The destruction of indoleacetic acid. I. Action of an enzyme from Omphalia flavida

i\RCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

64, 175-192 (1956)

The Destruction of Indoleacetic Acid. I. Action of an Enzyme from Omphaliu fravidd Peter M. Ray2 and Kenneth

V. Thimann

From the Biological Laboratories, Haward University, Cambridge, Massachusetts Received

February

9, 1956

The destruction of auxin by fungi has been known since at least 1935, when Ronsdorf (22) reported auxin inactivation by cultures of Pythium mammillatum. More recently, Sequeira and Steeves (24) found that cultures of the agaricacious fullgus Omphalia jlavida produced an indoleacetic acid-inactivat’ing principle which appeared to be an enzyme. Tonhazy and Pelczar (28) studied an enzyme, formed by liquid cultures of Polyporus versicoIor, which oxidized indoleacetic acid (IAA). The present study of Omphalia was undertaken to explore the possibility that its auxin-dest’roying enzyme might offer advantageous material for the study of IAA oxidat,ion and might yield information useful in the interpretation of IAA oxidation in higher plants. Since the enzyme is released by the fungus into the simple liquid culture medium, it can be obtained without homogenizing any tissue and thus affords a preparation relatively free of other cell constituents. This report deals with some general properties of the Omphalia enzyme and its reaction. Preliminary findings concerning the oxidation product (26) and the reaction intermediate (20) have been reported. The changes in light absorption which occur during IAA oxidation, and the mechanism of a&ion of the enzyme, are considered in the following paper (21). 1 This work was supported by Grants from the Committee on Growth, acting for the American Cancer Society, and from the National Science Foundation (NSF-GlS2). Part of the work was carried out during the tenure of a National Science Foundation Predoctornl Fellowship by I’. RI. Ray. 2 Society of Fellows, Harvard Universit?-. 175

176

P. M. RAY AND

K. V. THIMANN

EXPERIMENTAL

Analytical

Methods

IAA analysis with t,he Salkowski reagent was carried out following Gordon and Weber (9). Color intensity was det.ermined after 1 hr. either with a Klett-Summerson photoelectric calorimeter using a #54 filter, or with a Beckman I)IJ spect.rophotometer at 530 rnp. Manometric measurements were made with a Warburg apparatus at 25’C. I~)adding IAll (as Na salt) from the side arm to enzyme preparation in 0.05 J1 citric acid buffer, pH 3.5. Oxygen was determined in the presence of 0.2 ml. of 10% KOH in the center well; CO, was determined by the “direct” method (29). In the absence of IAA, enzyme preparations showed no 02 uptake or CO, production whatever. When final IAA concentrations greater than about 3 X 10-a 211were used, it was found convenient t,o add 1% digitonin to prevent crystallization of the highly supersaturated solution of IAA. Even 5% digitonin had no effect on either the total gas exchange or the rate of oxygen uptake. Addition of 5% polyoxyethylene sorbitan monooleate (Tween 80) also prevented crystallization but irkhibited the rate of oxygen uptake about 50%. As will be discussed in a subsequent paper (21), it was found that when the co,,centration of enzyme or substrate was low, the addition of a catalytic amount of H?O? (much less than 1 mole/mole IAA) hastened the &art of the reaction. Where this procedure mas employed in the present experiment,s, it is explicitly stated.

Enzyme Preparation About 50 stilboids (gemmae) of Omphalia JEavida, strain T4B of Seyueirs (23), were harvested from a potato-dextrose agar culture and inoculated into 100 ml. of sterile medium of the composition given by Sequeira and Stecves (24), contained in a 500-ml. flask. Cultures were incubated at 26°C. in the dark without shaking, because it was found that still cultures produced as much enzyme as shaken OIIW but did not excrete mucilage as do the latter. The IAA-destroying act,ivit,y, as measured with the Salkowski reagent, increased in the culture medium for about 3 weeks and then declined unless the medium was decanted and new, sterile medium added. Bfter two or three such changes of medium, a dense pad of mycelium had been formed, and the IAA-destroying activity of the medium was much increased. The pale yellow medium from such a cult,ure was decanted, filtered through Whatmnn No. 30 paper, and placed in a dialysis casing which was rotated slowly in lo-20 vol. of glass-distilled water, at 3°C. Five or six changes of dialyznte were made during a 4-day period. Some of the yellow color of the medium was rcmoved by dialysis, but, the cant,ents of the bag remained pale yellow. The liquid was finally filtered through sintered glass to remove a slight flocculent precipitate, and stored on ice or in a deep freeze, where t,he activit,y was stable for at least 4 weeks. A typical preparation contained 66 pg. X/ml., md had a (20,(N) of 560 J. OJhr./mg. N, measured manometrically. Partial purification of the enzyme was accomplished at 4°C. by slowly dissolving solid (NHa)xSO1 to 90 or 100% saturation. Upon centrifugation, the yellolvbrown pellet contained almost all of the activity-. The precipitate was redis,~olvctl

DESTRUCTION

OF INDOLEACETIC

ACID.

I

17i

in cold glass-distilled water, and the solution was then redialyzed. This procedure afforded a two- to fourfold enrichment, of activit,y, on a dry-Iv-eight basis. over that of the dialyzed culture medium.

The reaction misture contained initially 10 mg. MA/l. (57#j in 0.005 M citric acid buffer, pH 3.7, plus an amount of enzyme and catalytic ILO,: [e.g. 3 pill, cf. (21)] sufficient to cause complete oxidation of the IAA in 15-20 min. Chromatograms of the IAA oxidation product were prepared from 10 ml. of this mixture. This was extracted three times with an equal volume of chloroform; the latter m-as then concentrated in cacuo to 0.05 ml. and applied to the paper strip (Whatman So. 1 paper). After equilibration overnight over the solvent, the chromatogram~ were developed by the ascending technique. The two chromatographic solvents employed were chosen as a result of trials with a wide variety of solvent mixtures. The best solvent, termed solvent M, was a modification of that used by Mason and Berg (16) and had the composition bcnzene-n-butanol-methanol-water (2:2:4:1 parts by volume). Solvent G was that used by Manning and Galston (15), and had the composition isopropyl alcohol-28y0 ammonia-water (2:l:l parts by volume). For the oxidation products it was superior to the usual isopropyl alcohol-ammonia solvents (3, 25) in that, unlike the latter, it did not streak these compounds; however, with this solvent IA-4 had the same R, value as one of the oxidation products. Gross samples of oxidation product were prepared using a reaction misture of the same initial composition as that above, except that the buffer concentration was 0.0075 M. After 30 min., at which time the MA had been entirely destroyed, the mixture was shaken vigorously to oxygenate it, and a further 10 mg./l. IAA (as Na salt), together with the same amount of enzyme and Hz02 as initially, wad added. A similar addition was made every 30 min. until the desired amount of IAA (100-200 mg.) had been destroyed. In this way it was possible to obtain a relatively large amount of product without using digitonin (cf. above) and despite a tendency of the enzyme to become inactivated during the reaction. The final mixture was made to pH 7.5 by adding saturated P;a&03, extracted four times with one half its volume of chloroform, and the extract concentrated in WLCUO at 19-23°C.

Labeled IAA IAA labeled with Cnin the a-carbon atom of the ring was synthesized by Pichat et al. (18). It had a specific activity of 0.45 pc./mg. Chromatograms of radioactive materials were counted as follows : The paper strip was drawn through a counting chamber at constant rate, by means of a pulley turned by a synchronous motor. The counting chamber was sealed with rubber strips where the paper entered and left it, and was kept under slight positive pressure of counting gas. A 2-mm-wide slit oriented transverse to the long axis of the paper st,rip allowed a section of the paper to be exposed to the counting element. A gas-flow counter at 1800 v. Teas used. Counting pulses were amplified 2000.fold and integrated by means of a damped counting-rate monitor. The counting rate was recorded on a l-ma. Esterline-Angus recorder. Counting efficiency of the system was 14%. Reproducibility

178

P. M. RAY AND K. V. THIMANN

of the total count was of the order of 107, on separate samples of material separately chromatographed. With the damping employed in integration, statistical fluctuation in the trace approximated 5oj, of its amplitude. The major outline of the trace was found to be reproducible in form , and to within 7y0 in amplitude, and was considered to indicate the distribut,ion of radioactivity on the paper.

PROPERTIES OF THE ENZYME

A number of properties of the MA-inactivating principle of Omphalia cultures support the conclusion of Sequeira and Steeves (24) that it is an enzyme: 1. The activity is heat-labile, being entirely abolished after 5 min. at 90°C. 2. The activity is nondialyzable. In fact, activity usually increases slightly upon dialysis; the effect is small (15 %) compared with the dialysis-sensitive inhibitions of IAA destruction which have been studied in homogenates of peas (5) and pineapple (10). 3. The rate of IAA destruction by dialyzed culture medium is maximal at about pH 3.5 (Fig. 1). The dependence of activity on pH is similar to that reported for the IAA-oxidizing enzyme from pineapple (10). The Polyporus enzyme was stated t,o have an optimum at pH 4.5 (28).

PH

FIG. 1. Effect

of pH on destruction of IAA by Omphalia enzyme. Dialyzed culture medium (1 ml.) plus 0.8 pmole IAA in total volume of 1.4 ml., 0.05 M in citrate and phosphate at the indicated pH (glass electrode) ; incubated at 26°C. in the dark. Curve 1, IAA destroyed after 30 min.; curve 2, after 60 min.

179

DESTRUCTION OF INDOLEACETIC ACID. I TABLE Inhibition

of IAA

1

Destruction

by HCK

Figures indicate the amount of IAA (in pg.) destroyed, during each time interval, by 1 ml. of dialyzed culture medium in 5 ml. of 0.005 M citric acid buffer, pH 3.7, containing 52.5 pg. IAA initially. Time

HCN concentration, 10-s

0

dl 10-4

2 x

lo-’

?lGL

40 90

6 19

31 52

TABLE Stoichiometry

0 2

0 0

II

of 1AA Oxidation

All data in micromoles. Gases determined manometrically using 2 ml. of dialyzed culture medium and 9.2 pmoles of IAA in a total volume of 3.3 ml. 0.05 M citric acid buffer, pH 3.5. IAA determined in 0.0%ml. aliquots of a similar reaction mixture which was shaken in the water bath along with the Warburg vessels. Time min.

26 62 125

:;~x: 4.0 6.7 8.3

CO2 production

IAA disappearance

4.0 6.6 8.5

4.3 6.8 8.5

4. The IAA-destroying principle is precipitated by saturated (NH&SO, and recovered nearly quantitatively in the small yellow-brown precipitate. Centrifugation after treatment with less than 80 % saturated (NH&S04 gave no precipitate or only a very slight, inactive one. Not much activity could be precipitated with acetone or alcohol. 5. HCN inhibits the destruction of IAA by dialyzed culture medium (Table I) at concentrations comparable with those which inhibit metalloenzymes. This is a property common to all IAA-oxidizing enzymes which have been investigated. The rate of IAA destruction was almost linearly proportional to enzyme concentration, whether measured manometrically or colorimetrically. THE STOICHIOMETRY

OF

THE REACTION

No destruction of IAA occurred when it was added to the enzyme in t,he absence of air. In the presence of air, oxygen was consumed at nearly the same rate at which IAA disappeared, and CO2 was given off in equal amount (Table II). Oxygen uptake ceased when 1 mole O2 had been consumed per mole IAA initially present. Therefore, the reaction

180

P. M. RAY AND K. V. THIMANN

IAA + 0:: -+ co2 +- product The same stoichiometry has been found for most other IAA-oxidizing enzymes which have been reported on (10, 12, 27, 28, 30). EFFECTS OF MANGANESE

AND PHENOLS

The effects of manganous ion on IAA oxidation by t’he Omphalia enzyme (Fig. 2) were similar to those of MntZ on IAA oxidation by horse-radish peroxidase (12). At lo- 5 J! the initial rate of oxygen uptake was augmented, and the subsequent decline in rate, often observed in t,he absence of Mn++, was prevented. At low4 and lo-” M Mn++, increasingly long induction periods preceded t,he initiation of oxygen uptake. At 1O-3 M the rate of oxygen u$take never reached that in the controls, but also did not fall off with time, so that eventually Dhe total 02 uptake surpassed that of the control. At each Mn++ concentration the rate of oxygen uptake decreased sharply when the stoiochiometric amount (1 Oz/mole IAA) had been

TIME IN HRS. FIG. 2. Effect of manganese on IAA oxidation.

O~~phalia enzyme (prepared by (NHa)zSOa precipitation and dialysis) and 4 pmoles IAA in 2 ml. of 0.05 M citrate buffer, pH 3.5, containing4 mg. digitonin, and MnSOa in the following concentrations: curve 1,O; curve 2, lo-& M; curve 3, 1O-4M; curve 4, 10v3 M.

DESTRUCTION OF INDOLEACETIC ACID. I

181

consumed; however, slow further oxygen uptake continued, especially at the higher Mn++ concentrations, finally reaching approximately 1.5 On/mole IAA at 10-a M Mn++ (Fig. 2). The rate of this subsequent oxygen uptake was insufficient to account for the stimulation in initial rate of O2 uptake found with 10m5J1 Mn++. Kcnten (12) reported that oxidation of IAA by horse-radish peroxidase was stimulated by Mn++ only near pH 6, and only in the absence of citrate. He felt this latter result was due to chelation, possibly of Mn+++, by citrate. However, with the Omphalia enzyme the observed stimulation by Mn++ occurred at pH 3.5 in citric acid buffer. At pH 3.5, where the undissociated acid and the monoanion are the principal forms of citric acid, chelat,ion may not occur. Alternatively, it is possible that a mechanism different from that of horse-radish peroxidase is involved. At concentrations from 10m3to 10Y5 41, 2,4-dichlorophenol did not affect t’he rate of oxygen uptake. The Omphalia preparation thus differs markedly from horse-radish peroxidase (12) and the pea enzyme (8), which are stimulated by this and other monophenols, and more nearly resembles the IA4-oxidizing enzymes with acidic pH optima, which do not show stimulation by phenols (10, 28). Di- and polyphenols inhibit the enzyme strongly. This effect will be considered in detail in a separate paper. SUBSTRATE SPECIFICITY

Tndolepropionic (IPA) and indolebutyric (IBA) acids are oxidized at relatively slow but significant rates (compared to IAA) by preparations from peas and beans (30) and Polyporus (28). It seemed likely that substances as similar to IAA as IPA and IBA would serve as substrates also for the Omphalia enzyme. However, no appreciable amount of oxygen was taken up when eit’her acid was added to t,he enzyme under conditions where rapid oxygen uptake occurred with IAA. Furthermore, no significant decrease in intensity of the yellow Salkowski reaction of these substances occurred in the presence of the enzyme. Since Kenten (13) reported that horse-radish peroxidase did not oxidize IPA or IBA except in the presence of Mn++, and that citrate inhibited the reaction, the influence of Mn++ and buffer was studied. The Omphalia preparation gave a very slow 02 upt’ake from IPA in 1O-4 M Mn++ (Table III). This result was obtained in citric acid buffer (pH 3.5). Even though this buffer did not inhibit the positive effects of Mn++ on IAA oxidation by the Omphalia enzyme (unlike t,he case with

182

P.

M.

RAY

AND

H.

V.

THIMANN

TABLE III of Omphalia Enzyme Vessels contained 1 ml. of ammonium sulfate-precipitated Omphalia enzyme in 2 ml. 0.05 M citric acid buffer, pH 3.5, containing 4 pmolcs of the substrate (added as Na salt), and manganese where indicated. Substrate

Specijkity

Oxygen uptake Substrate

MnSO4 , 10-r af

hr. wmlles

7 hr. pmoles

Indoleacetic acid Indoleacetic acid Indoleisobutyric acid Indoleisobutyric acid Indolepropionic acid Indolepropionic acid

+ + +

1.9 3.2 3.6 4.2 0.0 0.1

3.6 4.3 4.4 4.8 0.1 0.4

2

peroxidase), the possibility that the buffer inhibits IPA oxidation was examined by using acetic acid buffer, pH 4.3 (0.05 M), which would not be expected to chelate manganese. In this medium no enzymatic oxidation of IPA was observed either with or without 1O-4M M&O4 , whereas IAA was oxidized regardless of the presence of Mn++. It seems evident that IPA is an extremely poor substrate for the Owzphalia enzyme. Indoleisobutyric acid (cr’,oc’-dimethylindoleacetic acid, ERA), on t’he other hand, was rapidly oxidized by the preparation, both in the absence and presence of Mn++ (Table III). The latter (low4 LV) had much the same effect on IiBA oxidation as it had on IAA oxidation. approximately 1 mole O2 was taken up per mole of IiBA, and an equivalent amount of COZ was given off. A white precipitate and pale orange color appeared in the reaction mixture. IiBA was stable in the absence of the enzyme. No oxygen uptake was obtained with tryptophan, skatolc, indoleacetamide, or ethyl indoleacetate in t’he presence of the Omphalia enzyme. No spectroscopic changes (20) occurred when the enzyme ivas added to indoleacetonitrile, skatole, indolealdehyde, or hr-formylaminoacetophenone. A slow reaction was observed spectroscopically with 5- and ‘i-hydroxyindoleacetic acids. Figure 3 shows the influence of the enzyme on part of the ultraviolet spectra of these compounds, compared with that on IAA. The IAA was destroyed in 40 min. and its spectrum ceased to change after 90 min. With the hydroxy derivatives the changes in absorption occurred much more slowly, and the spectra were still changing after 4 hr. It is important that absorption rose or remained steady at 272 and 285 rnp with the hydroxy acids, while it fell sharply with IAA. This indicates that the reaction products are not the same.

183

__ 5-hydroxy-IAA

__ 7-hydroxy-IAA

30

A

in

mp

FIG. 3. Changes in ultraviolet spcctrn of IAA derivatives, in the presence of the Owzphalia enzyme. Left, IAA; rrnter, 5-hydroxyindoleacet.ic acid; right, Thydrosyindoleacetic acid. Circles show initial spectra, triangles the spectra after 4 hr. Enzyme, buffer, and 0.02j~mole 11~02 in 3.0 ml. containing 0.18 pmole of the substrate. Optical densiCes determined against a blank cont,aining all components but the substrate.

The substrate range of t)he Om~phalia enzyme thus seems to be remarkably narrow. The preparation, unlike that from Poll~porus [(as), cf. (a)], shows no polyphenoloxidase activit’y. It does act peroxidatively on a variety of phenolic compounds; the relationship of this to IAA oxidation will be considered elsewhere. THE

PRODIXT

OF IAA

OXIDATION

The product of IAA oxidation by the Omphalia enzyme consists principally of neutral mat,erial soluble in ether or chloroform. Table IV shows the recovery of isotope from chloroform extracts of a solution of labeled IAA (C l4 in the 2-carbon of the indole ring), and of the same solution after oxidation by the enzyme. Most of the label appears, as expected, in the acid fraction of the starting material (some neutral impurities were present in it), while after oxidation most of the label is in the neutral fraction (soluble in chloroform at pH 10 or 3.5). Furthermore, within the error of the method the tracer is almost quantitatively recovered (cf. also Fig. 6), indicat)ing that the 2-carbon of IAA is retained in the oxidation products. From this fact it seems highly likely that the COZ produced during the reaction comes from t.he carboxyl of TAA. Spectroscopic properties of the oxidation product have been briefly

184

I’.

M.

RAY

.4ND

I<.

TABLE

V.

THIMANX

IV

Isotope Distribution upon Extraction of Solution and of Its Enzymatic Oxidation Product

of IAA*

Solution contained 0.2 mg. IAA*, or an equivalent amount of its oxidation products, in 20 ml. of 0.005 M citric acid buffer, pH 3.7. Extracts were concentrated, applied to planchets, and evaporated to dryness. Sample

1. 2. 3. 4.

IABa IAA* Productj” Product.*

In chloroform extracts Total’= Neutral* Acidb Counts per minute, in thousands

73

Left i;haaqsueeous

3 7

,37

5

57

9

11 3

.5s

Total

76 sn 69 (i!)

0 Extracted five times, at pH 3.7, with twice the volume of CHCI, * Extracted five times at pH 10, then five times at pH 3.5.

reported on previously (2G). Both infrared and ultraviolet spectra show conclusively that the product cannot contain appreciable amounts of indolealdehyde, or derivatives of o-aminoacetophenone or quinolinc. The infrared and ultraviolet spect,ra markedly resemble those of oxindole derivatives, and notably the spectra of 3-methyldioxindole (I), a aompound satisfying t,he observed stoichiometry.

H

0

The ultraviolet spectrum of the IAA oxidation products, observed in the reaction mixture before any preparatory procedures are applied, is shown in Fig. 4 and is compared wit,h the spectra of indolealdehyde, formylaminoacetophenone, and 3-methyldioxindole. Figure 5 presents the infrared spectra of a preparation of IAA oxidation products and of 3-methyldioxindole; they show particularly the strong peak at 5.80 P, which is characteristic of oxindoles and of the carbonyl group in a fivemembered ring (32). So far it has not proved possible to isolate any substance of certain purity out of the products of IAA inactivation. Materials of rather diverse properties have been obtained from the reaction mixture. The

Ik

4. o-

(3 cl 3, 9

5-

3. O-

I

220

260

300 X

in

340

mp

spectra of (1) IAA; (2) IAB oxidation product, as observed FIG. 4. Ultraviolet in the reaction mixture; (8) 3-methyldioxindole; (4) indole-3-aldehyde; and (6) o-formylaminoacetophenone.

404ot

I II ’

20 20~

3-inethyldmxindole 3-inethyldmxindole

2

3

4

5

6

-! , 7

, 8

, 9

1

A in p FIG.

5.

Infrared spectra of IAA oxidation product (above) and 3-methyldioxindole (below). Approximately 10 mg./ml. in chloroform. 185

186

P. M.

RAY

AND

K.

TABLE

V.

THIMANN

V

Materials Isolated from IAA Oxidation

Product

As described in text, 179 mg. IAA was oxidized enzymatically and extra&cd with chloroform. A (very slightly sol. ethanol, ether) precipitated from concentrated CHCl, extract on standing in the cold. It was removed, and washed with CHCla and EtOH. The now red CHC13 extract was evaporated to dryness and the red residue taken up in CHC13 ; more A was removed, and the solvent was evaporated to give B (very sol. ethanol, ether, CHC!II)

=

Isolate

A B

Color

Melting point

White 1 Deep red

-

Softens 230”; melts 270” to a tar 50” (unsharp)

=

Yield, mg.

Mol. wt.

C

--

H

.__

Found

Calcd.

Found

C&d.

5.76

5.52b

5.00

5.26d

18

! -a

66.5

66.2*

i-1

2ooc 62.8

63.2d

a Insoluble in camphor. b Calculated for CgH90,N. c Approximate only, due to the deep red solution d Calculated for C18H1805N2 .

i

in camphor.

properties and composition of two such preparations are given in Table V. Substance A, which has the same composition as 3-methyldioxindole, differs from t’he latter in being not more than slightly soluble in alcohol, chloroform, ether, water, or boiling NaOH; it is probably polymeric. Its ultraviolet spectrum (curve 4 of Fig. 6) exhibits a double maximum near 250 rnp, and no other peaks. Substance B is not polymeric but is deep red (oxindoles are generally colorless) and differs significantly in composition from what would be expected from the reaction stoichiometry. Its composition could result from further oxidation of the product by +dO/mole, which would seem to fit the observation that concentrated solutions of the products in organic solvents slowly change color from yellow-brown to deep red.3 Despite its color, the preparation showed no absorption maxima above 250 rnp (curve 3 of Fig. 6). The two other materials whose spectra are shown in Fig. 6 were isolated in small yields. In an effort to determine how many substances might be present in the products of IAA oxidation, chromatograms of labeled IAA and its enzymic oxidation products were examined (Fig. 7). A double or multiple peak of radioactivity was always found in the Rf region of the oxidation 3 It should oxindoleacetic

be noted that some oxindole acid, are highly unstable.

derivatives,

e.g., oxindolealanine

and

DESTRUCTION

OF INDOLEACETIC

I

I

260

300 ?\ in m

ACID.

I

187

I

340

Y

FIN. 6. Ultraviolet spectra of materials isolated from products of IAA oxidation: (1) Light yellow solid, m.p. 120-123”, from CCL ; 0.1 mg. in 10 ml. 1% EtOH. (2) White solid, m.p. (138”?) 146-148”C., obtained in very small yield from CCL after decolorizing with charcoal; turned pink on standing in air; ca. 0.1 mg. in 3 ml. 3oJ0EtOH. (S) Isolate R (Table V), 0.45 mg. in 30 ml. 10% EtOH. (I) Isolate B (Table V), satd. soln. in EtOH diluted tenfold. Ordinates: log of measured optical density.

products (R, O&0.8), showing the presence of more than one component. By comparing the color reactions of chromatograms with the distribution of isotope on them, several components (Table VI) could be distinguished. Component b appears to be the principal constituent of t,he isolated material B of Table V. It is not clear how the other component,s are related to t,he subst’ances of Fig. 6. Component d, which gave

188

I’. M. RAY AND K. V. THIMANN

640

640

640 320 0 640 320 0 0

5

IO DISTANCE

I5 IN

20

25

CM.

FIG. 7. Enzymatic

oxidation of labeled IAA. Counting records of chromatograms prepared from extracts of a reaction mixture at the times shown; developed with solvent M. Abscissae indicate distance from the starting point; F, solvent front. Total count given at left for each chromatogram. The peak at RI npproximately 0.4 is IAA.

no color with the Salkowski or Ehrlich reagent, ran at the same Rf as IAA in solvent G, and was dete@d as a peak of radioact,ivity associated with no color reactions. Components a and c, which gave pink colors with the Salkowski re-

DESTRUCTION

OF

INDOLEACETIC

TABLE Components

of IAA

ACID.

VI Oxidation

Product

Color reactions Substance (a)

0)) (cl (4 IAA

Native color

Yellorr Red

Fluorescenci!

Yellow Blue

189

I

Salkowski

Pink -

Ehrlich

Pink

Orange-red Yellow Ornnge-red

Red

Violet

Mean RI in solvent XI

G

0.78 0.69 0.66 0.62 0.37

0.73 0.67 O.SS 0.63 0.63

agent, gave pink colors also with 1% Fe& and with the nitrite reagent of Fischer (3). They may be identical with the two materials formed from IAA by the pea enzyme which were observed by Manning and Galston (15) to give a pink color with FeCla . The R, values in solvent G, given by these workers, were higher than found here, but so was that of IAA. However, the FeC13 color led Manning and Galston to conclude t’hat these compounds were phenolic. The fact that the pink colors in the present case are also formed wit,h nitrite shows clearly t,hat our colors with FeC13 cannot be considered evidence for a phenolic function. The color develops slowly (1-5 min.) and appears to be a true Salkowski reaction. Moreover, the same spots give no color react’ion with the phenol reagent of Folin and Denis (4), nor with diazotized sulfanilic acid, to which phenols typically respond (7); compounds of structure analogous to that proposed by Manning and Galston are known to give colors wit’h the latter reagent (14). It is unlikely, therefore, that the Omphalia enzyme causes oxidation of the benzene ring of IAA. Also, since 5- and 7-hydroxyindoleacet’ic acids were destroyed by the enzyme much more slowly than IAA, and were converted to products differentfrom those obtained from IAA, these compounds can hardly be intermediates in the reaction catalyzed by the Omphalia enzyme. Manning and Galston (15) also felt that t,he orange and red colors, obtained from the pea enzyme product with the Ehrlich reagent, indicated that the product possessed aryl amino groups. The presence of the 2-carbon of IAA in the oxidation products formed by the Omphalia enzyme shows that even had a formylaminoacetophenone derivative been formed, deformylation could not have taken place. In addition, none of the products is diazotizable, and therefore none possesses a free 4 This observation also indicat,es the absence of osindole derivatives hydrogen atom in the Y-position. Note, however, t,hnt X-methyldiosindole not respond to the Folin-Deni? reagent.

with a (I) does

190

P.

M.

RAY

AND

K.

V.

THIMANN

aromatic amino group. In the present case, therefore, the formation of orange and red colors with the Ehrlich reagent (Table VI) cannot be due to the presence of aryl amino groups. While there is no reason to take for granted that the products of the Omphalia and the pea enzymes are identical, they do resemble one another in respect to those properties for which bot,h have been studied. The spectra of materials eluted from chromatograms of the pea enzyme product (15) also agree with spectra of the Omphalia enzyme products in having no maxima at wavelengths above 300 mp. It is to be noted that maxima above 300 rnp are characterist,ic of aminoacetophenone derivatives and analogous compounds (1, 11, 17, 26), including hydroxylated ones. Wiltshire (31) reported observing an indole-type spectrum after oxidation of IAA by a pea preparation followed by deproteinization of the solution. Although he felt that t)his indicated side-chain degradation of IAA, in fact neither indolealdehyde nor indoleglyoxylic acid, the only such compounds which fit the oxygen st,oichiometry, possesses an indoletype spectrum. Curve 1 of Fig. 4 shows the indole-type spectrum, while curve 4 shows the spectrum of indolealdehyde, which also resembles that, of indoleglyoxylic acid. The view that indolealdehyde is the product of IAA-oxidizing enzymes of peas (30) has recently been revived (19). The latter report, indicated, however, only a 5 % over-all yield of the aldehydc, as judged by the 2,4-dinitrophenylhydrazine reagent. IAA itself gives a weak positive reaction on paper to this reagent, so the possibility should perhaps be considered that neutral, indolic constituents of the product other than indolealdehyde might be responsible for the reported results. The two Salkowski-positive components, a and c, found in the products of IA‘1 oxidation by the Omphalia enzyme may be indolic, but neither gave the Van Eck aldehyde reaction nor t’he Ehrlich color of indolealdehyde. Bs mentioned above, the ultraviolet absorption spectra of the Omphalia enzyme products definitely exclude the presence of appreciable amounts of indolealdehyde, because the spectra not only lack any maximum near 300 rnp comparable with the prominent peak of indolealdehyde, but do not even shorn a shoulder or inflection in this region such as n-ould be observed if as much as 5 % of indolealdehyde were present (see Fig. 4). In this connect,ion, the rapid oxidation of indoleisobutyric acaid by the enzyme, \I-it.h the same stoichiomet)ry as IAA, is significant because it seems irnprobable that this compound could be converted t,o an nldehyde

1)I:dTRT(“NO\;

OF

ISI)OLE.~(‘ETIC

.iCID.

1

I!91

by side-chain oxidation, nor pass through intermediate steps comparable with the postulated indoleglycolic and indoleglyoxylic acids. The multiple nature of the Omphalia product probably cannot be ascribed to the operation of more than one enzyme in the preparation, because other evidence (20, 21) indicates that only one enzymatic rewtion is occurring. On the contrary, the fact that the initial product of enzyme action is unstable (21) may allow the formation of more than one final product,, as happens in other enzymatic react.ions of simila,l coharactw.

The authors wish to thank J)r. J. 1’. Sits& for making the labeled IAA available; Mr. 14:.15’. Samuel for designing and constructing the counting apparatus for chromatograms; Dr. B. B. Stone for ~ynthcais of indolealdehyde, formylaminoacet,ophenone, and 3-methJ,ldioxindolc, and for determination of infrared spectra; Jjr. A. Ek and J>r. 13. Witkop for samples of 5. and T-h~dros~indolcacctic acids; and Prof. A. W. Galston for making available the dat,:t of Manning and Calston (15) before publication. Indolcisobutyric acid was kindly supplie(l by l’rof. 11. liurstr6m; it had been synthwizrd by A. ,Jiinsson.

1. The IAA-destroying principle produced by Omphalia jlavida is :I soluble, oxidat,ire enzyme. It can bc exhaustively dialyzed, and precipitated with ammonium sulfate, without loss of activity. It shows optimal activity at pH 3.5, and at this pH its activity is not enhanced by 2,3 dichlorophenol and is moderately stimulated by Mn++. It is highly sensitive to HCN. Mn++ prevents the enzyme activity from declining during the reaction. 2. The Ik4-destroying reaction is stoichiometric, consuming 1 mole 02 and releasing 1 mole CO2 per mole IAA destroyed. 3. The enzyme does not act on indolepropionic or indolebutyric acid, 11oron any of a wide variety of indole compounds except indoleisobutyric acid, which it oxidizes rapidly, and 5- and 7-hydroxyindoleacetic acids, which it attacks slowly. 4. The ultimate reaction product is a mixture of at least four conlponents, all of which contain the 2-carbon of IAA. Two give a positive Salkowski reaction, and several give yellow to red colors with the Ehrlich reagent. Neither indolealdehyde nor derivatives of o-aminoacetophenone 11or quinoline can be present in appreciable amounts. The products arc not phenolic and do not possess free aryl amino groups. At least one com-

192

1’. M. RAY Al\;n

E(. V. THIM.INS

ponent is unstable and appears to undergo spontaneous slow oxidation and/or to give rise to polymeric material. The major constituents of the product appear to contain the oxindole nucleus. Some, but, not, all, of t,heir properties agree with those of 3-methyldioxindok. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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