PLANT PHYSIOLOGY

3. BRoWN"-, A. H. Determination of pentose in thepresence of lalge quantities of glucose. Arch. Bio-chem. 11: 269-278. 1946.

4. BRYSON, J. L. and MITCHELL, T. J. Impiroved spIray-ing reagents for the detection of sugars on paperchromatograms. Nature 167: 864. 1951.

5. BuSCH, H., HURLBERT, R. B. and POTTER, V. R.Anion exchange chromatography of acids of thecitric acid cycle. Jour. Biol. Chem. 196: 717-727.1952.

6. GAUCH, H. G. and DUGGER, W. M., JR. The role ofboron in the translocation of sucrose. Plant Physiol.28: 457-466. 1953.

7. HO.AGLA.ND, D. R. and ARNON. D. I. The water-cul-tuirie method for growing plants without soil.Univ. of Calif. Agr. Expt. Station CircIlar' 347.1938.

8. KHYM, J. X. and ZILL, L. P. The separation ofsugars by ion exchange. Jou1r. Am. Chem. Soc. 74:2090-2094. 1952.

9. LIEBERMAN, S. Carbon dioxide assimilation into or-ganic acids by Peniicillium chrysogenum. Ph.D.thesis, University of Wisconsin, Madison 1956.

10. LINDSTROM, E. S., NEWTON, J. W. and WILSON, P. W.The relationship between photosynthesis and nitro-gen fixation. Proc. Nat. Acad. Sci., U.S. 38: 392-396. 1952.

11. MAGEE, W. E. Nitrogen fixation by excised nodulesand by cell-free preparations from Azotobacterviznelandii. Ph.D. thesis. University of Wisconsin,Madison 1955.

12. MORRIS, D. L. Quantitative determination of carbo-hydrates with Dreywood's anthone reagent. Sci-ence 107: 254-255. 1948.

13. NELSON, C. D. an(d GORHAM, P. R. Uptake atndtranslocation of C"4-labelled sugars applied to pri-mary leaves of soybean seedlings. Can. Jour. Bot.35: 339-347. 1957.

14. NELSON, C. D. and GORHAM, P. R. Translocationof radioactive stugars in the stems of soybean seed-lings. Can. Jour. Bot. 35: 703-713. 1957.

15. NOMURA, M., TAKAHASHI, H. and SAKAGUCHI, K.Ethylene oxide-a,/3-dicarboxylic acid (fumarylgly-cidic acid) production by microbes. Jour. Chem.Soc. Japan 28: 376-382. 1954.

16. SATTLER, L. and ZERBAN, F. W. New spray reagentsfor paper chromatography of reducing sugars.Anal. Chem. 24: 1862. 1952.

17; SEN, S. P. and LEOPOLD, A. C. Influience of light anddarkness upon carbon dioxide fixation. PlantPhysiol. 31: 323-329. 1956.

18. STUTZ, R. E. and BURRIS, R. H. Photosynthesis andmetabolism of organic acids in h-sigher plants.Plant Physiol. 26: 226-243. 1951.

19. VERNON, L. P. and ARONOFF, S. Metabolism ofsoybean leaves. IV. Translocation from soybeanleaves. Arch. Biochem. Biophys.36: 383-398. 1952.

20. VIRTANEN, A. I., MoisIo, T. and BURRIS, R. H. Fix-ation of nitrogen by nodules excised from illumi-nated and darkened pea plants. Acta Chem. Scand.9: 184-186. 1955.

21. YOUDEN, WY. J. Statistical Methods for Clhemists.Pp. 1-126. John Wiley and Sons, New Yorki 1951.

22. ZILL, L. P., KHYM, J. X. and CHENIAE, G. M. Fur-thei studies on the separation of the bor-ate com-plexes of sugars and related compounds by ionexehange chromatography. Jour. Am. Chem. Soc.75: 1339-1342. 1953.

THE PROPERTIES OF CYTOCHRONIE OXIDASE IN CHOLATEEXTRACTS FROMAl SOYBEAN ROOTS 1

GXENE W. MILLER, HAROLD J. EVANS AN-D EDWVARD SISLERNORTH CAROLINA STATF, COLLEGE. RALEIGH. NORTH CAROLINA

Cytochrome oxidase preparations have been ob-tained from many different organisms includinghigher plants (1, 4, 21). Since cytochrome oxidaseis firmly bound to the particulate material, fraction-ation and purification by usual techniques has notbeen possible. Surface active agents including sod-ium cholate or sodium deoxycholate have been shownto disperse particulate cytochrome oxidase fromheart muscle resulting in a clear extract. Some puri-fication from this material has been obtained (3, 19,20). Smith (16) reported a 20-fold purification ofcytochrome oxidase from a heart muscle extract byuse of sodium cholate treatment, trypsin digestion

1 Receiv-ed October 25, 1957.2 Contribution from the Faculty of Botany, North

Carolina Agricultural Experiment Station and publishedwith the approval of the Director as paper No. 849.

and then fractionation by ammloniumiii sulfate. Noinformation has been found in the published literatureconcerning the puirification of cytochrome oxidlasefrom higher plant sources.

In a previous paper (11) concerning the stimula-tory influence of salts on the activity of particuilatecytochrome oxidase, it was suggested that electrolytesmay be necessary either for the transfer of electronsthrough the lipoprotein particulates or for the main-tenance of the particulate enzyme in the proper ori-entation to allow access to substrates. If thesepostulations were correct, one possibly might expectthe salt stimulation to disappear if sufficient disper-sion of the particulates could be obtained by use ofsurface active agents. In this paper the propertiesof cytochrome oxidase are described in two types ofextracts prepared from soybean root plarticulates by

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use of sodlium cholate. The experimental results tobe pre-sented show that salts failed to stimulate theactivity of either of two cholate dispersed fractions.A preliminary report (12) of these findings alreadyhas been l)resented.

AIATERIALS AND -METHODSPARTICULATE PREPARATION: Roots from soy-bean

seedlings (Glycine max 'Merr.) grown as previouslvindicated (11) were homogenized for five minutes in

two weights (in respect to weight of roots) of cold0.1 M potassium phosphate buffer at pH 7.4. Thehomogenization was accomplished by use of an Omnimixer (Iv-an Sorvall Inc., Norwalk, Conn.) and theentire preparatory operation was conducted at tem-peratures between 0 and 4° C. The homogenate was

strained through two thicknesses of cheesecloth (frac-tion I) and the liquid adjusted to pH 5.6 with cold1 M acetic acid. The precipitate, collected by centri-fugation at 25,000 x G for 15 minutes, was suspendedin a volume (equal to one fourth the original volumeof the homogenate) of 0.1 MI phosphate buffer at pH7.4. A glass Ten Broeck homogenizer was used tosuspend the precipitate. This preparation was usedas the particulate suspension and is referred to as

fraction II.

PREPARATION OF CHOLATE EXTRACT: A quantityof a 20 % solution of sodium cholate at pH 7.4 was

mixed with the particulate suspension to make thefinal concentration 2 %. The stock solution of sod-ium cholate in 0.1 M phosphate buffer at pH 7.4 was

prepared from cholic acid (purchased from FisherScientific Co.) and adjusted to pH 7.4 by use ofNaOH. In certain experiments the mixture of sod-ium cholate and extract was digested with trypsin(18) or with papain, but this treatment had no ap-parent effect on the fractionation procedure andtherefore was not routinely used.

After centrifugation of the cholate extract for 10minutes at 25,000 x G, the sediment was discarded.The supernatant liquid is referred to as fraction III.Sufficient solid (NH4)2S04 was added to the super-natant to obtain a 30 % saturated solution. Theprotein from this precipitation was collected by cen-

trifugation and dissolved in a volume of 0.1 MI

K2HPO4 buffer at pH 9.0 equivalent to 20 % of thevolume of the supernatant (this is referred to as frac-tion IV). A protein fraction also was collected between30 and 65 % saturation with (NH4)2SO4 and wasdissolved in a volume of 0.1 M K2HPO4 buffer at pH9.0 equivalent to 20 % of the volume of the super-natant (referred to as fraction V 3 in table I). Bothof the extracts precipitated by (NH4)2SO4 were ad-justed with NaOH to a pH of 7.4. Centrifugationfor one hour at 25,000 x G of either fraction IV or Vrresulted in no loss of activity. Centrifugation for one

hour at an average force of 66,000 x G decreased theactivity of fraction IV to one third that of the orig-inal, but an equivalent centrifugation had no effectupon the activity of fraction V. Fraction IV was

yellow in color and contained 9.9 % cholate as de-termined by the method outlined by Reinhold andWilson (15). The activity of this extract on a pro-tein basis as determined by Folin's phenol reagent(8) was about twice that of the crude extract. Frac-tion V was colorless and contained 11.0 % cholate.The activity of this extract on a protein basis was

about 8 times that of the crude extract. Both ex-

tracts were optically clear and retained their activi-ties for several weeks when stored at - 15° C. Theessential data of the average of three purification ex-

periments are shown in table I. These data are typ-ical of many replications of the purification procedure.

ENZYME ASSAY PROCEDURES: In the standardcytochrome oxidase assay procedure, enzyme activ-ity was determined spectrophotometrically at 550 m,uwith a Beckman DU spectrophotometer (2, 11). Thereaction mixture in a volume of 1 ml contained; 85micromoles of potassium phosphate buffer at pH 7.0,0.05 micromoles of cytochrome c of 90 % purity(prepared and assayed as previously described (2,11)) and a quantity of enzyme sufficient to obtain thedesired activity. Enzyme activity in this procedureis indicated as the decrease in optical density at 550m/A during the 15-second interval between 10 and 25seconds after the enzvme was added to start the reac-

tion. In the purification procedure (table I) it was

3 In referring to fraction V an enzyme reaction is in-ferred; however, it is concluded that the nature of thereaction has not been established.

BLE IPURIFICATION OF CYTOCHROME OXIDASE FROM SOYBEAN ROOTS

FRACTION VOLUME TorAL RECOVERY PROTEIN SPECIFICACTIVITY REOEY PRTI APCTIFICY

ml units % mg/ml units permg protein

I Crude extract 480 399 ... 1.8 0.4II Acetic acid ppt. 125 199 54.2 1.8 0.8

III Cholate extract of II 138 262 71.3 1.8 0.95IV 0 to 30 % (NH4)2S04 ppt. of III 15 30 8.2 2.1 0.75V 30 to 65 % (NH4)2SO4 ppt. of III 18 70 19.0 1.2 3.2

* One unit is defined as a decrease in optical density of 1 per minute during the initial 15 second interval usingthe assay procedure described in the text.

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necessary to compare activities of fractions exhibit-ing nonlinear rates with those exhibiting linear rates.In these experiments, therefore, the standard pro-cedure was used with the exception that the opticaldensity change for the initial 15-second interval wasdetermined instead of the change during the 10- to25-second interval. This determination was cor-rected for the optical density change resulting indilution by the addition of enzyme.

The data presented in figure 4 were obtained byuse of the colorimetric method for determination ofcytochrome oxidase (17) which involved the meas-urement of the rate of oxidation of the leuco form of2,3',6-trichloroindophenol (obtained from the East-man Kodak Co.) at 645 m,u. The oxidation of thereduced dye in these experiments was dependent on

catalytic concentrations of oxidized cytochrome c.The addition of crystalline catalase (kindly sup-

plied by Dr. Sam Tove) to the various cytochrome cpreparations failed to influence activity and there-fore it was concluded that hydrogen peroxide was notpresent in the cytochrome c solutions.

Other enzymes assayed include DPNH oxidasewhich was determined spectrophotometrically by therate of decrease in optical density at 340 m,u (6) andascorbic acid oxidase activity which was assayed bymeasurement of the decrease in optical density at 265mju (14).

ABSORPTION SPECTRA: The difference between thespectra of the extracts in the reduced and oxidizedforms was obtained by use of a Warren Spectracord.The enzyme was reduced by addition of sodium hy-drosulfite to a cuvette of 1 cm light path which con-tained the enzyme (3). The spectrum in each ex-periment wi-as recorded within 90 seconds after ad-dition of the reducing agent.

RESULTSKINETICS OF REACTIONS: The curves in figure 1

illustrate the course of reactions catalyzed by frac-tion II (acetic acid precipitated particulates), frac-tion III (cholate extract of particulates) fraction IV(0 to 30 % (NH4)2SO4 precipitate (ppt.) from frac-tion II) and fraction V (30 to 65 % (NH4)2SO4 pre-cipitate (ppt.) of fraction III). From these curvesit is apparent that the rates of reactions catalyzed byfractions II and IV were linear with time. The re-

action rate with the crude extract (fraction I) alsowas linear; however, this data is not presented. Itis generally reported that cytochrome oxidase reac-

tions follow first order kinetics (2, 21) however usingthe assay system described and the particulate frac-tions indicated zero-order reactions were consistentlyobtained. These results are in accord with those ofa previous report (11). The rate of the reaction cata-lyzed by fraction III was diphasic decreasing slightlyin the initial phase of the reaction then remaininglinear for the remainder of the experimental period.The activity of fraction V fell off rapidly with timeand as indicated by the curve (fig 1) most of thereaction took place in the initial 25 seconds. When

the logarithml of this decreasing rate was plottedlagainst time the curve failed to indicate a first-orderreaction. After 115 seconds the addition of reducedcytochrome c to the reaction mixture containingfraction V failed to accelerate the reaction. Theaddition of enzyme to a comparable reaction after100 seconds, however, resulted in a marked decreasein optical density for the ensuin, 25-second interval.This suggests that the fraction V preparation pre-sumed to be an enzyme was inactivated by some sub-stance and this point receives further considerationunder studies of inhibitors.

Figures 2 and 3 provide evidence that the initialvelocities of the reactions catalyzed by both fractionIV and V were proportional to enzyme concentration.The reaction mixtures containing fraction V (fig 3)showed high activity in the first 25 seconds andvirtual inactivation after one minute. This phenom-enon was observed regardless of enzyme or cyto-chrome c concentration. The evidence indicating thevalidity of assay used with this fraction is indicatedin figure 3.

HEAT LABILITY: Cytochrome oxidase in fraction IV(0 to 30 % ppt.) was completely inactivated by ex-posure for one minute in a boiling water bath but

300' -e- FRACTION NlOn _o_ FRACTION x

FRACTION U

, 250 FRACTION m

In 200

F _+ OO~~~~~~~~~~~~DITIONAL REDUCED6150 ~~~~~~~~ADDITIONAL CYTOCHIROME C

0 30 60 90 120 150TIME (SECONDS)

FIG. 1. Reaction kinetics of various cytochrome oxi-dase preparations. The standard assay procedure wasused with the exception that reactions were allowed toproceed for the time periods indicated. The protein inmg added in fractions II, III, IV and V were respec-tively: 0.03, 0.02, 0.013 and 0.02. In the experiments in-volving fraction V two identical experiments were con-ducted for 100 seconds, however, since the curves werealmost identical up to this point only one curve is shown.At 100 seconds additional enzyme (0.02 mg of fractionV) was added and the rising broken curve represents thecourse of the reaction. After 115 seconds in the otherexperiment involving fraction V additional cytochrome c(0.05 micromoles) was added as indicated. When addi-tions were made O.D. changes were corrected for dilution.

The multiplication factor in the ordinate of this fig-ure and in figures 2, 3, 4 and 5 applies to optical densitychange only.

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MIILLER ET AL-CYTOCHRONIE OXIDASE

n0

x

zt

2

0

IC)

4

z

z

4

0)

300

250

200 [

o50 [

100

50

0

300

0

o 200

4

o 150

z

, 100

z

, 50

20 40 60 80TIME (SECONDS)

100

20 40 60 80 100TIME IN SECONDS

FIG. 2. Reaction kinetics of fraction IV at variousenzyme concentrations. The standard assay procedurewas used except for the time factor. The extract con-tained 0.27 mg of protein per ml.

FIG. 3. Reaction kinetics of fraction V at variousenzyme concentrations. The standard assay procedurewas used except for the time factor. The extract con-tained 0.30 mg of protein per ml.

the activity of fraction V decreased only 40 % underthe same experimental conditions. Fraction V (30 to65 % ppt.) however was inactivated by an exposurefor five minutes in a boiling water bath. As previ-ously indicated fraction III (figure 1) catalyzed adiphasic reaction. Since fractions IV and V were de-rived from fraction III (cholate extract of precipi-tated particulates) it was of interest to determine theheat lability of the latter. After exposure of frac-tion III to boiling water for one minute a reaction

was catalyzed at a rate very similar to that indicatedfor fraction V in figure 1. This information indicatesthat two different factors were involved in the di-phasic reaction catalyzed by fraction III. FractionsIV and V must then represent the two factors.

INHIBITORS: Experiments (fig 1) indicated thatthe cytochrome oxidizing, factor in fraction V was in-activated by some substance and pointed to the cyto-chrome c preparation as the source of the inhibitor.Preincubation of fraction V with different concentra-tions of oxidized cytochrome c for various periods oftime showed that the degree of inhibition was corre-lated with both of these variables (table II). Pre-incubation of the enzyme for five minutes with thecytochrome c concentrations ranging between 1.2 x10-5 and 5 x 1O5 MI resulted in complete inactiva-tion in every case. The activity of fraction IV, onthe other hand, was not appreciably affected by pre-incubation with oxidized cytochrome c at the variousconcentrations tested.

Since there was a possibility that the cytochromec preparation contained some inhibitor, solutions ofcytochrome c were prepared by reducing with eitherhydrogen and platinized asbestos or sodium hydro-sulfite and these were dialyzed for 12 hours againstthree liters 0.05 M phosphate buffer at pH 7.0. As-says containing the dialyzed reduced cytochrome so-lutions still exhibited the characteristic inactivationwhen fraction V was utilized as the oxidizing factorbut no inactivation was observed when fraction IVwas utilized. The substitution in the assay mediumof reduced cytochrome c prepared from pyrogenfree cytochrome c (Sigma Chemical Co., St. Louis)resulted in the characteristic sharp decrease in ae-tivity after the initial 25 seconds. Several differentpreparations of both horse heart and beef heart

TABLE IIEFFECT OF OXIDIZED CYTOCHROME C ON THE ACTIVITY OF

DISPERSED CYTOCHROME OXIDASE FROMSOYBEAN ROOTS

PREINCUBATION TIMECONCENTRATION WITH CYTOCHROME C

EXTRACT OF OXIDIZED (MINUTES)CYTOCHROME C

0 1 3 5

M Change in O.D. in 16seconds at 650 m,

Fraction IV 0.0 0.019 0.024 0.022 0.022Fraction V 0.0 0.072 0.071 0.069 0.071Fraction IV 1.2 x 10' 0.019 0.020 0.022 0.020Fraction V 1.2 x 10- 0.063 0.025 0.020 0.000Fraction IV 2.5 x 10' 0.020 0.020 0.021 0.021Fraction V 2.5 x 10' 0.045 0.020 0.005 0.000Fraction IV 5.0 x 10' 0.020 0.022 0.020 0.019Fraction V 5.0 x 10' 0.038 0.015 0.000 0.000

The standard assay procedure was used. Fraction IVcontained 0.08 mg and fraction V 0.02 mg of protein.

Fraction IV was prepared by 0 to 30 o (NH4)2SO4precipitation and fraction V by 30 to 65 % (NH4)2SO4precipitation.

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0.20 ml ENZYME

0.10 ml ENZYME

0.05 ml ENZYME

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cytochrome c from commercial sources were testedwith the fraction V preparation and all behaved in asimilar manner. This information suggests thereforethat oxidized cytochrome c or an impurity in it in-hibited the oxidation.

The possibility that the oxidizing factor was in-activated by the reduced form of cytochrome c aswell as the oxidized form was investigated by usingthe leuco dye method outlined by Smith and Stotz(17). Fractions IV and V were found to activelycatalyze the oxidation of reduced 2,3',6-trichloroin-dophenol, ascorbate, and DPNH in the absence ofcytochrome c. After treatment of fractions IV andV for one minute in a boiling water bath both ex-tracts failed to catalyze the direct oxidation of ascor-bate, DPNH, and leuco dye. Addition of cytochromec to an assay medium containing leuco dye and theheat treated fraction V resulted in oxidation of theleuco dye. This cytochrome dependent leuco dyeoxidation by fraction V was inhibited by the samecompounds that were inhibitory to this preparationwhen measured by the standard procedure (tableIII). This cytochrome dependent leuco dye oxida-tion was not manifested with fraction IV extractsthat had been exposed to boiling water. Catalyticconcentrations of cytochrome c, however, increasedthe rate of leuco dye oxidation using the unboiledfraction IV. The rate of oxidation of leuco dye bythe heat treated fraction V preparation decreased

TABLE IIIINHIBITION OF DISPERSED CYTOCHROME OXIDASE

BY VARIOUS COMPOUNDS

INHIBITIONINHIBITOR CONCENTRATION

FRACTION IV FRACTION V

NaN3 104M 78 0NaN3 10-2M 97 7KCN 10-8M 89 1KCN 10-2 M 92 33CO (dark) * 19 to 1 62CO (light) * 19 to 1 25Antimycin A 5.5 x 10- M 0 35Antimycin A 9.1 x 1O1 M 3 52

* Manometric assay utilized. All other tests involvedthe use of the standard spectrophotometric assay.

In all reaction mixtures except those involving COthe standard assay procedure was used after the enzymein buffer was preincubated for 1 minute with the inhibi-tors indicated. These reaction mixtures contained 0.02mg of protein from fraction IV and 0.01 mg of proteinfrom fraction V.

For the determination of CO inhibition the proce-dure of Webster (21) was used with the exception that75 micromoles of ascorbic acid was added to each flaskinstead of hydroquinone. In these experiments 2 mg ofprotein from fraction IV and 1.3 mg of protein fromfraction V was included in the assays. Manometricassays were corrected for the oxidation in absence ofadded cytochrome c. Fraction IV was prepared by 0 to30% (NH4)2SO4 precipitation and fraction V by 30 to65% (NH4)2S04 precipitation.

z00 20

20~~~~~X

o W4b 2100

1l lez ~~~~~~~~60210 40

O ~~~~~~~~ 0220

10 20 30 40 60 60TIME (SECONDS)

2 4 6 8 10 12 14 IS lISCYTOCHROUE C CONCENTRATION X IO (M)

FIG. 4. (Major graph) Cytochrome oxidase activityat various concentrations of cytochrome c using theleuco dye method for assay. The assay medium in afinal volume of 1 ml contained the following in micro-moles: 0.5 of leuco dye; 50 of phosphate buffer at pH7.0; and the concentrations of cytochrome c as indi-cated. The fraction V extract added to reaction mix-tures was heated for one minute in a boiling water bathand contained 0.03 mg protein. Cytochrome oxidaseactivity was determined by the change in O.D. at 645m,u in the 15-second interval between 10 and 25 seconds.

(Inset) Time course of the reaction using the leucodye method for assay. The assay medium in a final vol-ume of 1 ml contained the following in micromoles: 0.5of leuco dye; 50 of phosphate buffer at pH 7.0; and0.005 of cytochrome c. The amount and kind of extractadded was identical to that indicated for the majorgraph of figure 4.

sharply with time (inset in fig 4). This curve is verysimilar to that indicated in figure 2 where reducedcytochrome c oxidation as catalyzed by fraction Vwas measured directly. The effect of various concen-trations of cytochrome c on the enzyme activitymeasured by the leuco dye method is presented inthe major graph of figure 4. Since there were con-siderably greater concentrations of leuco dye thancytochrome c in all reaction mixtures and since leucodye and cytochrome c were mixed together before theenzyme was added, the reduced dye maintained thecytochrome c in the reduced state throughout the ex-perimental period. Using this assay method, a con-centration of 5 x 10-6 M cytochrome c resulted inthe highest enzyme activity. Since higher concentra-tions of cytochrome c resulted in decreased enzymeactivity, it seems probable that reduced as well as oxi-dized cytochrome c inhibited oxidation by fractionV. The possibility that the cytochrome c preparationcontained an inhibitor as a contaminant, however, hasnot been ruled out.

Smith (16) reported that high concentrations ofcholate inhibited cytochrome oxidase from animalsources. In an effort to determine whether the ob-served differences in the rates of reactions catalyzed

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by fractions IV (0 to 30 % (NH4)2SO4 ppt.) and V(30 to 65 % (NLH4)2SO4 ppt.) preparations were dueto differential inhibition of the two fractions by cho-late, samples of both fractions were dialyzed for 14hours against each of the following cold solutions: 3liters of 0.1 M phosphate buffer at pH 7.0; 3 litersof 0.02 M TRIS buffer at pH 7.0; and 3 liters of 0.1M sucrose. Assays of the extracts after dialysis in-(icated that each contained concentrations of cholateof approximately 0.06 %. After the dialyses bothextracts were assayed for cytochrome oxidase activ-ity by the standardI procedure. Fraction IV sampless4till catalyzed the oxidation of reduced cytochrome cat linear rates ancl fraction V extracts resulted in cy-tochrome c oxidation at the characteristic curvilinearrates. Extracts dialyzed against 13 clc (NH4)2SO4solutions (18) behaved in a manner similar to thosedialyzed against the buffer or sucrose solutions. Theeffect of Antimycin A, KCN and NaN3 on cytochromeoxidase activity of fractions IV and V is shown intable III. Antimyrcin A had no effect upon fractionIV reactions but at low concentrations inhibitedfraction V reactions markedly. KCN at 1(3 AI al-most completely inhibited activity of fraction IV.Oxidation by fraction V was insensitive to 10 3AIKCN and was inhibited only 33 % at 102' AI KCN.Usino the manometric assay it was demonstrated thatfraction IN was inhibited by a 19 to 1 mlixtuire of COand oxygen and thatt the inhibition w*as markedlyreversed by light. Since the reaction resulting fromthe fraction V preparation did not, continue for pe-riods greater than one mlinute, oxygen uptake by thisfraction could not be demonstrated. It was shown bv

x

in

on.

zt)w'A

z

0.10 0.15CONCENTRATION OF KCI (N)

FIG. 5. Effect of various concentrations of KCl on

cytochrome oxidase activity of cholate extracts fromsoybean roots. The procedure was that of the standardassay with v-ariations in concentration of KCI as indi-cated. The quantities of protein in mg added to assays

indicated by curves 1, 2, 3, and 4 are respectively: 0.04,0.04, 0.025 and 0.05. Extracts used in reactions indicatedby curves 1 and 2 were dialyzed for 14 houIs against 3liters of 0.02 M TRIS buffer at pH 7.4.

zZ ,(Gos / \

\_,/ ~~~FRACTION x

060

425 450 475 500 525 550 575 600 650

X (me)

FIG. 6. Difference sI)ectra (reduced muinuis Oxi(liZe()of cholate extracts fromii soybean roots. Fractions I1rand V were prepared as indicated in Materials andMethods. To obtain the reduced minus oxidized differ-ence spectra. sodium lhydrosulfite was added to onecuvette and the spectra plotted within 90 seconds by useof a Warren Spectracord. Fractions IV and V contained1.6 and 2.0 mg protein respectively.

use of a cuv-ette with a Thunberg atta.chnment that a19 to 1 CO-O2 mixture did not inhibit the oxidationof reduced cytochrome c when fraction V was uti-lized as the enzyme. To date no oxygen requirementhas been demonstrated for the reaction involving thefraction V preparation by use of a cuvette with aThunberg attachment. Since the technique used wasadequate for the (lemonstration of an oxygen require-ment for cytochrome oxidase from other sources thequestion of involvement of oxygen in the fraction Vreaction remiains open.

EFFECT OF KCL ON- ENZY-ME ACTIV-ITY: The ac-tivity of particulate cytochrome oxidase was mark-edly stimulated by salts as previously reported (11).The adldition of KCl to assays containing either frac-tions I\- (0 to 30% (NH4)2SO4 ppt.) or V (30 to65 % (NH4)2SO4 ppt.) failed to stimulate the ac-tivity. In order to remove salts from the prepara-tions both fractions IV and V were dialyzed over-night against three liters of a solution of 0.1 'M stu-crose or an equal volume of a solution of 0.02 M\TRIS buffer, pH 7.4 The cholate content was ap-proximately 0.04 % after dialysis against sucrose.The cholate content of fraction V' after clialvsisagainst TRIS btuffer w'as approximately 0.07 % andaipproximately 0.09 % in that dialyzed against su-crose. Sodium introduticed into the reaction mixturewas less than 2 x 10-3 M. Assay mixtures containingthe dialyzed extracts showed no enzyme activation aIsa result of the addition of KCl at final concentra-tions ranging up to 0.2 M (fig 5). Both dispersedfractions, however, were inhibited by NaHCO3 asprevioul.y- reported for particulate preparations (10).

SPECTRA: Difference spectra of fractions IV and Vin the range 400 to 620 mU are shown in figure 6.The spectra were plotted with the oxidized pigmentsin the reference cuvette and dithionite reduced pig-ments in the 2nd cuvette. Peaks w-ere observed in

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the difference spectrum of fraction IV near 428, 527,557 and 602 m/u. A peak at 426 m,u was evident forfraction V. Certain other preparations of fraction VexhibitedI adlditional peaks aroundl 560 and 600 my,but the magnitudes of these peaks were much lessthan those observed with fraction IV. The differencespectrum of fraction III was similar to that of frac-tion I\. The oxidized extracts of all fractions ab-sorbed strongly near 413 mu.

DISCUSSIONThe purification of particulate cytocbrome oxidase

fromii soy-bean roots has been attempted in an effortto proVide evidence of the mechanism of action spe-cificallv with reference to the mechanism wherebysalts stimulate the enzyme activity in particulate ex-tracts. Two dispersed fractions containing cyto-chrome oxidizing factors were prepared by use ofsodium cholate.

The 1st preparation was obtaine(d by precipita-tion of a.protein fraction from the extracts of cholatetreated particulates by adding (NH4)2SO4 until thesolution was 30 % saturated. The fraction possessedall the major characteristics which have been estab-lished for classical cytochrome oxidase (5, 7). Theactivity of this preparation was strongly inhibited byboth KCN and NaN3 and CO inhibition in the darkwas reversed by light in manometric assays. Initialrates of the reactions catalyzed by this fraction ap-proached linearity, an observation which is consistentwith a previous report (11) with similar material.These results, however, are not in agreement withthose obtained with the enzyme prepared from ani-mal sources (18). Our assay svstem contained aboutthree times the concentration of recluced cytochromec used by other workers (2) and this may be an im-portant factor contributing to the observed kinetics.A difference spectrum of the fraction IV (0 to 30 %(NH4)2S04 ppt.) preparation revealed evidence ofreduced absorption bands characteristic of cyto-chrome oxidase and also evidence that cvtochromepigments other than the oxidase were contained inthis extract. It seems clear that this preparationcontained the classical cytochrome oxidase system.The extent of purification of the enzyme in the frac-tion was too small to be of appreciable significance.

The 2nd preparation referred to as fraction V andprepared by increasing the (NH4)2SO4 concentrationfrom 30 to 65 % saturation in a cholate extract ofparticulates behaved differently than that of fractionIV. Activity was not appreciably inhibited by KCN,NaN3 or CO. It was inhibited, however, by a verylow concentration of Antimycin A, a compound thathad no effect on the fraction IV extract. It is wellknown (6, 13) that this antibiotic inhibits the stepbetween cytochromes b and c, however, no similarinhibition between cytochrome c and oxygen has beenreported. The difference spectrum of fraction V re-vealed no clear cut evidence of cvtochrome bandswith the exception of that near 426 m,L. Furtherpurification will be necessary before the significance

of this can be evaluated. Since reactions involvingthis extract ceased in a period usually less than oneminute and since additional reduced cytochrome cfailed to renew the oxidation (fig 1) the question ofwhether or not an enzyme was involved in the reac-tion is obviously presented. It was demonstratedthat the oxidizing factor was non-dialyzable, labileto boiling for five minutes and required cytochromec for the oxidation of reduced 2,3',6-trichloroindophe-nol. Evidence is presented indicating that fractionV oxidation was inhibited by both oxidized and re-duced cytochrome c. Smith and Conrad (18) havemade similar observations with preparations fromheart muscle. These properties certainly would notbe expected if a non-enzymatic oxidation were in-volved. Since all efforts to demonstrate an oxygenrequirement for the fraction V oxidation have failed,obviously the exact nature of the reaction remains forfuture investigation. It seems probable that thefraction V preparation represents some fragment ofan electron transport system that has some pecul-iarly high affinity for cytochrome c. The possibilityof preparatory procedures creating a fragment of aparticle with an affinity for cytochrome c seems rea-sonable. It will be necessarv to test the inhibition ofthe fraction V activity by highly purified cytochromec preparations before it can be concluded with cer-tainty that. cytochrome c per se and not a contami-nent is responsible for the inhibition.

It is of interest to consider that the cytochromeoxidase activity in particulate preparations is strik-ingly stimulated by salts but no such stimulation isobserved with the cholate dispersed preparations.It would be logical to expect that sodium cholatewould serve as a non-specific salt activator. Reac-tion mixtures including the dialyzed extracts con-taining approximately 2 x 10 3M with respect tosodium cholate and 0.025 A with respect to TRISchloride were not stimulatedl by the addition of KClat concentrations ranging between 0.05 and 0.2 A.On the basis of previous studies (11) of the influenceof salts on cytochrome oxidase activity in particu-lates, the combined sodium cholate and TRIS chlor-ide concentrations in reactions involving the dispersedfractions would not have been sufficient for an ap-preciable salt stimulation. It seems possible that thestimulatory influence of salts on particulate cyto-chrome oxidase is manifested as a result of somephysical effect on the enzyme that improves the ac-cessibility of it to substrates and that the surface ac-tive properties associated with cholate salts makethem highly effective in small concentrations. Thepossibility that an electrolyte may be required in theflow of electrons from one reactant to another insome structural unit of cells already has been dis-cussed by Lundegardh (9) and briefly considered byMiller and Evans (11). From this viewpoint, itwould seem logical that the destruction of the nor-mal physical orientation within particulates by useof dispersing agents might allow direct interaction ofreactants and therefore electron flow without a rela-

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MILLER ET AL-CYTOCHRONIE OXIDASE

tivelyIhigh electrolyte concentration. Apparentlythere is some specific effect of cholate salts not char-acteristic of other dispersing agents since Sisler (inpreparation) has prepared by use of digitonin op-tically clear extracts of tobacco root particulateswhich contain cytochrome oxidlase that is markedlystim-luate(l by salts.

SUMMARY

A dispersed preparation of cytochrome oxidasehas been prepared by adding sodium cholate to par-ticulates isolated from roots of soybean seedlings. Byuse of (NH4)2SO4 the dispersed extract was frac-tionated into two parts. The addition of either frac-tion to a solution containing reduced cytochrome cresulted in rapid oxidation. The properties of thetwo fractions however differedl greatly.

One fraction, which was obtained by 30 % satura-tion of the dispersed extract with (NH4)2SO4, cata-ly3zed reduced cytochrome c oxidation at a linearrate for a short period of time and exhibited all thewell known properties of cytochrome oxidase thathave been described for the enzyme from varioussources.

The 2nd fraction, obtainecl by increasing the(NH4)SO4 saturation in the dispersed extract to65 %, caused an oxidation of reduced cvtochrome cat a curvilinear rate, then the reaction quicklyceasedl. The evidence presented indicates that oxi-dized cytochrome c or a contaminate in this prepa-ration inhibited the oxidation and therefore is re-sponsible for the inactivation. Also the activity ofthe 2nd fraction was strongly inhibited by AntimycinA but was not appreciably inhibited by NaN3 or CO.These properties are in direct contrast to those ex-hibited by the 0 to 30 % (NH4)2SO4 precipitatedfraction. The 0 to 30 % (NH4)2S04 fraction cata-lyzed the uptake of oxygen; however, all efforts todemonstrate an oxygen requirement for the 30 to65 % (NH4)2SO4 fraction have failed.

Samples of the dispersed extracts were subjectedto dialysis and afterward assayed for activities withand without added KCl. Concentrations of KCl thatmarkedly stimulated the activity of particulate pre-parations hadl no appreciable effect on the dispersedfractions.

The results are discussed with respect to the prop-erties of the two fractions and to the mechanismwhereby salts activate particulate preparations andfail to activate cholate dispersed preparations.

The authors gratefully acknowledge the technicalassistance of _Mrs. Thea Schostag.

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10. MILLER, G. WZ and EVANS, H. J. Inhibition of plantevtochrome oxidase by bicarbonate. Nature 178:974-976. 1956.

11. MILLER. G. W. and EVANS, H. J. The influence ofsalts on the activity of particulate (ytochrome oxi-dase from rioots of higher plants. Plant Physiol.31: 357-364. 1956.

12. MILLER, G. W. and EVANS. H. J. Comparison ofcertain properties of particuilate cytochrome oxi-dase with a purified solubilized cytochrome oxi-dase from soybean roots. Plant Physiol. 31 Suppl.:xxv. 1956.

13. POTTER, V. R. and RIEF, A. E. Inhlibition of anelectron transport compound by Antimycin A.Jour. Biol. Chem. 194: 287-297. 1952.

14. RACKER, E. Spectrophotometer measurements ofthe metabolic formation and degradation of thiol-esters and enediol compounds. Biochim. Biophys.Acta 9: 577-578. 1952.

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17. SMITH, F. G. and STOTZ, E. A colorimetric methodfor the determination of cvtochrome oxidase.Jour. Biol. Chem. 179: 891-902. 1949.

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