B-Acetaldehyde at pH 7.8 . 67 18. Effect of Manganese Ions and 2,4—Dichloro- phenol on the Oxidation of Indole-B-Acetic Acid and Indole-B-Acetaldehyde . . . . . . . . 69 i9. Ultraviolet Absorption Spectra of Compound Eluted from Spots on Thin-Layer Chroma- 1325813111.. 00.000000000000000 73 20. Ultraviolet Absorption Spectra of Compound X2 Eluted from Spots on Thin-Layer Chroma- togram0.000000000000000... 73 21. Ultraviolet Absorption Spectrum of the Purified Compound x2 0 e e e e e e e e o e o o 77 22. Infrared Absorption Spectra of Compound X2 and LIV-Hydroxyquln01ine o o e e e e o e c e e o 79 23. Thin-Layer Chromatogram of Indole-B-Carbox- aldehyde Obtained by Oxidation of Indole-3- Acetaldehyde O O O O O O O O O O O O O O O 0 O 82 24. Ultraviolet Absorption Spectrum of Compound X1 0 O O O O O O O 0 O O O O O O O O O O O O O 85 25. Changes of Ultraviolet Absorption Spectrum During Enzymatic Oxidation of 1-Methylindole- 3-A06taldehyde................ 89 vii Figure 26. 27. 28. Page Thin-Layer Chromatogram of 1-Methylindole- 3-Carboxaldehyde Obtained by Oxidation of 1~Methylindole-3-Acetaldehyde . . . . . .'. . . 91 Effect of Hydrogen Peroxide, Indole-B- Acetic Acid and Sodium Bisulfite on the Absorption Spectra of Horseradish Peroxidase . 95 Effect of Hydrogen Peroxide and Sodium Bisulfite on the Absorption Spectra of Horseradish PerOXIdaSe e o e e o o o e o e o o 9? viii LIST OF ABBREVIATIONS u-HQ HRPO IAA IAAld IAld 1-Methyl-IAAld 1 -Methyl-IAld TLC IAAld-NaHSO3 1-Methy1-IAA1d-NaH303 IPyA 2,4-D 4-Hydroxyquinoline Horseradish Peroxidase Indole-B-acetic Acid Indole-B-acetaldehyde Indole-B-carboxaldehyde 1-Methylindole-3-acetaldehyde 1~Methylindole-3-carboxaldehyde Thin-layer Chromatography Indole-B-acetaldehyde Sodium Bisulfite Adduct 1-Methylindole-B-acetaldehyde Sodium Bisulfite Adduct Indole-B-Pyruvic Acid 2,h-Dichlorophenol ix Dedicated to My Wife Ching-Chu ili‘N‘ INN N NIT {NIT 1 Ill} '1'. INTRODUCTION INTRODUCTION Literature_Survey In 1934 K3gl, Haagen-Smit and Erxleben (52) demon- strated that indole-3-acetic acid (IAA), which was isolated from urine, had a striking effect in promoting cell elonga; tion in.§z§n§ coleoptile sections. It was not until 1946 that Haagen-Smit and Went (34) definitely showed the presence of IAA in Zea mays by isolating this compound in pure form. Most of their characterization of IAA was based on chromatographic analysis. IAA has been found to be widely distributed in the plant kingdom (8, 28, 88, 107). In addition to the wide occurrence of IAA in plants, the presence of other indolic compounds has been reported exten- sively (26, 27, 53, 60, 79, 117). All of these compounds, which are either the precursors of the active auxins or the end products of hormonal metabolism, constitute a pool of auxin growth regulators in plants. The presence of indole-3-acetaldehyde (IAAld) in plant tissues was first reported by Larsen (57) two years before KOgl, Haagen-Smit and Erxleben's publication (52) on the identification of IAA. Evidence for the occurrence of this neutral auxin derivative was also confirmed by other investigators (29, 30, 55). Several investigators (55, 99, 108) suggest two metabolic pathways for the formation of ‘l lllll ll. ‘ 3 IAAld: one involves the decarboxylation of trytophan to tryptamine followed by oxidative deamination to indole-3- acetaldehyde; and the other involves an oxidative deamina- tion to indole-3-pyruvic acid (IPyA) with subsequent decarboxylation to IAAld. Direct evidence (16) for the tryptamine pathway was provided by the finding that in watermelon tissue tryptophan could be transformed easily into tryptamine. Furthermore, the evidence that a puri- fied amine oxidase obtained from pea seedlings (13) could convert tryptamine readily into IAAld lends support to this pathway. On the other hand, the presence of IPyA in plant tissues was not confirmed (40). However, it has been demonstrated that in aqueous solution IPyA can be decom- posed readily to form IAAld (109). Observations that IAA can be formed easily from IPyA during chromatographic analysis (40, 88) give further support to the IPyA pathway. The suggested biochemical pathways leading to the formation of IAAld are summarized in Figure 1. It is apparent from eXperiments (87) that auxin- stimulated growth depends not only on the biosynthesis of auxins, but also on their metabolism. The presence in plants of a so-called auxin oxidase system (93, 94, 105), which catalyzes the oxidation of IAA and its derivatives, has been recognized extensively (10, 35, 76), but the chemistry of this "auxin oxidase" is controversial. Galston, Bonner, and Baker (22) postulate that "auxin oxidase" is actually a combination of enzymes: an oxygenase which is (I721? CH-COOH Tryptophan O ‘ CHZCHZNHZ N H Tryptamine O ; CHZCONHZ .I N H Indoleacetamide Indole-3—pyruvic Acid N H Indole-3-acetonitrile xvi CHZCHO l ‘ CHO ‘ ~ 9 N’J N H Indole-3-acetaldehyde 4-Hydroxyquinoline j/ C HZCOOH N COOH H I N H Indole-3-acetic Acid Indole-B-carboxaldehyde i Indole-3-carboxylic 2 O H 3-Methylene Oxindole Figure 1. Pathways for the Metabolism of IAAld 5 a light-activated flavoprotein and a peroxidase which can use the hydrogen peroxide generated from the flavoprotein moiety to oxidize IAA. On the other hand, Kenten and Stutz (43, 91) could not identify the requirement for a light-activated flavoprotein factor in bean and lupine plants. They implied that an."auX1n oxidase" is simply a peroxidase which can also act as oxidase in the presence of certain heat-stable cofactors such as monophenols and manganese ions. The belief that "auxin oxidase" is an oxidase-peroxidase is based on the following observations: 1. "Auxin oxidase" appears to be a hemoprotein since it can be inhibited by-cyanide or by car- bon monoxide. The inhibition by carbon monox- ide is light—reversible (75). 2. "Auxin oxidase" is inhibited by catalase and stimulated by hydrogen peroxide (5, 43, 78). 3. Tissue extracts containing "auxin oxidase" can replace peroxidase for peroxidase substrates (75, 84. 91). 4. Highly purified horseradish peroxidase can also replace "auxin oxidase" to catalyze the oxidation of IAA (22, 42, 43, 105). 5. The seasonal changes and the activity of "IAA oxidase? manganese oxidase, and peroxidase are well correlated (70). 6. In vitro oxidation of IAA with purified horse- radish peroxidase yields the same products as "IAA oxidase" (37). ‘ NIT [1. 6 7. The oxidation of IAA consumes one molecule of oxygen and evolves one molecule of carbon dioxide per molecule of IAA oxidized (94). Obviously, a wealth of evidence suggests a close similarity between "IAA oxidase" and peroxidase. The purification of peroxidase from horseradish was first reported by Keilin and Mann (4i) and crystallized by Theorell (97). It was the fourth hemoprotein to be crys— tallized, succeeding hemoglobin, myoglobin, and catalase. Other crystalline peroxidases from plant sources have been obtained from turnips, Japaneses radishes, wheat germ, and pineapples. Theorell and Akeson (96) have determined the amino acid and carbohydrate composition of horseradish peroxidase. Analyses indicate that the enzyme contains carbohydrate. Similarly, Japanese radish peroxidase also has a high content of carbohydrate residues: mannose (35), xylose (11), and arabinose (4) (69). The heterogeneity of horseradish peroxidase has been studied extensively (95). The multiple components of horse- radish peroxidase with identical Spectra and activity are separable by salt precipitations, paper or starch electro- phoresis, or chromatography. Theorell (98) reported the existence of a variable component, para-peroxidase or horse- radish peroxidase I, which moves in a direction opposite to the major component, horseradish peroxidase II, during elec- trophoresis. In many aspects, horseradish peroxidase resembles methemoglobin in absorption spectrum and in com- plex formation with hydrogen peroxide. 7 As far as the kinetics of the enzyme reaction are concerned, the contribution of Chance (i4, 15) should be mentioned. Mild treatment of horseradish peroxidase with hydrochloric acid cleaves the heme group of the enzyme, and the nondenaturated apoprotein with some of the original activity of the enzyme is obtained. This observation was reported earlier by Maehly (63). Recently Siegel and Galston (85) observed that the cleaved protein without the heme group did not oxidize IAA. However, IAA was oxidized in the presence of a monophenol cofactor and manganese ions. The cleavage of horseradish peroxidase was foundto be reversible; the acid treated peroxidase could be restored to an active form by neutralization with alkali. Numerous chemical compounds react with peroxidase to form stable compounds which can be detected Spectroscop- ically (83). Many of these substances inhibit enzymatic activity. Cyanide, fluoride azide, hydroxylamine, and nitric oxide are several compounds known to be inhibitors of peroxidase activity. Some investigators (5, 23) suggest that these o-polyphenols act as inhibitors since they are capable of being oxidized rapidly by peroxidase with the subsequent result of a deficiency of hydrogen peroxide in the reaction mixutre. However, it is possible that the inhibiting effect of o-polyphenols is due to the enzyme catalyzed formation of the condensation products between IAA and the polyphenols (59). The stimulating effect of some naturally occurring monophenols may account for the 8 fact that these compounds are oxidizable in the presence of a peroxidase system (43). A cyclic mechanism was advanced by Kenten and Mann (45) to explain the oxidase character of the peroxidase. The suggested cyclic mechanism is illus- trated by the following diagram: Phenol Oxidized Phenol Mn3+ or Mnu+ Mn2+ O2 3202 In addition to the monophenols, maleic hydrazide (5, 43) was found to be a promoter in the oxidation of IAA, but its mode of action is unknown. Recently Klebanoff (49, 50, 51) reported that bisulfite or sulfite anions stimu- lated the oxidation of pyridine nucleotides by horseradish peroxidase and that thyroxine was a stimulator in the oxida- tion of adrenaline, uric acid, and ascorbic acid. Some compounds act either as accelerators in certain reactions or as inhibitors in other reactions catalyzed by the same enzyme system. Thyroxine is an example of such a compound. It is a strong accelerator in the peroxidase oxidation of adrenaline, uric acid, and ascorbic acid but a strong inhibitor of peroxidase oxidation of iodide and pyrogallol. Among other compounds which are found to have a significant effect on peroxidase were ammonia, hydrazine, pyridine and imidazole (17). 9 Peroxidase catalyzes the reaction of hydrogen perox- ide by electron donors other than hydrogen peroxide. A wide variety of chemical compounds have been found to be 'active as hydrogen donors in the oxidation and reduction systems. Therefore, the reaction involved in peroxidase- catalyzed reactions shows great versatility. Peroxidase can act as an oxidizing agent, halogenating agent, and hydroxylating agent as well. Peroxidase can also partici- pate in polymerization, isomerization, and decarboxylation reactions. These observations on the versatility of perox- idase reactions have been reviewed by Mason and Saunders (65. 83). Recently, IAAld was found to be an excellent sub- strate for rabbit muscle homogenates (81), liver homogenates (115), and pure horseradish peroxidase (116). Apparently, the peroxidase reactions are not only involved in the regu- lation of the hormonal level in plant tissues, but are closely related to the hormonal metabolism in mammalian tissues. The interconversion of steroid hormones in mam- malian tissue depends greatly on the peroxidase system (47). The mechanism of enzymatic degradation of auxins has for many years been a subject attractive to many investiga- tors. The publications in this field are voluminous. The early report by Tang and Bonner (94) indicated that the oxidation of IAA consumed one molecule of oxygen and evolved one molecule of carbon dioxide per molecule of IAA. The proposed reaction product was indole-3-carboxaldehyde 10 (IAld). The results obtained by them demonstrated that the major feature of enzymatic degradation of IAA by "auxin oxidase" was decarboxylation of the side chain of the IAA molecule. However, later reports published by Manning and Galston (64) suggested that IAA could be oxidized in a manner analogous to the well-known kynurenine pathway of trypotophan metabolism in animal tissues, but they could neither identify the formation of indole-3-carboxaldehyde nor the production of formylkynurenine in the oxidation of IAA by pea homogenates. The formation of a small quantity of indole-3- carboxaldehyde, among other products of IAA oxidation, later was detected by Racusen (73), Pilet (71), and Stutz (90) in reaction mixtures containing plant tissue homogen- ates. The formation of IAld from IAA oxidation catalyzed by Japanese radish peroxidase was reported recently (68). Furthermore, Stutz (90) discovered that the addition of cytochrome c and a cytochrome oxidase containing homogenate of rat liver to the lupine enzyme converted IAA to IAld in 50% yield. Moreover, Stutz (92) reported IAld could be formed almost quantitatively from indole-3-glycolic acid. Because of the ultraviolet and infrared Spectra of the reaction product, Stowe, Hay, and Thimann (89) suggested that one of the IAA oxidation products might be 3-methy1 oxindole. By studying the oxidation of IAA by a highly purified enzyme from lupine seedlings, Stutz (91) found at least five oxidation products of IAA. 11 Following the observation of Galston, Bonner, and Baker (22) that horseradish peroxidase itself could oxi- dize IAA, research has been conducted mainly on the Oxida- tion of IAA in the presence of peroxidases alone. In the studies on the oxidation of IAA by Omphalia enzyme and horseradish peroxidase, Ray (77) observed the formation of an intermediate compound, and as a result he proposed a two-step reaction mechanism for the degradation of IAA by the enzyme system. This intermediate compound was regarded as the actual product of the enzymatic reaction. The com- pound in question was neutral and retained the indole struc- ture, but was neither oxindole nor indole-3-carboxaldehyde. This intermediate compound was called compound A by Bay. The second step of the reaction, a course to the final products, was regarded as a nonenzymatic reaction. Recently, Hinman and Lang (36) reported that the oxidation of IAA catalyzed by crystalline horseradish peroxidase yielded 3-methylene oxindole as a major product. They also showed that oxindole-3-carbinol was a precursor of 3-methy1ene oxindole. Consequently, oxindole-3-carbinol was regarded as the intermediate compound A in Ray's two- step reaction mechanism. Hinman and Lang (36) proposed a new mechanism for the degradation of IAA catalyzed by crys- talline horseradish peroxidase. The mechanism involved, as a first intermediate, an indolenine hydroperoxide which is converted through an indolenine epoxide to oxindole-3- carbinol, which in turn undergoes a Slow but Spontaneous 12 transformation into 3-methylene oxindole. This suggested mechanism is of great interest and will be discussed further. Recently, Latarjet and Caldas (58) made the interest- ing observation that certain bacteria can be made sterile by heavy exposure to ultraviolet light and then restored to activity by peroxidase. Kamerbeek (39) and Galston (20) reported, reSpectively, that there is remarkably greater peroxidase activity in dwarf varieties of beans or peas. These findings imply that the sui generig synthesis of peroxidase decides the height of certain variety of beans or peas . Mathan (66) reported that leaf shape or size of normal (La+/La+), Lanceolate (La+/La'), and several other leaf-shape mutants of tomato was determined by the content of peroxidase, catalase and phenoloxidase. Another theory advanced by Galston and Dalberg (21) suggested that the ageing process in plants is closely cor- related to the activity of peroxidase in plant tissues. This assumption was based on the observation that the IAA destroying activity in pea stem or root sections increased longitudinally from the apex (minimum activity) toward the point where growth ceased and cell maturation occurred. Siegel (84) noted that the lignification of vascular tis- sue involved the action of peroxidase. Apparently, other than the oxidation and reduction processes, the biologically Significant aSpects of the peroxidase catalyzed oxidation of auxins may be exhibited in the areas of growth, ageing, morphogenesis, and protection 13 or adaptability of plants to the Specific conditions they may encounter. The observations of Saunders §t_§l. (83) indicate that the versatility of peroxidases may be related to the biosynthesis of certain complex organic compounds in plants. Recently, Galston and co-workers (19), and Key and Ingle (46) mentioned that growth processes controlled by auxin were closely related to the synthesis of RNA, especi- ally messenger RNA, since auxin could enhance the synthesis of RNA in excised plant tissue. Such processes involved the combination of IAA to the RNA fraction, but the combin- ation could only be shown in the presence of peroxidase. Statement of Problem Several investigators (6, 29, 55) propose that IAld is an immediate precursor of IAA. Indeed, the presence of an enzyme system which will catalyze readily the oxidation of IAAld to IAA has been reported (29, 55). However, a satisfactory stoichiometric relationship between IAAld dis- appearance and IAA formation has never been obtained. Therefore, some wOrkers (6, 44) suggest that in plant tis- sues IAAld and its analogs may be metabolized in a manner other than by oxidation or dismutation to the corresponding acids (56). Larsen (56) studied the metabolism of IAAld and naphthalene-1-acetaldehyde by Aggna coleoptile sap, and found that at least one-half of the aldehydes added to the coleoptile sap disappeared so rapidly that they did not give 14 rise to the formation of IAA. Consequently, he suggested that the conversion of IAAld to IAA might involve a dismu- tation reaction in which equivalent amounts of alcohol and acid are formed. ' However, Kaper and Veldstra (40), who studied the metabolism of tryptophan by.Agrobacterium tumefaciggg, did not find the 1:1 ratio between IAA and tryptophol formation reported by Larsen (56). The ratio of IAA to tryptophol depended on the experimental conditions used. Ashby (6) also studied the metabolism of naphthalene-1-acetaldehyde by Artemisia-seedling root sap and found less than 1 mole of acid per 2 moles of aldehyde. These facts do not sup- port Larsen's (56) dismutation theory for the explanation of the extra loss of the aldehydes in plant tissues. Apparently, no Single enzyme called aldehyde mutase is present in both plant and animal tissues. The purified aldehyde mutase from beef liver was found by Hacker (72) to be actually a mixture of two separable enzymes, an alcohol dehydrogenase and aldehyde dehydrogenase. The enzymes, which will catalyze the transformation of IAAld and its analogs to the correSponding acids or alcohols, are illustrated in.the following scheme: 15 NADH +\ H+ CH 2CHZNH2 CH CHO /j?0 fiHZCHZOH @Alcohol Monoamine Oxidase‘ N \Dehydrogenasem Peroxidase Aldehyde dehydrogenase New FMNH‘ “\ \NADH + H+ Pathway Aldehyde OX1dase\__i]-CHZCOOH mb/JV ON H The lack of a stoichiometric relation between alde- hyde disappearance and acid formation suggests that there are at least two metabolic pathways which are involved in the metabolism of IAAld or its analogs. One catalyzes the transformation of IAAld to IAA and the other catalyzes the direct metabolism of the active auxin precursors. These facts indicate a balance between these two enzymatic systems according to the stage of develOpment of the living organ- isms. Kenten and Mann (45) studied the oxidation of B- phenylethylamine by pea-seedling extracts and found that the total oxygen uptake was in excess of that required for the oxidation of the amine to B-phenylacetaldehyde. They sug- gested that a so-called aldehyde oxidase was present in the extracts. Further studies on this pea-seedling "aldehyde oxidase" by Kenten (44) indicated that the rate of oxygen 16 uptake required for the oxidation of B-phenylacetaldehyde can be inhibited to the extent of 85% when a high concen- tration of catalase is added. But for a Simple flavopro- tein, the inhibition of catalase would, at most, reduce the reaction rate to half of the total oxygen uptake. From this and other evidence, Kenten (44) concluded that the "aldehyde oxidase" catalyzed reaction is very similar to the oxidase-peroxidase reaction, and that the oxidase- peroxidase-catalyzed oxidation of B-phenylacetaldehyde does not yield phenylacetic acid but phenylcarboxaldehyde. Some observations (44) suggest that the wide distribution of oxidase-peroxidase systems in nature may be responsible for the rapid disappearance of IAAld and its analOgs in various plant tissues. Recently, Bartholini gg,g;. (7) noted the presence of enzyme systems in platelets and erythrocytes which can readily catalyze the transformation of IAAld to its corres— Tponding alcohol and acid. However, a lack of stoichiometric relationship between aldehyde disappearance and acid forma- tion was also observed in these eXperiments. Udenfriend 22,21. (103) reported that only 30% of the serotonin metabo- lized by rat liver homogenate could be recovered as 5- hydroxyindole-3-acetic acid, whereas Wu (111) could not detect any IAA when rat liver mitochondria was employed as a catalyst for tryptamine oxidation. Similar results (115) were observed when IAAld was directly used as substrate for the oxidation studies by liver homogenates. Evidence has 17 accumulated to support the existence of an alternative path- way other than IAA formation for the metabolism of the hormonal substances such as IAAld and its analogs in both plant and animal tissues. Moreover, several investigators (3, 4, 9, 12, 32, 54) have studied the effect of IAAld on various auxin tests. Results of these tests indicate that IAAld becomes active by being oxidized enzymatically into IAA. Moreover, another noteworthy observation is that in all of these tests, the relative activity of IAAld is not always the same. Accord- ing to Larsen (54), the differences found in the relative activity of IAAld in different tests may be due to test objects with different properties in absorbing IAAld or to conversion of IAAld into IAA. Indirectly, this finding also suggests the existence of an alternative pathway for the metabolism of IAAld. Because of the dual role of horseradish peroxidase to, act both as an."IAA oxidase" and an "aldehyde oxidase," the present work on this enzyme was taken as a model to study this new metabolic pathway of IAAld. Attempts were made to identify the oxidation products of IAAld catalyzed by this crystalline enzyme. Studies were made also on 1-methyl- IAAld to give further information concerning the mechanism of horseradish peroxidase-catalyzed oxidation of indole aldehydes. MATERIALS AND METHODS MATERIALS AND METHODS Synthesis of Compounds A number of different methods were selected for the preparation of indole derivatives for this thesis. Detailed procedure for synthesis of the compounds is outlined below: Indole-3-acetaldehyde.--Indole-3-acetaldehyde was synthesized from tryptophan by treatment with dilute sodium hypochlorite solution by the method of Gray (32). Three grams of DL-tryptophan were dissolved in 100 ml of a 10% sodium hydroxide solution which was then adjusted to a pH of 8.5 with 4 N,hydrochloric acid. The solution was poured at once into a 4,000 ml Erlenmeyer flask containing 1,200 ml of distilled water and 700 to 800 ml of benzene: then 200 ml of a 0.52% sodium hypochlorite solution (1 part Clorox with 9 parts water), was added. Immediately after the addition of the hypochlorite solution, the flask and contents were shaken with a swirling motion in hot water bath. The temperature was raised to 50° over a period of 20 minutes and then held between 50 and 510 for an addi- tional 15 minutes. During this operation, the benzene layer became yellow to orange in color indicating a successful synthesis. While still warm, the benzene layer was separated from the water phase and concentrated to a volume of 65 ml 19 20 under vacuum while maintaining the temperature below 50°. A crystalline product was obtained by shaking the result- ing benzene solution with 50 ml of saturated sodium bisul- fite solution. The crystals were collected on a Bfichner funnel and washed several times with 95% ethanol and finally with absolute ethanol and ether. The product was recrystallized by dissolving IAAld-NaHSO3 in a minimum amount of water and then diluting with absolute ethanol to make a final concentration of 90% ethanol. The yield was 1.9 to 2.0 g of pure IAAld-NaHSOB. To obtain the free aldehyde, 200 mg of IAAld-NaHSO3 was dissolved in 8 ml of distilled water and a few drops of concentrated Na2003 was added. The aldehyde was then extracted with peroxide-free ether. After evaporation of the ether solution to dryness under vacuum, 82 ug IAAld was attained. 1-Methylindole-3-acetaldehyde.--The title compound was made by the same procedure described by Gray (32) for indole-3-acetaldehyde and was also obtained in the form of bisulfite salt. Since the bisulfite salt of 1-methy1-IAAld is very soluble in 95% ethanol and cannot be recrystallized by the method outlined for IAAld-NaHSOB, it was recrystal~ lized by the fOIIOWing procedure. First, the salt was dissolved in a minimum amount of water to make a saturated solution, and then it was filtered. A small amount of 180» propanol was added to the clear filtrate to induce cloudi- ness. Then the mixture was set in a refrigerator overnight 21 or longer for crystallization to occur. One and one-half grams of white needles of 1.methyl-IAAld-NaHSO3 was obtained from 4 g of 1-methyltryptophan. The free 1-methyl-IAA1d was obtained in the same manner as described for indole-3-acetal- dehyde (32). 1-Methyltryptophan.--1-Methy1tryptophan was obtained from the sodium salt of tryptophan with methiodide in liquid ammonia according to a process outlined by Yamada g§.gl. (112). 1 Metallic Na (3.2 g, 0.14 atomic mass units) was added in small pieces to a mixture of about 450 ml of liquid ammonia containing 0.2 g of ferric nitrate monohydrate while the mixture was being stirred. After the sodium metal was com— pletely dissolved, 12.3 g of DL-tryptophan which was sus- pended in an appropriate amount of anhydrous ether, was added to the mixture while it was being stirred. After 30 minutes 11.4 g of methyl iodide was added dropwise over a 15 minute period and stirring was continued until all ammonia was com- pletely evaporated. The residue was then slowly transferred to a mixture containing 350 ml of ethanol and 350 ml of H20. This mixture was then evaporated under vacuum to dryness. The residue was added to 350 ml H20, which was heated to dissolve the mass and then filtered. The warm solution was adjusted to pH 5.0 with glacial acetic acid and diluted with 350 ml of 95% ethanol. The mixture was then placed in a refrigerator overnight. The resulting white precipitate was collected on a Bachner funnel and washed successively with 22 40 ml of H20, 40 ml of 50% aqueous ethanol, 40 ml absolute ethanol, and finally with 40 ml of dry ether. This precipi- tate, which was recrystallized from 50% aqueous ethanol, consisted of colorless needles of melting point 2690 (lit., mp 269°) (112) and gave a positive ninhydrin test. 1-Methylindole:3-carboxaldehyde.--The title compound was synthesized from l-methylindole by reaction of dimethyl- formamide on indole in the presence of phOSphorus oxychloride by a method described by Tyson and Shaw (102). To a flask protected from atmospheric moisture, fitted with a mechanical stirrer, and containing 1.18 mole of dimethylformamide cooled to -5°, 2.0 mole of phOSphorus oxychloride was added. During this procedure the mixture was stirred constantly to prevent the temperature from rising above 10°. After phosphorus oxychloride was added, 1.0 mole of i-methylindole was added in small quantities while a tem- perature between 23 and 270 was maintained. Stirring was continued at 25° for 0.5 hour. Then 4.0 g of finely divided calcium carbonate was added to the mixture and the tempera- ture was gradually raised from 30 to 35°. From this point on the temperature was controlled by cooling the flask with ice from time to time until the temperature reached 55 to 600 in 0.5 hour. The Slurry was cooled externally to 100 with ice and then 20.0 ml of 30% solution of sodium acetate was poured into the flask as the mixture was stirred. Then the mixture was diluted with water to a volume of 1.000 ml. After 1.18 moles of sodium hydroxide was added and the mixture was 23 refluxed for 3 hours, dimethylamine formed copiously. The mixture was diluted with water so that its volume reached 3.5 liters. Then it was heated to reflux and filtered. After the resulting filtrate was treated as described above, it yielded colorless needles of melting point 69° (lit. mp 69°) (82). 1—Methyl-4-hydroxyguinoline.--1-Methyl-4-hydroxy- quinoline was made by using the method reported by Simpson and Wright (86) for the synthesis of 6-nitro-1-methyl-4- quinoline. Methyl sulfate (2.2 ml) was added to a solution of 1.5 g of 4-hydroxyquinoline in 48 m1 of aqueous potassium hydroxide (1.9%) at 50°. At the end of the reaction, excess alkali was added, and the 1-methy1-4-hydroxyquinoline (2.1 g) extracted with chloroform. The product was recrystallized from benzene, and gave colorless prisms of melting point 150 to 152° (lit. 150-152°) (1). The chloride of 1-methyl-4- hydroxyquinoline was obtained by dissolving the 1-methyl-4- hydroxyquinoline in diluted hydrochloric acid solution. After the solution was evaporated to dryness, the residue was recrystallized from isopropanol. The crystals melted at 164 to 166° (lit. 164-166°) (67). IdentificatiOn of Compounds 1-Methylindole-3-carboxaldehyde.--The Synthesized aldehyde gave positive tests with 2,4-dinitrophenylhydrazine 24 and xanthydrol, but reSponded poorly to Schiff's and Ehrlich's color reaction. Its crystals were colorless needles of 69° (lit. 69°) (82). 1:2: 1780. 1720, 1650, 1560, 1470, 1400, 1380, 1375. 1327, 1260, 1190, 1125, 1075. 1032, 1012, 787, 749, and 730 cm'l. The NH stretching band could not be EtOH Shown. Amax 262 me The infrared Spectrum of 1-methylindole-3-carboxalde- hyde was very similar to indole-3-carboxaldehyde. The two infrared Spectra are Shown in Figures 2 (A) and 2 (B) respec- tively, for comparison. 1-Methyl-4-hydroxyguinoline.--According to AlliSon 23,31. (1), 1-methyl-4-hydroxyquinoline has the following reasonance structures: 0H 01 \ \ e—AQO , e I < | 01' I I CH 3 CH3 - CH I II III The quinolinone, structure II, shows no ketonic properties and is unaffected by methiodide in boiling methanol, indicating that the ionic "aromatic" form II makes the major contribution to the reasonance structure. The free 1-methy1-4-quinolinone crystals were color- less needles of melting point 150 to 152° (lit. 150-152°) (1). The chloride melted at 164 to 166°(lit. 164-166°) (68) Afifigfl 313. 329 mu. 25 .aopoaoposaoapooam man cmaxoom m an eoSHaaOpop Ohms mapooam one .poaaoa oodaoan aaammmpoa SH Amv oeanoeamaonHSOImuoaopad dam A(; o :1::...... m_:_.__._ _...__.._. 22:2: ._ Z 1...; fl .... On on 3‘ 3.. Co 27 Igdgle-3-acetaldehyde.--The title aldehyde was iden- tical to the one reported by Gray (32). It was a colorless syrupy liquid and was positive to Schiff's and 2,4-dinitro- phenylhydrazine reactions for aldehydes and to Salkowski, Ehrlich, and xanthydrol color reactions for the indole ring. The infrared spectrum of the freshly released indole- 3-acetaldehyde was the same as that reported by Brown (12) and Gray (32). It showed strong absorption bands at 3400, 1710, 747 cm.1 and medium strong bands at 1465, 1430, 1350, and 1100 cm'l. The infrared spectrum of the crystalline sodium bisulfite addition product of IAAld in a KBr pellet showed no carbonyl stretching band at 1710 cm-1. Strong to medium-strong absorption bands were noticed at 3400, 3200, 2930, 1620, 1490, 1460, 1345, 1300, 1242, 1050, 1005, 880, 870, 845, 800, 740, 700, and 645 cm'l. The ultraviolet absorption spectrum of the free IAAld in ethanol had absorption maxima at wavelengths of 273, 281, and 289 mu The infrared Spectra of IAAld-NaHSO3 and free IAAld are given in Figures 3 (A) and 3 (C), respectively. 1-Methylindole-3-acetaldehyde.--The free 1-methy1- indole-3-aceta1dehyde was a colorless syrupy liquid. It gave positive tests to Schiff's and 2,4-dinitropheny1hydrazine reactions for aldehydes: and to Salkowski, Ehrlich and xanthydrol reactions for the indole ring. The bisulfite salt of 1-methyl-IAAld was as stable as that of IAAld-NaHSOB. The aldehyde was stable enough for 28 Figure 3. Infrared absorption Spectra of indole-3- acetaldehyde sodium bisulfite salt (A), 1-methylindole-3-acetaldehyde sodium bisulfite salt (B), indole-3-acetaldehyde (C) and 1~methylindole-3-acetaldehyde (D). 29 VAMIII u m 30 chemical analysis, but it is highly recommended that the aldehyde be stored under an atmosphere of nitrogen for longer periods. The aldehyde is not stable in acidic or alkaline solution. During chromatography on thin-layer plates the formation of small Spots of 1-methyl-3-carbox- aldehyde, IAAld and an unknown substance were detected. Apparently, the aldehydic carbon became unstable due to the presence of a methyl group on the indolic nitrogen atom. The infrared spectrum of the freshly released 1- methyl-IAAld bears a great similarity to that of IAAld except that the N-H stretching band is not present at 3390 cm-1. The aldehyde showed strong to medium-strong bands at 1720, 1470, 1430, i370, 1335. 1250, 1160, 1130, 1070,- 1015, and 740 cm'l. The infrared Spectrum of the aldehyde was made as a thin film on a sodium chloride plate. The infrared Spectrum of the crystalline sodium bisulfite addition product of 1-methy1-IAAld in a potassium bromide pellet showed strong to medium-strong bands at 3450, 3050, 2850, 1610, 1550, 1470, 1420, 1370, 1330, 1220-1175. 1100, 1050, 1015. 735. 695, and 670 cm'l. The carbonyl stretching band at 1720 cm"1 could not be located. The spectrum closely resembles that of IAAld-NaHSOB. The ultraviolet absorption Spectrum of the l-methyl- IAAld in ethanol showed absorption maximum at wavelength of 288 mu. The bisulfite addition product in water gave a Similar ultraviolet absorption curve with a single maximum 31 at the same wavelength. The infrared Spectra of the aldehyde and its bisul- fite salt are shown in Figures 3 (D) and 3 (B), respectively. Analysis of C11H12°ANSNa: Calcd. C, ”7.653 H9 “0363 N0 5.05% Found. C, 46.04: H. 4.86: N. 4.93% 1-Methyltryptophan.--The synthesized i-methyltrypto- phan gave a positive ninhydrin reaction. It crystallized as colorless prisms with a melting point of 269°, which is in agreement with that reported by Yamada 22.3l. (112). Methods for Determination of Substrate Concentrations The concentrations of IAA and IAAld were determined by xanthydrol reaction with Slight modifications of the method described by Weissbach.§£.§l. (106). Before each determination a fresh solution of 0.1% xanthydrol in glacial acetic acid was prepared. The following procedure was used. To 1.0 ml of the sample in water was added 0.8 ml of 12 N hydrochloric acid and 2.0 ml of the xanthydrol reagent. Five minutes later 1.0 ml of acetone was added. After each addition of reagent, the solution was mixed well. The intensity of the pink color, which developed, was measured within 5 to 10 minutes at 520 mg in a Spectrophotometer. The standards and blanks were treated in the same manner. The xanthydrol reaction is very sensitive to compounds having 32 an indole ring. AS little as 8 ug of IAA could be detected,~ and the optical density was found to be proportional to IAA concentration over the entire range from 8 to 200 ug. The calibration curves for IAA and IAAld were determined as previously described. The data are shown in Figure 4. Enz me The crystalline horseradish peroxidase (HZ = 3), which was procured from Worthington Chemical Company, was stored at 00 immediately after arrival. Enzyme with RZ less than 3 was also employed, but had no effect on the results reported in this thesis. Enzyme Reactions For the enzymatic studies the reaction mixture con- tained the following: 3 ml of substrate (1 x 10'“ M.of IAA or IAAld) in 0.05 fl.of buffer solutions (acetate buffer for pH below 5.2 or phOSphate buffer for pH above 5.6), 0.1 ml of enzyme solution (usually contained 6 ug to 0.3 mg enzyme protein), and 0.1 ml of hydrogen peroxide (1 x 10"2 M). Other cofactors (manganese, 2,4-dichlorophenol, and bisul- fite), if used, were added to the substrate solutions. Total reaction mixture per cuvette was 3.2 ml. After reac- tion was started by the addition of hydrogen peroxide, the rate of the reaction was followed in a Beckman DK-2 Ratio Recording Spectrophotometer to determine the change of the absorbancy which was due either to the oxidation of the substrate or to the formation of the reaction products. 33 Figure 4. Calibration curves for estimating IAA and IAAld by xanthydrol reaction. 0.7 0.6 Absorbancy (5 20 Mu) O .0 45 01 .0 OJ 0.2 0.I _....J- . 5 CcOcentrcIiOn x 0.038 JIM 35 Incubation Conditions for Biosynthesis Of a New Oxidation Product 4-H drox uinoline.--The new oxidation product, 4- hydroxyquinoline, was isolated from a reaction mixture con- sisting of 2,000 ml of 200 mg of IAAld-NaHSO 40 mg of 3. peroxidase, and 0.05 M,of phOSphate buffer solution at pH 7.8. The reaction was started by addition of 160 ml of 2 x 10'3 M_hydrogen peroxide and the rate of reaction was determined by the xanthydrol reaction and also by the changes of the optical density at 316 and 330 mu (a typical absorp- tion maximum of 4-hydroxyquinoline) in the spectrophotometer. After changes in the structure of the substrate were com- pleted, the same amount of IAAld-NaHSO was added together 3 with 20 mg of peroxidase. This procedure was repeated in the same way until finally 800 mg of IAAld-NaHSO and 100 mg of peroxidase were added. In a similar manner :60 ml of 2 x 10""3 ,M of hydrogen peroxide was added dropwise to the reaction mix- ture until a total of 650 ml of the hydrogen peroxide solution was added. After the reaction was completed, the solvent was evaporated under vacuum to dryness. The residue was extracted with isopropanol and the crude material (267.9 mg) in the isopropanol extract was further purified by fractionation through a silicic acid column. Silicic Acid Column Chromatography The silicic acid (Fisher Scientific Company, 200 mesh, chromatographic grade) was suspended with stirring in a 36 sufficient quantity of pure benzene to make a slurry. The supernatant was decanted and the silicic acid was washed twice in the same manner with a mixture of 5% benzene in hexane. After the final wash, the silicic acid was resus- pended in 5% benzene in hexane. This suSpension was stirred continually while it was transferred to a glass column, 2.0 cm in diameter, to give a silicic acid column having a length of 20 cm. Samples were.applied in amounts of about 200 mg. After the sample, which was obtained from the enzyme reac- tion mixture, was applied to the top of the adsorbent in the column, the adsorbent and sample were first developed with a solvent system consisting of hexane and ethyl acetate (50:100) and then with isoprOpanol. About 250 ml of the hexane-ethyl acetate mixture was used for the first development. Samples were collected with an automatic fractionation collector and fractions having similar absorption patterns were combined for further purification. Thin-Layer Chromatography For routine work the thin-layer plates were prepared by the method of Randerath (74). For the results formally recorded in this thesis, Silica Gel F25,+ (from Brinkmann Instruments, Inc.) and Eastman Chromatogram Sheet (Type K 301, R2 Silica Gel withOut fluorescent indicator) were used. The following solvent systems were employed: (1) n-propanol- hexane (1:4); (2) chloroform-acetic acid (95:5): (3) benzene- ' 37 acetic acid-water (4:1:1): and (4) n-butanol-hexane (18% n-BuOH). IAld and 1-methyl-IAld were identified by Spraying the adsorbent on the plates with 2,4-dinitrophenylhydrazine reagent, which was prepared according to Randerath (74). Since both IAld and 1-methyl-IAld responded poorly to Erhlich's reagent, the latter reagent was not employed for identification of these two compounds. For identification of 4-hydroxyquinoline or 1-methyl-4-hydroxyquinoline ultra- violet light was used as detecting agent with plates made of fluorescence background. glpraviolet and Infrared Spectra Ultraviolet Spectra were determined in a Beckmann DK-2 Ratio Recording Spectrophotometer either in 95% ethanol solution or in water solution. Infrared Spectra were made in a Beckmann IR-5 Infrared Spectrophotometer. For liquid compounds such as IAAld and i-methyl-IAAld, the Spectra were obtained as a thin film and for other crystalline compounds as potassium bromide pellets. RESULTS RESULTS Spectrophotometric Studies on the Oxidation of IAAI IAAld and IAAld-NaHSO3 The changes in ultraviolet Spectra during the peroxidase reactions were followed by using the Beckman DK-2 Ratio Recording Spectrophotometer. The incubation conditions were the same as those described under the section on methods. The series Of ultraviolet Spectra of IAA, IAAld, and IAAld- NaHSO3 are shown, reSpectively, in Figures 5, 6, and 7. For the three different substrates used, it can be. clearly seen that the breakdown of IAA, IAAld, and IAAld- NaHSO3 by the pure horseradish peroxidase followed different patterns of oxidative pathways. The conversion of IAA to 3-methylene oxindole is clearly noted by the appearance of the typical intense double peaks at Xmax 247 and 253 mu (Figure 5) parallel with the disappearance of the indole absorption at 277 and 285 mu. These changes in spectrum, which were observed when IAA was used as substrate under the present conditions, are very similar to the observations previously reported by Hinman 23.31. (36, 37) and Ray (77). The SXperiments shown in Figure 5 were carried out at pH 3.9 acetate buffer solution without addition of any cofactor such as manganese, 2,4-dichlorophenol, and sulfite or bisulfite anion. It is clearly observed that at 265 mp, 39 40 N N _I am no” a n no: 0 m do pose sad a tea a A he: s : <dhpaz SH mowamso .m madmam 41 It]!!! Sal-:2.) a: :30 «ca .CNEJOOM it. C: .25 .35 c1583.- 1!: 42 absorbancy increases at first and then decreases; this behav- ior was Shown by Bay to be evidence for the formation of an intermediate A. A Similar increase and decrease phenomenon at 295 mm, as reported by Bay, was also observed in Figure 5. The appearance of isosbetic points at 233, 262, and 296 mu was also noted in Figure 5. It took 45 to 60 minutes for such isosbetic points to appear. This was further evidence for Ray's support of his sequential IAA to'A and B transfor- mation mechanism. Qualitatively, the Spectroscopic changes during the course of oxidation of IAA showed little difference within the pH range from 3.7 to 4.5, but within this pH range the rate of reaction was reduced quantitatively as the pH increased. At pH above 6.1 only a slight change of IAA Spectrum could be produced by the peroxidase system. The changes of spectrum of IAAld during the course of oxidation under the same condition used for IAA (pH 4.58 acetate buffer) are shown in Figure 6. Obviously there are significant differences in the spectroscopic changes between Figures 5 and 6. The action of peroxidase on IAAld caused the immediate changes of the Spectrum of IAAld at 244, 246, and 298 to 300 mu regions, but not at 253 and 247 mu regions (characteristic absorption maximum for 3-methylene oxindole). The changes of the Spectrum during the course of the reaction finally reached a steady state as is shown by the shape of the plotted curve. This absorption behavior shows no similar- ity with that of the typical absorption of the 3-methylene- 43 .Sda H S“ empoaaaoo was choose some A.sas oma sec moa .ma .06 .ms .om .ma .0 no sense cams mobaso .13 men as Gamma who: meaoomm .Ha N.m mos oppo>sO Hog chapxaa SoapomOH on» mo madaob Hence em_esoa x m as: moms ac use» use m.enoa x a mos campxaa Soapomoa on» mo UH¢50\w1 o.nav manage Ha H.o .mw.h m9 pd mommleddddH ho Sodpmddxo OHPMEhNflm madman azapooam Soapaaomnm poaoabmapad SH mownmso .5 oaswam 48 ., 2n. .1, F: L11: .5 1.3 , .3300! (.1; is... r..- 5.5.... S!- 530 3.0 .CflEv-OOM 49 Figure 8. Effect of pH on the oxidation of IAA, IAAld, IAAld-NaHSOB. The rate of the enzymatic reaction was followed by the change of absorbance at 244 mu per hour. Reaction mixture contained 1 x 10-“ M_substrates, IAA or IAAld or IAAld-NaHSO in 0.05 M 3 buffer solution, 6 pg of enzyme (R2 2 2.0) and 1 x 10'4'M_H 0 . For pH values rang- 2 2 ing from 3.6 to 5.2 and from 5.6 to 7.8 acetate and phOSphate buffers were used, respectively. 60- 10.0. 244 mp 40 30 20 IO I l i I I I J 1 1 J J £16 4%) 4A»¢18 512 5£SISO 6!) 68 THE 7AI'18 |__AOEIATE ___J L___PHOSPHATE BUFFER pH . BUFFER 51 was clearly seen: the oxidation of IAA and IAAld at a pH above 6.2 decreased continually as the pH was increased, whereas the oxidation of IAAld-NaHSO3 increased profoundly as the pH was raised above 6.2. Apparently, the competition between the hydroxide ions and sodium bisulfite may account for the differential behavior of the enzymatic system toward these three different substrates. It is possible that the increase of the concentration of hydroxide ions inactivated the enzymatic system, whereas the presence of sodium bisul- fite prevented the inhibitory effect of the hydroxide ions and restored the activity of the enzymatic system at the elevated pH values. Hochster and Quastel (38) believe that the inactivation of the enzyme system toward oxidation of IAA at high pH values may be due to the formation of manganese carbonate at the higher pH values which lowers the effective manganese concentration below optimum. .However, since there were no manganese ions present in the reaction mixtures, the lowering of the effective manganese concentration does not account for the reduced activity of the reaction system at the elevated pH values. The effect of pH on the Spectroscopic changes of IAA, IAAld, and IAAld-NaHSO has been described previously and, therefore, will not be3elucidated further. However, one should recall that the presence of sodium.bisulfite had a profound effect on the enzymatic system, whereas at the lower pH conditions sodium bisulfite exhibited, to some extent, an inhibitory effect on the activity of the enzyme. At higher pH values the enzymatic action was enhanced (Figure 9). 52 Figure 9. Effect of sodium bisulfite on the oxidation of IAAld at different pH values. Data were taken from Figure 8. .4) E). 876.5432l098765432l0 I I I I I Iazsem . 3.03903 5055330. 54 - -2 Effect of H803 or SO Ions on the Oxidation of IAA and IAAld The effect on sodium bisulfite on the oxidation of IAA has been reported by Ray (77). He found that sodium bisulfite prevented further changes of optical density of IAA oxidized by the peroxidase system. Similar results were also obtained by Tonhazy and Pelczar (100) fromlg‘ versicolor enzyme preparation. But it is noteworthy to add that in Ray's experiments the pH of the reaction mix- -tures was 3.7, whereas the optimal pH for P. versicolor was 4.5. These results correspond well with those observed in the present work (Figure 9). It has been noted pre- viously that at pH between 3.7 and 4.4 the effect of bisulfite ions on the enzymatic system was inhibitory rather than stimulatory in contrast to that observed at high pH values. ’ The effect of bisulfite ions on the activity of enzyme system toward IAA and IAAld is shown in Figures 10 and 11. respectively. Curve 1, Figure 10 Shows the effect of sodium bisulfite on the oxidation of IAAld and curve 2 shows the activity in the absence of bisulfite anions. In curve 1, Figure 11. the experiment was made by the addition of sodium bisulfite to the reaction mix- ture containing IAA at the beginning of the reaction. whereas in curve 2 the sodium bisulfite was added 60 minutes after incubation was started. These results clearly indicate that the oxidative degradation of both Figure 10. Figure 11. 55 " Effect of NaHSO3 on the oxidation of IAAld. The incubation condition was similar to that mentioned for IAA in Figure 11. Curve 1 was obtained in the presence of 0.1 ml of 3 x 10"3 MNaHSO3 (final concentration was 4 0.9 x 10' M): curve 2 was obtained in the absence of NaHSOB. Effect of NaHSO3 on the oxidation of IAA. Reaction mixture contained 3 ml of 1 x io'u'M,IAA solution in a 0.05 fl’phOSphate buffer, pH 7.8, 0.3 mg of enzyme (R2 = 1.5) in 0.1 ml water solution, and 0.1 -2 ml of 1 x 10 M‘HZOZ. Total volume was 3.2 ml per cuvette. Curve 1 was obtained by addition of 0.1 ml of 3 x 10"3 m NaHSO3 to the reaction mixture at the beginning of the incubation. Curve 2 was obtained when 0.1 ml of the NaHSO3 solution was added 60 min. after incubation. Reaction rate was followed at 244 mu. 4O 30 ACID. 0I244mp ' 20 IO .40 30 ACID. at 244 my. 2C) I 1 Curve! NOH803 Curve 2 x X X”T I J ‘ J 30 60 90 Min. ' 59 /////x _ Curve! x x/ /x Curve 2 57 IAAld and IAA at the high pH values requires the presence .of bisulfite ions. Although in the absence of bisulfite ions there was little observable change in the Spectrum, the addition of a small amount (1 x 10'“ M) of sodium bisulfite solution to the reaction mixture enhanced the oxidative degradation of both IAAld and IAA immediately. The complete reaction system required only the presence of an active enzyme, hydrogen peroxide and sodium bisul- fite. The addition of manganese ions (Mn2+) and 2,4- dichlorophenol had no effect on the optical density of the substrates at pH 7.8. The-Spectroscopic change of IAAld during the course of oxidation by the addition of sodium bisulfite to the reaction mixture was identical to that observed for the oxidation of IAAld-NaHSO3 at the same pH conditions (Figure 12. A). It is noteworthy that the oxidation of IAA at the same alkaline pH value, in the presence of bisulfite ions gave the same pattern of Spectroscopic changes as that observed for the oxidation of IAAld-NaHSO3 under similar conditions (Figure 12, B). Apparently, the presence of bisulfite ions during the oxidation of both IAA and IAAld by the enzyme system results in the same oxidation product along with the formation of the typical double peak in 316 and 330 mu regions. A comparison of the spectroscopic changes during the oxidation of IAA and IAAld at pH 7.8 in the presence of bisulfite ions are illustrated in Figure 12. Figure 12, A. Figure 12, B. 58 Spectroscopic changes during the oxida- tion of IAAld at pH 7.8 in the presence of NaHSOB. Enzyme (0.3 mg per cuvette HZ a 1.5). 3 ml of 1 x 10.)+ u_of IAAld in 0.05 §,phosphate buffer solution. pH 7.8, and 0.1 ml of 1 x 1.0"2 n_H202. Reaction mixture contained 0.9 x 10'“ g of NaH803 (0.1 ml of 3 x 10"3 _1o_1_ NaHSOB per cuvette of 3.2 m1). Spectroscopic changes during the oxida- tion of IAA at pH 7.8 in the presence of NaHSOB. The incubation condition was similar to that used in A, except the reaction mixture contained 1.35 x 10"“ y; of NaHSOB (0.15 ml of 3 x 10‘3 g; NaHSO3 per cuvette of 3.2 m1). 59 Figure 12, A. Flare 12 . Be 58 Spectroscopic changes during the oxida- tion of IAAld at pH 7.8 in the presence of NaHSOB. Enzyme (0.3 mg per cuvette HZ a 1.5), 3 ml of 1 x 10‘“ n_of IAAld in 0.05 fl,phosphate buffer solution, -2 pH 7.8, and 0.1 ml of 1 x 10 fi_H202. Reaction mixture contained 0.9 x 10'“ g of NaHSO3 (0.1 ml of 3 x 10'"3 g NaHSO per cuvette of 3.2 ml). 3 Spectroscopic changes during the oxida- tion of IAA at pH 7.8 in the presence of NaHSOB. The incubation condition was similar to that used in A, except the reaction mixture contained 1.35 x 10"“ p; of NaHSO (0.15 ml of 3 x 10"3 g; 3 NaHSO3 per cuvette of 3.2 m1). 59 60 The results indicate that the 503'2 or 11303"2 ions and not the Na+ ions acted as catalysts since increasing the ionic strength by raising the buffer concentration with sodium phosPhate had little effect on the enzyme activity. No increase or decrease of activity was observed for the 304"“ ion toward the enzyme system. The oxidation of IAAld at an elevated pH was found to be proportional to the concentration of bisulfite ion added. The values plotted for increase in the reaction velocity caused by the addition of SO ion against the concentration 3 of N32803' formed a rectangular hyperbolic curve (Figure 13). Accordingly. a constant similar to the Km value was calcu- 2 lated for 803' Burk (61) and found to be 3.5 x 10'5 n.1n the presence of ion by a method described by Lineweaver and 1 x io'“'h, concentration of the IAAld at pH 7.8. The stoichiometric relation between the rate of oxidation and concentration of bisulfite ions suggests that the salt acts as a catalyst in the enzymatic system. Effect of Enzyme, SubstrateI Hydrogen Peroxide. Bisulfite Ion and Sulfite Ion Concentrations on the Formation of a Com ound with a S ectral Absorption of gig and E30 mg In the presence of bisulfite ion, the changing of optical density at 2h“ mu due to the oxidative breakdown of IAA and IAAld no longer represents the overall reaction. Hence, the rate of the reaction was measured at 316 mm to determine whether the formation of the new product having Figure 1 3 o 61 Effect of Na2303 concentration on the oxidation of IAAld. Reaction mixture contained 1 x 10"” :1 of IAAld, pH 7.8; concentrations of Na2803 varied from 0.25 x 10'“ fl_to 2.0 x 10")+ 5, Reaction rate was followed at 316 mu. O\ (\3 Io— . 9? M no.0. s- (“I/c - ‘ . Of3|6fl1p7~ / 6L- e/C’ 5.. 4- r 3... 2.— ‘a... , J I L l I l I 1 0.25 0.5 0.75-4|.O |.25 |.5 L75 2.0 xlO M NOZSO3 P.3- AOYD" 3'6 mp. 0,2.- Km _, SLOPE = 0.0337 = -—v_:- 0.1 / Km = 3.5 x IO'5M V = IO.6 A 0.D./hr. |.O 2.0 3.0 4.0 63 the typical double peak, which appeared at 316 and 330 mu regions, involved any enzymatic reaction. The effect of IAAld concentration on the rate of for- mation of the compound bearing an absorption at 316 and 330 mu is shown in Figure 1#. The experiments were made in the 3 was obtained by the addition of 3.57 x 10‘5 :1 solution of sodium bisulfite; curve 2 was obtained at a 3.57 x 10-415 presence of 80 ion at pH of 7.8. Curve 1 in Figure 14 solution of sodium bisulfite. The Km values for IAAld determined by the reciprocal plots (61) were found to be 1.02 x io-u‘fl’and 0.008 x 10'“ g, respectively. in the presence of the two different concentrations of sodium sulfite. Obviously an increase in the concentration of sodium sulfite in the reaction mixutre had a profound effect on both the Vmax and the Km of the reaction system. The ten- fold increase in the amount of 803"2 ions doubled the maxi- mum velocity of the reaction as determined from the spectral -2 3 ions concentration produced a twentyfold decrease in Km for changes at 316 mu. In contrast, a tenfold increase in SO IAAld. It is clear that the presence of sodium bisulfite had significantly enhanced the affinity of the enzyme system toward the substrate. The effect of enzyme concentration on the formation of a compound with an absorption Spectra of 316 and 330 mm from the oxidation of IAAld-NaHSO3 is shown in Figure 15. The pH of the reaction mixture was 7.8. The formation of the double peak at 316 and 330 mu regions was found to Figure 14. Figure 15. 64 Effect of IAAld concentration on the forma- tion of a compound with Spectral absorption at 316 and 330 mu. Reaction mixture con- tained 15.7 us enzyme (RZ 2 2.9) per m1, 7 x 10'“'g_H 02, 0.05 g phOSphate buffer 2 solution, pH 7.8, substrate and bisulfite. Substrate concentrations were varied from 4 to 2.17 x 10'”'g, Curve 1 0.438 x 10‘ was obtained in the presence of 3.57 x 10'5finNa2803: curve 2 in the presence of 3.57 x 10"“ g NaZSOB‘ Initial velocity (£50.D./min.) was followed at 316 mu in a DU-Beckman Spectrophotometer. Effect of enzyme concentration on the forma- tion of a compound with Spectral absorption at 316 and. 330 We A00” 3|6mp. 4H0 (I x IO‘5M) 99.0.900009 c 3.57 x 10’5 3.3 N02803 .2 - l _. Or- 7 Curve! 9 _ 3.57 x IO'4M N02303 .8 - .7 Curve2 .6 .5 4 3 2 | .. ‘ I L I J O.I 0.2 0.3 0.4 ‘é‘ (X442 x I0"_M_) 5 #30 '0 ” 25 °/. YIELD .5 - 20 .0 - .s < I I I . 2.62 7.85 l3.5 38.3 ENZYME CON CN. (fig/ml. of Reaction Mixture) 66 increase as the concentration of enzyme increased. Extrapo- lating the enzyme concentration-velocity curve to zero- enzyme concentration it is clearly shown that only a negli~ gible amount of auto-oxidation of the substrate was observed from the intercepting point. The presence of hydrogen peroxide in the reaction system is required. At the elevated pH values in the presence of bisulfite ions IAAld was oxidized rapidly when a stoichio- metric amount of hydrogen peroxide was present. An amount less than the stoichiometric quantity caused incomplete oxi- dation of the substrates. These results are shown in Figure 16. At pH of 7.8, little formation of the compound with an absorption Spectra of 316 and 330 mm was observed in the absence of either one of the components: enzyme, hydrogen peroxide, or bisulfite ion. Heat-treated or trichloroacetic acid-denaturated enzyme had no activity toward the formation of this compound. These results are given in Figure 17. Effect of 2,4-Dichlorophenol and Man anese Ions on he Oxidation of §IK and IAAld ‘ The effect of 2,4-dichlorophenol and manganese on the oxidation of IAA and IAAld is shown in Figure 18. The experi» ments were made in the absence of hydrogen peroxide at pH 0.0. At a pH greater than 6 the presence of 2,0-dichlorophenol and manganese had little effect on the peroxidase-catalyzed oxida- tion of IAA and IAAld, even when hydrogen peroxide was present Figure 160 Figure 17. 67 Effect of hydrogen peroxide concentration on the oxidation of IAAld-NaHSO . Reaction 3 mixture contained 0.6 u mole of IAAld, 0.33 H mole of Na2803, 0.05 fl,phOSphate buffer solution, pH 7.8. Curve -x-x- con- tained 0.1 0 mole of H202, 0.2 u mole of H202, -6;A- contained 0.3 u -o-o- contained mole of H202 and -o-o- contained 0.” u mole of H202. Total volume 3.2 ml. Effect of horseradish peroxidase on the oxidation of IAAld at pH 7.8. Complete system contained 8.3 as enzyme (R2 = 2.9) per cuvette of 3.2 ml, 1 x 10'4'!_IAAld, -4 -4 3 x 10 3 H202 and 1 x 10 fl'NaZSOB. 30 - AOD EHGMp. 04 . . /g:::—:2 O. 3 20 - 3 8/ / -——-€'-——-—o 0.2 0&0 /g_/ '0 _ flit—r 4““ X--——-x 0.: 8 . 1 l g l J 30 50 90 '20 I50 MINUTES 30- A00 3|6 My, 203 A/ IO - / . . . /x WITHOUT H202 COMPLETE SYSTEM A/ A / ‘ /§ 8 WITHOUT $02 / fi—u—fl / é‘f"; .-——-—-- , WITHOUT ENZYME -««—-?::2.’£g:_.._.. .—-——---l---—f 4 HEATED ENZYME 3O 60 90 I20 ISO IBO MINUTES ”‘33.”... Figure 18. 69 Effect of 2,4-dichlorophenol and manganese on the oxidation of IAA and IAAld. Reac- tion mixture contained 1 x 10"4 E’substrate (either IAA or IAAld), 1 x 10'4 fl,MnCl 4 2. 1 x 10' g_2,h-dichlorophenol and 0.2 m1 of enzyme (8.3 us per cuvette, R2 3 2.9). Total volume of the reaction mixture was 3.2 ml per cuvette. Blank contained all reaction components except without addition of enzyme. Reaction mixtures were made in 0.05 §_acetate buffer solution, pH ”.0. 244mn f‘“ ,U 0 +Mn*2+E+IAA 0 +E + IAA 0 2,4 0+E 3:--~_8-—:8 0 flan-w? IAAld + 2,4-0+Mn 2++E 20 30 4O 50 60 MINUTES 71 in the reaction mixture. In these studies the reaction was followed spectrophotometrically at the 204 mu region. It was observed that at pH “.0 the oxidation of IAA was remark- ably stimulated in the presence of 2,4-dichlorophenol and manganese, and also in the absence of these cofactors IAA appeared to be an excellent substrate for peroxidase activ- ity. In contrast to what was observed for IAA, the oxida- tion of IAAld by the same enzyme system was not influenced, to any extent, by the presence of these two cofactors. Under these conditions IAAld was not oxidized. These observations are contradictory to what is expected. One should recall the observation previously described that IAAld was an excellent substrate for peroxidase activity in the presence of hydrogen peroxide at pH 0.0. Apparently, the diverted behavior of the enzyme toward IAA and IAAld was a result of the slight difference in the chemical structure of these two auxin deriva- tives. The lack of reactivity of IAAld by the system con- sisting of 2,0—dichlorophenol, manganese ions, and peroxidase was not simply due to the deficiency of the hydrogen peroxide, because the function of manganese and 2,0—dichlorophenol are closely related to hydrogen peroxide formation required for the peroxidase catalyzed reaction (#5). Identification of the Oxidat on Products Some Preliminary Studies with Thin-Layer Chromatography.=- Preliminary studies with thin-layer chromatography indicate 72 that as a result of the oxidation of IAA, IAAld and IAAld- NaHSO several spots could be detected under ultraviolet 3 light. Among them two or three Spots could be regarded as common products. One of these common products was called compound X1, having Rf values of 0.91 (Spot A1) and 0.92 (Spot A2) which were obtained from the oxidative breakdown of IAA and IAAld, respectively. Spot A1 and spot A derived 2 from compound x resembled each other not only in the Rf 1 values, but also in the absorption spectrum under ultra- violet light (Figure 19). These spots had absorption maxima at 200, 260, and 290 mu regions. Originally. 1t was sus- pected that the compound was a derivative of an oxindole, Since oxindole was known to be an established end product resulting from the action of peroxidase on IAA. However, analysis indicated that it was not oxindole. Another common product which was isolated from the ff peroxidase oxidation of either IAAld or IAAld-NaHSO had R 3 f values of 0.73 (Spot B1 from IAAld) to 0.77 (Spot B2 from IAAld-NaHSOB). The two Spots, B and B2, obtained from 1 IAAld and IAAld-NaHSO3, respectively, gave similar spectra with a characteristic double peak at 316 and 330 mu regions (Figure 20). In the preliminary eXperiments the pH of the reaction mixture was made at 6.1 since the reaction rate of the oxida- tion of the IAAld and IAA was the lowest at this pH, conse- quently, additional products may be produced in this reaction. A significant conclusion which may be drawn from these 73 .Aom.mmamdv coaxonohs seasoaadiaoamaohmlNtopmpeom HhSpm .aopmhm psobaom .0 How moaaam ”Hohmaisane .H.@ was chapxaa Soapomma can we mm 629 .haobap loommoa .Amm poamv mommSZIoaddH use Aam poamv caddH Beam dosampno mposooaa Soapmvaao who; mpomm omega .Nm none can a H m poam aoam oepSHo N ossoaaoo mo mapooam soapmaomnm wadedbmapao .om easwam .Aomsmmumev oeawoaoaa sadnesss IHosmaoamrmnopmpeow Hanna ”ampmhm pae>aom no How moaaam aheamHI:a£B .H.w mmz campwaa soap lemon 0:» we ma .haobdpoommoa .oH4dH one 44H mo szooxmoan obapmoaxo on» Sony ooaaeppo ones .N H macaw chose a noun one 4 poam aoah oopsao ax osdoaaoo we mapooam Soapaaomnm peaodbmhpao .mH madman :2 £55653 ovm omm 00m omm Ohm omm 0mm OWN 0mm CNN 0 _ . _ _ _ q _ _ _ _ _ _ I _ l o_ in sesmvoES om: \ 1 ans .oseeOmIoz- ES: oml 1 own 1 on! 1 o 114 _ _ _ _ _ _ _ _ _ _ o_1 1 _ ~ .oNI A4 334;: L on: awfosmequH 1 ovl 1 ear 1 0_ ON on ow 0_ ON on 0v om Asunciosqv 75 preliminary studies, is that the presence of sodium bisulfite does not appear to be absolutely necessary for producing compound X from the IAAld, since the peroxidase-catalyzed 2 oxidation of IAAld in the absence of bisulfite ion also gave rise to the same product with the typical double peak at 316 and 330 mu. The presence of bisulfite ion only stimulated the yield of the X Whether the same is true in case of 2. IAA is unknown, although it was noted above that the presence of bisulfite ion stimulated the formation of X from IAA. 2 The X1 and X2 were later identified as IAld and h-hydroxy- quinoline, respectively. Furthermore, for each substrate (IAA, or IAAld, or IAAld-NaHSO3) oxidized at least 5 Spots could be detected. In the present work only two of them were identified. Isolation and Purification of the Compound With a Spectral Absorption of 216 and :30 EB by Column Chromatography.»- For the isolation of the new oxidation product having a typim cal double peak at 316 and 330 mu regions, the biosynthetic product was placed on top of the adsorbent in a Silicic acid column packed according to the method previously described. The column packing was first developed with a solvent system consisting of 250 m1 of hexane and ethyl acetate mixture (50:100): and Second, with isopropanol. The samples were collected in two m1 portions by an automatic fractionation collector: Portions having similar absorption patterns were combined; The compound having the typical double peak at 76 316 and 330 mu remained at the top of the adsorbent during first development and was isolated in a fairly purified form from the second development with isopropanol. It was crys- tallized in an acetone-ethyl acetate mixture and finally recrystallized several times in pure acetone until a constant melting point was determined. Identification of Compound X2 as 4-Hydronguinoline.-- The ultraviolet and infrared spectra of compound X2 are shown in Figure 21 and Figure 22 (A), respectively. The ultra- violet absorption of the compound showed a double peak at 316 and 330 mu and a broad peak at 234 mu. Infrared Spectrum of the crystalline compound in.a.KBr pellet Showed strong to medium-strong bands at 3250,13050, 2560, 1650, 1620, 1580, 1500, 1480, 1440, 1360, 1320, 1200, 1140, 1100, 830, 800, 775, 760, 725 and 675 cm'l. The ultraviolet and infrared Spectra of the unknown compound were the same as the Spectra of an authentic sample of 4-hydroxyquinoline. The infrared Spectrum of 4-hydroxyquinoline is shown in Figure 22 (B). The 4-hydroxyquinoline, which was purchased from .Aldrich Chemical Company, was a trihydrate having a melting point of 200-2020. The 4-hydroxyquinoline isolated from the peroxidase reaction mixture was melted at 207-2080 (uncor- rected): However, after the samples were dried over phos- phorous pentoxide, both the authentic and the isolated com- pound melted at 207-208° (uncorrected) and a mixture of both compounds showed no depression of the melting point. The 77 .a.:1oa N m.o mm: soapmnpzoosoo .sodpsHom Hopes ca vocaeppo was sappoenm one .mx .0569800 no asapooam sodpaaomnm poaoabmapao .am oaswam 3.2 .szm4w><>> own own com 0mm Ohm com com O¢N . 0mm CNN 0 . _ _ _ _ _ _ _ _ _ _ - _ _ o 0:1 40. ON I. now on .. . . on O? I 1 ov on 1 1 on 00 1 J 00 on T 1 0h NN.dSSoQEOU cm 1 1 om om I . , . . i om oo_ 7 , oo. Roundicsqv 79 .mpoHHoQ coaaoan asdmmmpoa opza coma one: moHaamm .Am adhpooamv enaaosaswhxoaussld use A4 asapooamv «x ossoaaoo no sanctum soapaaomnc UoHSHmGH .NN shaman 80 8s _._. 8n: _:. 022v: :— I»O:w:>(3 o I; .2. __ .6: 32. 1 u ailaztil ___:___ .._ 8: I. 8. .— .1 00a. 81 molecular weight of the oxidation product determined by mass Spectrophotometric method or by a freezing-point depression in acetone was found to be 145.15 and is the same as the calculated value. The compound gave the following percent age on elemental analysis: C 74:47, H 4.90, N 9.68, 0 10.95. All available evidence indicates that the oxidation product with the typical double peak in the 316 and 330 mu regions was 4-hydroxyquinoline which has the following percentage composition: Calcd. C 74.47, H 4.82, N 9.65, 0 11.06. The yield of 4-hydroxyquinoline under the experimental condi- tions used was 20% of the substrate (IAAld) oxidized. ThingLazer Chromatogpaphical Identification of Compound £1 as IAld.--The results of thin-layer chromatography eXperi- ments with four different systems are shown in Figure 23. The enzymic oxidation of IAAld was made at pH 4.4. It is evident that the R1. values of the reaction product (compound X1) on the chromatograms in the different solvent systems correspond to the IAld (Figure 23 and Table 1). The compound X1 was further identified by ultraviolet Spectra which also corresponded to the IAld. The absorption Spectrum of the aldehyde isolated from thin-layer chromatography and that of IAld are given in Figure 24. The isolated IAld gave positive testswith 2,4-dinitro- phenylhydrazine and xanthydrol, but responded poorly to Schiff's and Ehrlich's test. These tests correspond with those observed for IAld. 82 .Hoamspo ad osdnchoba Iahaozaoapaaaoid.m uncommon waahmhmm .s@: we ootha one: oohsooampeoSIMIoHoos« mo soapmoawo soap mpoAm on» use :4: mm dogmas ones mpOAm oaasosps4 .Aaosmpsnia Rmav osmxenlaocmpznla Asv dam ”Aflzaaav nonexioaom oapeomloSonacD Amy “Am.mmv pace oapooSIanMOHOHSO Amy ”Adaav osmxosuaoscaoHAIS AHV ones maopmhm pambaom .AaopmoaUSa peoomonosam unozpaz How coaaam mm .aom a cease ocean snawoadsoaso seasons “mopmam .ooasooampobmnmicaooad mo aoapmdawo an doaampno «N canoaaoo no amawopmaohso Hoheaaaana .mm seamed r .... a... . 1. “1.. ..n . I, .r .. ~.(v.-).Ww “u“-- \ -.Q. .M.‘6 ....M. 11.7.. ...H ......H: .....n .. A ~&*. g 0“ rlqi O~.\$‘_ w...“ ”mama... dimer? ‘7 . ,6 84 AHoSdpsst away emmm Hoe soaaan 05.0 111 0m.0 oSdNom1Hocmpam1a _AHo:dpsm1: fimav 111 No.0 No.0 esmwom1aoscpsm1a e AH.H.:V 111 05.0 00.0 acpm31oaod oapoodroaomaom e Am.mm0 1:1 mm.o om.o oao4 oaaoodraaouoaoaao e A:.Hv peosm 111 mu.0 05.0 oedwomiaoacmoam1: seawopmaoaso seapmem . 0H4H 0H¢¢H dopmaomH 0H4H aopmhm unobaom mepmam Hohmq1adse m dddH dopmHOmH 0G6 0H m ma manna 85 am such a m.o mos soapmapaoozoo .zoapsHom hopes ad donaaacpou has adhpooam soapaaomnm one .dadH one «x caSomaoo can go sapwooam soapaaomnm poaoabmapas .sm shaman 32 .IPGZMJMZS,» es... on 8m 08 ea 08 com SN 3% 8m O [—11— _. a — u u q _ — u 1 — d o 0.7 -o_ on- -om OM11. \\\I.IIIII 10m \\ / 0V fil \\ ’0 [CW 1 s\s / {C I v \I I.” \ . I I 7,... 00 so; \xs a/I\\ I” \\\ a; on can , -ee “14H es- -oa om- Lem em- _ Joe oo. , . oo_ 7401100103 1'17 87 The yield of IAld obtained by the peroxidation of IAAld was approximately 50%. About 0.5 x 10-4’§.of IAld was formed by the oxidation of 1 x 10’“ EDof IAAld. The yield was calculated from the Spectral changes at 289 mu when the reaction reached the final steady state. Oxidation of 1-Meth l-IAAld 52 Horseradish FeroxIEase The Spectrophotometric changes of 1-methylindole-3- acetaldehyde during the course of oxidation was more compli- cated than that observed for IAA and IAAld. Both the rate of the reaction and the pattern of oxidation product were greatly influenced by the pH of the reaction mixture. The optimal pH for the oxidation of 1-methy1-IAAld was in the range of about 4.0, and the reaction rate decreased as the pH was increased above 6.5. A similar stimulatory effect of bisulfite ions on the oxidation of 1-methylindole-3- acetaldehyde at high pH values was observed. One of the major oxidation products was found to be lamethyl-IAld. Its formation from the peroxidase oxidation of 1-methyl-IAAld was identified by thin-layer chromatog- raphy in different solvent systems. However, the formation of 1-methyl-IAld from 1-methyl-IAAld could not be shown spectrophotometrically under ultraviolet light, since 1-methyl- IAAld and 1-methy1—IA1d have very similar ultraviolet absorp- tion Spectra. 1-Methy1-IAld has a single absorption maximum at 288 mu while 1-methy1-IAAld has lmax at 292 no. The 88 distinguishable difference between these two absorption Spectra is the absorption minimum. 1-Methyl-IAAld shows a trough at 247 mm and 1-methyl-IAld has a trough at 261 mu. During the course of peroxidase oxidation of 1-methyl-IAA1d, some new peaks were found to appear at 249, 260 and 330 mu regions which neither represent the formation of IAld nor that of 1-methy1-4-hydroxyquinoline. Apparently, other than 1-methyl-IA1d and 1-methy1-4— hydroxyquinoline, other oxidation product or products were formed as a result of the oxidative degradation of 1-methy1- IAAld catalyzed by the peroxidase system. The spectroscopic changes of 1~methy1-IAAld during the course of the oxidation is shown in Figure 25. Because the characteristic absorption peak appeared in the region 240 to 250 mu, the formation of a derivative of oxindole is not improbable. However, addi- tional work will be required to identify the structural characteristics of this unknown substance. The enzymatic oxidation of 1-methyl-IAAld was made at pH 4.4. The results from the thin-layer studies are summar- ized in Figure 26 and Table 2. It is clear that the Rf values of the reaction product correspond quite well with lumethyl-IAld. The spots were identified by Spraying the thin-layer plates with 2,4-dinitropheny1hydrazine reagent. Furthermore, if the reaction was made at higher pH values in the presence of bisulfite ion, the formation of 1-methyl-4- hydroxyquinoline was not shown by thin-layer chromatography on a plate having a fluorescence background. The possibility 89 am sued a n not «own co our» use .fl :Ioa H.H mm: 0H4¢H1Hh£pca1u no nodpmnpSooaoo Hmaam .opsada a :« copoaaaoo mezoaoooa Some .mopSSHE 0NH one moa .om .ms .oc .me .om .om .ma .oH .m .o as Scam» egos mobaso .18 can um semen one: monooem .oppcbso you He N.m mm: eHSpHaa Soapomch can no ossaot Hobos .soaasaou NoN 2 ea 2 a co as H.o m1 and .mm.: ma .aoassaou acumen oasaoos_a mo.o ca easaHuaasoosua_m each a so.a co as m .aaa N.m co oppobso Hon w: 5H0 cahuao Ha «.0 .A0H44H1thpoanav we»:coacpoom1m1eaooaaah£pea:a mo Soapmoawo oapmahnso weaned anypooam aoauaaomnm poaoabeapas ad mowamso 1555......rr13: .. -.A ., K. . e—fi .mm oaswam 90 .121... 5... :25 «in III-i latte?) chsxoam . : z, ....r .a 91 .HoSSSpe ca oaanmhohnahaonnothSau 1:.N apaomcoa waahcaam .ama he venues one: oohsooadpooc1m1oaoosdahnpoa1a mo aoapcvawo scam npomm on» use eds mm defines one: ovhsouam 1Honamolm1oH00aathpoa1H mo macaw oapSoSpad .AHosmpsn1a xmav osmwoSIHoampsn1: A00 020 “Aaaaaav Hopm310aoc oapooe1oaowaop “my ”Amammv odom capoomlahomoaodso ANV ”Aiaav ocmNoQIHosmaoHQIQ Adv one: aopmhm paobaom .AHOpooadca pavemenosam psonpdz How moadam mm .Homu enhav poosm amawopmaoasU amapmmm aeoasam .ooaaooasacodnmuoaocsaaasooa1a mo soapmudxo an doaampno odhnovamwonamo 1m1eaccaaHhSpoaua mo amawopmaonzo aohma1aasa .mm shaman 93 xaossosmis away mm.0 111 0m.0 oscNmmIHosepsm1a emmm H00 Saddam Afloampsm1a Rmav 111 0w.o 05.0 ondflomldondufimln a Aaaaaav 111 00.0 00.0 aopczlcaod odpoodroSoNsom a Amammv 111 00.0 00.0 esmwcm1aaomoaodso a as.a0 ocean 111 00.0 00.0 oseNomIHoamaonmis amawopdaoaso smapmcm 0H¢ m .m canoe 94 of the formation of 1-methy1-4-hydroxyquinoline cannot be ruled. out if appropriate reaction conditions and better chromatographical technique can be developed. At this point, it should be mentioned that 1-methy1-IAAld was not as stable as IAAld during chromatographical analysis since it undergoes Spontaneous decomposition to form 1-methy1- IAld, and two other unidentified compounds. Therefore, it may be possible that because of the structural. character- istics of 1-methyl-IAAld, the formation of l-methyl-IAld and the other unidentified compounds are energetically more favorable than is 1-methy1-4-hydroxyquinoline. Possible Mechanism. of. Peroxidase Oxidation of and IAAld The effect of bisulfite ion on the formation of peroxide-peroxidase complexes is Shown in Figures 27 and 28, reSpectively.‘ The experiments presented in Figures 27 and 28 were made aerobically at pH 7.68 in 0.15 .11 phoSphate buffer solution. Curve A, Figure 27, represents the optical absorption of the normal peroxidase solution which had a maximum peak at 402 mu. Curve B is the absorption curve of complex II which had a maximum absorption at 418 m and was formed when a small amount of hydrogen peroxide was added to the free enzyme solution (14, 15). Curve C was obtained when a small amount of IAA was added ,to the solution contain- ing the peroxidase and hydrogen peroxide. The transformation of the hydrogen peroxide-peroxidase complex, curve B, to free w:- m 1"...” T.- Figure 27. 95 Effect of hydrogen peroxide, indole-3- acetic acid, and sodium bisulfite on the absorption spectra of horseradish peroxidase. 0.15 g phOSphate buffer solution, pH 7.68. A. Free enzyme. 2. c. B plus 0.05 ml of IAA (2 mg) . B. A plus 0.05 ml of 0.3% H20 D. 0 plus 0.03 ml of 1 x 10"3 _1«_1_ NaHSOB. Enzyme: 0.85 mg per cuvette (3 ml). Absorbancy \O O\ .00 I00 90.- ~90 00" -00 E 701- 77° 60F- ~60 507- -50 4o ‘40 301- ~30 20' ~20 10/ ~10 o 1 1 1 1 L 340 360 370 400 428 450 4780 WAVELENGTH, Mu Figure 28 o 97 Effect of hydrogen peroxide and sodium bisulfite on the absorption spectra of horseradish peroxidase. 0.15 5 phosphate buffer solution, pH 7.68. A. Free enzyme. B. A plus 0.05 ml of 0.3% 11202. c. B plus 0.03 ml of 1 x 10"3 p NaHSOB. D. 30 min. after the addition of NaHSOB. E. Two hours later after the addition of NaHSOB. Enzyme: 0.85 mg per cuvette (3 m1). Absorbancy '70 6() 5C) 3 O 01 0 2() N} 380 I I 390 400 WAVELENGTH,Mp '70 150 450 99 enzyme, curve C, occurred instantaneously. Apparently the addition of IAA had reduced the complex II back to free enzyme and prevented its further formation. Furthermore, it is also interesting to note that further addition of a small amount of sodium bisulfite to the solution represented by curve C would convert the free enzyme (curve C) to complex II (curve D) again. At this point the enzyme complex mixture did not remain in a steady manner, but the solution underwent further transformation as represented by curve E in a consecutive manner which was different from the instantaneous change of B to C. It is possible that the transition of B to C is due to the inhibitory effect of IAA toward the formation of complex II, whereas the transformation of D to E is an oxidation and reduction process. Because bisulfite ion is also the sub- strate of the enzyme system (18) it reversed the inhibitory effect of IAA under the alkaline pH condition and induced the formation of the complex II which is essential for the further oxidation of IAA. The observation that complex II is essential for the peroxidase-catalyzed reactions is in agreement with that reported previously by Chance (14, 15). Figure 28 shows the direct effect of bisulfite ion on the formation of complex II in the absence of IAA. Curve A of the Figure represents the optical absorption of the free enzyme and curve B is that of complex II. In the absence of IAA the addition of sodium bisulfite, at first, enhanced complex II formation and then reduced complex II through a 100 redox process back to the original free enzyme (Curve E). In Figure 28, Curve C was made immediately after the addition of bisulfite, Curve D was taken 30 minutes after the addition of bisulfite, and Curve E was obtained 2 hours later. These data indicate that during the course of bisul- fite oxidation, complex II was reduced in a consecutive man- ner. Consequently, the free enzyme was released. This con- . '1?!- E trasts with the case when IAA was oxidized under the same tit? condition. When IAA was oxidized at pH 7.68 in the presence as of bisulfite ion, the final absorption of enzyme was also changed, possibly indicating the change of the enzyme confor- mation due to the interaction of the enzyme molecule with IAA. Yamazaki and Souzu (114) reported that IAA can inhibit the oxidation of guaiacol by peroxidation. As reported (114), the inhibition of oxidation of guaiacol by IAA could only be observed under anaerobic conditions. However, the inhibitory effect of IAA toward the formation of complex II was found under aerobic conditions at alkaline pH values. It was assumed by the same authors that the inhibitory effect of IAA toward the oxidation of guaiacol possibly was due to the inactivation of the enzyme by interaction of the enzyme molecule with the free radicals generated from IAA. If the same eXplanation can be applied in the case of IAA oxidation, it is possible that the inactivity of peroxidase toward IAA at pH above neutrality is due to the inhibitory effect of IAA toward the formation of complex II as was observed in Figure 101 27. This seems to constitute a typical example of substrate inhibition of enzyme action. It is interesting to note that at the physiologically important pH range the oxidation of IAA by nauxin oxidase" is controlled by auxin itself. in» t 1. DISCUSSION FT??? id‘s-v.93 1- .- DISCUSSION The possibility that 4—hydroxyquinoline is produced as an oxidation product of IAA has been discussed by Manning and Galston (64). They believed that 4-hydroxyquinoline was not formed as an oxidative product in the "auxin oxidase" catalyzed oxidation of IAA. However, our work definitely shows that 4—hydroxyquinoline, which has typical absorption at 316 and 330 mu, can be formed from peroxidase-catalyzed oxidation of either IAA or IAAld. However, in order to detect its formation, specific reaction conditions must be employed. Apparently there exist, simultaneously, at least two pathways for the oxidative breakdown of IAA or IAAld catalyzed by the peroxidase system. These pathways compete with each other and the inhibition of the one may result in the stimulation of the other. Although 4-hydroxyquinoline is not formed as a major product in peroxidase oxidation of IAA, the probability of such a transformation seems to be always present. Once favorable conditions are established, its formation can be detected easily. Our work partially supports Kenten's viewpoint (44), 1.6., that the oxidation of IAAld catalyzed by peroxidase may result in the formation of IAld as one of the major products. However, the results indicate that the peroxidase- catalyzed reactibns depended mainly on the reaction conditions. 103 a‘! 7‘ FT". 104 Varying the pH and the presence or absence of certain cofac- tors may change the pathways. These observations that the change of pH will result in the change of the reaction path- ways have not been reported previously for any other enzyme. The effect of the bisulfite ion on the enzymatic system might be accomplished in several different ways. First, like hydrogen peroxide, it might participate directly in the reaction as one of the enzyme substrates; second, it might change the structural character of the side chain of the indole compounds either of the substrate, IAAld or the product, IAld: and finally, the bisulfite ion might have a profound effect on the structural characteristics of the enzyme. Because of the large differences between the pH- activity curves in the presence and absence of the bisulfite ion, it seems that the effect of the ions might have a direct influence on the catalytic behavior of the enzyme. This was found to be the case. In this thesis, direct evidence has been obtained to illustrate how the peroxidase molecule is involved in the bisulfite-stimulated oxidation of IAA (see Figures 25 and 26). As far as the pH effect is concerned, Maclachlan and Waygood (62) has proposed a free radical mechanism to eXplain the inactivity of the peroxidase system above neutral- ity. The proposed reaction mechanism is Shown by: ’l’i 3“‘_-1 f ' ‘ ~11 ‘ I L 105 ICHZCOO" + Mn3+ > 10112000“ + Mn2+ (1) 10112000' ./. 1032' + 002 (2) ICHZ’ + 02 _7\ 1011200' (3) 1011200' + ROH Penn“? 101120011 17 R0 (£1) 110' + an+ + 3* ‘ > ROH + Mn3+ (5) From the suggested mechanism it is evident that Waygood attributed the lack of reaction above neutrality to the insta- bility of the free radical intermediates, since, according to the mechanism, the generation of the free radicals (RO’) involves the utilization of hydrogen ions. In our reaction system no manganese was added: thus Waygood's eXplanation is unlikely. Furthermore, there is some degree of uncertainty involved in the proposed mechanism: first, Waygood's mechan- ism does not involve hydrogen peroxide (see equation 3). Second, Waygood's mechanism is based almost entirely on the initial rate of nonenzymatic reactions (equations 1, 2, 3) and consequently neglects the importance of the involvement of the enzymatic process. The general mechanism of peroxidase- catalyzed reactions has been studied extensively by Chance (14, 15). He has shown that the peroxidase-catalyzed reac- tions depend on the formation of chemical complexes between peroxidase and H O 2 2' to Show how the peroxide-peroxidase complexes are involved in In the present work we were also able the enzyme-catalyzed reaction. It is apparent that the inhibitory effect of IAA toward the formation of compound II may account for, at least partially, the instability of the 106 enzymatic system at pH values above neutrality. Since Way- good's mechanism was also criticized by Ray (76), there is no need to repeat in detail all of his statements here. Recently, Fridovich (17) reported that certain nitro- genous compounds such as ammonia, pyridine, imidazole, and hydrazine can stimulate the peroxidase-catalyzed oxidation of anisidine. In contrast to stimulators of the monophenol type, activation of the enzyme system by these ligands resulted in changing the pKa of the enzyme protein from 6.5 to 9.5. Similar results were observed in the present inves- tigation. It is known that the optimal pH for the Mn3+-mono- phenol system is in the range of 3.5 to 4.5. However, the optimal pH for the thyroxine activated oxidation of epineph- rine and norepinephrine is between 7 and 8 (50), and thyroxine at pH 5.6 was inhibitory to the oxidation of iodide catalyzed by the same peroxidase system. Another interesting observa- tion reported by Kirdani and Layne (47) was that the oxida- tiOn of norethynodrel by horseradish peroxidase was increased as the pH was increased from 6.0 to 8.5. The conclusion drawn from these observations is that the optimal pH for peroxidase-catalyzed reactions depends on both the chemical properties of the substrates and those of the stimulators. Yamazaki and Souzu (114) has classified peroxidase substrates into two groups: redogenic substrates and oxidogenic substrates. The redogenic substrates are characterized by their capacity to reduce ferricytochrome c in the presence of hydrogen peroxide and peroxidase, a . '5 w“ - r - 107 property not possessed by the oxidogenic group. By analogy, it seems that peroxidase substrates and its stimulators as well can be divided into two groups according to the optimal pH at which they react. One group reacts with an alkaline enzyme system, whereas the other reacts with an acidic one. The results are possibly the consequence of the amphoteric character of the enzyme molecules. Thus, we may say that peroxidase may assume two conformational forms: Form I is predominant in acidic medium and Form II in alkaline medium. Compounds like thyroxine, bisulfite, and nitrogenous ligands are a group which can activate the enzyme system with alkaline Form II, whereas the Mn3+-phenol group is active toward the acidic Form I. From this assumption, it is not unreasonable to pre- dict that a compound which stimulates enzyme Form I might inhibit enzyme Form II. The phenomena was apparently observed in the present experiments. Therefore, it appears that at the physiologically important pH range (near neutral), the action of the peroxidase system requires the presence of an appropriate cofactor such as bisulfite ion, ammonia, thy- roxine, etc. The presence of the one of such cofactors may induce the enzyme protein to assume a proper conformational form essential for the enzyme activity toward the Specific substrate. However, in our work it has been shown that peroxidase activity directly involves the heme group of the enzyme '— ill I: 108 molecule. The inhibitory effect of IAA toward the formation of compound II at alkaline pH under aerobic condition may involve an electron transfer process in which the hematin- peroxide complex is an essential intermediate. Recently Yamazaki ppflgl. (113) established that free radicals participate in certain peroxidase-catalyzed reac- tions. In fact, by using the technique of.paramagnetic pm resonance, they were able to detect the formation of free I radicals of dihydroxyfumarate, aScorbate, hydroquinone, and 5+ IAA from peroxidase-catalyzed reactions. The proposed free radical mechanism of Iamazaki 23,2l, (113) is as follows: Peroxidase + H202 ____5l__9.Compound I (1) k2 Compound I + AH2 ; Compound II + AH° (2) k3 Compound II + AH2 > Peroxidase + AH' (3) k 2AH° d \ 7,A + AH2 (or HA-AH) (4) In the above mechanismAH2 is the substrate electron donor, AH° the free radical intermediate, and A is the oxidized product. I From the above formulation, the free radical concen- tration at steady state should be [...-1 . Di [E] 1101 “2 S 1 k 1 d 109 The fundamental concept of this formulation is based on two propositions: that the free radicals are formed from sub- strates, and that the formation of free radicals depends on an enzymatic process. The results obtained by Yamazaki from electron paramagnetic resonance Spectra indicate that the concentration of free radical formation is proportional to [311/2 in accordance with the mechanism indicated above. The same mechanism was also suggested by George (24). The results obtained by Yamazaki are completely inconsistent with Waygood's formulation which depends basically on the auto-oxidation process. It is evident that auto-oxidation processes are not necessarily related to the enzyme-catalyzed reactions. Aerobic oxidation of sulfite ions has been used by Alyea and Backstrom (2) and also by Fridovich and Handler (18) as a sensitive detector for the occurrence of free radicals. In the presence of their reSpective substrates, the enzymes, xanthine oxidase, aldehyde oxidase, and horse- radish peroxidase, were found capable of initiating the oxidation of bisulfite ion. The mechanism involved in bisulfite oxidation was nearly identical to that found in bioluminescence (101) caused by xanthine oxidase acting on xanthine in the presence of luminol or lucigenin, in that both of these two processes depend on the generation of free radicals. AS reported (18), the oxidation of the bisulfite ion initiated by horseradish peroxidase required the presence of both hydrogen peroxide and a peroxidizable 110 substrate. Either substrate by itself caused no oxidation of the sulfite. Accordingly, since no free radical can be generated in the transfer of an electron pair, they sug- gested that the free radical was not generated from the interaction of hydrogen peroxide with peroxidase, but from the oxidation of a peroxidizable substrate through a succes- sive univalent electron transfer. . fit-9'0 all ""C... In view of the important observations reported by Alyea and Backstrom (2), Fridovich and Handler (17, 18), L. George (24, 25), and Yamazaki gp_gl. (113, 114), it is clear that the stimulatory effect of the bisulfite ion toward the oxidation of IAA and IAAld by the peroxide-peroxidase system occurs because the bisulfite anions are capable of being oxidized through a chain reaction which is initiated by the enzyme system. This type of reaction is essential for the production, maintenance, and propagation of the free radicals required for the catabolism of the substrates at the elevated pH values. Furthermore, during such free radical transfer processes the formation of peroxide-peroxidase intermediate is essential for the overall reaction sequence. In other words, the overall process of the free radical chain is under the strict control of the enzymatic system. This is reflected in the changes of Vmax and Km of the enzymatic system in the presence of bisulfite anions. In addition, the fact that the presence of bisulfite ion can change the oxidative products may also attribute to the free radical mechanism, Since the change of the environmental factors such as pH or enzyme 111 conformation may have changed the distribution of the Single electron about the substrate molecules. Such environmental changes may lead to the increased formation of a product which originally was only produced in a small quantity. The observation by Stutz (90) that cyto- chrome c and a cytochrome oxidase containing homogenate of rat liver can stimulate the formation of IAld from IAA is ,q another example to support such a hypothesis. At this point, 3 it should be recalled that Yamazaki and Souzu (1111) have 1. divided the peroxidase substrates into two groups based on their capacity to reduce ferricytochrome c in the presence of hydrogen peroxide and peroxidase: and that Fridovich and Handler (18) noted that in the presence of ferrocytochrome c cytochrome oxidase can initiate the oxidation of sulfite ion vigorously. Hence the presence of the cytochrome c and cyto- chrome oxidase may have changed the electron wave function of the IAA free radical, thereby facilitating the elimination of the carboxyl carbon and stimulating the yield of IAld. A Similar eXplanation may be applied to 4-hydroxyquinoline since its formation is greatly stimulated by the existence of a strong free radical generating system, i.e., a bisulfite- peroxide-peroxidase system. If Yamazaki 21; 5;. (113) mechanism is correct, in case of IAA oxidation, the following reactions may be suggested: k HRPO + H202 1 >_ Complex I (1) k2 Complex I + IAA >» Complex II + IAAI (2) k3 Complex II + IAA )7 HRPO + IAA' (3) k IAA' + Fe3+ d >. IAA” + Fez+ . (4) In the above mechanism IAA' represents free radicals of IAA and IAA" represents IAA oxidation products. Apparently, this mechanism is only applicable in the acidic conditions, at pH values above neutrality the oxidation of IAA is inhibited because of the inhibitory effect of the IAA toward the formation of the compound II. In other words, at alka- line pH values IAA itself inhibits reactions (1) and (2) but not reactions (3) and (4). However, when bisulfite anions are also present, these ions are also the substrate of the peroxide-peroxidase system, so they can act as a bridge to induce the reproduction of the compound II, thereby activat- ing the overall system. Such an idea is depicted as follows: k HRPO + 3202 1 >- Complex I (1) - k' Complex I + 11303 2 g; Complex II + (11803 ) (2) 1‘3 Complex II + IAA 5» ano + IAA' (3) 3+ kd 2+ IAA' + Fe > IAA" + Fe (4) In.the above formulation, (HSO3‘) Stands for bisulfite free radicals. Since bisulfite itself can also be oxidized 1... r: 1m I 113 through the same mechanism, the oxidation of bisulfite by the peroxidase system may follow the scheme: k. HRPO + 11202 1 2; Complex I (1) .k' Complex I + 3303 2 ; Complex II + (1130;): (2) - k' Complex II + 11303 3 _; HRPO + (HSOB-)‘ (3) I ' 3 k' 2+ (11303")1 + Fe + d > 11304” + Fe (11) Since at this alkaline pH k' is much smaller than k3 (the 3 transition of D to E in Figure 25 took only 30 minutes, but it took more than 3 hours for the transition of B to E in Figure 26), the presence of bisulfite results in the stimu- lation of the oxidation of IAA. Although the formation of a small amount of IAld has been reported from the oxidation of IAA by peroxidase from various plant sources, the major oxidation product was not IAld but 3-methy1ene oxindole. In our work 3-methylene oxindole rather than IAld was formed as one of the major products in the peroxidase-catalyzed oxidation of IAA. How- ever, in contrast to the observation noted for IAA, the oxidation of IAAld by the same system yielded as high as 50% of IAld in the reaction mixture. These results suggest that IAA and IAAld were degraded by the same peroxidase sys- tem in different ways and that IAA was not an intermediate product in the course of peroxidase-catalyzed oxidation of IAAld: Apparently there is a common factor in the peroxidase- 1;”;th h I‘Fr. I 114 catalyzed oxidation of either IAA or IAAld. As a result of oxidative breakdown of IAA and IAAld, the oxidation products in general are one carbon atom less than the original com- pound, and the missing carbon atom is generally derived from the terminal carbon atom of the Side chain. Thus it appears that there is a general principle which is governing the course of the enzymatic reaction. This principle may be closely related to the chemical property of the free radical generated during the course of the catalyzed reaCtion. A two-step sequential reaction mechanism (IAA to A to B) has been suggested by Ray (78) to explain some results which he had obtained in a series of spectroscopic studies on the course of peroxidase oxidation of IAA. Furthermore, based on some results obtained in the present work, the author suggests that Ray's intermediate compound A, either generated from IAA or from IAAld may have a structural fea- ture in common: Recently, Hinman and Lang (36) suggested an "epoxide mechanism” to SXplain the oxidation of IAA to 3-methylene oxindole or indole-3-carboxaldehyde as catalyzed by highly purified horseradish peroxidase. Reviewing the results obtained in the present work, it is worthwhile to point out the following about the mechanism proposed by Hinman and Lang. 1. They proposed that the aldehydic oxygen in the IAld molecule is derived directly from H202, whereas the oxygen at 2-position of 3-methylene F17“?— '3??vn"£€“‘A a! ... I . . 719.0343 fiHH.I...—m.le..1 ..hnrr J7 3. 115 oxindole is derived from the water molecule in the reaction mixture. According to the proposed mechanism of Hinman and Lang, they further proposed that in the formation of 3-methy1ene oxindole from the hypothetical epoxide precursor, the epoxide oxygen is finally lost to the reaction mixture again. It appears that the major feature of this scheme designed for W‘72 Lao-=- an 3 3-methy1ene oxindole is simply a sequence of the two steps of hydration and dehydration of the epoxide intermediate. Finally, their eXperiments indicated that the immediate precursor for the formation of 3-methylene oxindole is oxindole-3-carbinol. According to their observations, the formation of oxindole-3-carbinol is an enzyme-catalyzed process, whereas the trans- formation of oxindole-B-carbinol to 3-methylene oxindole is nonenzymatic. In their experiments it took 76 minutes for the oxindole-3-carbinol absorption peak to appear, but 23 hours for the spontaneous transformation of this oxindole-3- carbinol to the typical abSorption of 3-methylene oxindole. Under our experimental conditions the formation of 3-methylene oxindole is not a Slow reaction. The typical peak of 3-methy1ene oxindole can be observed within one or two hours and the 116 rate of its formation depends on the enzyme concenu tration. The more enzyme present the faster is the appearance of this peak. The inconsistency between the observation of the writer and the latter investigators may be that the enzyme used by Hinman and Lang had a rather low activity (RZ = 0.64), whereas the RZ of the horseradish peroxi- dase used in our experiments was between 2.0 and 3.0. Nevertheless, there are at least two paths in the formation of 3~methylene oxindole: one by the spontaneous dehydration of oxindole-3-carbinol (a slow process): and the other by direct addition of one oxygen atom to the free radical intermedi- ate compound A (a fast process). The chemical nature of Ray's intermediate compound A is unknown. However, our evidence indicates that during the course of enzyme—catalyzed oxidation of either IAA or IAAld there may be a transient existence of a 3-methylene indolenine-like structure as indicated below: / jch ...COOH (IT CH2...CHO l 2 . \ / N/ N ’ Indole-3-acetic Acid Indole-3-acetaldehyde Yamazaki and Sauzu (114) noted that electron density of the IAA free radical generated from the peroxidase- 117 catalyzed oxidation of IAA was highest at the 3-position, therefore the above diagrams of IAA and IAAld free radicals are only hypothetical. However, a single electron in a molecule does not have a localized charge, but has a die» tributed wave function. A change in the environment may change the distribution of the electron within the molecule. Consequently, a Single electron of the IAA or IAAld free radical could be distributed among three possible sites in the molecule. One site is located at the methylene carbon of the side chain, and the other two at the 2- and 3- positions of the indole ring. The course of the reaction is then determined by the following factors: 1. The nature of the carbonyl carbon.--Since the carboxyl group of the acetic acid Side chain of IAA is more stable than the aldehydic carbonyl group of IAAld, the carboxyl group stabilizes the methylene hydrogens of the side chain of IAA, whereas the same hydrogens are activated by the aldehydic group in IAAld. Consequently, because of this activation, the methylene group of IAAld becomes more susceptible to attack by the oxidiz- ing agent (in this case peroxide-peroxidase com- plex) and thereby results in the formation of IAld as the major product. 2. The polarity of the -N=C- group in the indolenine intermediate.-~According to Witkop and Patrick (110), the polarity of the -N=C- group of the indolenine ring is determined by the acidity of 1“““1 118 the reaction medium. The presence of HI will enhance the additive ability of the -N=C- group, whereas the hydroxide anions will reverse this effect. Thus under alkaline conditions the 2- position of the pyrrole rings of either IAA or IAAld becomes less active toward the approaching oxidizing agent (in this case peroxide-peroxidase complex), consequently leaving the other two positions as possible oxidation Sites. Indeed, under alkaline conditions as a result of the oxidation of IAAld, the major products were found to be a mixture of 4-hydroxyquinoline and indole- 3-carboxaldehyde. The evidence for the existence of the indolenine-like free radical is as follows: 1. From the peroxidase-catalyzed oxidation of the IAA reaction mixture, Hinman and Lang (36) have iso- lated an ester which had the following structure and possibly is formed by addition of IAA to 3- methylene-indolenine: CH COOH CH -C0-0-CH ‘\ .307. CW 2 (EFT 2 arm 1 J N N N/ H ,H H 3-Methylene Indole-3- Ester Indolenine acetic Acid 2. The methylene Side chain of the intermediate 3- methylene indolenine is also the side chain of 119 3-methy1ene oxindole, which is a major oxidation product from IAA. 3. The functional group which can stabilize the indolenine structure greatly stimulates the a- carbon elimination process. For instance, during Chromatographical analyses, 1-methyl-IAA1d can undergo spontaneous auto-oxidation to give 1- methyl-IAld at room temperature in the presence of atmospheric oxygen. Therefore, the possible routes for the oxidation of IAA or IAAld are the following: CH N 0 N 0 H H Oxindole-3-carbino1 3-Methy1ene R\\\\ ///27 Oxindole If 3-Methylene Indolenine / )7 \ 011 W CHZCO-O-CHZ [p GTE/[CH0 (1:1 Ester Indole-3- 4-Hydroxy- carboxaldehyde quinoline 120 Goutarel gp_§l, (31) suggests that two naturally occurring alkaloids cinchonamine and cinchonine are biochem- ically related. A hypothetical pathway for production of cinchonamine and cinchonine is Shown below: CH CH-COOH CH CH20H I 21 I 2 N N N H NH2 H Tryptophan Cinchonamine CHZCHZOH CHZCHCOOH ‘\N O \ Cinchonine / Tyrosine Recently, Van Tamelen and Haarstad (104) suggested that cinchonamine could be the precursor of cinchonine. Using 2-methyl-IAA1d as a model intermediate, they suggested a sequence of reactions for the possible manner of transfor- mation of cinchonamine into cinchonine. The suggested reac- tion scheme follows: X+ OH I X CCTV (1‘1 . " R I If R \ N/ B H H Cinchonamine COR COR x CH0 A g \ =0 / A NH OH OH 2 H H COR H \ A \ N’ ’ ’ Cinchonine Based on the above working hypothesis, they were able to synthesize 4-acetquuinoline directly from 2-methyl tryptophan by treatment of the tryptophan derivative with two equivalents of sodium hypochlorite. The key hypothetical intermediate of the proposed reaction scheme is a 2-methyl- indole-3-acetaldehyde derivative. The natural occurrence of quinamine constitutes support for the proposed electrophilic attack at the 3-position. In the above scheme, XI represents the agent OH+ or an equivalent. OH 00 H Quinamine 122 The sequence of the reactions proposed is very similar to the well-known kynurenine pathway for the tryptophan metabolism except that the cleavage of the indole ring occurs at 1, 2-position and no carbon is lost during the ring expan- sion process. In the transformation of IAAld to 4-hydroxyquinoline a Similar mechanism, suggested by Van Tamelen and Haarstad (104), may be followed with a net loss of carbon atom at 2-position. In view of the results obtained in the present work, it appears that a common feature of peroxidase-cata- lyzed oxidation of IAA and IAAld is the elimination of the first carbon of the Side chain. If this is also true in the case when 4-hydroxyquinoline is produced, an alternative pathway of transformation of IAAld into 4-hydroxyquinoline iS suggested. An alternative mechanism which has an analogy among other organic reactions is the rearrangement shown below: HI 0 J, 23 OH 032 C a CH' \ ...—9 :9H' ‘2 / N N 3-Methy1ene 4-Hydroxy- Indolenine quinoline Since no direct evidence has been available to support either mechanism proposed, further studies of this problem are of great interest. The formation of 1-methyl-4-hydroxyquinoline from 'i» '1'“ “LA“. p‘ . . . 123 lamethyl-IAAld could not be verified in the present work. However, this naturally occurring alkaloid was first iso- lated from Echipses by Greshoff (33) in 1900 as the chloride. Consequently it was called echipsin. Possibly, the procur- sor of echipsin is either IAAld or 1-methyl-IAAld. From results obtained in our work we suggest that echipsin may be directly produced from methylation of 4-hydroxyquinoline rather than from 1-methyl-IAAld. This discussion shows that it is possible that plant peroxidase may have played an important role in biosynthesis of some physiologically important plant alkaloids as cinchonine, quinine, and echipsin. All these processes may require the participation of certain cofactors such as bisulfite ion, ammonia or other normal metabolites present in plant systems. Recently it was reported that abnormally large quan- tities of IAld have been detected in the urine of schizo- phrenic patients (80). The presence of peroxidase system in nervous systems has been reported (11). It is possible that peroxidase is also involved in these biologically important processes. The reason why manganese and 2,4-dichlorophenol have no effect on the peroxidase-catalyzed oxidation of IAAld in the acidic pH range is unknown. Further studies of this aspect are required. Klapper and Hackett (48) demonstrated that commercial horseradish peroxidase contains several isozymes. However, the five purified enzyme components have been shown to have 124 identical catalytic activities and the amino acid compositions of three of the five purified isozymes are similar. It is unlikely that the formation of 4-hydroxyquinoline is a result of structural differences between these isozyme components. SUMMARY SUMMARY In a series of Spectrophotometric studies on the oxida- tion of indole-3-acetic acid (IAA), indole-3-acetaldehyde (IAAld), and indole-3-acetaldehyde sodium bisulfite addition product (IAAld-NaHSOB) catalyzed by horseradish peroxidase, it was found that these substances each followed a different pattern of oxidative pathway. Either qualitatively or quan- titatively the peroxidase-catalyzed oxidation of IAA and its analogs are pH dependent. At a pH range from 3.7 to 4.5, the conversion of IAA'to 3-methy1ene oxindole, as reported previously, was confirmed in the present work; whereas the oxidation of IAAld under the same conditions gave rise to as high as 50% yield of indole-3-carboxaldehyde (IAld). The oxidation of IAAld-NaHSO3 at pH between 3.7 and 4.4 followed the same pattern as that observed for free IAAld. AS the pH was increased above 6.2, a differential behavior of the enzy- matic system was noted, i.e., the oxidation of IAA and IAAld decreased continually as the pH was increased, whereas the oxidation of IAAld-NaHSO3 increased greatly as the pH increased above 6.2. One of the interesting observations is that at alkaline pH values as result of oxidative breakdown of IAAld-NaHSOB, a new product having a double peak at 316 and 330 mu regions was always observed. The presence of bisulfite ion was found 126 127 to be stimulatory toward the oxidation of the auxins catalyzed by the horseradish peroxidase. In the presence of 1 x 10"“ p of IAAld Km calculated for bisulfite ion was found to be 3.5 x.10'5 5. Increasing bisulfite concentration from 3.57 x 10'5 to 3.57 x 10‘“ fold and reduced the Km of the System twentyfold. Apparently §,increased the V of the system two- the presence of bisulfite ion greatly enhanced the affinity of horseradish peroxidase toward its substrates at elevated pH values. The present work definitely shows that 4-hydroxy- quinoline is the oxidation product in horseradish peroxidase- catalyzed oxidation of IAAld. However, a change of pH and the presence or absence of a cofactor greatly influenced the peroxidase-catalyzed oxidation of IAA and IAAld. In the present work the formation of 4-hydroxyquinoline from peroxi- dation of IAAld was established. The evidence was obtained by the direct isolation of the oxidation product in crystal- line form. The mechanism of the bisulfite ion stimulated oxidation of IAA and IAAld was also studied. The results indicated that at alkaline pH values the presence of the bisulfite ion stimu- lated the formation of peroxidase complex II which is essen- tial for peroxidase-catalyzed reactions. Another interesting observation is that during the course of peroxidase oxidation of IAA the indole ring was altered by the oxidizing agent, whereas when IAAld was oxi- dized the ring remained intact. This is not true when the bisulfite ion is added to the reaction mixture. At alkaline pH values in the presence of bisulfite ion the oxidation of 128 IAAld will result in the SXpansion of a five-membered indole ring to a six-membered quinoline ring. Whether this is true in case of the oxidation of 1~methylindole-3-acetaldehyde (1-methy1-IAAld) is unknown. No definite results have been obtained to indicate that 1-methyl-4-hydroxyquinoline is an oxidation product of 1-methyl-IAAld. Apparently, there is no single rule which governs the peroxidase-catalyzed oxida- tion of auxins, although a common feature in the peroxidase- catalyzed oxidation of IAA, IAAld, and 1-methyl-IAA1d was the elimination of the terminal carbon of the side chain.) 1-Methyl-IAld was found to be one of the major products in the peroxidase oxidation of 1-methyl-IAAld. The reason why the presence of bisulfite can divert the oxidation pathway in the course of peroxidase-catalyzed oxidation of IAA and IAAld is unknown. Some possible mechan- isms are discussed. The inhibitory effect of IAA toward oxidation of guaiacol was recently reported. Our investigations are similar to the previous observation, i.e., IAA is inhibitory to the peroxidase system. Furthermore, our work indicates that the inhibitory effect of IAA toward the enzymatic sys- tem is directly related to the formation of peroxide-peroxi- dase complexes. 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