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The effect of 2,2-diphenyl-1-picrylhydrazyl and p-cresol on the oxidative degradation of indole-3-acetate
ARCEIVK3
OF
HIOCHBMISTKY
AND
RIOPHYSICS
143,
276-285 (1971)
The Effect of 2,2-Diphenyl-1-picrylhydrazyl on the Oxidative
Degradation
R. W. MILLER Cell Biology Research Insiilute
ANIl
and p-Cresol
of Indole-3-acetate’ E. V. PARUI’S
and Plant Research Inelitule, Research Branch, Agriculture, Ottawa J, Canada
Received October
13, 1970; accepted
January
Canada Department
of
13, 1971
A stable free radical compound, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and an oxidogenic substrate of peroxidase, p-cresol, boOh markedly stimulate t.he catalytic activity of 10-8 M horseradish peroxidase in the oxidation of indole-3-acetate. However, at higher enzyme concentrations (lO+ M), the oxidative degradation of the plant growth regulator is inhibited or preveuted by these same compounds. The new finding that the overall effect of these agents is a function of enzyme concentration is interpreted in terms of the concentration-dependent reactivit.y of a reduced, oxygenated enzyme iutermediate, compound III, iu a second reaction wit.h the promoting agents. Under some conditions the essential intermediate is destroyed rapidly in a second reaction with p-cresol or 1)PPH. However, formation of the enzyme compound is apparently accelerated at both high and low enzyme concentrations. IAA reacts directly with peroxidase and oxygen in au initial rapid reaction to cause accumulation of a mixt,ure of reduced enzyme intermediates which exhibit absorbance maxima uear 418 and 430 rnp. However, oxygen is consumed in a second reaction which is presumed to be catalyzed by compound III (oxyferroperoxidase) which contains the bound superoxide anion. At, low enzyme concentrations the first reaction is rate-limiting since it. appears that compound III must. accumulate before the oxygen-consuming reactions may proceed. Under these condit.ions, DPPH and pcresol stimulate the initial process and the overall reaction. At, higher enzyme levels the essential int,ermediat.e is formed more rapidly but is removed by the promoting agents leading to overall inhibition of IAA degradation.
A previous communication report,ed a marked enhancement by 2, Z-diphenyl-lpicrylhydrazyl (DPPH)* of the rate of oxidative degradation of indole-3-acetate (IAA) in the presence of horseradisb peroxidase (HRP) (1). Stable free-radical compounds, such sa DPPH, might promote IAA oxidation by abstracting a hydrogen at,om (2) from the substrate or might, alt.ernatively, convert the enzyme t,o a form highly reactive 1 Contribution No. 681 from the Cell Biology Research Institute and Contribution No. 778 from the Plant Research Institute. 3 Abbreviations: The abbreviations used are: IAA, sodium indole-3-acetate; HRP, horseradish peroxidaae II; DPPH, 2,2-diphenyl-l-picrylhydrazyl radical.
with IAA (which is ordinarily regarded as a slow substrate of the enzyme). Phenolic substrates of peroxidase which accelerate the degradat.ion of IAA also might, act t.hrough either of these possible mechanisms. In the case of the hydrogen-abstraction pathway, prior enzymic oxidation of the phenol t.o a semiquinone would precede reaction wit.h IAA (3). p-Cresol is such a substrate and has the advantage of having no absorbance above 400 w at the concentrat.ions which were used as compared with the highly absorbing DPPH. The present work shows that the agents which promote IAA degradation at low enzyme concentration, inhibit the same process at the higher enzyme levels which are 276
OXIDATIVE
DEGRAI)ATTOS
used in spectroscopic experiments. A possible explanation of this phenomenon would be t,hat, at low enzyme concentration t)hese agents circumvent or increase the velocity of a rate-limiting reaction while at, higher concentrations, t,hey also remove an essential enzyme intermediate from solution. The equilibrium posit.ion of this latter reaction must require high concentrations of both the reactants, i.e., compound III and the promot.ing agent. In order to obt.ain more information about. the role of promot,ing compounds during the primary events in the oxidat,ive degradation of IAA, &opped-flow spectroscopic studies were carried out, w&h purified horseradish peroxidase. Our attention was focused on differences in initial spectroscopic changes in the enzyme in the presence and in the absence of agents which can give appreciable free radical concentrat.ions in aqueous solution. Chemical modification of peroxidase by IAA and the part,icipat.ion of enzyme compounds in the oxidaGve degradation of the substrat.e have been discussed by Yamazaki and his colleagues (‘2, 4) and by Ricard and co-workers (5, 6). The data reported here support, the view t,hat compound III forms in the presence of IAA. This compound, report,edly diamagnet,ic (7)) contains an oxygen molecule which carries three oxidizing equivalents. Some authors assume that compound III contains superoxide but, since no dissociat,ion occurs, this has not been confirmed directly. It is generally accepted that t.his it1t;ermediat.e is reactive wit.h additional molecules of IAA and that it is responsible for the cat,alytic degradation of the growt,h regulator. In addition, HRP is converted relatively slowly by IAA to a green compound having an absorbance maximum at, 670 mE.c.This form of t.he enzyme is referred t.o here as P-670 aft,er Yamazaki et al. (4). The role of t,he superoxide anion (or hydroperoxyl radical) free in solution is examined in the present work through the use of superoxide scavenging agent.s (8, 9). MATERIALS Indole-3-acetic electrophoretically peroxidase were
AND
METHODS
acid and purified type VI or purified t.ype VII horseradish obtained from Sigma Chemical
OF
ISI)OLE-3-4CETATIS
277
Co. The enzyme had a measured RZ value of 3.16 when dissolved in 0.05 M sodium citrate buffer at pH 5. This preparation yielded the visible absorbance spectrum expected for high-spin peroxidase II of horseradish wit.h a Soret peak at 403 rnp. The enzyme exhibited a catalytic activity of 250 purpurogallin units per mg protein. 2,2Diphenyl-1-picrylhydraryl and p-cresol were supplied by J. T. Baker Co., while dithionite was a product of Eastman Kodak Co. Oxygen concentrat,ions were determined with a Gilson Model K Oxygraph. A Clark electrode in a jacketed glass vessel having a 1.5-ml capacity was thermostated at 26”. In order to compare oxygenconsumpt,ion data with the slower spectroscopic changes which were observed 2-5 set after mixing the enzyme with a substrate it was necessary to characterize t.he response of the electrode system. Dithionite, 2 X lW3 M, was prepared with oxvgenfree water and 56 ~1 were inject.ed into the closed polarographic vessel. The response of the electrode to t.he result.ing rapid change in oxygen coucentration was recorded at a chart speed of 5 cm/set. The resulting dead time (no response) and t,he times for lO(>A and 90yc of t,he final electrode response were then measured and found to be 1.3, 2.1, and -l.3 set, respectively. Therefore, a significant change in oxygen concentration was observable after about. 2 set and data obt.ained after this elapsed react,ion time may be compared qualitatively with the relatively slow spectroscopic changes which were observed in the stopped flow apparatus. St,opped-flow kinetic experiments were carried out with the Gibson-Milnes mixing apparatus w manufactured by the Durrum Instrument Co. HRP (2 X 10-j M) was mixed with various concentrat,iona of IAA and other reagents. Changes in transmittance were recorded from an oscilloscope screen with a Polaroid camera. Transmittance scales and wavelengt.hs are shown for each oscilloscope trace in the figures and estimated transmitt.ance values before mixing are given in the legends to t.he figures. Kxperiments represented by the traces of the figures were repeated a minimum of three times. The monochromator was calibrated with ferrocytochrome c at 550 m. The input signal from the photomultiplier was applied to an RC net,work having a time constant of either 0.1 or 0.03 msec for traces recorded on the ranges, 2-5 set/cm or 20-100 msec/cm, respectively. Initial oxygen concentrations of the enzyme and substrate solutions were controlled at three different levels by equilibrating the reactants with pure oxygen, air, or helium in glass tonometer reservoirs prior to filling of the mixing syringes of the spectrophotomet,er. All reaction mixtures for polarographic, spec-
-MILLER
AND PARUPS
troscopic, and colorimet,ric studies were buffered at pH 5.0 with 0.05 M sodium cit.rate. Absorbance spectra of HRP and P-G70 were obtained with a Beckman DK-1 spcctrophotometcr. In experiments requiring catalytic quantities of peroxidase, unreacted IAA was determined as a funct,ion of time with t.he calorimetric procedure of Gordon and Yaleg (10). The initial concentrat,ion of the substrate was estimated prior to the addition of peroxidase. Oxygen-deficient reaction media were prepared by evacuation and flushing with helium or nitrogen. Erythrocuprein was prepared from beef blood according to the procedure of McCord and Fridovich (8). The activity of the enzyme was determined with a spectrophotometric system in which cytochrome c is directly reduced by photochemically generated superoxide anion (11). The identity of superoxide anion as the reduct,ant in this system has been established by ESR spectroscopy (12). In this assay, the details of which will be published elsewhere, t,he reduction of cytochrome c is linear wit,h time under standardized conditions of temperature and light intensity. Erythrocuprein inhibit.6 the reduction of cytochrome e in proportion to its activity in destroying superoxide anion. The preparation of McCord and Fridovich was highly active in this assay. Total inhibition of cytochrome c reduction was caused by 0.01 mg under the condit.ions of superoxide generation of Massey el al. (11). RESULTS
Promotion of IAA oxidation at low enzyme concentration. Figure 1 illustrates in two sets of experiments the requirement for oxygen and a promoting agent for rapid degradation of IAA under catalyt,ic conditions. Curve 4 of Part. A was obtained in the presence of low4 M IAA, 10m8M HRP, and 10-j M DPPH and 230 PM oxygen. When t.he reaction vessels were made oxygen deficient (<3 PM) the DPPH-promoted reaction was markedly inhibited as in curve 3. Controls lacking t,he enzyme (curve 1) or DPPH (curve 2) showed a much slower rat.e of degradation of the substrate under aerobic condit,ions. Curve 10 of Part B, Pig. 1, represents a repeat, of curve 4 for assessment.of the reproducibilit,y of the resu1t.swith DPPH. pCresol, lo-” M, also accelerated aerobic IAA degradation under identical experimental conditions as shown in curve 11. At a lower concentration of p-cresol (10m6M, curve 8) there is still appreciable stimulation of t.he
60 XIAA
40
0
2
4
6
a
10-O
2
4
6
H
10
1. Dependence of t.he rate of degradat.icln of IAA by lo-8 M peroxidase on the presence of promoting compounds, oxygen, and an inhibitor. Reaction mixtures containing 10-’ M IAA in 0.05 JI sodium cit.rate buffer, pH 5, were prepared as listed. The degradat.ion of the substrate wa.9 started by addition of 10-B M HRP unless the enzyme was deleted. The percentage of IAA remaining was determined ae a function of time by I he method of Gordon and Paleg (10). FIG.
CUIW
IIRP
no.
1 2 3 4
0 .OOl .OOl ,001
5 G
0 ,001 .@I1 .OOl .OOl .OOl .OOl
7
8 !I 10 11
1)PPH ..-._-
)-Crew1 _-x10-%I
Part A 1.0 -0 1.0 1.0 Part B 1 .o 1.0 -1.0 -0.1 1.0 -1.0 .1.0
-
Tiron
Oxygen _. .._-
-_-
23 23 <0.3 23
1.0 0.20
23 23 <0.x 23 23 23 23
--
_
rate of IAA degradat,ion as compared with :I react,ion mixture lacking a degradation-promot.ing compound (curve 2, Part A). Curve 7, which was obtained with lo-” XXp-cresol under anaerobic conditions, shows t.hat the p-cresol effect. is, as expected, also oxygendependent. Catechols are reportedly oxidized to benzoquinones and other products by compound III (5). In eit.her event,, catechols compete
OSIDATIVE
DEGRADATIOS
with slow substrates for oxidizing equivalents and, therefore, inhibit t.he degradation of IAA. l ,%Dihydroxy-benzene-3, ci-disulfonate (tiron) is also known to inhibit the reduction of cybochrome c and other accept,ors by enzymically or photochemically generated superoxide anion (9). In the study of Fig. 1 this catechol effectively inhibited the DPPH-promot,ed oxidation of IAA when added at 2 X lo-” 11 (curve 9) or 10-j 11 (curve 6). The catechol is oxidized and hence the inhibition which is observed in curve 9 decreaseswith time. However, excess erythrocuprein, an enzyme which catalyzes the very rapid disproportionation of free superoxide anion (S), had no inhibitory effect on any of the oxidative or spectroscopic processesreported here when added to reaction mixtures at. a concentration of 50 pg/ml. It is unlikely, therefore, that free superoxide anion is involved in the oxidative degradation of IAA. However, an enzyme-bound form of the oxygen radical (compound III) may be accessible to the catechol inhibitor but not. to the erythrocuperin. The dat:a summarized by Fig. 1 are in agreement with previous reports. However, Pnrups (1) showed that the acceleration of IAA oxidat.ion by DPPH was optimum at about. 1.25 X 1O-s 11.Higher concentrations of the radical were lesseffect.ive. In the present work it was found t.hat if the 10d5M promoting agent (DPPH or p-cresol) was added at high (10-I) ;\I) enzyme concentration, a net, inhibition in IAA oxidation and oxygen consumption resulted as compared with the enzyme alone. This is t,he reverse of t,he stimuh&on of IAA degradation shown in Fig. 1. Spectroscopickinetic studiesat h.igh enzyme concentration. Figure 2 illustrat.es rapid and slower absorbance changes in the spectrum of 10m5AI HRP when reacting wit,h a lo-fold excess of IAA. The left-hand side of the figure showsthat rapid, concurrent increases in absorbance at 418 rnp (curve 1) and at 560 rnp (curve 4) occur which are followed by a steady-state period during which lit,tle additional change at these wavelengths can be observed. This period lasts several hundred milliseconds. The great.est increase in absorbance after mixing occurred near 418
OF INDOLE-3-ACETATE
3'79
FIG. 2. Effect of p-cresol on the initial rate and kinetic course of accumulation of peroxidaae int,ermediates. HRP, at a concent,raLion of 2 X 10-6 M, dissolved in 0.05 M sodium citrate buffer, pH 5, was mixed in a 2-cm cuvette with an equal quantity of a solution of 2 X 10-4 Y IAA in the same buffer. The concentrations of all reactants were halved on mixing. Initial oxygen concentration was 240 WM and the reaction temperature was 26”. p-Cresol was incorporated in the substrat,e solution at a concentration of 2 X lO+ M for the experiments represented by curves 2 and 5 at 2 X 10-d M for curve 3. Vertical dashed lines indicate a change of t,ime scales ss noted in the Figure. Transmitt.ance scales are shown for each trace. Init.ial t,ransmibtance values before mixing were as follows: curve 1, 21yo; curve 2, 15%; curve 3, 10yO; curve 4, 5, 9171. The transmit,tance levels before mixing represent the transmittance of the fully reacted enzyme. A decrease in t,ransmitt.ance (absorbance increase) is shown as an upward movement of the voltage trace on the oscilloscope screen. The dashed portion of the curve represents a change (increase) in absorbance due to filling of the 2-cm cuvette with fresh reactants. The actual starting point. of the reaction in all cases is higher in absorbance than t,hat of the final react,ion mixt.ure.
rnp with a smaller maximum at higher wavelengths as shown below. The effect, of p-cresol on the rapid changes at 418 rnB is illustrated by curves 2 and 3 of Fig. 2. At a concentration of lo-” JI p-
280
MILLER
AND PARUPS
mixt.ure of intermediates within bhe first few milliseconds of the reaction. It, should be noted t.hat when lo-’ JI p-cresol and W5 M HRP were reacted in the absenceof IAA, a curve similar to curve 3 was obtained. This confirms t,he presence of a fast reaction between the enzyme and p-cresol. This pattern is repeated at 560 rnp asindicat.ed by curves 4 and 5 of Fig. 2. In the absenceof p-cresol, the enzyme is converted to a relatively stable new form. Wit.h an equimolar concentrat,ion of p-cresol, the int,ermediate state is unstable and decays rapidly. Figure 3 shows a difference spectrum of WAVE LENGTH the absorbance changes in t.he enzyme beFIQ. 3. Rapid changes induced in peroxidase tween 408 and 432 rnp. The spectrum exspectrum by IAA. Air-saturated 2 X 10-S M HRP hibits two maxima; one is located near 418 was mixed with and diluted by a factor of 2 with rnccwhile the other is much higher, in the a lo-fold excess of IAA according to the condiregion of 430 mp. Since no known single tiona of Fig. 2 with an absorbance increase reprecompound of peroxidase has two maxima in sented by an upward movement of the trace. the Soret region, it. is probable that, t.his specDead time of the apparat.ua was approximately 3 msec in the stopped-flow apparatus. Oscilloscope trum represent,s a mixt,ure of intermediate traces of the rapid reactions were recorded aa in enzyme products resulting from the aerobic the left-hand portion of curve 1 of Fig. 2. The reaction bet,ween IAA and HRP. The region maximal increase in absorbance was determined of 418 rnp is reportedly isosbestic for the infrom a point immediately after mixing (unreacted terconversion of ferro- and ferriperoxidase reagents) to a point 300 maec after mixing. Little and is near the absorbance maximum for further increase or change in absorbance at any wavelength occurred on this time scale after 300 oxyferroperoxiase (compound III, 5). One, mBec since after this time the reaction mixture is of the products of reaction is, t,herefore probably compound III. This supposition in a relatively static condition. Subsequent deis supported by t.he fact that at. low oxygen creaseB in absorbance were recorded on a slower time scale aa shown in the right-hand portion of concentration, no persistant absorbance curve 1 of Fig. 2. The rapid absorbance changes change at 418 rnp occurs. The formation and calculated from the initial reaction data and maintenance of a steady-state concentration plotted in Fig. 3 represent the difference spectrum of t,he enzyme intermediate which absorbs of the mixture of enzyme intermediates over the at t.his wavelength is, t.herefore, dependent indicated wavelength range. Vertical dashed on the presence of molecular oxygen. lines in Part A, Fig. 2 indicate the change in abThe right-hand side of I’ig. 2 illustrates sorbance which occurred on introduction of fresh reactants into the 2-cm cuvette. This change is t,he slower decreasesin absorbance at 415 rnp and X0 rnp which follow the initial rapid innot included in the data of Fig. 3. crease at these wavelengt.hs. In the absence cresol, the initial absorbance increase was of p-cresol, the absorbance drops off to a second steady st,ate. During this period the reversed before it reached the level of curve 1. However, the initial rise in absorbance at substrate is degraded and oxygen is con418 rnp was more rapid with p-cresol. The sumed at, a relatively const,ant rate as shown half-time of t.his iniOia1 pseudo-first-order in subsequent experiments. The reaction process was estimated to be 40-50 msec in mixt,ure does not return to the final absorbthe absence of p-cresol; 25 msec with 10-j 1\1 ance level until all of the IAA is degraded. p-cresol; and about 10 msec with 1W4 or In the presence of lo+ 31cresol (final conaft,er mixing) a second rise in abp-cresol. The added compound clearly elicits centration a profound effect. on the kinetics of forma- sorbance at 418 and 560 rnp occurs after Con and removal of some int.ermediate or about 10 sec. This rise corresponds in time
OXIDATIVI!:
403 mp 403 mp
DINRADAT10N
2
11= 5;-
-y3
-
AT
iFT$gq TIME
FIG. 4. Comparison of slow changes in the spectrum of peroxidsse with oxygen consumption in the presence and absence of p-cresol and DPPH. In Part A, 2 X 1W M HRP was mixed with 2 X 10-4 M IAA under aerobic conditions. The reaction t,emperature was 26”. Spectroscopic changes were recorded SB a function of time at the indicated wavelengths. Absorbance increases are indicated by upward movement of the trace. Oxygen and p-cresol concentrations after mixing are listed below.
CUlW no.
p-Cred
DPPH
Oxygen
I 1 2 3 4 5 0 7
’
x10-%l
0 1.0 0 0 0 10.0 1.0
iI O
1.0 0 0 0 0 0 0 0
24 24 <0.3 24 24 24 24 I 24
8 8 8 60 13 -
-
-
-
In Part B, 10-5 HRP in 0.05 M citrate buffer at pH 5.0 was placed in a polarographic cell together with the concentrat.ion of p-cresol listed above. IAA was added from a syringe to give a concentration of 10-d M at the time indicated by the arrow at the left hand side of the figure. The time at which the electrode could show a response to
OF
ISI)OLE-S-ACETATE
2Sl
with t,he second st,eady-stat.e period of curve 1. Apparent.ly after a few seconds the pcresol (originally stoichiomet,ric with HRP) has been oxidized by t,he enzyme and the 418 rnp-absorbing intermediates are allowed to accumulate with concomit.ant degradation of IAA. With a lo-fold excess of p-cresol over peroxidase, IAA degradation is suppressed completely and the second rise in absorbance at 41s rnp is virtually eliminated. The absorbance of the fully react.ed mixt.ures at 415 rnp dropped to a value below the initial absorbance (which was observed immediately after mixing). The effect is due t.o the part.ial conversion of the enzyme P-670 as discussed below. Figure 4A shows the slower spectral changes which occur at 403 rnp (curve 5) and 670 rnp (curve 4) during oxygen consumption. The initial decrease at, 403 rnp appears to precede any appreciable oxygen uptake (Fig. 4B, curve S), taking into accou?bt the time lag in the response of the Clark Electrode. The decrease in absorbance at 403 rnp corresponds in time wit,h t,he decrease in absorbance at 418 and 560 rnp to the second more lengthy st,eady-state period. The second st.eady-st,ate period, in turn, corresponds in time with the maximal rate of oxygen uptake. The steady-stat.e period shown in curve 5, Fig. 4 indicates a relatively const,ant, concentration of a mixture of enzyme intermediates which disappears on exhaustion of IA,4. Essential features indicated in curve 5 of l;igure 4A are the reversibility of the major part of t,he MA-induced transmittance increase at 403 rnp and the irreversibility of a smaller portion of the change. The magnit,ude of the irreversible portion is represented by the transmittance difference be-
any change in oxygen concentration is indicated by the vert.ical dashed line in Part B. Between 2 and 3 set after this time, au electrode response of 90% would be expected aa shown in curve 9. In this control experiment, 70 PM dithionite was introduced at the time indicated by the arrow. The dashed vertical line on curve 9 indicates initiation of electrode response to a nearly instantaneous change in oxygen concentration.
2x2
MILLER
ASD
tween the starting point and the point immediately after mixing. Curve 4 of Fig. 4 shows a slow, irreversible increase in absorbance at 670 rnp which begins during the second steady-state phase and reaches a maximum on reappearance of most of t,he original absorbance at 403 rnp. This change is due to t.he partial conversion of the enzyme to P-670 as confirmed in separate experiments, where spectra were taken with low5 M HRP after reaction wit,h lOA M IAA in the presence of excessoxygen. Each time an aliquot waz added t,o the enzyme, a portion of it was converted to a form having an absorbance maximum at 670 rnp in agreement with the results of Yamazaki el al. (4). The effect of anaerobiosis on the slow 403-u absorbance changes is evident from curve 3 of Fig. 4 which shows that, they are greatly suppressedat very low (less than 3 ~11)oxygen concentration. p-Cresol, lo4 >f, also prevents any of the slow spect,roscopic changes at 403 rnp (curve 2) as expected from t,he results of Fig. 2. Curve 1 of Fig. 4 was obtained in the presence of lo-” Y DPPH, lo+ M peroxidase, and lo4 M IAA and also shows a complete lack of reaction. In Fig. 4B, oxygen upt.ake is eliminat.ed (curve 6) and inhibited (curve 7) by lo4 hi and 1O-5 h.1p-cresol, respectively. Curve X represents the oxidat,ion of 10e4 Y of IAA which is complete in 20 set in the absenceof additional reagents. The net effect of t.he pcresol or DPPH on the reaction is clearly inhibitory at this high enzyme concent,ration. Curve 9 of Fig. 4B illustrates the method which was used for determination of t,he responseof the elect.rode to a rapid decreasein oxygen concent,ration. When the 10-j h1enzyme was converted nearly completely to P-670 by a single addit,ion of a SO-fold excess of IAA in a sealed polarographic vessel, followed by reaeration, subsequent. determination of oxygen consumption due to IAA degradat,ion according to the conditions of curve 8 of Fig. 3B showed no inactivat,ion of the system. Therefore P-670 apparently, although a chemically modified form of HRP, remains act,ive in IAA degradation. Variation of IAA corlcentrat,ions yielded
PARUPS
FIG. 5. Effects of different. concentrations of oxygen and IAA on the slow absorbancechanges of HRP at 403 m. HRP, 2 X 10-e M, was mixed with an equalvolume of IAA to give the substrate concentrations specified below. Both reactant
solutions were 0.05 M with respect to citrate buffer at pH 5.0. Oxygen concentration waq varied by equilibration of bot.h the enzyme and reactant solutions with air, pure oxygen, or helium at 25’. Absorbance increases are indicated by movements of the oscilloscope tract. -IAA ‘y. Transmittance at Oxygen Curve
no.
1 2 3 4 5
x10--6x -.--_-
24 <3 24 <3
1140
403 m(r before
-.
1.0 1.0 100 100 100
(fully ..~__.
mixing reacted)
8 8
12 8 12
t,he effects on the slow absorbance change at, 403 mp shown in Fig. 5. In curve 1, 10-j 11 IAA is seen to give a slower (relative to results obtained with lOA 31 IAA) reversible conversion of HRP to a 41X-rnp absorbing species.The reaction is inhibited completely in t,he absenceof oxygen (curve 2). The time course of the reaction is in agreement, with the resulbs of Ricard et al. (6) which also were obtained wit.h roughly equimolar concentrat,ions of HRP and IAA. Wit,h a lOO-fold excess of IAA (lo4 hI, curve 3, Fig. 5) the course of events is accelerated (aa confirmed by oxygen consumpt.ion data). Here oxygen (0.24 X 10” Y), rather than IAA, is the limiting reagent
OSI1)ATIVK
I)EGRAlMTION
and absorbance at 403 rnp returns to a point near the initial value when all the oxygen is utilized. The result. confirms that the reaction of IAA and HRP as observed at 403 rnp is oxygen-dependent. The resulting intermediates cannot form or exist under anaerobic conditions sinw, in the absence of oxygen, little reaction is observed on a mow sensitive transmittance scale (curve 4). In an oxygen-sat.urat et1 medium (curve 5) t h t? enzyme remains in t,he steady state (low absorbance at. 403 mp) for a longer time. This result further confirms that, the length of time that the second steady-state period per-
OF INI)OI,E-3-rlCETATI~
mediate appears to contain molecular oxygen since it is not observed 300 msec after mixing IAA and HR,P in the absence of air. It may be a ferrous form of the enzyme complexed with molecular oxygen but. is probably not simply fcrropcroxidase since this form is know-n to br highly unstable and not observable spectroscopically under aerobic condit.ions (2). The finding that IAA, HKI’, and oxygen react relatively slowly to yirltl products which remain in a steady state for several hundred milliseconds supports ;I view that the init,ial events shown in Reaction 1 only occur aerobic-
r
HRP4 I Fez+-- 0 2
IAA: + HRP-Fe3+
+ 0, ---*
sists depends on oxygen concentration well as on IAA concent.ration.
as
The kinetic course of the aerobic reaction between IAA and peroxidase may be considered on rapid (O-500 msec) and slow (l-20 set) time scales. In t.he initial rapid period, HRP is converted to intermediate forms. The spectrum of Fig. 3 reflects the absorbance of these comp0nent.s 300 msec after mixing, during a steady-stat.e period of little absorbance change. Since the maximum absorbance level at, all of the wavelengths indicated in the figure is stable for several /--\
H3cv
OH + HRP-Fe3+
+ 0, ----t
hundred milliseconds, it may be presumed t.hat, an equlibrium mixture is present and that the rate of interconversion of intermediates is faster than t.he reactions visualized here by the stopped-flow method. The 418-rnp absorbing species may be identified as compound III from the oxygen requirement and spectral dat.a and from previous reports (5, 13). Identification of the 430-rnp absorbing species is less positive. However, this inter-
2S3
1
ally. [HRP-l~e3+--O;?] represent,s compound III which cont.ains one more electron than HRP and molecular oxygen. This compound is assumed to be the activated form of the enzyme which degrades IAA (2, Fj). DPPH and p-cresol clearly exert an effect on the initial, rapid react.ion between HRP and IAA. Figure 2, curve 2, shows that t,he rate of the initial absorbance rise at, 41s rnp (rate of compound III accumulation) increases with stoichiometric amounts of HRP and p-cresol. h lo-fold excess of p-crexol (lo-* 11) over the enzyme gives an ewn faster rise in absorbance (Kg. 2, curve 3). Reaction 2 is much more rapid than rwc-
IHRPI -1Fez+- 0 2 I tion 1 and this may explain the stimulation of IAA degradat,ion by DPPH and p-cresol at, low- enzyme concent.rations where formation of compound III is a rate-limiting process. The data are consistant wit.h the view that the compounds convert the enzyme, rather than JAA, to a catalytically active form. The subsequent, course of the degradation of IAA might, occur by two rapid cornpIt!mentary pathways. Reaction 3 shows the
284
MILLER
ASI)
+ IAA: ----em’
[HRP-F~~+-o;]
single elect,ron oxidation of IAA by cornpound III while React.ion 4 represents the HRP-Fe3+
PARUPS
+ IAA’
+ HRP--FeSt
3.
postulated t,o occur according bo Reaction ,i. The products of oxidation of cresol are +
IAA
4.
[ HRP - Fe3+- Oi]
,T
H&<&O’
+ O,----+[HRP-Fe3+-Oi]
single electron oxidation of monodehydro indole-3-acetate by HRP (2). In these reactions IAA:, IAA., and IAA represent the unreacted, monodehydro and the two electron-deficient forms of indole-3-acetate, respectively. These reactions would be expected to be more rapid than Reaction 1 since free radical species are involved. React.ions 3 and 4 taken t,ogether comprise a possible free radical chain propagation reaction. At low enzyme concentration (low8 M), the number of parallel chains would be small and chain length would be relatively long. With only a lo-fold excess of IAA over enzyme (lo+ >I), chain numbers would be much longer and termination reactions, such as disproportionation of IAA., might effectively short,en chain length. The involvement of a chain reaction in the oxygen-consuming degradat,ion of IAA also might. explain the lag in oxygen consumption shown in curve S of Fig. 4B and the lag in the decrease of 41%rnp absorbance t.o the second steady-state level (right-hand portion, curve 1, Fig. 2). If this mechanism is correct, both compound III and monodehydro indole-3-acetate are required to be present. in appreciable concentrat.ions for the rapid catalytic degradation of IAA. Otherwise, regeneration of compound III can occur only through the slower React)ion 1. The inhibition of IAA degradation at high enzyme concentrations may be considered taking into account React,ions 14. The results can be viewed as t.he consequence of the equilibrium position of a secondary reaction between p-cresol or DPPH and a member of either Reaction 3 and 4. Since the data of I:ig. 2 indicate that p-cresol acts to prevent attainment, of the first steady absorbancy at, 418 mF, this process may be
+ H202
-
j
r mixed 1osgl
1 1 +
I H,;,
+
HRP
Fe3+
5.
classically mixtures of catechols, hydroquinones, quinones, etc. (14). The result of this process is to make compound III unavailable effectively for participation in IAA degradation. Because of the unfavorable equilibrium position of Reaction 5, a higher concentration of compound III is required to initiat,e it. This may also be viewed as a chain terminat.ion process. Once initiated, this reaction may form catechoi products which are known inhibitors of the degradation of IAA by peroxidase. The experimental data of Fig. 2 support t.he conclusion that monodehydro-p-cresol and DPPH act to inhibit the oxidation of IAA at high enzyme concentration by reacting with compound III. Curve 2 shows that t.he inhibitor, originally equal in concentrat,ion with the enzyme, is consumed because after 10 set absorbance at 418 rnp rises a second time and the oxidat,ive degradation of IAA proceeds. Apparently, the enzyme species P-670 is formed relatively slowly (curve 4, Fig. 4) and is the product of a side reaction. This reaction probably does not affect the initial react.ion of IAA with HRP which is the site of action of p-cresol and DPPH. ACKNOWLEDGMENT The authors express their appreciation for helpful discussions of the work with Miss Dorothy Dow. We &JO thank Mr. W. Richards and Mr. W. Adams for their technical assistance. ~-_~~~~~~~~ .~-~~ comaetent -.~ .
OXIl)ATIVE
I~EGRAIMTIO~
REFERENCES 1. PARUPS,
E. v.,
YAMAZAKI,
I.,
Can. J. Biochem.
YOKOI~A,
R.,
“S;‘,
McCom, J. M., .\ND F~rnovrc~r, I.. J. Biol. Chem. 244, 6049 (1969). 9. ~~LLEB, R. W., Cwt. J. &X~WI. 46, 935 (1970) . 10. (;ORDON, S. A., .\xI) PALEG, L. G., ~‘k~ysid. l’lanl. 10, 39 (1967). 8.
47, 220
(1989). 2.
OP KI)C)LE-3-ACETATE
AND
N~K.LIIM.\,
R., in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. I, p. 503. Wiley, New York (1965). 3. ONISHI, T., YAMhZAKI, I-I., ~YANIGI, J., N,~KAMUM, J., AND Y,ULIZAKI, I., Biochim. Biophys. Ada 173, 357 (1969). 4. YAM.4ZAK1, H., &IISHI, 8., AND YAM.~Z.\IiI, I., Arch Biochem. Biophys. 136, 41 (1970). 5. RICARD, J., AND N~tr, J., Biochim. Biophys. Acta 133, 321 (1967). 6. RICARD, J., Jou, D., S~NTIMONE, &I., ASD C.~MBE, II., C. R. H. Ad. Sci. 269, 1415 (1969). 7. BLUMBEHG, W. E., PEISACH, J., WITTENHERG, B. A., AND WITTENBERG, J. B., J. Biol. Chem. 243, 1854 (1968).
11.
kkRSEy,
I'..
It.
cr.,
hI.LOC,
G.,
&I.,
Riophya. I).,
Biochem. (1969).
P.\LMER,
Hiopkys.
8.,
bfUI.\YHEW,
1’. C.,
I':NGEL,
khIUx\N,
Hiorhem. (19(i9). 12.
,STlHCKL.\XD,
L.
IIo~EL~~,
s. (;.,
MATTHEWS,
AND k$ULLIVAN, P. A., Res. Cotnnucn. 36, 891 c;.,
Res.
.4ND
%1.4SSEY,
Comnwn.
v.,
36, 898
13. NICHOI.I.S, l’., i,n “Oxygenases” (0. Hayaishi, ed.), p. 280. -4cademic Press, New York (1962). 14. BREWSTER, It. Q., “Organic Chemistry,” p. 609. Prentice-Ilall, Englewood Cliffs, New Jersey (1953).
OF
HIOCHBMISTKY
AND
RIOPHYSICS
143,
276-285 (1971)
The Effect of 2,2-Diphenyl-1-picrylhydrazyl on the Oxidative
Degradation
R. W. MILLER Cell Biology Research Insiilute
ANIl
and p-Cresol
of Indole-3-acetate’ E. V. PARUI’S
and Plant Research Inelitule, Research Branch, Agriculture, Ottawa J, Canada
Received October
13, 1970; accepted
January
Canada Department
of
13, 1971
A stable free radical compound, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and an oxidogenic substrate of peroxidase, p-cresol, boOh markedly stimulate t.he catalytic activity of 10-8 M horseradish peroxidase in the oxidation of indole-3-acetate. However, at higher enzyme concentrations (lO+ M), the oxidative degradation of the plant growth regulator is inhibited or preveuted by these same compounds. The new finding that the overall effect of these agents is a function of enzyme concentration is interpreted in terms of the concentration-dependent reactivit.y of a reduced, oxygenated enzyme iutermediate, compound III, iu a second reaction wit.h the promoting agents. Under some conditions the essential intermediate is destroyed rapidly in a second reaction with p-cresol or 1)PPH. However, formation of the enzyme compound is apparently accelerated at both high and low enzyme concentrations. IAA reacts directly with peroxidase and oxygen in au initial rapid reaction to cause accumulation of a mixt,ure of reduced enzyme intermediates which exhibit absorbance maxima uear 418 and 430 rnp. However, oxygen is consumed in a second reaction which is presumed to be catalyzed by compound III (oxyferroperoxidase) which contains the bound superoxide anion. At, low enzyme concentrations the first reaction is rate-limiting since it. appears that compound III must. accumulate before the oxygen-consuming reactions may proceed. Under these condit.ions, DPPH and pcresol stimulate the initial process and the overall reaction. At, higher enzyme levels the essential int,ermediat.e is formed more rapidly but is removed by the promoting agents leading to overall inhibition of IAA degradation.
A previous communication report,ed a marked enhancement by 2, Z-diphenyl-lpicrylhydrazyl (DPPH)* of the rate of oxidative degradation of indole-3-acetate (IAA) in the presence of horseradisb peroxidase (HRP) (1). Stable free-radical compounds, such sa DPPH, might promote IAA oxidation by abstracting a hydrogen at,om (2) from the substrate or might, alt.ernatively, convert the enzyme t,o a form highly reactive 1 Contribution No. 681 from the Cell Biology Research Institute and Contribution No. 778 from the Plant Research Institute. 3 Abbreviations: The abbreviations used are: IAA, sodium indole-3-acetate; HRP, horseradish peroxidaae II; DPPH, 2,2-diphenyl-l-picrylhydrazyl radical.
with IAA (which is ordinarily regarded as a slow substrate of the enzyme). Phenolic substrates of peroxidase which accelerate the degradat.ion of IAA also might, act t.hrough either of these possible mechanisms. In the case of the hydrogen-abstraction pathway, prior enzymic oxidation of the phenol t.o a semiquinone would precede reaction wit.h IAA (3). p-Cresol is such a substrate and has the advantage of having no absorbance above 400 w at the concentrat.ions which were used as compared with the highly absorbing DPPH. The present work shows that the agents which promote IAA degradation at low enzyme concentration, inhibit the same process at the higher enzyme levels which are 276
OXIDATIVE
DEGRAI)ATTOS
used in spectroscopic experiments. A possible explanation of this phenomenon would be t,hat, at low enzyme concentration t)hese agents circumvent or increase the velocity of a rate-limiting reaction while at, higher concentrations, t,hey also remove an essential enzyme intermediate from solution. The equilibrium posit.ion of this latter reaction must require high concentrations of both the reactants, i.e., compound III and the promot.ing agent. In order to obt.ain more information about. the role of promot,ing compounds during the primary events in the oxidat,ive degradation of IAA, &opped-flow spectroscopic studies were carried out, w&h purified horseradish peroxidase. Our attention was focused on differences in initial spectroscopic changes in the enzyme in the presence and in the absence of agents which can give appreciable free radical concentrat.ions in aqueous solution. Chemical modification of peroxidase by IAA and the part,icipat.ion of enzyme compounds in the oxidaGve degradation of the substrat.e have been discussed by Yamazaki and his colleagues (‘2, 4) and by Ricard and co-workers (5, 6). The data reported here support, the view t,hat compound III forms in the presence of IAA. This compound, report,edly diamagnet,ic (7)) contains an oxygen molecule which carries three oxidizing equivalents. Some authors assume that compound III contains superoxide but, since no dissociat,ion occurs, this has not been confirmed directly. It is generally accepted that t.his it1t;ermediat.e is reactive wit.h additional molecules of IAA and that it is responsible for the cat,alytic degradation of the growt,h regulator. In addition, HRP is converted relatively slowly by IAA to a green compound having an absorbance maximum at, 670 mE.c.This form of t.he enzyme is referred t.o here as P-670 aft,er Yamazaki et al. (4). The role of t,he superoxide anion (or hydroperoxyl radical) free in solution is examined in the present work through the use of superoxide scavenging agent.s (8, 9). MATERIALS Indole-3-acetic electrophoretically peroxidase were
AND
METHODS
acid and purified type VI or purified t.ype VII horseradish obtained from Sigma Chemical
OF
ISI)OLE-3-4CETATIS
277
Co. The enzyme had a measured RZ value of 3.16 when dissolved in 0.05 M sodium citrate buffer at pH 5. This preparation yielded the visible absorbance spectrum expected for high-spin peroxidase II of horseradish wit.h a Soret peak at 403 rnp. The enzyme exhibited a catalytic activity of 250 purpurogallin units per mg protein. 2,2Diphenyl-1-picrylhydraryl and p-cresol were supplied by J. T. Baker Co., while dithionite was a product of Eastman Kodak Co. Oxygen concentrat,ions were determined with a Gilson Model K Oxygraph. A Clark electrode in a jacketed glass vessel having a 1.5-ml capacity was thermostated at 26”. In order to compare oxygenconsumpt,ion data with the slower spectroscopic changes which were observed 2-5 set after mixing the enzyme with a substrate it was necessary to characterize t.he response of the electrode system. Dithionite, 2 X lW3 M, was prepared with oxvgenfree water and 56 ~1 were inject.ed into the closed polarographic vessel. The response of the electrode to t.he result.ing rapid change in oxygen coucentration was recorded at a chart speed of 5 cm/set. The resulting dead time (no response) and t,he times for lO(>A and 90yc of t,he final electrode response were then measured and found to be 1.3, 2.1, and -l.3 set, respectively. Therefore, a significant change in oxygen concentration was observable after about. 2 set and data obt.ained after this elapsed react,ion time may be compared qualitatively with the relatively slow spectroscopic changes which were observed in the stopped flow apparatus. St,opped-flow kinetic experiments were carried out with the Gibson-Milnes mixing apparatus w manufactured by the Durrum Instrument Co. HRP (2 X 10-j M) was mixed with various concentrat,iona of IAA and other reagents. Changes in transmittance were recorded from an oscilloscope screen with a Polaroid camera. Transmittance scales and wavelengt.hs are shown for each oscilloscope trace in the figures and estimated transmitt.ance values before mixing are given in the legends to t.he figures. Kxperiments represented by the traces of the figures were repeated a minimum of three times. The monochromator was calibrated with ferrocytochrome c at 550 m. The input signal from the photomultiplier was applied to an RC net,work having a time constant of either 0.1 or 0.03 msec for traces recorded on the ranges, 2-5 set/cm or 20-100 msec/cm, respectively. Initial oxygen concentrations of the enzyme and substrate solutions were controlled at three different levels by equilibrating the reactants with pure oxygen, air, or helium in glass tonometer reservoirs prior to filling of the mixing syringes of the spectrophotomet,er. All reaction mixtures for polarographic, spec-
-MILLER
AND PARUPS
troscopic, and colorimet,ric studies were buffered at pH 5.0 with 0.05 M sodium cit.rate. Absorbance spectra of HRP and P-G70 were obtained with a Beckman DK-1 spcctrophotometcr. In experiments requiring catalytic quantities of peroxidase, unreacted IAA was determined as a funct,ion of time with t.he calorimetric procedure of Gordon and Yaleg (10). The initial concentrat,ion of the substrate was estimated prior to the addition of peroxidase. Oxygen-deficient reaction media were prepared by evacuation and flushing with helium or nitrogen. Erythrocuprein was prepared from beef blood according to the procedure of McCord and Fridovich (8). The activity of the enzyme was determined with a spectrophotometric system in which cytochrome c is directly reduced by photochemically generated superoxide anion (11). The identity of superoxide anion as the reduct,ant in this system has been established by ESR spectroscopy (12). In this assay, the details of which will be published elsewhere, t,he reduction of cytochrome c is linear wit,h time under standardized conditions of temperature and light intensity. Erythrocuprein inhibit.6 the reduction of cytochrome e in proportion to its activity in destroying superoxide anion. The preparation of McCord and Fridovich was highly active in this assay. Total inhibition of cytochrome c reduction was caused by 0.01 mg under the condit.ions of superoxide generation of Massey el al. (11). RESULTS
Promotion of IAA oxidation at low enzyme concentration. Figure 1 illustrates in two sets of experiments the requirement for oxygen and a promoting agent for rapid degradation of IAA under catalyt,ic conditions. Curve 4 of Part. A was obtained in the presence of low4 M IAA, 10m8M HRP, and 10-j M DPPH and 230 PM oxygen. When t.he reaction vessels were made oxygen deficient (<3 PM) the DPPH-promoted reaction was markedly inhibited as in curve 3. Controls lacking t,he enzyme (curve 1) or DPPH (curve 2) showed a much slower rat.e of degradation of the substrate under aerobic condit,ions. Curve 10 of Part B, Pig. 1, represents a repeat, of curve 4 for assessment.of the reproducibilit,y of the resu1t.swith DPPH. pCresol, lo-” M, also accelerated aerobic IAA degradation under identical experimental conditions as shown in curve 11. At a lower concentration of p-cresol (10m6M, curve 8) there is still appreciable stimulation of t.he
60 XIAA
40
0
2
4
6
a
10-O
2
4
6
H
10
1. Dependence of t.he rate of degradat.icln of IAA by lo-8 M peroxidase on the presence of promoting compounds, oxygen, and an inhibitor. Reaction mixtures containing 10-’ M IAA in 0.05 JI sodium cit.rate buffer, pH 5, were prepared as listed. The degradat.ion of the substrate wa.9 started by addition of 10-B M HRP unless the enzyme was deleted. The percentage of IAA remaining was determined ae a function of time by I he method of Gordon and Paleg (10). FIG.
CUIW
IIRP
no.
1 2 3 4
0 .OOl .OOl ,001
5 G
0 ,001 .@I1 .OOl .OOl .OOl .OOl
7
8 !I 10 11
1)PPH ..-._-
)-Crew1 _-x10-%I
Part A 1.0 -0 1.0 1.0 Part B 1 .o 1.0 -1.0 -0.1 1.0 -1.0 .1.0
-
Tiron
Oxygen _. .._-
-_-
23 23 <0.3 23
1.0 0.20
23 23 <0.x 23 23 23 23
--
_
rate of IAA degradat,ion as compared with :I react,ion mixture lacking a degradation-promot.ing compound (curve 2, Part A). Curve 7, which was obtained with lo-” XXp-cresol under anaerobic conditions, shows t.hat the p-cresol effect. is, as expected, also oxygendependent. Catechols are reportedly oxidized to benzoquinones and other products by compound III (5). In eit.her event,, catechols compete
OSIDATIVE
DEGRADATIOS
with slow substrates for oxidizing equivalents and, therefore, inhibit t.he degradation of IAA. l ,%Dihydroxy-benzene-3, ci-disulfonate (tiron) is also known to inhibit the reduction of cybochrome c and other accept,ors by enzymically or photochemically generated superoxide anion (9). In the study of Fig. 1 this catechol effectively inhibited the DPPH-promot,ed oxidation of IAA when added at 2 X lo-” 11 (curve 9) or 10-j 11 (curve 6). The catechol is oxidized and hence the inhibition which is observed in curve 9 decreaseswith time. However, excess erythrocuprein, an enzyme which catalyzes the very rapid disproportionation of free superoxide anion (S), had no inhibitory effect on any of the oxidative or spectroscopic processesreported here when added to reaction mixtures at. a concentration of 50 pg/ml. It is unlikely, therefore, that free superoxide anion is involved in the oxidative degradation of IAA. However, an enzyme-bound form of the oxygen radical (compound III) may be accessible to the catechol inhibitor but not. to the erythrocuperin. The dat:a summarized by Fig. 1 are in agreement with previous reports. However, Pnrups (1) showed that the acceleration of IAA oxidat.ion by DPPH was optimum at about. 1.25 X 1O-s 11.Higher concentrations of the radical were lesseffect.ive. In the present work it was found t.hat if the 10d5M promoting agent (DPPH or p-cresol) was added at high (10-I) ;\I) enzyme concentration, a net, inhibition in IAA oxidation and oxygen consumption resulted as compared with the enzyme alone. This is t,he reverse of t,he stimuh&on of IAA degradation shown in Fig. 1. Spectroscopickinetic studiesat h.igh enzyme concentration. Figure 2 illustrat.es rapid and slower absorbance changes in the spectrum of 10m5AI HRP when reacting wit,h a lo-fold excess of IAA. The left-hand side of the figure showsthat rapid, concurrent increases in absorbance at 418 rnp (curve 1) and at 560 rnp (curve 4) occur which are followed by a steady-state period during which lit,tle additional change at these wavelengths can be observed. This period lasts several hundred milliseconds. The great.est increase in absorbance after mixing occurred near 418
OF INDOLE-3-ACETATE
3'79
FIG. 2. Effect of p-cresol on the initial rate and kinetic course of accumulation of peroxidaae int,ermediates. HRP, at a concent,raLion of 2 X 10-6 M, dissolved in 0.05 M sodium citrate buffer, pH 5, was mixed in a 2-cm cuvette with an equal quantity of a solution of 2 X 10-4 Y IAA in the same buffer. The concentrations of all reactants were halved on mixing. Initial oxygen concentration was 240 WM and the reaction temperature was 26”. p-Cresol was incorporated in the substrat,e solution at a concentration of 2 X lO+ M for the experiments represented by curves 2 and 5 at 2 X 10-d M for curve 3. Vertical dashed lines indicate a change of t,ime scales ss noted in the Figure. Transmitt.ance scales are shown for each trace. Init.ial t,ransmibtance values before mixing were as follows: curve 1, 21yo; curve 2, 15%; curve 3, 10yO; curve 4, 5, 9171. The transmit,tance levels before mixing represent the transmittance of the fully reacted enzyme. A decrease in t,ransmitt.ance (absorbance increase) is shown as an upward movement of the voltage trace on the oscilloscope screen. The dashed portion of the curve represents a change (increase) in absorbance due to filling of the 2-cm cuvette with fresh reactants. The actual starting point. of the reaction in all cases is higher in absorbance than t,hat of the final react,ion mixt.ure.
rnp with a smaller maximum at higher wavelengths as shown below. The effect, of p-cresol on the rapid changes at 418 rnB is illustrated by curves 2 and 3 of Fig. 2. At a concentration of lo-” JI p-
280
MILLER
AND PARUPS
mixt.ure of intermediates within bhe first few milliseconds of the reaction. It, should be noted t.hat when lo-’ JI p-cresol and W5 M HRP were reacted in the absenceof IAA, a curve similar to curve 3 was obtained. This confirms t,he presence of a fast reaction between the enzyme and p-cresol. This pattern is repeated at 560 rnp asindicat.ed by curves 4 and 5 of Fig. 2. In the absenceof p-cresol, the enzyme is converted to a relatively stable new form. Wit.h an equimolar concentrat,ion of p-cresol, the int,ermediate state is unstable and decays rapidly. Figure 3 shows a difference spectrum of WAVE LENGTH the absorbance changes in t.he enzyme beFIQ. 3. Rapid changes induced in peroxidase tween 408 and 432 rnp. The spectrum exspectrum by IAA. Air-saturated 2 X 10-S M HRP hibits two maxima; one is located near 418 was mixed with and diluted by a factor of 2 with rnccwhile the other is much higher, in the a lo-fold excess of IAA according to the condiregion of 430 mp. Since no known single tiona of Fig. 2 with an absorbance increase reprecompound of peroxidase has two maxima in sented by an upward movement of the trace. the Soret region, it. is probable that, t.his specDead time of the apparat.ua was approximately 3 msec in the stopped-flow apparatus. Oscilloscope trum represent,s a mixt,ure of intermediate traces of the rapid reactions were recorded aa in enzyme products resulting from the aerobic the left-hand portion of curve 1 of Fig. 2. The reaction bet,ween IAA and HRP. The region maximal increase in absorbance was determined of 418 rnp is reportedly isosbestic for the infrom a point immediately after mixing (unreacted terconversion of ferro- and ferriperoxidase reagents) to a point 300 maec after mixing. Little and is near the absorbance maximum for further increase or change in absorbance at any wavelength occurred on this time scale after 300 oxyferroperoxiase (compound III, 5). One, mBec since after this time the reaction mixture is of the products of reaction is, t,herefore probably compound III. This supposition in a relatively static condition. Subsequent deis supported by t.he fact that at. low oxygen creaseB in absorbance were recorded on a slower time scale aa shown in the right-hand portion of concentration, no persistant absorbance curve 1 of Fig. 2. The rapid absorbance changes change at 418 rnp occurs. The formation and calculated from the initial reaction data and maintenance of a steady-state concentration plotted in Fig. 3 represent the difference spectrum of t,he enzyme intermediate which absorbs of the mixture of enzyme intermediates over the at t.his wavelength is, t.herefore, dependent indicated wavelength range. Vertical dashed on the presence of molecular oxygen. lines in Part A, Fig. 2 indicate the change in abThe right-hand side of I’ig. 2 illustrates sorbance which occurred on introduction of fresh reactants into the 2-cm cuvette. This change is t,he slower decreasesin absorbance at 415 rnp and X0 rnp which follow the initial rapid innot included in the data of Fig. 3. crease at these wavelengt.hs. In the absence cresol, the initial absorbance increase was of p-cresol, the absorbance drops off to a second steady st,ate. During this period the reversed before it reached the level of curve 1. However, the initial rise in absorbance at substrate is degraded and oxygen is con418 rnp was more rapid with p-cresol. The sumed at, a relatively const,ant rate as shown half-time of t.his iniOia1 pseudo-first-order in subsequent experiments. The reaction process was estimated to be 40-50 msec in mixt,ure does not return to the final absorbthe absence of p-cresol; 25 msec with 10-j 1\1 ance level until all of the IAA is degraded. p-cresol; and about 10 msec with 1W4 or In the presence of lo+ 31cresol (final conaft,er mixing) a second rise in abp-cresol. The added compound clearly elicits centration a profound effect. on the kinetics of forma- sorbance at 418 and 560 rnp occurs after Con and removal of some int.ermediate or about 10 sec. This rise corresponds in time
OXIDATIVI!:
403 mp 403 mp
DINRADAT10N
2
11= 5;-
-y3
-
AT
iFT$gq TIME
FIG. 4. Comparison of slow changes in the spectrum of peroxidsse with oxygen consumption in the presence and absence of p-cresol and DPPH. In Part A, 2 X 1W M HRP was mixed with 2 X 10-4 M IAA under aerobic conditions. The reaction t,emperature was 26”. Spectroscopic changes were recorded SB a function of time at the indicated wavelengths. Absorbance increases are indicated by upward movement of the trace. Oxygen and p-cresol concentrations after mixing are listed below.
CUlW no.
p-Cred
DPPH
Oxygen
I 1 2 3 4 5 0 7
’
x10-%l
0 1.0 0 0 0 10.0 1.0
iI O
1.0 0 0 0 0 0 0 0
24 24 <0.3 24 24 24 24 I 24
8 8 8 60 13 -
-
-
-
In Part B, 10-5 HRP in 0.05 M citrate buffer at pH 5.0 was placed in a polarographic cell together with the concentrat.ion of p-cresol listed above. IAA was added from a syringe to give a concentration of 10-d M at the time indicated by the arrow at the left hand side of the figure. The time at which the electrode could show a response to
OF
ISI)OLE-S-ACETATE
2Sl
with t,he second st,eady-stat.e period of curve 1. Apparent.ly after a few seconds the pcresol (originally stoichiomet,ric with HRP) has been oxidized by t,he enzyme and the 418 rnp-absorbing intermediates are allowed to accumulate with concomit.ant degradation of IAA. With a lo-fold excess of p-cresol over peroxidase, IAA degradation is suppressed completely and the second rise in absorbance at 41s rnp is virtually eliminated. The absorbance of the fully react.ed mixt.ures at 415 rnp dropped to a value below the initial absorbance (which was observed immediately after mixing). The effect is due t.o the part.ial conversion of the enzyme P-670 as discussed below. Figure 4A shows the slower spectral changes which occur at 403 rnp (curve 5) and 670 rnp (curve 4) during oxygen consumption. The initial decrease at, 403 rnp appears to precede any appreciable oxygen uptake (Fig. 4B, curve S), taking into accou?bt the time lag in the response of the Clark Electrode. The decrease in absorbance at 403 rnp corresponds in time wit,h t,he decrease in absorbance at 418 and 560 rnp to the second more lengthy st,eady-state period. The second st.eady-st,ate period, in turn, corresponds in time with the maximal rate of oxygen uptake. The steady-stat.e period shown in curve 5, Fig. 4 indicates a relatively const,ant, concentration of a mixture of enzyme intermediates which disappears on exhaustion of IA,4. Essential features indicated in curve 5 of l;igure 4A are the reversibility of the major part of t,he MA-induced transmittance increase at 403 rnp and the irreversibility of a smaller portion of the change. The magnit,ude of the irreversible portion is represented by the transmittance difference be-
any change in oxygen concentration is indicated by the vert.ical dashed line in Part B. Between 2 and 3 set after this time, au electrode response of 90% would be expected aa shown in curve 9. In this control experiment, 70 PM dithionite was introduced at the time indicated by the arrow. The dashed vertical line on curve 9 indicates initiation of electrode response to a nearly instantaneous change in oxygen concentration.
2x2
MILLER
ASD
tween the starting point and the point immediately after mixing. Curve 4 of Fig. 4 shows a slow, irreversible increase in absorbance at 670 rnp which begins during the second steady-state phase and reaches a maximum on reappearance of most of t,he original absorbance at 403 rnp. This change is due to t.he partial conversion of the enzyme to P-670 as confirmed in separate experiments, where spectra were taken with low5 M HRP after reaction wit,h lOA M IAA in the presence of excessoxygen. Each time an aliquot waz added t,o the enzyme, a portion of it was converted to a form having an absorbance maximum at 670 rnp in agreement with the results of Yamazaki el al. (4). The effect of anaerobiosis on the slow 403-u absorbance changes is evident from curve 3 of Fig. 4 which shows that, they are greatly suppressedat very low (less than 3 ~11)oxygen concentration. p-Cresol, lo4 >f, also prevents any of the slow spect,roscopic changes at 403 rnp (curve 2) as expected from t,he results of Fig. 2. Curve 1 of Fig. 4 was obtained in the presence of lo-” Y DPPH, lo+ M peroxidase, and lo4 M IAA and also shows a complete lack of reaction. In Fig. 4B, oxygen upt.ake is eliminat.ed (curve 6) and inhibited (curve 7) by lo4 hi and 1O-5 h.1p-cresol, respectively. Curve X represents the oxidat,ion of 10e4 Y of IAA which is complete in 20 set in the absenceof additional reagents. The net effect of t.he pcresol or DPPH on the reaction is clearly inhibitory at this high enzyme concent,ration. Curve 9 of Fig. 4B illustrates the method which was used for determination of t,he responseof the elect.rode to a rapid decreasein oxygen concent,ration. When the 10-j h1enzyme was converted nearly completely to P-670 by a single addit,ion of a SO-fold excess of IAA in a sealed polarographic vessel, followed by reaeration, subsequent. determination of oxygen consumption due to IAA degradat,ion according to the conditions of curve 8 of Fig. 3B showed no inactivat,ion of the system. Therefore P-670 apparently, although a chemically modified form of HRP, remains act,ive in IAA degradation. Variation of IAA corlcentrat,ions yielded
PARUPS
FIG. 5. Effects of different. concentrations of oxygen and IAA on the slow absorbancechanges of HRP at 403 m. HRP, 2 X 10-e M, was mixed with an equalvolume of IAA to give the substrate concentrations specified below. Both reactant
solutions were 0.05 M with respect to citrate buffer at pH 5.0. Oxygen concentration waq varied by equilibration of bot.h the enzyme and reactant solutions with air, pure oxygen, or helium at 25’. Absorbance increases are indicated by movements of the oscilloscope tract. -IAA ‘y. Transmittance at Oxygen Curve
no.
1 2 3 4 5
x10--6x -.--_-
24 <3 24 <3
1140
403 m(r before
-.
1.0 1.0 100 100 100
(fully ..~__.
mixing reacted)
8 8
12 8 12
t,he effects on the slow absorbance change at, 403 mp shown in Fig. 5. In curve 1, 10-j 11 IAA is seen to give a slower (relative to results obtained with lOA 31 IAA) reversible conversion of HRP to a 41X-rnp absorbing species.The reaction is inhibited completely in t,he absenceof oxygen (curve 2). The time course of the reaction is in agreement, with the resulbs of Ricard et al. (6) which also were obtained wit.h roughly equimolar concentrat,ions of HRP and IAA. Wit,h a lOO-fold excess of IAA (lo4 hI, curve 3, Fig. 5) the course of events is accelerated (aa confirmed by oxygen consumpt.ion data). Here oxygen (0.24 X 10” Y), rather than IAA, is the limiting reagent
OSI1)ATIVK
I)EGRAlMTION
and absorbance at 403 rnp returns to a point near the initial value when all the oxygen is utilized. The result. confirms that the reaction of IAA and HRP as observed at 403 rnp is oxygen-dependent. The resulting intermediates cannot form or exist under anaerobic conditions sinw, in the absence of oxygen, little reaction is observed on a mow sensitive transmittance scale (curve 4). In an oxygen-sat.urat et1 medium (curve 5) t h t? enzyme remains in t,he steady state (low absorbance at. 403 mp) for a longer time. This result further confirms that, the length of time that the second steady-state period per-
OF INI)OI,E-3-rlCETATI~
mediate appears to contain molecular oxygen since it is not observed 300 msec after mixing IAA and HR,P in the absence of air. It may be a ferrous form of the enzyme complexed with molecular oxygen but. is probably not simply fcrropcroxidase since this form is know-n to br highly unstable and not observable spectroscopically under aerobic condit.ions (2). The finding that IAA, HKI’, and oxygen react relatively slowly to yirltl products which remain in a steady state for several hundred milliseconds supports ;I view that the init,ial events shown in Reaction 1 only occur aerobic-
r
HRP4 I Fez+-- 0 2
IAA: + HRP-Fe3+
+ 0, ---*
sists depends on oxygen concentration well as on IAA concent.ration.
as
The kinetic course of the aerobic reaction between IAA and peroxidase may be considered on rapid (O-500 msec) and slow (l-20 set) time scales. In t.he initial rapid period, HRP is converted to intermediate forms. The spectrum of Fig. 3 reflects the absorbance of these comp0nent.s 300 msec after mixing, during a steady-stat.e period of little absorbance change. Since the maximum absorbance level at, all of the wavelengths indicated in the figure is stable for several /--\
H3cv
OH + HRP-Fe3+
+ 0, ----t
hundred milliseconds, it may be presumed t.hat, an equlibrium mixture is present and that the rate of interconversion of intermediates is faster than t.he reactions visualized here by the stopped-flow method. The 418-rnp absorbing species may be identified as compound III from the oxygen requirement and spectral dat.a and from previous reports (5, 13). Identification of the 430-rnp absorbing species is less positive. However, this inter-
2S3
1
ally. [HRP-l~e3+--O;?] represent,s compound III which cont.ains one more electron than HRP and molecular oxygen. This compound is assumed to be the activated form of the enzyme which degrades IAA (2, Fj). DPPH and p-cresol clearly exert an effect on the initial, rapid react.ion between HRP and IAA. Figure 2, curve 2, shows that t,he rate of the initial absorbance rise at, 41s rnp (rate of compound III accumulation) increases with stoichiometric amounts of HRP and p-cresol. h lo-fold excess of p-crexol (lo-* 11) over the enzyme gives an ewn faster rise in absorbance (Kg. 2, curve 3). Reaction 2 is much more rapid than rwc-
IHRPI -1Fez+- 0 2 I tion 1 and this may explain the stimulation of IAA degradat,ion by DPPH and p-cresol at, low- enzyme concent.rations where formation of compound III is a rate-limiting process. The data are consistant wit.h the view that the compounds convert the enzyme, rather than JAA, to a catalytically active form. The subsequent, course of the degradation of IAA might, occur by two rapid cornpIt!mentary pathways. Reaction 3 shows the
284
MILLER
ASI)
+ IAA: ----em’
[HRP-F~~+-o;]
single elect,ron oxidation of IAA by cornpound III while React.ion 4 represents the HRP-Fe3+
PARUPS
+ IAA’
+ HRP--FeSt
3.
postulated t,o occur according bo Reaction ,i. The products of oxidation of cresol are +
IAA
4.
[ HRP - Fe3+- Oi]
,T
H&<&O’
+ O,----+[HRP-Fe3+-Oi]
single electron oxidation of monodehydro indole-3-acetate by HRP (2). In these reactions IAA:, IAA., and IAA represent the unreacted, monodehydro and the two electron-deficient forms of indole-3-acetate, respectively. These reactions would be expected to be more rapid than Reaction 1 since free radical species are involved. React.ions 3 and 4 taken t,ogether comprise a possible free radical chain propagation reaction. At low enzyme concentration (low8 M), the number of parallel chains would be small and chain length would be relatively long. With only a lo-fold excess of IAA over enzyme (lo+ >I), chain numbers would be much longer and termination reactions, such as disproportionation of IAA., might effectively short,en chain length. The involvement of a chain reaction in the oxygen-consuming degradat,ion of IAA also might. explain the lag in oxygen consumption shown in curve S of Fig. 4B and the lag in the decrease of 41%rnp absorbance t.o the second steady-state level (right-hand portion, curve 1, Fig. 2). If this mechanism is correct, both compound III and monodehydro indole-3-acetate are required to be present. in appreciable concentrat.ions for the rapid catalytic degradation of IAA. Otherwise, regeneration of compound III can occur only through the slower React)ion 1. The inhibition of IAA degradation at high enzyme concentrations may be considered taking into account React,ions 14. The results can be viewed as t.he consequence of the equilibrium position of a secondary reaction between p-cresol or DPPH and a member of either Reaction 3 and 4. Since the data of I:ig. 2 indicate that p-cresol acts to prevent attainment, of the first steady absorbancy at, 418 mF, this process may be
+ H202
-
j
r mixed 1osgl
1 1 +
I H,;,
+
HRP
Fe3+
5.
classically mixtures of catechols, hydroquinones, quinones, etc. (14). The result of this process is to make compound III unavailable effectively for participation in IAA degradation. Because of the unfavorable equilibrium position of Reaction 5, a higher concentration of compound III is required to initiat,e it. This may also be viewed as a chain terminat.ion process. Once initiated, this reaction may form catechoi products which are known inhibitors of the degradation of IAA by peroxidase. The experimental data of Fig. 2 support t.he conclusion that monodehydro-p-cresol and DPPH act to inhibit the oxidation of IAA at high enzyme concentration by reacting with compound III. Curve 2 shows that t.he inhibitor, originally equal in concentrat,ion with the enzyme, is consumed because after 10 set absorbance at 418 rnp rises a second time and the oxidat,ive degradation of IAA proceeds. Apparently, the enzyme species P-670 is formed relatively slowly (curve 4, Fig. 4) and is the product of a side reaction. This reaction probably does not affect the initial react.ion of IAA with HRP which is the site of action of p-cresol and DPPH. ACKNOWLEDGMENT The authors express their appreciation for helpful discussions of the work with Miss Dorothy Dow. We &JO thank Mr. W. Richards and Mr. W. Adams for their technical assistance. ~-_~~~~~~~~ .~-~~ comaetent -.~ .
OXIl)ATIVE
I~EGRAIMTIO~
REFERENCES 1. PARUPS,
E. v.,
YAMAZAKI,
I.,
Can. J. Biochem.
YOKOI~A,
R.,
“S;‘,
McCom, J. M., .\ND F~rnovrc~r, I.. J. Biol. Chem. 244, 6049 (1969). 9. ~~LLEB, R. W., Cwt. J. &X~WI. 46, 935 (1970) . 10. (;ORDON, S. A., .\xI) PALEG, L. G., ~‘k~ysid. l’lanl. 10, 39 (1967). 8.
47, 220
(1989). 2.
OP KI)C)LE-3-ACETATE
AND
N~K.LIIM.\,
R., in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. I, p. 503. Wiley, New York (1965). 3. ONISHI, T., YAMhZAKI, I-I., ~YANIGI, J., N,~KAMUM, J., AND Y,ULIZAKI, I., Biochim. Biophys. Ada 173, 357 (1969). 4. YAM.4ZAK1, H., &IISHI, 8., AND YAM.~Z.\IiI, I., Arch Biochem. Biophys. 136, 41 (1970). 5. RICARD, J., AND N~tr, J., Biochim. Biophys. Acta 133, 321 (1967). 6. RICARD, J., Jou, D., S~NTIMONE, &I., ASD C.~MBE, II., C. R. H. Ad. Sci. 269, 1415 (1969). 7. BLUMBEHG, W. E., PEISACH, J., WITTENHERG, B. A., AND WITTENBERG, J. B., J. Biol. Chem. 243, 1854 (1968).
11.
kkRSEy,
I'..
It.
cr.,
hI.LOC,
G.,
&I.,
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P.\LMER,
Hiopkys.
8.,
bfUI.\YHEW,
1’. C.,
I':NGEL,
khIUx\N,
Hiorhem. (19(i9). 12.
,STlHCKL.\XD,
L.
IIo~EL~~,
s. (;.,
MATTHEWS,
AND k$ULLIVAN, P. A., Res. Cotnnucn. 36, 891 c;.,
Res.
.4ND
%1.4SSEY,
Comnwn.
v.,
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13. NICHOI.I.S, l’., i,n “Oxygenases” (0. Hayaishi, ed.), p. 280. -4cademic Press, New York (1962). 14. BREWSTER, It. Q., “Organic Chemistry,” p. 609. Prentice-Ilall, Englewood Cliffs, New Jersey (1953).