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Flavoprotein and peroxidase as components of the indoleacetic acid oxidase system of peas
Flavoprotein and Peroxidase as Components of the Indoleacetic Acid Oxidase System of Peas Arthur W. Galston, James Bonner and Rosamond From
the Kerckho$
Laboratories of the Technology, Pasadena
Biology, California
S. Baker Institute
of
4, California
Received June 29, 1950 Revised mahuscript received December
1, 1952
Earlier papers have described an enzyme system found in the tissues of higher plants, a system whose activity consists in the oxidative inactivation of the plant growth hormone indoleacetic acid (MA). Recause this system results in the uptake of one molecule of oxygen pert molecule of IA4 oxidized, we shall refer to it’as the IAA-oxidase system. The facts known at present concerning this system as it occurs in pea seedlings suggest that it consists of two portions, a heavy metal enzyme (1,2) to which is coupled .a light-activatable flavoprotein constituent (3,4). The presence of a heavy metal-containing component has previously been inferred from the fact that the system is inhibited by cyanide, and the present paper will present evidence indicating t,hat this heavy metal-containing component is, in fact, a peroxidase. The flavoprotein component of indoleacetic oxidase was originally suspected because of the fact that the enzyme is light-activated, the action spectrum for such light activation agreeing very closely with the absorption spectrum for riboflavin (3). The photorecept,or for the light
activation
is nondialyzable
and
heat
labile,
suggesting
that
a
flavoprotein rather than a nonprotein-bound form of riboflavin is involved (4). The light activation of IAA oxidase was further shown to consist of the reversal of the effect of an inhibitor which occurs together with the enzyme in the plant. Thus, crude preparations of the enzyme may be rendered more active either by physical removal of the inhibitor or by illumination. results in no significant
Illumination
of an inhibitor-free
preparation
additional activation. The action of the naturally 456
INDOLEACETIC
ACID
OXIDASE
457
SYSTEM
occurring inhibitor can be duplicated by the Mn++ ion (lo-” M) or by C!u++ and Fe++ at higher concentrations ( 10ey n/r). In an attempt to integrate t’hese various fart’s into a single coherent picture of the nature of this enzyme system, we have been guided by two further observations: (a) Goldacre (5) has found that. cat,alase inhibits the action of indoleaeetic acid oxidase. Since cat’alase is essentially specifics for H202, this indicates that H+& may be involved as an intermediary in the action of the system; (6) Bernheim and Dixon (6) have shown that the flavoprotein enzyme, xanthine oxidase, which produces H& upon its reoxidation, is light-activated. This flavoprotein system may be coupled through H20z to a peroxidase, the Hz02 produced by the flavoprot.ein system being utilized by t,he peroxidase to oxidize its own suitable substrate. activated I~educrd R:~voprot,ein
+
I
()?
---+
by light oridized tkivoprotein
I I
unknown reducing agent (IAA?)
FIG. 1. The proposed enzyme constit,uents
+
interrelat,ion of the of t,he indoleacetic
H203
peroxidasc, IAA -- ~- ~- *
block hrre by ratshe or 1)~ Mn++ flavoprot,ein acid osidase
+
oxidized HZ0
IAA
block her<, 1)) cx and heavy metal system of peas.
The fart’s concerning indoleacetic acid oxidase may he formally a+ counted for by the scheme shown in Fig. 1. The present, paper will he tle\voted to a critical consideration of this proposed scheme, and itI will present evidence which, it is believed, demonstrates the c*orre(%ness of this formulation in its essential outline. MATERIALS
AND
METHODS
Alaska peas were grown for 7-O days in vermiculite, in a dark room maintained :it 25 i 1 Y‘. .4t t.hc time of harvest, the illumination necessary for manipulation of thr plants was supplied 1,~ a weak blue light (less than 1 ft.-candle). The epic.otyls were cut off at the ground level, cut rapidly into small pieces, and ground in :I minimal volume of water in a Waring Blendor kept, in a room at 0°C. The resulting brei was passed t.hrough cheesecloth to remove coarse fragments. and t hc crudr ensymp was precipitatrll tly thr addition of #I ml. ~01~1 acctonc: per I00
458
GALSTON,
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ml. of brei. This preparation was further fractionated after it had stood overnight in the cold room in the dark. The supernatant liquid was carefully decanted and discarded, and the precipitate was centrifuged down. The precipitate was then suspended overnight in one-half the original volume of ice-cold phosphatecitrate buffer, pH 6.6, and centrifuged clear. The clear solution, which could he stored for several weeks in the frozen state, was used as the IAA-oxidase preparation. Upon rethawing of a frozen preparation, a small precipitate usually appeared. This could most easily be removed by Seitz filtration. All measurements of the activity of the enzyme were made by calorimetric determination of residual indoleacetic acid (IAA) with Salkowski reagent (I). TABLE I Activity by Catalase, and Reversal of the Inhibition by Light in the text. Initial IAA concentration = 25 pg./ml., corresponding to an initial Salkowski color of 220
Inhibition Description
of IAA-Oxidase
=
Cat&we added per 4 ml. of reaction mixture
IX
Salkowski reaction
color after 35 minutes of in the dark and light (arbitrary units)
Dark
IAA destroyed
Light
Dark
0.1 0.3 1.0 1.6
Light
dml.
w. 0.0
’
126 144 188 222 229
92 .99 100 125 151
-
12.7 10.4 4.3 0.0 0.0
P&-M.
17.3 16.2 16.1 12.3 9.5
’ i
RESULTS
The Inhibiting E$ect of Catalase The observation of Goldacre (5) that catalase inhibits the action of IAA oxidase has been confirmed and extended to show that the inhibition may be reversed by light. For these experiments, crystalline beef liver catalasel was used. Varying quantities of a stock solution containing approximately 1 mg. of catalase/ml. were added to tubes containing 2 ml. IAA oxidase and 0.4 ml. IAA (250 pg./ml. stock solution) in a total
volume
of 4 ml. Duplicate
tubes
were
run in the dark
and light,
the exposure to light being under a battery of daylight fluorescent lamps with an illumination at the reaction mixture of approximately 300 ft.-candles. The results of such an experiment are .shown in Table 1 Obtained
from the Worthington
Biochemical
Laboratory,
Freehold,
N. J.
INDOLEACETIC
ACID
OXIDASE
459
SYSTEM
I. It is clear that catalase inhibits the destruction of IAA by IAA oxidase, and that this inhibition is partially reversed by light. To determine which spectral regions are most effective in reversing the catalase inhibition, thin layers of the reaction mixture were exposed to approximately equal total energies of red, green, and blue light transmitted by various Corning glass filters. Comparison samples were also run in complete darkness and in higher intensity fluorescent light previously described. The data, summarized in Table II, clearly show that of the visible radiation, blue light is most effective in reversing catalase inhibition. In this respect, the light reversal of the catalase inhibition resembles that obtained for Mn++ and for the natural inhibitor. TABLE The E$ect
of Light
Details
Quality
in the Light
text.
II
on the Reversal The
starting
of Catalase IAA
light
of IA&Osidase
was 30
Salkowski color of aliquot taken after 90 min. of reaction
condition
Dark....... Red. Green... Blue....... White (fluorescent intensity)
Inhibition
concentration
241 255 556
240 242 242 189
-
142
pg./ml. IAA
destroyed
1.0 0.7 0.7 9. 1
of higher
The E$ect of Hz02 on the Activity
15.6
oj the Enzyme
Since it is readily possible to achieve complete inhibition of IAAoxidase activity in the dark by adding catalase (Table I>, it would seem possible that this system as a whole may be limited by available H& That this is true is shown by the fact that the addition of low c’oncentrations of Hz02 to the IAA-oxidase system incsreases the rate of enzymatic IAA destruction in the dark. It was first necessary t,o study the etfect of tI& on the direct (nonenzymat,ic) oxidation of IA.4, and to choose a level of H& which could permit, observation of t,he effects of the enzyme. For this study, tubes were set, up contraining 25 eg/ ml. IAA plus varying concentrations of II&. At. int,ervals after mixing the reagents, l-ml. aliquots were removed and added to 1 ml. of Salkowski reagent. It was noticed that in tubes containing H,Oy the pink color characteristic of IAA developed immediately and faded quickly, whereas in the tubes lacking HZ& the color developed slowly, reaching :I maximum after 20 min. This fading effect
460
GALSTON,
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AND
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of Hz02 is probably due to the masking of the Salkowski color complex by residual H202 (12). Therefore, the procedure was altered to remove Hz02 after the completion of the reaction. After 10 min. of reaction time, 1 ml. of catalase (Armour No. 30, 1 mg./ml.) was added to each tube and permitted to react for 5 min. Aliquots were then removed for calorimetric analysis. The colors developed with this procedure were still somewhat unstable but were sufficiently persistent to permit quantitative determinations to be made. The results clearly indicated interference with the Salkowski test by concentrations of Hz02 in excess of 0.3a/o, but negligible interference by concentrations less than 0.03%.
In the experiments which follow, an Hz02 concentration of O.O03oj, (8.8 X 1O-4M) was used. It was observed, as shown in Table III, that in the dark the system enzyme + H202 destroys more IAA than either TABLE
III
Enhancement of Enzymatic IAA Destruction in the Dark Total volume = 10 ml.; initial IAA concentration Starting Salkowski color = 220 __Reaction
IAA oxidizing
mixture Salkowski
HtOz (0.003%)
IAA oxidase Dark _-____-
color after of reaction , -
-__
42 min.
Light
of Hz02
by Addition
= 25 pg./ml.
activity Per cent destruction
of IAA
Dark
Light
0 8.7 28.2 48.0
50.4 51.6
ml.
W&l.
0
0
1 0 1
0 3 3
--___
!
221 216 182 131 -_____
I-
) I
128 125
1
component alone. This indicates then that the IAA oxidase contains a peroxidase which utilizes H202 for the destruction of IAA. The data further indicate that in the dark the destruction of IAA oxidase may be limited
by available
peroxide.
The E$ect of Crystalline Horseradish Root Peroxidase on the Oxidation of IAA The fact that available peroxide limits the rate of the over-all system in the dark but not in the light (Table III) suggested that in the light a peroxidase
component
of the system
could
be rate-limiting.
This
sup-
position is confirmed by the fact that in the light, but not in the dark,
INDOLEACETIC
ACID
OXIDASE
461
SYSTEM
the rate of IAA destruction by IAA oxidase may be increased by addition of further peroxidase. Crystalline horseradish root peroxidase? was made up in a stock solution containing 1 mg. enzyme/ml. redistilled water. Varying quantities of the enzyme were then added to an IAA-Ihh oxidase reaction misture, and aliquots were removed periodically for analysis of residual [Ah. The data (Table IV) clearly show an enhancement of IAA disappearance in the light due to the addition of peroxidase. These results suggested the furt’her possibility that crystalline horseradish root peroxidasc might be able to itself oxidize MA in the preseuw of H&. To test this, reaction mixt,ures were made up to contain either perosidase alone, Hz02 alone, or both. The results (Table T’) show clearly TABLE The Effect
ofCr@alline
Reaction amounts
IV
Horseradish Root Peroridase in the Light and Dark
on the dctivify
ofZ;l:t-O.ridax(,
mixture contained 3 ml. IAA-oxidase , 25 pg./ml. IA-4 of peroxidase in a total volume of 10 ml. Starting Salkowski Salkowski
Peroxidnse
r,q. 0.0
0.1 0 .:3 1 .o
ulded
1
~~
color
~.
after
45 min
_~
nnd varying color = 220
Per cent destruction
of I.AA
-
Dark
Light
Dark
Linht
195 195 192 193
1x5 155 125
12.x 12.5 14.4 14.0 -~
1X.8 35.2 51.2 72.0
does attack
alld dest,roJ
I I
I -1
~~-
~~~
5% ~~-~
that crystalline horseradish root peroxidase IAA in the presence of Hz02.
It. should be ment,ioned that preparations of crude horwr:tdish root peroxid:tw made I)y the t,echniquc of Keilin :tnd Mnnn (7) m:iy shop. IAA-oxid:tsr :tc*t,ivit \. oven in the :bhsence of added H?O,, such :tctivity being ilthil,itnl)l~ 1ly cat:&w or RIn*+ ions (Table VI). These inhibitions WC :tlso I):u-tially rrwrsihlr 1)~ 11111~ light, possibly indicating t,hat light-activated H~O?-genpr:ttirlg fl:rv~,protc~ir~s :IIX’ :Issociat,ed with peroxidase in t,he crude preparations. This same wtivity is ~lsr) found ait,h ccrt& “pure” cryst:tlline horseradish root peroxidnsr prcp:~r:ltiorw Lvhich, aft.er :t suitable incuhLttion period in the presence of MA, suddenly Iwpill 1.0 dest,roy IAA without thr :iddit.ion of I-I&. Such prelimimrry Intent lwriwls, presumably due to the slow production of HrO? in t hr lwepwations, WC gr’wt 1~ .-__--~ 2 Ol)t,:Lin(ld from the Deltu Chemical Clomp:rll~~. SPW York. S. 1’.
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GALSTON,
shortened light.
BONNER
by exposure to blue light
(Table
TABLE The Destruction
of IAA
AND
by Crystalline in the Presence
BAKER
VII),
but are also affected by red
V Horseradish
Root
Peroxidase
of Hz02
All tubes contained 25 rg./ml. IAA and 1 ml. 0.067 M (M/15) pH 6.6 phosphatecitrate buffer in a total volume of 10 ml. Aliquots for Salkowski determinations were taken after 45 min. Starting Salkowski color = 220. 0.003%
Hz02
Crystalline
ml.
Salkowski color after 45 min.
peroxidase mg.
3.0 0.0 0.1 0.3 1.0 3.0
0.0
1.0 1.0 1.0 1.0 1.0
Oxidation
0.0
220 216 207 195 170 131 TABLE
The
Per cent destruction of IAA
8.7 14.1 21.4 31.4 53.2
VI
of IAA by Crude Horseradish Root Peroxidase in the Absence and Its Inhibitabili.?y by Catalase and Mnif
of HzO?
Each tube contained 3 ml. peroxidase + 1 ml. 0.067 M (M/15) pH 6.6 phosphatecitrate buffer + 25rg./ml. IAA in a total volume of 10 ml. Experiment conducted in the dark.
=
Salkowski
color
Per cent destruction
I
Time
Control
1.4 mg. Armour No. 30 catalase
-___
10-4 M 1 Mn++ ___-
’
Control
of IAA
cJf$;‘,“,,
With Mn++ ___-~~
0.0 3.3 4.5
0.0 0.9 4.0
-~
min.
0 15 30
225 118 83
241 233 230
227 225 218
,
0.0 59.2 73.2
Fractionation of the IAA Oxidase from Peas The evidence presented above indicates that the IAA-oxidase system of peas may contain a flavoprotein which produces HzOz, and a peroxidase which utilizes this H202 to oxidize IAA. It was therefore desirable to attempt to demonstrate the presence of these constituents in the enzyme system directly, to separate them with loss of IAA-oxidase activity, and to recombine them with restoration of activity.
INDOLEACETIC
ACID
OXIDASE
463
SYSTEM
ICtiolated pea epicotpls (3.8 kg. fresh weight) were cut into small segments and ground in a Waring Blendor with 60 ml. of ice-cold redistilled water. The resulting slurry was filtered through cheesecloth to remove the coarse fragments. Approximately 3 I. of yellow-green fluorescent juice was obtained. Forty ml. acetone was now added for each 100 ml. juice, and the mixture was allowed to stand overnight in a cold room at O-PC>. The supernatant was then carefull) tiecanted and discarded, and the precipitate was washed three times with a 1: 10 :tcet,one-water mixture, the wash solut,ions being discarded. The washed precipitatr was t.hen suspended in 300 ml. of ice-cold 0.35 saturated (NH,)?SO, previousl>, adjusted to pH 6.6 with concentrated NH&OH. This suspension was stirred fr+ quent,l!. :mtl i hen pcrmitt,ed to stand overnight, in the colt1 room. The follo\vin~ TABLE Oxidation All (U/15) indicate
1’11
oj Z,L1 by C’rystalline Horseradish Root Z’eroridase 0.f H& After Suitable Period of Incubation in (‘ontact tubes contained 1 mg. pH 6.6 phosphate-citrate end of “lag period.”
peroxidase, huf?er
25 pg./ml. IAA in a total volumr
Glkowski
color after
and 0.5 ml. 0.067 .I1 of 5 ml. Dotted lincas
incubation
in
Time Red light
Dark
60
236
Blue
in the Absenrt with IA.4
light
’
High intensity white light
245
147
12X
160
112
102
98
83
7s
I 105
220
1x5
124
I
morning, t,he solution was cent,rifuged clear, and thr prrcipitalr was discarclt~d. The supernatant liquid was :I very active 1A.4.oxidase preparation. Sep:trat,ion of the system into several components \vas nest. ac~complisheti following the technique originally suggested t)y Warburg ant1 Christian (8) for I’(‘moval of t,he prosthetic group of flavoproteins. The cold enzyme in (SH,),SO, w:~s brought to pH 2.5 by the dropwisc addition of 0.1 .V H?SO,. The solution, which was now 0.22 saturated with respect to (NHI),SO,, was pc~rmittrd to stand in an ic.(a l)at,h for30min.. after which time a copious whit,e precipitate had scttlrtl to the t)ottom of the beaker. This precipitat,e. which was cylntrifugrtl oft’ :tnd t,:lkcll up in the usual 0.067 IV (M/15) pH 6.6 I’hosphatc-c.itr.~ltr t~uffer had no IB.\-oxi~ clasp activit,y. The supernatant liquid of thta acid precipitation was :t clear yellow solution possessing a green fluorescrncc. It was :tclJustrcl to pII 6.X5 \Vith c,ollcc~tltt,:ttt,(l
464
GALSTON,
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AND
BAKER
NH,OH and then dialyzed for 72 hr. against ice-cold 0.35 saturated (NH&SO,. The dialyzate was very fluorescent, while the residual material in the dialysis bag was essentially nonfluorescent. The dialyzate was without IAA-oxidase activity; the nondialyzable materials had considerable peroxidase activity and slight IAA-oxidase activity which was greatly augmented by the addition of 0.093% H202. The peroxidase fraction was very active against a wide variety of substrates in conventional peroxidase tests (Table VIII). These tests were performed by mixing 1.0 ml. of enzyme, 0.2 ml. of a 4% solution of substrate in 95% ethanol, and 0.2 ml. of 3% H20*. The only conventional peroxidase substrate which showed no evidence of reaction was tyrosine. The dialyzate fraction was concentrated to small volume in ZKXUO, saturated with phenol, and permitted to stand overnight in a separatory funnel. The top oily (phenolic) layer contained all of the yellow pigment and the fluorescence. TABLE The
Activity
of the
Peroxidase
Fraction Peroxidase Description
VIII of Pea Substrates in the
Enzyme
Toward
Conventional
text
Substrate
Nature of the react ion
Pyrogallol Guaiacol Hydroquinone Benzidine Catechol p-Cresol o-Cresol m-Cresol Tyrosine
Red color Red-brown color Deep wine red color Blue color Brown color Milky white suspension Brown color Yellow color None
This pigment in phenolic solution has an absorption peak at 3600 A., but no peak in the visible spectrum. We have not succeeded in obtaining a satisfactory flavin spectrum for this yellow pigment, presumably because of the presence of pigmented impurities. The pigment can be caused to migrate from the phenolic to the aqueous layer by acidification of the phenol layer with 0.1 N HZSOI. This behavior is in accord with that to be expected of free riboflavin (9). Assay of the crude material contained in the water layer for riboflavin by the Lactobacillus casei method (10) showed it to contain approximately 25 mg. of riboflavin per gram dry weight of assayed material.
Reconstitution
Experiments
It has been demonstrated above that the IAA oxidase of peas can be resolved into three parts, a protein insoluble at pH 2.5 in 0.22 saturated ammonium sulfate at O”C., a dialyzable fraction containing riboflavin,
INDOLEACETIC
ACID
OXIDASE
-465
SYSTEM
and a nondialyzable portion possessing peroxidase activity. It ivas next of interest to attempt to reconstitute enzymatic activity by recombining these fractions. A difficulty experienced in such experiments is the fact, that the peroxidase fract’ion by itself is never eriCrely devoid of IAAoxidase activity, presumably due to the persist’enre in it of some H.& generating system. It is however readily possible to reconstitute IAAoxidase activity by combining the first inactive (T\‘H4)iS04-insoluble precipitate \vith the supernatant containing the flavin and peroxidase fract’ion. It appears to be essential that the protein precipitated at pH 2.5 be immediately taken up in pH tj.6 phosphat,e-citrate buffer and combined \vith the neutralized supernatant cont,aining t,he peroxidasc TABLE Il’econstitulion Precipirate ,411
IS
of ZAA-Ozidase Activity by Combining Solutions of‘ the First ,with the Supernntant Liquid Containing Fluorescent Flavin Peroridase
Protein and
tubes contained 35 rg./ml. IAA, were buffered at pH 6.6, :md had :t totit volume of 5 ml. Initial Salkowski color was 208 Reaction
Protein
solution
mixture
contains Supernalant liquid containing flavin and peroxidase
Ml.
Salkowski min.
30
color after of reaction
Per cent of IA.4 destroyed
rd.
0
299 287 299 165 103
1
0 0
3
1
3
1
and flavin prosthetic group fractions. If stand at pH 2.5 for any ext’ended period activity caannot, be demonstrated. Typical of enzymatic activity are shown in Table (‘oupliny
Xanthine
O~idasc
0.0 3.8 0.0 44.6 65.5
the precipitate is permitted to of time, t’hen reconstit,ution of data indicating reconstitut,ion IX. to I’woxidasc
It is of interest to note than an analog of t,he Ir2A-osidase system catI be formed by roupling crystalline horseradish root, perosidase Cth a completely different flavoprotein, xanthine osidase. An acstive santhille osidase was prepared from cream according to the tecbhniqut of Ball (I 1). ht~ experiment was then cAonduct,editr 1vhic.h sntlt~hine oxidase,
466
GALSTON,
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AND
BAKER
hypoxanthine, and peroxidase were added separately and in combination to IAA in buffer. Aliquots were removed periodically for Salkowski analysis of residual IAA. The data of Table X show that xanthine oxiTABLE X of an Analog of the IAA-Oxidase System by the Combination of Oxidase wilh Crystalline Horseradish Root Peroxidase All tubes contained 25 pg./ml. IAA and 1 ml. of 0.1 M pH 7.2 phosphate buffer in a total volume of 6 ml. Zero-time Salkowski color = 435, the unusually high color being due to pigmentation of the xanthine oxidase preparation. Tubes were incubated-in the dark. The
Synthesis Xanthine
Reaction Xanthine
oxidase
mixture
contains
0.05 N hypoxanthine
Peroxidase
ml.
ml.
WT.
3 3 0 3
0.0 0.9 0.0 0.9
0.0 0.0
Salkowski color after 65 min.
Decrxg;
423 372 430 327
0.1 0.1
in
7 63 5 108
TABLE XI The Effect
of Various Known on the Activity
Flavoprotein Enzyme of the IAA-Oxidase
Substrates and Other System from Peas
Compounds
All tubes contained 25 ag./ml. IAA and 3 ml. IAAoxidase in a total volume of 10 ml. Addendum
Hypoxanthine, 506 pg./ml. Xanthine, 500 pg./ml. Uric acid, 506 pg./ml. Adenine sulfate, 500 pg./ml. Adenine sulfate, 50 pg./ml. nL-a-Alanine, 400 pg./ml. Reduced coenzyme I, 406 pg./ml. Formaldehyde, 406 pg./ml. Succinic acid, 400 pg./ml.
Effect
on enzyme
activity
Complete inhibition Complete inhibition Complete inhibition Complete inhibition No effect No effect No effect Effect unknown because of interference with Salkowski color No effect
dase itself brings about an appreciable destruction of IAA, presumably due to nonenzymatic effects of the HeOn produced in hypoxanthine oxidation. The addition of crystalline peroxidase to the system results, however, in an increased rate of IAA disappearance.
INDOLEACETIC
ACID
OXIDASE
SYSTEM
467
This system differs from IAA oxidase in at least one important respect. The inactivation of IAA in the analog system does not occur unless some substrate for the xanthine oxidase, such as hypoxanthine, is added. Peroxidative destruction of IAA is thus directly dependent upon the oxidation of hypoxanthine. With the IAA-oxidase system, on the contrary, no second substrate seems to be required. Substrate for the Flavoprotei,n h’nzyme The previous experiments have yielded no information relative ,to the nature of the subst’rate for the flavoprotein part, of the IAA-oxidase syst,em. This problem was attacked by attempting to add to the enzyme-K4 syst,em in the dark (H202 limited) various known flavoprotein substrates. If the substrate were utilized, then more H,O: should be produced and the activity of the system should be increased. The various substances added to the system and their effects on JAA destruction are tested in Table XI. Plv’one of the added compounds caused any increase in the rate of disappearance of IAA, and, in fact, all of the purines added were markedly inhibitory at levels of approximately 500 pg./ml. The substrate for the flavoprotein enzyme, therefore, remains obscure, but, certain facts brought out below suggest that it may he IAA itself. DISCUSSION The evidence presented in this paper, together with that of earliel &dies on the inhibition (1,2) and light activat,ion (3,q) of the IAAosidase system of peas, suggests that a flavoprotein and a peroxidase may caooperate in the destruction of the IAA molecule. The data are further consistent with the view that the flavoprotein may oxidize an as yet undetermined substrate with the product’ion of Hz&, and that t,he peroxidase acromplishes the final oxidation of IAA using the HZOz produred in the first reaction. The participation of I&O, is indicated by the inhibitory effects of catalase, Mn++, and other ions which decompose Hz02 as well as by the stimulatory effect of low concentrations of Hz02 on t’he rate of enzymatic IAA destruc*tion in the dark. The presence of a funrtional flavoprotein is indicated by the action spectrum for the light act,ivat,ion. The participat,ion of peroxidase is indicat,ed by t,he (1N-inhibition, by the effect of peroxidase in increasing the reaction rate of the system in the light, and by the in vitro destruction of IAA by crystalline peroxidase. The entire arguments is strengthened by the fact t,hat’ the artificial system santhine oxidase and peroxidase operates in a
438
GALSTON,
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AND
BAKER
manner somewhat similar to the IAA-oxidase system itself, and also by the fact that other enzyme systems, such as the tryptophan oxidase of liver (13) and the triphosphopyridine nucleotide (TPN)-oxidase of plants (14) show similar behavior and have been explained by analogous mechanisms. It should be emphasized that the scheme as outlined. represents merely one possible integration of the data, and other formulations are certainly not excluded. One possible alternative stems from the observation of, Swedin and Theorell (15) that peroxidase acts as an oxidase toward dioxymaleic acid. In the presence of peroxidase, this substrate spontaneously produces enough HZ& to Enitiate the peroxidative action of the enzyme. The fact that all onr preparations of crystalline peroxi.dase oxidize IAA after a suitable lag period (Table VII) would lend support to the concept that IAA in the presence of peroxidase may similarly produce a peroxide which then permits the peroxidative destruction of other IAA molecules. Further support for this idea lies in other similarities between the IAA and dioxymaleic acid oxidase systems, such as inhibition by catalase and cyanide, irregular inhibition,by CO, and large effects of the Mn++ ion. If such a system were operating, the peroxidase would be the enzyme specific for IAA destruction, and the flavoprotein merely subsidiary as a nonspecific producer of Hz02 for the peroxidase. ..,;With regard to CO, Tang and Bonner (1) originally reported a lightreversible CO inhibition, while Wagenknecht and Burris (2) could find no CO inhibition at all. This difference of experience can also be accounted for in terms of the dioxymaleic oxidase type of reaction. Swedin and Theorell (15) reported that the production of peroxide from substrate apparently involves a ferrous g ferric transformation of the peroxidase and is light-reversibly inhibited by CO. Once peroxide is present, the enzyme acts as a peroxidase, and since this action involves no reduction of the ferric nucleus, CO is without effect. Thus, one may postulate that Tang-Bonner experiments were carried out in systems where peroxide formation was the rate-limiting step, while the Wagenknecht-Burris experiments were limited by peroxidative action. Such a possibility will be examined further in the future. With regard to the physiological significance of LAA oxidase, previous views on its function in the plant (16) must be revised in view of the fact that we now recognize the system to be activated by blue light and to be .present in green as well as in etiolated plants (4). The activation
INDOLEACETIC
ACID OXIDASE
SYSTEM
469
of the IAA-oxidase system by light occurs only in the presence of an inhibitor such as catalase or Mn++ ions, whose action is reversed by light,. It must be anticipated therefore that inactivation of IAA by the IAA-oxidase system in the plant may also be a process accelerated by light, and specifically by blue light. Among the processesmediated in the plant by blue light’ is phototropic curvature, which is known to be clue t,o changes in auxin level induced by light (17). It will therefore be of great interest to determine to what extent the light activation of ILL1 oxidase may be responsible for the phototropic response. SUMMARY
I. Several lines of evidence indicate that the TAB-osidasc system of peas consists of a light,-artivatable flavoproteill enzyme coupled through H20L’to a peroxidase. (n) The system is inhibited by rat’alase, the inhibit,ion being reversed by blue light. (b) The activity of the system is increased by the addition of Hz& ill the dark, and by the addition of crystalline horseradish root, peroxidase in the light. This indicates that H,O, limits t’he react#ion in the clurk; in the light, H202 production is incareasedand the peroxidasc lw c*omcslimiting. (r) Crystalline horseradish root perosidase readily oxidizes IAB in thcapresence of H20Z. (4) The IAA-oxidase syst,emhas been separated int#oa peroxidase ant1 3. fiavin-c,ontaining component. The peroxidase component is active in the presence of H202 in oxidizing IAA and wnventional peroxidase suhstrates. Successful reconstitution of the IAA-oxidase system from itJs fragments has been arhieved. The substjrat8efor the flavin enzyme is tl~tknowri, but may he IAA itself. (12)_4n analog of the IAA-oxidase syst’em has been made by combinilig the santhine osidase of cream with the crystBalline peroxidase of horseradish root. The analog system diRers from IAA osiclasein requirillg n substrate other than L\A for the flavoprotein. 2. I’ossihlc alt8ernati\-e schemeshave been esaminrcl.
470 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
GALSTON,
BONNER
AND
BAKER
GOLDACRE, P. L., Australian J. Sci. Research Ser. B 4, 293 (1951). BERNHEIM, F., AND DIXON, M., Biochem. J. 22, 113 (1928). KEILIN, D., AND MANN, T., Proc. Roy. Sot. (London) Bl22, 119 (1937). WARBURG, O., AND CHRISTIAN, W., Biochem. 2. 298, 150 (1938). CRAMMER, J. L., Nature 161, 349 (1948). SNELL, E. E., AND STRONG, F. M., Ind. Eng. Chem., Anal. Ed. 11, 346 (1939) BALL, E. G., J. Biol. Chem. 128,51 (1939). SIEGEL, S. M., AND WEINTRAUB, R. L., Physiol Plantarum 6, 241 (1952). KNOX, W. E., AND MEHLER, A. H., J. Biol. Chem. 187, 419 (1950). CONN, E., KRAEMER, L. M., LILT, P-N., and VENNESLAND, B., J. Biol. Chem. 194, 143 (1951). 15. SWEDIN, B., AND THEORELL, II., Nature 146,71 (1940). 16. TANG, Y. W., AND BONNER, J., Am. J. Botany 36, 570 (1948). 17. GALSTON, A. W., Botan. Rev. 16,361 (1950).
the Kerckho$
Laboratories of the Technology, Pasadena
Biology, California
S. Baker Institute
of
4, California
Received June 29, 1950 Revised mahuscript received December
1, 1952
Earlier papers have described an enzyme system found in the tissues of higher plants, a system whose activity consists in the oxidative inactivation of the plant growth hormone indoleacetic acid (MA). Recause this system results in the uptake of one molecule of oxygen pert molecule of IA4 oxidized, we shall refer to it’as the IAA-oxidase system. The facts known at present concerning this system as it occurs in pea seedlings suggest that it consists of two portions, a heavy metal enzyme (1,2) to which is coupled .a light-activatable flavoprotein constituent (3,4). The presence of a heavy metal-containing component has previously been inferred from the fact that the system is inhibited by cyanide, and the present paper will present evidence indicating t,hat this heavy metal-containing component is, in fact, a peroxidase. The flavoprotein component of indoleacetic oxidase was originally suspected because of the fact that the enzyme is light-activated, the action spectrum for such light activation agreeing very closely with the absorption spectrum for riboflavin (3). The photorecept,or for the light
activation
is nondialyzable
and
heat
labile,
suggesting
that
a
flavoprotein rather than a nonprotein-bound form of riboflavin is involved (4). The light activation of IAA oxidase was further shown to consist of the reversal of the effect of an inhibitor which occurs together with the enzyme in the plant. Thus, crude preparations of the enzyme may be rendered more active either by physical removal of the inhibitor or by illumination. results in no significant
Illumination
of an inhibitor-free
preparation
additional activation. The action of the naturally 456
INDOLEACETIC
ACID
OXIDASE
457
SYSTEM
occurring inhibitor can be duplicated by the Mn++ ion (lo-” M) or by C!u++ and Fe++ at higher concentrations ( 10ey n/r). In an attempt to integrate t’hese various fart’s into a single coherent picture of the nature of this enzyme system, we have been guided by two further observations: (a) Goldacre (5) has found that. cat,alase inhibits the action of indoleaeetic acid oxidase. Since cat’alase is essentially specifics for H202, this indicates that H+& may be involved as an intermediary in the action of the system; (6) Bernheim and Dixon (6) have shown that the flavoprotein enzyme, xanthine oxidase, which produces H& upon its reoxidation, is light-activated. This flavoprotein system may be coupled through H20z to a peroxidase, the Hz02 produced by the flavoprot.ein system being utilized by t,he peroxidase to oxidize its own suitable substrate. activated I~educrd R:~voprot,ein
+
I
()?
---+
by light oridized tkivoprotein
I I
unknown reducing agent (IAA?)
FIG. 1. The proposed enzyme constit,uents
+
interrelat,ion of the of t,he indoleacetic
H203
peroxidasc, IAA -- ~- ~- *
block hrre by ratshe or 1)~ Mn++ flavoprot,ein acid osidase
+
oxidized HZ0
IAA
block her<, 1)) cx and heavy metal system of peas.
The fart’s concerning indoleacetic acid oxidase may he formally a+ counted for by the scheme shown in Fig. 1. The present, paper will he tle\voted to a critical consideration of this proposed scheme, and itI will present evidence which, it is believed, demonstrates the c*orre(%ness of this formulation in its essential outline. MATERIALS
AND
METHODS
Alaska peas were grown for 7-O days in vermiculite, in a dark room maintained :it 25 i 1 Y‘. .4t t.hc time of harvest, the illumination necessary for manipulation of thr plants was supplied 1,~ a weak blue light (less than 1 ft.-candle). The epic.otyls were cut off at the ground level, cut rapidly into small pieces, and ground in :I minimal volume of water in a Waring Blendor kept, in a room at 0°C. The resulting brei was passed t.hrough cheesecloth to remove coarse fragments. and t hc crudr ensymp was precipitatrll tly thr addition of #I ml. ~01~1 acctonc: per I00
458
GALSTON,
BONNER AND BAKER
ml. of brei. This preparation was further fractionated after it had stood overnight in the cold room in the dark. The supernatant liquid was carefully decanted and discarded, and the precipitate was centrifuged down. The precipitate was then suspended overnight in one-half the original volume of ice-cold phosphatecitrate buffer, pH 6.6, and centrifuged clear. The clear solution, which could he stored for several weeks in the frozen state, was used as the IAA-oxidase preparation. Upon rethawing of a frozen preparation, a small precipitate usually appeared. This could most easily be removed by Seitz filtration. All measurements of the activity of the enzyme were made by calorimetric determination of residual indoleacetic acid (IAA) with Salkowski reagent (I). TABLE I Activity by Catalase, and Reversal of the Inhibition by Light in the text. Initial IAA concentration = 25 pg./ml., corresponding to an initial Salkowski color of 220
Inhibition Description
of IAA-Oxidase
=
Cat&we added per 4 ml. of reaction mixture
IX
Salkowski reaction
color after 35 minutes of in the dark and light (arbitrary units)
Dark
IAA destroyed
Light
Dark
0.1 0.3 1.0 1.6
Light
dml.
w. 0.0
’
126 144 188 222 229
92 .99 100 125 151
-
12.7 10.4 4.3 0.0 0.0
P&-M.
17.3 16.2 16.1 12.3 9.5
’ i
RESULTS
The Inhibiting E$ect of Catalase The observation of Goldacre (5) that catalase inhibits the action of IAA oxidase has been confirmed and extended to show that the inhibition may be reversed by light. For these experiments, crystalline beef liver catalasel was used. Varying quantities of a stock solution containing approximately 1 mg. of catalase/ml. were added to tubes containing 2 ml. IAA oxidase and 0.4 ml. IAA (250 pg./ml. stock solution) in a total
volume
of 4 ml. Duplicate
tubes
were
run in the dark
and light,
the exposure to light being under a battery of daylight fluorescent lamps with an illumination at the reaction mixture of approximately 300 ft.-candles. The results of such an experiment are .shown in Table 1 Obtained
from the Worthington
Biochemical
Laboratory,
Freehold,
N. J.
INDOLEACETIC
ACID
OXIDASE
459
SYSTEM
I. It is clear that catalase inhibits the destruction of IAA by IAA oxidase, and that this inhibition is partially reversed by light. To determine which spectral regions are most effective in reversing the catalase inhibition, thin layers of the reaction mixture were exposed to approximately equal total energies of red, green, and blue light transmitted by various Corning glass filters. Comparison samples were also run in complete darkness and in higher intensity fluorescent light previously described. The data, summarized in Table II, clearly show that of the visible radiation, blue light is most effective in reversing catalase inhibition. In this respect, the light reversal of the catalase inhibition resembles that obtained for Mn++ and for the natural inhibitor. TABLE The E$ect
of Light
Details
Quality
in the Light
text.
II
on the Reversal The
starting
of Catalase IAA
light
of IA&Osidase
was 30
Salkowski color of aliquot taken after 90 min. of reaction
condition
Dark....... Red. Green... Blue....... White (fluorescent intensity)
Inhibition
concentration
241 255 556
240 242 242 189
-
142
pg./ml. IAA
destroyed
1.0 0.7 0.7 9. 1
of higher
The E$ect of Hz02 on the Activity
15.6
oj the Enzyme
Since it is readily possible to achieve complete inhibition of IAAoxidase activity in the dark by adding catalase (Table I>, it would seem possible that this system as a whole may be limited by available H& That this is true is shown by the fact that the addition of low c’oncentrations of Hz02 to the IAA-oxidase system incsreases the rate of enzymatic IAA destruction in the dark. It was first necessary t,o study the etfect of tI& on the direct (nonenzymat,ic) oxidation of IA.4, and to choose a level of H& which could permit, observation of t,he effects of the enzyme. For this study, tubes were set, up contraining 25 eg/ ml. IAA plus varying concentrations of II&. At. int,ervals after mixing the reagents, l-ml. aliquots were removed and added to 1 ml. of Salkowski reagent. It was noticed that in tubes containing H,Oy the pink color characteristic of IAA developed immediately and faded quickly, whereas in the tubes lacking HZ& the color developed slowly, reaching :I maximum after 20 min. This fading effect
460
GALSTON,
BONNER
AND
BAKER
of Hz02 is probably due to the masking of the Salkowski color complex by residual H202 (12). Therefore, the procedure was altered to remove Hz02 after the completion of the reaction. After 10 min. of reaction time, 1 ml. of catalase (Armour No. 30, 1 mg./ml.) was added to each tube and permitted to react for 5 min. Aliquots were then removed for calorimetric analysis. The colors developed with this procedure were still somewhat unstable but were sufficiently persistent to permit quantitative determinations to be made. The results clearly indicated interference with the Salkowski test by concentrations of Hz02 in excess of 0.3a/o, but negligible interference by concentrations less than 0.03%.
In the experiments which follow, an Hz02 concentration of O.O03oj, (8.8 X 1O-4M) was used. It was observed, as shown in Table III, that in the dark the system enzyme + H202 destroys more IAA than either TABLE
III
Enhancement of Enzymatic IAA Destruction in the Dark Total volume = 10 ml.; initial IAA concentration Starting Salkowski color = 220 __Reaction
IAA oxidizing
mixture Salkowski
HtOz (0.003%)
IAA oxidase Dark _-____-
color after of reaction , -
-__
42 min.
Light
of Hz02
by Addition
= 25 pg./ml.
activity Per cent destruction
of IAA
Dark
Light
0 8.7 28.2 48.0
50.4 51.6
ml.
W&l.
0
0
1 0 1
0 3 3
--___
!
221 216 182 131 -_____
I-
) I
128 125
1
component alone. This indicates then that the IAA oxidase contains a peroxidase which utilizes H202 for the destruction of IAA. The data further indicate that in the dark the destruction of IAA oxidase may be limited
by available
peroxide.
The E$ect of Crystalline Horseradish Root Peroxidase on the Oxidation of IAA The fact that available peroxide limits the rate of the over-all system in the dark but not in the light (Table III) suggested that in the light a peroxidase
component
of the system
could
be rate-limiting.
This
sup-
position is confirmed by the fact that in the light, but not in the dark,
INDOLEACETIC
ACID
OXIDASE
461
SYSTEM
the rate of IAA destruction by IAA oxidase may be increased by addition of further peroxidase. Crystalline horseradish root peroxidase? was made up in a stock solution containing 1 mg. enzyme/ml. redistilled water. Varying quantities of the enzyme were then added to an IAA-Ihh oxidase reaction misture, and aliquots were removed periodically for analysis of residual [Ah. The data (Table IV) clearly show an enhancement of IAA disappearance in the light due to the addition of peroxidase. These results suggested the furt’her possibility that crystalline horseradish root peroxidasc might be able to itself oxidize MA in the preseuw of H&. To test this, reaction mixt,ures were made up to contain either perosidase alone, Hz02 alone, or both. The results (Table T’) show clearly TABLE The Effect
ofCr@alline
Reaction amounts
IV
Horseradish Root Peroridase in the Light and Dark
on the dctivify
ofZ;l:t-O.ridax(,
mixture contained 3 ml. IAA-oxidase , 25 pg./ml. IA-4 of peroxidase in a total volume of 10 ml. Starting Salkowski Salkowski
Peroxidnse
r,q. 0.0
0.1 0 .:3 1 .o
ulded
1
~~
color
~.
after
45 min
_~
nnd varying color = 220
Per cent destruction
of I.AA
-
Dark
Light
Dark
Linht
195 195 192 193
1x5 155 125
12.x 12.5 14.4 14.0 -~
1X.8 35.2 51.2 72.0
does attack
alld dest,roJ
I I
I -1
~~-
~~~
5% ~~-~
that crystalline horseradish root peroxidase IAA in the presence of Hz02.
It. should be ment,ioned that preparations of crude horwr:tdish root peroxid:tw made I)y the t,echniquc of Keilin :tnd Mnnn (7) m:iy shop. IAA-oxid:tsr :tc*t,ivit \. oven in the :bhsence of added H?O,, such :tctivity being ilthil,itnl)l~ 1ly cat:&w or RIn*+ ions (Table VI). These inhibitions WC :tlso I):u-tially rrwrsihlr 1)~ 11111~ light, possibly indicating t,hat light-activated H~O?-genpr:ttirlg fl:rv~,protc~ir~s :IIX’ :Issociat,ed with peroxidase in t,he crude preparations. This same wtivity is ~lsr) found ait,h ccrt& “pure” cryst:tlline horseradish root peroxidnsr prcp:~r:ltiorw Lvhich, aft.er :t suitable incuhLttion period in the presence of MA, suddenly Iwpill 1.0 dest,roy IAA without thr :iddit.ion of I-I&. Such prelimimrry Intent lwriwls, presumably due to the slow production of HrO? in t hr lwepwations, WC gr’wt 1~ .-__--~ 2 Ol)t,:Lin(ld from the Deltu Chemical Clomp:rll~~. SPW York. S. 1’.
462
GALSTON,
shortened light.
BONNER
by exposure to blue light
(Table
TABLE The Destruction
of IAA
AND
by Crystalline in the Presence
BAKER
VII),
but are also affected by red
V Horseradish
Root
Peroxidase
of Hz02
All tubes contained 25 rg./ml. IAA and 1 ml. 0.067 M (M/15) pH 6.6 phosphatecitrate buffer in a total volume of 10 ml. Aliquots for Salkowski determinations were taken after 45 min. Starting Salkowski color = 220. 0.003%
Hz02
Crystalline
ml.
Salkowski color after 45 min.
peroxidase mg.
3.0 0.0 0.1 0.3 1.0 3.0
0.0
1.0 1.0 1.0 1.0 1.0
Oxidation
0.0
220 216 207 195 170 131 TABLE
The
Per cent destruction of IAA
8.7 14.1 21.4 31.4 53.2
VI
of IAA by Crude Horseradish Root Peroxidase in the Absence and Its Inhibitabili.?y by Catalase and Mnif
of HzO?
Each tube contained 3 ml. peroxidase + 1 ml. 0.067 M (M/15) pH 6.6 phosphatecitrate buffer + 25rg./ml. IAA in a total volume of 10 ml. Experiment conducted in the dark.
=
Salkowski
color
Per cent destruction
I
Time
Control
1.4 mg. Armour No. 30 catalase
-___
10-4 M 1 Mn++ ___-
’
Control
of IAA
cJf$;‘,“,,
With Mn++ ___-~~
0.0 3.3 4.5
0.0 0.9 4.0
-~
min.
0 15 30
225 118 83
241 233 230
227 225 218
,
0.0 59.2 73.2
Fractionation of the IAA Oxidase from Peas The evidence presented above indicates that the IAA-oxidase system of peas may contain a flavoprotein which produces HzOz, and a peroxidase which utilizes this H202 to oxidize IAA. It was therefore desirable to attempt to demonstrate the presence of these constituents in the enzyme system directly, to separate them with loss of IAA-oxidase activity, and to recombine them with restoration of activity.
INDOLEACETIC
ACID
OXIDASE
463
SYSTEM
ICtiolated pea epicotpls (3.8 kg. fresh weight) were cut into small segments and ground in a Waring Blendor with 60 ml. of ice-cold redistilled water. The resulting slurry was filtered through cheesecloth to remove the coarse fragments. Approximately 3 I. of yellow-green fluorescent juice was obtained. Forty ml. acetone was now added for each 100 ml. juice, and the mixture was allowed to stand overnight in a cold room at O-PC>. The supernatant was then carefull) tiecanted and discarded, and the precipitate was washed three times with a 1: 10 :tcet,one-water mixture, the wash solut,ions being discarded. The washed precipitatr was t.hen suspended in 300 ml. of ice-cold 0.35 saturated (NH,)?SO, previousl>, adjusted to pH 6.6 with concentrated NH&OH. This suspension was stirred fr+ quent,l!. :mtl i hen pcrmitt,ed to stand overnight, in the colt1 room. The follo\vin~ TABLE Oxidation All (U/15) indicate
1’11
oj Z,L1 by C’rystalline Horseradish Root Z’eroridase 0.f H& After Suitable Period of Incubation in (‘ontact tubes contained 1 mg. pH 6.6 phosphate-citrate end of “lag period.”
peroxidase, huf?er
25 pg./ml. IAA in a total volumr
Glkowski
color after
and 0.5 ml. 0.067 .I1 of 5 ml. Dotted lincas
incubation
in
Time Red light
Dark
60
236
Blue
in the Absenrt with IA.4
light
’
High intensity white light
245
147
12X
160
112
102
98
83
7s
I 105
220
1x5
124
I
morning, t,he solution was cent,rifuged clear, and thr prrcipitalr was discarclt~d. The supernatant liquid was :I very active 1A.4.oxidase preparation. Sep:trat,ion of the system into several components \vas nest. ac~complisheti following the technique originally suggested t)y Warburg ant1 Christian (8) for I’(‘moval of t,he prosthetic group of flavoproteins. The cold enzyme in (SH,),SO, w:~s brought to pH 2.5 by the dropwisc addition of 0.1 .V H?SO,. The solution, which was now 0.22 saturated with respect to (NHI),SO,, was pc~rmittrd to stand in an ic.(a l)at,h for30min.. after which time a copious whit,e precipitate had scttlrtl to the t)ottom of the beaker. This precipitat,e. which was cylntrifugrtl oft’ :tnd t,:lkcll up in the usual 0.067 IV (M/15) pH 6.6 I’hosphatc-c.itr.~ltr t~uffer had no IB.\-oxi~ clasp activit,y. The supernatant liquid of thta acid precipitation was :t clear yellow solution possessing a green fluorescrncc. It was :tclJustrcl to pII 6.X5 \Vith c,ollcc~tltt,:ttt,(l
464
GALSTON,
BONNER
AND
BAKER
NH,OH and then dialyzed for 72 hr. against ice-cold 0.35 saturated (NH&SO,. The dialyzate was very fluorescent, while the residual material in the dialysis bag was essentially nonfluorescent. The dialyzate was without IAA-oxidase activity; the nondialyzable materials had considerable peroxidase activity and slight IAA-oxidase activity which was greatly augmented by the addition of 0.093% H202. The peroxidase fraction was very active against a wide variety of substrates in conventional peroxidase tests (Table VIII). These tests were performed by mixing 1.0 ml. of enzyme, 0.2 ml. of a 4% solution of substrate in 95% ethanol, and 0.2 ml. of 3% H20*. The only conventional peroxidase substrate which showed no evidence of reaction was tyrosine. The dialyzate fraction was concentrated to small volume in ZKXUO, saturated with phenol, and permitted to stand overnight in a separatory funnel. The top oily (phenolic) layer contained all of the yellow pigment and the fluorescence. TABLE The
Activity
of the
Peroxidase
Fraction Peroxidase Description
VIII of Pea Substrates in the
Enzyme
Toward
Conventional
text
Substrate
Nature of the react ion
Pyrogallol Guaiacol Hydroquinone Benzidine Catechol p-Cresol o-Cresol m-Cresol Tyrosine
Red color Red-brown color Deep wine red color Blue color Brown color Milky white suspension Brown color Yellow color None
This pigment in phenolic solution has an absorption peak at 3600 A., but no peak in the visible spectrum. We have not succeeded in obtaining a satisfactory flavin spectrum for this yellow pigment, presumably because of the presence of pigmented impurities. The pigment can be caused to migrate from the phenolic to the aqueous layer by acidification of the phenol layer with 0.1 N HZSOI. This behavior is in accord with that to be expected of free riboflavin (9). Assay of the crude material contained in the water layer for riboflavin by the Lactobacillus casei method (10) showed it to contain approximately 25 mg. of riboflavin per gram dry weight of assayed material.
Reconstitution
Experiments
It has been demonstrated above that the IAA oxidase of peas can be resolved into three parts, a protein insoluble at pH 2.5 in 0.22 saturated ammonium sulfate at O”C., a dialyzable fraction containing riboflavin,
INDOLEACETIC
ACID
OXIDASE
-465
SYSTEM
and a nondialyzable portion possessing peroxidase activity. It ivas next of interest to attempt to reconstitute enzymatic activity by recombining these fractions. A difficulty experienced in such experiments is the fact, that the peroxidase fract’ion by itself is never eriCrely devoid of IAAoxidase activity, presumably due to the persist’enre in it of some H.& generating system. It is however readily possible to reconstitute IAAoxidase activity by combining the first inactive (T\‘H4)iS04-insoluble precipitate \vith the supernatant containing the flavin and peroxidase fract’ion. It appears to be essential that the protein precipitated at pH 2.5 be immediately taken up in pH tj.6 phosphat,e-citrate buffer and combined \vith the neutralized supernatant cont,aining t,he peroxidasc TABLE Il’econstitulion Precipirate ,411
IS
of ZAA-Ozidase Activity by Combining Solutions of‘ the First ,with the Supernntant Liquid Containing Fluorescent Flavin Peroridase
Protein and
tubes contained 35 rg./ml. IAA, were buffered at pH 6.6, :md had :t totit volume of 5 ml. Initial Salkowski color was 208 Reaction
Protein
solution
mixture
contains Supernalant liquid containing flavin and peroxidase
Ml.
Salkowski min.
30
color after of reaction
Per cent of IA.4 destroyed
rd.
0
299 287 299 165 103
1
0 0
3
1
3
1
and flavin prosthetic group fractions. If stand at pH 2.5 for any ext’ended period activity caannot, be demonstrated. Typical of enzymatic activity are shown in Table (‘oupliny
Xanthine
O~idasc
0.0 3.8 0.0 44.6 65.5
the precipitate is permitted to of time, t’hen reconstit,ution of data indicating reconstitut,ion IX. to I’woxidasc
It is of interest to note than an analog of t,he Ir2A-osidase system catI be formed by roupling crystalline horseradish root, perosidase Cth a completely different flavoprotein, xanthine osidase. An acstive santhille osidase was prepared from cream according to the tecbhniqut of Ball (I 1). ht~ experiment was then cAonduct,editr 1vhic.h sntlt~hine oxidase,
466
GALSTON,
BONNER
AND
BAKER
hypoxanthine, and peroxidase were added separately and in combination to IAA in buffer. Aliquots were removed periodically for Salkowski analysis of residual IAA. The data of Table X show that xanthine oxiTABLE X of an Analog of the IAA-Oxidase System by the Combination of Oxidase wilh Crystalline Horseradish Root Peroxidase All tubes contained 25 pg./ml. IAA and 1 ml. of 0.1 M pH 7.2 phosphate buffer in a total volume of 6 ml. Zero-time Salkowski color = 435, the unusually high color being due to pigmentation of the xanthine oxidase preparation. Tubes were incubated-in the dark. The
Synthesis Xanthine
Reaction Xanthine
oxidase
mixture
contains
0.05 N hypoxanthine
Peroxidase
ml.
ml.
WT.
3 3 0 3
0.0 0.9 0.0 0.9
0.0 0.0
Salkowski color after 65 min.
Decrxg;
423 372 430 327
0.1 0.1
in
7 63 5 108
TABLE XI The Effect
of Various Known on the Activity
Flavoprotein Enzyme of the IAA-Oxidase
Substrates and Other System from Peas
Compounds
All tubes contained 25 ag./ml. IAA and 3 ml. IAAoxidase in a total volume of 10 ml. Addendum
Hypoxanthine, 506 pg./ml. Xanthine, 500 pg./ml. Uric acid, 506 pg./ml. Adenine sulfate, 500 pg./ml. Adenine sulfate, 50 pg./ml. nL-a-Alanine, 400 pg./ml. Reduced coenzyme I, 406 pg./ml. Formaldehyde, 406 pg./ml. Succinic acid, 400 pg./ml.
Effect
on enzyme
activity
Complete inhibition Complete inhibition Complete inhibition Complete inhibition No effect No effect No effect Effect unknown because of interference with Salkowski color No effect
dase itself brings about an appreciable destruction of IAA, presumably due to nonenzymatic effects of the HeOn produced in hypoxanthine oxidation. The addition of crystalline peroxidase to the system results, however, in an increased rate of IAA disappearance.
INDOLEACETIC
ACID
OXIDASE
SYSTEM
467
This system differs from IAA oxidase in at least one important respect. The inactivation of IAA in the analog system does not occur unless some substrate for the xanthine oxidase, such as hypoxanthine, is added. Peroxidative destruction of IAA is thus directly dependent upon the oxidation of hypoxanthine. With the IAA-oxidase system, on the contrary, no second substrate seems to be required. Substrate for the Flavoprotei,n h’nzyme The previous experiments have yielded no information relative ,to the nature of the subst’rate for the flavoprotein part, of the IAA-oxidase syst,em. This problem was attacked by attempting to add to the enzyme-K4 syst,em in the dark (H202 limited) various known flavoprotein substrates. If the substrate were utilized, then more H,O: should be produced and the activity of the system should be increased. The various substances added to the system and their effects on JAA destruction are tested in Table XI. Plv’one of the added compounds caused any increase in the rate of disappearance of IAA, and, in fact, all of the purines added were markedly inhibitory at levels of approximately 500 pg./ml. The substrate for the flavoprotein enzyme, therefore, remains obscure, but, certain facts brought out below suggest that it may he IAA itself. DISCUSSION The evidence presented in this paper, together with that of earliel &dies on the inhibition (1,2) and light activat,ion (3,q) of the IAAosidase system of peas, suggests that a flavoprotein and a peroxidase may caooperate in the destruction of the IAA molecule. The data are further consistent with the view that the flavoprotein may oxidize an as yet undetermined substrate with the product’ion of Hz&, and that t,he peroxidase acromplishes the final oxidation of IAA using the HZOz produred in the first reaction. The participation of I&O, is indicated by the inhibitory effects of catalase, Mn++, and other ions which decompose Hz02 as well as by the stimulatory effect of low concentrations of Hz02 on t’he rate of enzymatic IAA destruc*tion in the dark. The presence of a funrtional flavoprotein is indicated by the action spectrum for the light act,ivat,ion. The participat,ion of peroxidase is indicat,ed by t,he (1N-inhibition, by the effect of peroxidase in increasing the reaction rate of the system in the light, and by the in vitro destruction of IAA by crystalline peroxidase. The entire arguments is strengthened by the fact t,hat’ the artificial system santhine oxidase and peroxidase operates in a
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manner somewhat similar to the IAA-oxidase system itself, and also by the fact that other enzyme systems, such as the tryptophan oxidase of liver (13) and the triphosphopyridine nucleotide (TPN)-oxidase of plants (14) show similar behavior and have been explained by analogous mechanisms. It should be emphasized that the scheme as outlined. represents merely one possible integration of the data, and other formulations are certainly not excluded. One possible alternative stems from the observation of, Swedin and Theorell (15) that peroxidase acts as an oxidase toward dioxymaleic acid. In the presence of peroxidase, this substrate spontaneously produces enough HZ& to Enitiate the peroxidative action of the enzyme. The fact that all onr preparations of crystalline peroxi.dase oxidize IAA after a suitable lag period (Table VII) would lend support to the concept that IAA in the presence of peroxidase may similarly produce a peroxide which then permits the peroxidative destruction of other IAA molecules. Further support for this idea lies in other similarities between the IAA and dioxymaleic acid oxidase systems, such as inhibition by catalase and cyanide, irregular inhibition,by CO, and large effects of the Mn++ ion. If such a system were operating, the peroxidase would be the enzyme specific for IAA destruction, and the flavoprotein merely subsidiary as a nonspecific producer of Hz02 for the peroxidase. ..,;With regard to CO, Tang and Bonner (1) originally reported a lightreversible CO inhibition, while Wagenknecht and Burris (2) could find no CO inhibition at all. This difference of experience can also be accounted for in terms of the dioxymaleic oxidase type of reaction. Swedin and Theorell (15) reported that the production of peroxide from substrate apparently involves a ferrous g ferric transformation of the peroxidase and is light-reversibly inhibited by CO. Once peroxide is present, the enzyme acts as a peroxidase, and since this action involves no reduction of the ferric nucleus, CO is without effect. Thus, one may postulate that Tang-Bonner experiments were carried out in systems where peroxide formation was the rate-limiting step, while the Wagenknecht-Burris experiments were limited by peroxidative action. Such a possibility will be examined further in the future. With regard to the physiological significance of LAA oxidase, previous views on its function in the plant (16) must be revised in view of the fact that we now recognize the system to be activated by blue light and to be .present in green as well as in etiolated plants (4). The activation
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of the IAA-oxidase system by light occurs only in the presence of an inhibitor such as catalase or Mn++ ions, whose action is reversed by light,. It must be anticipated therefore that inactivation of IAA by the IAA-oxidase system in the plant may also be a process accelerated by light, and specifically by blue light. Among the processesmediated in the plant by blue light’ is phototropic curvature, which is known to be clue t,o changes in auxin level induced by light (17). It will therefore be of great interest to determine to what extent the light activation of ILL1 oxidase may be responsible for the phototropic response. SUMMARY
I. Several lines of evidence indicate that the TAB-osidasc system of peas consists of a light,-artivatable flavoproteill enzyme coupled through H20L’to a peroxidase. (n) The system is inhibited by rat’alase, the inhibit,ion being reversed by blue light. (b) The activity of the system is increased by the addition of Hz& ill the dark, and by the addition of crystalline horseradish root, peroxidase in the light. This indicates that H,O, limits t’he react#ion in the clurk; in the light, H202 production is incareasedand the peroxidasc lw c*omcslimiting. (r) Crystalline horseradish root perosidase readily oxidizes IAB in thcapresence of H20Z. (4) The IAA-oxidase syst,emhas been separated int#oa peroxidase ant1 3. fiavin-c,ontaining component. The peroxidase component is active in the presence of H202 in oxidizing IAA and wnventional peroxidase suhstrates. Successful reconstitution of the IAA-oxidase system from itJs fragments has been arhieved. The substjrat8efor the flavin enzyme is tl~tknowri, but may he IAA itself. (12)_4n analog of the IAA-oxidase syst’em has been made by combinilig the santhine osidase of cream with the crystBalline peroxidase of horseradish root. The analog system diRers from IAA osiclasein requirillg n substrate other than L\A for the flavoprotein. 2. I’ossihlc alt8ernati\-e schemeshave been esaminrcl.
470 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
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GOLDACRE, P. L., Australian J. Sci. Research Ser. B 4, 293 (1951). BERNHEIM, F., AND DIXON, M., Biochem. J. 22, 113 (1928). KEILIN, D., AND MANN, T., Proc. Roy. Sot. (London) Bl22, 119 (1937). WARBURG, O., AND CHRISTIAN, W., Biochem. 2. 298, 150 (1938). CRAMMER, J. L., Nature 161, 349 (1948). SNELL, E. E., AND STRONG, F. M., Ind. Eng. Chem., Anal. Ed. 11, 346 (1939) BALL, E. G., J. Biol. Chem. 128,51 (1939). SIEGEL, S. M., AND WEINTRAUB, R. L., Physiol Plantarum 6, 241 (1952). KNOX, W. E., AND MEHLER, A. H., J. Biol. Chem. 187, 419 (1950). CONN, E., KRAEMER, L. M., LILT, P-N., and VENNESLAND, B., J. Biol. Chem. 194, 143 (1951). 15. SWEDIN, B., AND THEORELL, II., Nature 146,71 (1940). 16. TANG, Y. W., AND BONNER, J., Am. J. Botany 36, 570 (1948). 17. GALSTON, A. W., Botan. Rev. 16,361 (1950).