- Email: [email protected]
Oxidation of catechol in plants
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Oxidation III. Purification
and Properties
247-257
166,
(1973)
of Catechol
in Plants
of the Diphenylene
Dioxide
2,3-Quinone-Forming
Enzyme System from Tecoma Leaves C. KANDASWAMI’ Department
of Biochemistry,
AND
Indian Received
C. S. VAIDYANATHAN
Fnstitute August
of Science, Bangalore-12,
India
15, 1972
In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricaleium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120.fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of t,he enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35’C. The K, for catechol was determined as 4 X lo-” M. The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activit.y. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show an\ peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.
The enzymatic conversion of catechol to diphenylene dioxide 2 ,3-quinone catalyzed by Tecoma leaf extracts was reported in an earlier communication (1). A partially purified enzyme system from Tecoma leaves was found to convert catechol to diphenylene dioxide 2,3-quinone (DDQ),2 stoichiometrically (Eq. 1). Interestingly, this preparation was found to be resistant to copperchelating agents. These results suggested 1 Senior Research Fellow of the Department of Atomic Energy, Government of India. 2 Abbreviation used: DDQ = Diphenylenedioxide 2,3-quinone.
the possibility of occurrence of a distinct enzyme, for the metabolism of catechol, in Tecoma leaves. Such an enzyme has been reported in spinach leaves by Nair and Vining (2). But nothing is known about the distribution of this enzyme in plants. It was worthwhile, therefore, to determine precisely the nature of the enzyme system which mediated the conversion of catechol to DDQ, in Tecoma leaves. To this end, further purification of the enzyme system from Tecoma leaves was made. The characterization of this enzyme system from Tecoma leaves is presented in this paper. 247
Copyright .411 rights
@ 1973 by Academic Press, of reproduction in nny form
Inc. reserved.
248
KANDASWAMI
AND
VAIDYANATHAN
OH
CATECHOL
DIPHENYLENE DIOXIOE-2,3 - qUlNONE
EQUATION
MATERIALS
AND
METHODS
Chemicals. Catechol was purchased from E. Merck Ag., Darmstadt, W. Germany. Protamine sulfate was obtained from Mann Research Laboratories, Inc., New York. Beef liver catalase, yeast alcohol dehydrogenase, bathocuproin sulfonate, NAD, NADP, NADH, ATP, FAD, FMN, GSH, etc. were procured from Sigma Chemical Co., St. Louis, MO. DEAE-Sephadex and Sephadex G-150 were obtained from Pharmacia, Uppsala, Sweden. Protein estimation. The protein content of the enzyme preparation was estimated by the method of Lowry et al. (3). Crystalline bovine serum albumin was used as standard. Determination of the approximate molecular weight of the enzyme. The method of Andrew (4) was used to assess the approximate molecular weight of the Tecoma leaf enzyme, Gel filtration was performed in a column (1.5 X GOcm) of Sephadex G-150, calibrated by using standard proteins. Disc electrophoresis. Polyacrylamide gel electrophoresis was done following the method of Davis (5) using Tris-glycine buffer, pH 8.3, and fl-alanine buffer, pH 4.3. Electrophoresis was carried out for 1 hr at room temperature with a current of 8 mA per tube. The gel was stained overnight with amido black and destained using 10% acetic acid. Enzyme assay. The enzyme activity was determined as follows. The reaction mixture consisting of 0.1 M sodium phosphate buffer, pH 7.1 (1.0 ml), 3 rmoles of catechol, enzyme solution (0.5 ml), and water in a total volume of 2 ml was incubated at 35°C for 15 min. The reaction was terminated by the addition of 0.2 ml of 0.5 N HCl and 3.0 ml of distilled chloroform were then added to the mixt,ure and the product was extracted into chloroform. DDQ formed was measured spectrophotometrically by measuring the absorbance at 410 nm. Catechol was estimated in the reaction mixtures following the calorimetric method of Nair and Vaidyanathan (6). A unit of activity is defined as the amount of enzyme which would catalyze the formation of 1 @mole of DDQ under the standard conditions of assay described. Estimation of dihydric phenols. The disappearance of o-dihydric phenols in the reaction mix-
tures was followed by the calorimetric method of Arnow (7). Assay of polyphenol oxidase. The assay of polyphenol oxidase was performed by the spectrophotometric method of Boston et al. (8), based on the increase in OD per minute at 470 nm, when the phenolic substrate is incubated with the enzyme preparation. The indirect spectrophotometric assay of Sisler and Evans (9) was also used to determine polyphenol oxidase activity. Preparation of potato phenolase. Purified preparations of pot,ato phenolase were obtained by adopting the purification procedure devised by Patil and Zucker (lo), up to the first DEAEcellulose column chromatographic stage. Estimation of copper in enzyme samples. For the estimation of copper in enzyme preparations, a calorimetric method of assay, using dithizone, as described by Sandell (11) was adopted. Estimation of microquantities of copper in the enzyme preparations was also done by the method of Stark and Dawson (12) as modified by Vaughan and Butt (13). Enzymatic determination of small quantities of Hg02. For the determination of small quantities of Hz02 produced, if any, during the conversion of catechol to DDQ, catalyzed by the Tecoma leaf enzyme, the micromethod described by Nakeno et al. (14) was used. Oxygen uptake. Oxygen uptake studies were carried out in Warburg flasks at 30°C as follows. The reaction mixture contained 1.5 ml of 0.1 M sodium phosphate buffer, pH 7.1, 4.5 pmoles of catechol, and 0.25 ml of water. The reaction was started by the addition of 0.75 ml of the Tecoma leaf enzyme from the side arm. Catechol disappearance in the reaction mixture was measured concurrently. RESULTS
Purification of the enzyme system from Tecoma leaves. All the operations were performed between stated otherwise.
0
and
5”C,
unless
Step 1, Preparation of crude extract. Fresh, leaves of Tecoma stans L, were
mature t,aken,
the
midribs
from
the
leaves
mere
OXIDATION
OF CATECHOL
detsc,hed and hvashed several times with glass-distilled water, and ground in a chilled mortar with acid-washed sand (9 g) and extracted with 175 ml of 0.01 M sodium phosphate buffer, pH .5.7. After squeezing through cheesecloth the greenish extract was cent’rifuged at 12,OOOgfor 15 mm. The clear supernatant fluid obtained was designated as the crude extract. Step 2,. T,reatnzent with protamine sulfate. Nine milliliters of 2% aq. protamine sulfate solution were added to the crude extract (180 ml) drop by drop wit,h gentle stirring. After 15 min the extract was centrifuged at 12,OOOg for 10 mm to get a clear supern&ant fraction. Step 3. Tricalcium phosphate gel fractionation. An S5-ml quantity of tricalcium phosphate gel (20 mg/ml) was centrifuged and to the residue obtained were added 188 ml of the supernatant fluid from Step 2 (pH 5.7) with stirring. After 20 min the mixture was centrifuged at 12,000g for 10 min. The gel supernatant fraction did not show any activity. The gel was washed twice with 40 ml of 0.02 RI sodium phosphate buffer (pH 6.0) and later the enzyme was eluted twice with 30 ml of 0.05 M sodium phosphate buffer, pH 7.0. The gel was removed by centrifugation and the eluate was processed further. Step 4. DEAE-Sephadex chromatography. DEAESephadex was equilibrated with 0.05 M sodium phosphate buffer and packed into a column (1.5 X 20 cm). The gel eluate obtained from Step 3 was concentrated to 8 ml by aquacide dialysis. The concentrate was loaded onto the column of DEAESephadex equilibrated with 0.05 M sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl. The column was initially washed with 40 ml of 0.05 M sodium phosphate buffer, pH 7.0. After this, the column was successively washed with 40-ml quantities of 0.05 M sodium phosphate buffer (pH 7.0) containing 0.15 and 0.2 nt NaCl, respectively. Finally the enzyme was eluted twice with 20-ml portions of 0.05 nc sodium phosphate buffer, pH 7.0, containing 0.25 M T‘JaCl. This eluate was designated as the purified enzyme. By following the purification procedure
IN PLANTS
245
III TABLE
I
SUMMARY OF PURIFICATION OF THE DDQ-FORMING ENZYME FROM Tecowaa LEAVES
Step Volume
I II III IV
180 188 60 40
Total activity (units) 105.30 157.10 70.22 62.86
Protein (w/ml)
4.50 2.20 0.44 0.10
Specific activity (units ‘mg protein)
-
0.13 0.38 2.66 15.72
outlined above, a 120.fold purified enzyme was obtained with nearly 60% recovery. A summary of the purification proccdurc is given in Table I. Electrophoresis of the puri$ed enzyme. The purified Tecoma leaf enzyme was analyzed by disc electrophoresis on polyacrylamide gel by the method of Davis (5). A prominent single band was observed in the gel which stained darkly. Only one prominent band was detected when electrophoresis was performed at pH 8.3 and pH 4.3 (fig. 1). Molecular weight of the Tecoma leaf DDQforming enzyme. The molecular weight. of the purified Tecoma leaf enzyme as determined by gel filtration of t#he protein on Sephadex G-150 and G-200 was approximately 200,000. E$ect oj pH on enzyme activity. Figure 2 shows the effect of pH on the purified Tecoma leaf enzyme. The optimum pH of the reaction was determined by using 0.1 M citrate-O.2 M sodium phosphate and 0.1 nr sodium phosphate buffers. Optimal act’ivity was observed around pH 7.1. There was no appreciable rate of activity at pH values above 7.8 and below 4.5. Eflect of substrate concentration. The optimal substrate concentration for the reaction was found to be 1.5 X 10d3 M. The ILL value for catechol was determined as 4 X 10e4 M from a Lineweaver-Burk plot. E$ect of enzyme concentration. The reaction rate was found to be linear up to a protein concentration of 50 pg. Effect of temperature. The optimum temperature for the enzyme activity was found to be 35°C. The activity fell off rapidly beyond 50°C. Time-course and stoichiometry of the
250
KANDASWAMI
AND
VAIDYANATHAN
presence of the purified Tecoma leaf enzyme is shown in Table III. The data indicated a requirement for 1.5 atoms of oxygen per mole of catechol oxidized when measurements were made after 5 min. Substrate spec$city. It was of interest to compare the substrate specificity of the purified Tecoma leaf enzyme with that of the plant polyphenol oxidases which oxidize a bewildering range of phenolic substrates (15). Various 0-dihydric phenols were tested as substrates for the purified Tecoma 0.60 I
FIG. 1. Polyacrylamide gel electrophoresis of the purified DDQ-forming enzyme from Tecoma leaves. Polyacrylamide gel electrophoresis of the purified protein was performed employing Trisglycine buffer, pH 8.3. Electrophoresis was carried out for 1 hr at room temperature with a current of 8 mA per tube. The gel was stained with amido black and destained using 10% acetic acid.
reaction. The effect of different periods of incubation on the formation of DDQ, catalyzed by the purified Tecoma leaf enzyme, showed a linear relationship between the enzyme activity and the incubation period up to 15 min. One mole of DDQ was formed for 2 moles of catechol metabolized, in presence of the purified Tecoma leaf enzyme. The stoichiometry of DDQ formation catalyzed by the purified Tecoma leaf enzyme is presented in Table II. Oxygen uptake. The conversion of catechol to DDQ, catalyzed by the purified Tecoma leaf enzyme, proceeded only under aerobic conditions. The oxygen consumption during the oxidation of catechol to DDQ, in
a 0;
6.2
6.6
7.0
7.4
7
PH
FIG. 2. Effect of pH on the purifigd Tecoma leaf enzyme. The reaction mixture consisbd of 1.0 ml of 0.1 M sodium phosphate buffer of different pH values, 3 pmoles of catechol, 50 rg of purified Tecoma leaf enzyme, and water to a total volume of 2 ml was incubated at 35°C for 15 min.
TABLE CONVERSION OF PURIFIED
II
CATECHOL
TO
Tecoma LEAF
DDQ
BY
THE
ENZYME
STOICHIOMETRY~
Time (min)
Catechol disappeared bole)
DDQ formed (wale)
5 10 15
0.31 0.63 0.89
0.15 0.30 0.42
(1 Standard assay conditions were used and the reaction was performed for various intervals of time.
OXIDATIOX TABLE
OF CATECHOL
III
OXYGEN CONSUMPTION DURING THE CONVERSION OF CATECHOL TO DDQ BY THE PURIFIED Tecoma LEAF ENZYME” Time (min)
Catechol oxidized Oxygen consumed (patorns) bole) ..~ .~~~
5 10 15
0.40 0.84 1.60
0.28 0.58 1.10
(LThe composition :he s:ame as described
of the reaction mixt,ure is for oxygen-uptake studies.
TABLE
IV
OXIDATION OF VARIOUS O-DIHYDRIC PHENOLS BY THE PURIFIED Tecoma LEAF ENZYME” Compound tested
Catechol DL-Dihydroxyphenylslanine Caffeic acid Chlorogenic acid 3,4-Dihydroxybenzoic acid 2,3-Dihydroxybenzoic acid Dihydrocaffeic acid Homoprotocatechuic acid
pmoles of compound disappeared 4.20 0.01 0 0.02 0.01 0.03 0 0
The composition of the reaction mixture was the same as described for the standard assay except that 5 X lo-’ M of each phenolic substrate xas utilized in t,he assay in each case.
leaf enzyme and the results are summarized in Table IV. ,4 perusal of the results presented in Table IV would show that the Tecoma leaf enzyme is highly specific for catechol. The ability of the purified Tecoma leaf enzyme to attack vie-trihydric phenols was studied. Pyrogallol and gallic acid did not serve as substrates for the purified Tecoma leaf enzyme. These substrates were tested by the indirect spectrophotometric method. The purified enzyme was feebly active with monohydric phenols like L-tyrosine and p-cresol. The unreactivity of the monohydric phenols was confirmed by oxygen uptake studies and by calorimetric assay met,hod. Obviously the Tecoma leaf enzyme did not possess hydroxylase activity toward monohydric phenols. The purified Tecoma leaf enzyme was not
IN
PLANTS
231
III
able to oxidize hydroquinone, t’hereby showing that the enzyme did not possess lactase activity (15). The Tecoma leaf enzyme failed to display any activity toward such flavonoids as phloridzin and quercetin. Comparison of the puriJied Tecoma leaf enzyme with potato polypkenol oxidase. The substrate specificity of the potato phenolase was examined in order to get a comparative picture with the Tecoma leaf enzyme. It is known that potato phenolase can attack many polyphenols in addition to monohydric phenols (15). It was earlier observed that the purified Tecoma leaf enzymes were incapable of at,tacking o-dihydric phenols like Dopa, caffeic acid, chlorogenic acid, protocatechuic acid etc. A partially purified preparation of pot’ato phenolase was obtained by following the method of Patil and Zucker (10). The different o-dihydric phenols were tested in presence of t’his preparation and the results are summarized in Table V. Chlorogenic acid was found Do be the most preferred substrate for the phenolase among the o-dihydric phenols Dested. The activity of the pot,ato phenolase towards di- and trihydric phenols and monophenols was tested by the indirect spectrophotometric met’hod (9). Potato phenolase was found to be very active with trihydric phenols like pyrogallol. E$ect of metal ions on the purified Tecoma leaj enzyme. The response of the Tecoma leaf TABLE
V
OXIDATION OF 0-DIHYDRIC PHENOLS BY THE PURIFIED POTATO PHENOLASE~ Substrate
Chlorogenic acid Catechol Caffeic acid DL-Dihydroxyphenylalanine 3,4-Dihydroxybenzoic a The reaction sodium phosphate of the enzyme, 1 tested, and water Incubations were The assay of the imetrically (Ref.
fimoles of substrate disappeared
acid
0.47 0.36 0.18 0.08 0.25
mixture consisted of 0.1 M buffer, pII 6.8 (1.0 ml), 0.5 ml Fmole of each substrate to be in a total volume of 2.0 ml. carried out at 30°C for 10 min. enzyme was performed color7).
252
KANDASWAMI
AND
enzyme to various metal ions was studied. Activity was not increased by supplementing the enzyme with any of the following salts (at lo4 M and 5 X lo4 M), CdS04, MgSCh , MnSOc , FeS04, and CuS04. Of the different metal ions tested HgCl, , FedSOd , and ZnS04 inhibited the reaction to 56,40, and 32% at 5 X lo4 M. E$ect of metal-chelating agents on the purified Tecoma leaf enzyme. The response of the Tecoma leaf enzyme to various metalchelating agents was studied and the results are shown in Table VI. Ferrous inhibitors such as o-phenanthroline and a-n’-dipyridyl did not inhibit the enzyme to any appreciable degree. The Tecoma leaf enzyme was found to be insensitive to diethyl dithiocarbamate and salicylaldoxime. The effect of different copper-chelating agents such as diethyldithiocarbamate, salicylaldoxime, potassium ethylxanthate, and bathocuproin sulfonate was tested at various concentrations (Table VII). They caused very little inhibition of the oxidation of catechol in presence of the purified Tecoma leaf enzyme. Estimation of copper in the purijed Tecoma leaf enzyme. T\Tocopper could be detected in the purified Tecoma leaf enzyme preparations by following different micromethods. TABLE
VI
EFFECT
OF METAL-CHELATING AGENTS ON THE PURIFIED Tecoma LEAF ENZYMES Final concenSupplement Catechol tration disappeared
(4 None (Y,or’-dipyridyl o-Phenanthroline S-Hydroxyquinoline Salicylaldoxime Diethyldithiocarbamate EDTA
10-d 5 x 10-d 10-d 5 x lo-4 lo-4 5 x lo-4 5 x 10-h 5 x 10-d 10-4
5 x
10-d
(Irmoles) 0.88 0.81 0.72 0.82 0.74 0.74 0.68 0.79 0.72 0.81 0.72
Each chelating agent was preincubated with the enzyme for 5 min prior to the addition of the substrate. Standard conditions of assay were employed.
VAIDYANATHAN TABLE
VII
EFFECT OF COPPER-CHELSTING AGENTS ON THE PURIFIED Tecoma LEAF ENZYME* Addition
Concentration (M)
None Salicylaldoxime
Diethyldithiocarbamate Bathocuproin sulfonate
Potassium xanthate
10-4 5 x 10-4 10-Z 10-d 5 x 10-d 10-a 10-4 5 x 10-4 lo-3
ethyl5 x
10-b 10-d 10-S
Catechol disappeared (pmoles) 0.87 0.81 0.78 0.75 0.78 0.71 0.69 0.85 0.79 0.77
0.79 0.72 0.70
a Each inhibitor was incubated with the enzyme for 5 min prior to the addition of the substrate. Standard conditions of assay were employed.
Effect of thiol inhibitors. The Tecoma leaf enzyme was found to be insensitive t’o thiol inhibitors like pHMB, iodoacetat’e, and N-ethylmaleimide. E$ect of reducing agents on the enzyme. An interesting property of the DDQ-forming enzyme, from Tecoma leaves, was its extreme susceptibility to reducing agents like GSH and cysteine. Cysteamine suppressed the reaction completely at 5 X lOA M.
E$ect of axide, sulfide, and other complexing agents on the purified Tecoma leaf enzyme. None of the copper-complexing agents such as cyanide, aside, sulfide, and citrate exhibited any significant effect on the purified Tecoma leaf enzyme. E$ect of dialysis of the puri$ed Tecoma leaf enzyme against copper-chelating agents. Extensive dialysis of the purified DDQforming enzyme from Tecoma leaves against copper-chelating agents and cyanide caused no reduction in the activity of the enzyme providing additional evidence t,hat the Tecoma leaf enzyme was not a copper protein. E$ect of cofactors on DDQ-forming enzyme from Tecoma leaves. The effect of various
OSIDSTION
OF CATECHOL
cofactors like l+lN, FAD, NAD, etc., on the purified Tecoma leaf enzyme was studied. NADI’ caused about 2S% stimulation of the enzvme activity. Other compounds testtld did not enhance the reaction rate appreciably. FAD and FRIN did not have any activating effect. The effect of atebrine 011 t.he purified Tecowa leaf enzyme was examined in order t’o know whet,her there was a flavin requirement, for the enzyme. Atcbrinc at, a cow centratJion of 5 X 10d4 31 had no effect on the oxidation of catcchol by the Teconza leaf enzyme. E.flect of carbon monoxide. The purified DDQ-forming enzyme from Tecoma leaves was insensitive to carbon monoxide. Efect of p-nitrophenol and sodium jluoride on the pu+ed Tecoma leaf enzyme. The activity of the Tecoma leaf enzyme n-as not affected by p-nitrophcnol or sodium fluoride. E#ect qf thiourea on the Tecoma leaf enzyme. The &ect of various concentrations of thiourea, on the Tecoma leaf enzyme was st’udicld. The results showed that t’hiourea did not cause any inhibition of the enzyme activity at a concent’ration of 10U3hi. Treatment of the Teconza leaf enzyme with denaturing agents. It has been reported that the tyrosinase from broad bean leaves is a latent, enzyme and that the act’ivity of the cbnzyme can be enhanced considerably by treatment with acid and alkali (16). The DDQ-forming enzyme from Tecoma lcaves was exposed for a limited time to pH 3.0-3.5 and 10-l 1. Such treatments did not activate the enzyme. On the other hand, prolonged t’reatment caused inhibition of the enzyme activit’y. The purified Tecoma leaf enzyme was found to low its activity under mild denaturing conditions in contrast to the broad bean tyrosinase (17). At a conce&ation of 6 YI urea half the enzyme activity was lost. The purified Tecoma leaf enzyme was t’reated with different concentrations of urea for varying intervals of time and the activity was assayed in each case. A IO-min treatment of the enzyme with S M urea resulted in nearly 60°jo loss in enzyme activity (Fig. 3). i3jeect of catalase on the Tecoma leaf enzyme. Beef liver catalase added to the
IN
01 0
PLANTS
I 5
25’ c
III
I I 10 15 Time (mins.)
I 20
FIG. 3. Effect of urea treatment on the purified DDQ-forming enzyme from Tecoma leaves. The purified Tecoma leaf enzyme was incubated with varying concentrations of urea for various intervals of time. The enzyme activity was assayed standard conditions described under under Materials and Methods.
reaction mixture (300 pg-1 mg) had nc inhibitory effect on the Tecoma leaf enzyme. It was observed that catalase did not alter the time-course of the DDQ-forming enzyme markedly. E’nxymatic determination of hydrogen peroxide. Attempts were made to estimate very small amounts of H202 produced, if any, during the conversion of catechol to DDQ by the purified Tecoma leaf enzyme. 50 Hz02 could be detected during the enzymat,ic conversion of catechol to DDQ. Intracellular distribution oj the Tecoma leaf enzyme. Table VIII shows the distribution of DDQ-forming enzyme in different subcellular fractions. Cellular fractionation of Tecoma leaves was carried out in saline medium by the procedure of Nair and Vaidyanathan (1s). The activity was mainly localized in the soluble fraction. DISCUSSION
A wide range of structurally diverse phenolic compounds found in higher plants and microorganisms-tannins, lignins, antibiotics, alkaloids, melanins-are recognized to be derived from the oxidative coupling of simple phenolic substances (15, 19). The formation of such natural products involve frequently carbon-carbon and carbon-oxygen linkages. The importance of the occur-
254
KANDASWAMI TABLE
AND
VIII
CELLULAR DISTRIBUTION OF THE DDQ-FORMING ENZYME FROM Tecoma LEAVES" Fraction
Chloroplast fraction Mitochondrial fraction Soluble fraction
Catechol oxidized (@moles) 0.12 0 0.88
a The assay conditions employed were standard except that the purified enzyme was replaced by 0 5 ml each of the above fractions in each case.
rence of the carbon-oxygen coupling during the formation of catechol melanins in higher plants and fungi has been emphasized (20). Nair and Vining (2) obtained a partially purified enzyme preparation from spinach leaves which converted catechol to DDQ. This preparation was not inhibited by diethyldithiocarbamate and was specific for catechol. The DDQ-forming enzyme isolated from Tecoma leaves was found to catalyze the stoichiometric conversion of catechol to DDQ. A perusal of the results shows that this enzyme is different from the polyphenol oxidases. At this stage it is necessary to interpret the results obtained with this enzyme system in the light of our present day knowledge concerning the specificity and catalytic activity of the phenol oxidases. A scrutiny of the substrate specificity of the purified DDQ-forming enzyme from Tecoma leaves would reveal the high specificity of the enzyme for catechol. This enzyme is not able to oxidize trihydric phenols and complex polyphenols like the flavonoids and thus differs markedly from the potato phenolase (21). The Tecoma leaf enzyme also lacks the hydroxylase activity towards monohydric phenols, which is a characteristic feature of the potato polyphenol oxidase (15). The tyrosinase isolated from the leaves of the broad bean (Vi&a faba L.) resembles the potato enzyme in its two basic catalytic activities, namely, the orthohydroxylation of monohydric phenols and the dehydrogenation of o-dihydric phenols (22). A significant feature in regard to the substrate pattern of the broad bean tyrosinase is that, substituted catechols containing a free carboxyl
VAIDYANATHAN
group, such as protocatechuic, caffeic, and chlorogenic acids, are not particularly good substrates, Dopa being a notable exception. The substrate specificity of the Tecoma leaf DDQ-forming enzyme seems to distinguish it from the broad bean tyrosinase. Another important aspect of the substrate specificity of the DDQ-forming enzyme from Tecoma leaves concerns its action toward hydroquinone. Hydroquinone is a preferred substrate for lactase whereas its oxidation is not catalyzed by tyrosinase (15) and this constitutes an important difference between tyrosinase and lactase (15). The DDQ-forming enzyme from Tecoma leaves is totally unreactive toward hydroquinone. It is evident, therefore, that the Tecoma leaf enzyme does not display lactase activity. The balance of evidence obtained on the DDQ-forming enzyme from Tecoma leaves indicates that it is not a copper protein. The purified Tecoma leaf enzyme was insensitive to a range of copper-chelating agents like salicylaldoxime, diethyldithiocarbamate, potassium ethylxanthate, and bathocuproin sulfonate. Moreover, the Tecoma leaf enzyme was not inactivated by dialysis against copper-chelating agents and cyanide. On the other hand, the dialysis of the mushroom tryosinase against cyanide (23) was sufficient to deprive the enzyme of practically all of its copper and its activity. Pomerantz (24) prepared the apoenzyme of tyrosinase from hamster melanoma by treatment with KCN or diethyldithiocarbamate. Omura (25) prepared the apoenzyme of Rhus vernicefera lactase by dialyzing the enzyme against cyanide or EDTA. Phenolic substances like p-nitrophenol and resorcinol form inactive complexes with copper proteins (26). These compounds had no effect on the Tecoma leaf enzyme. Bonner and Wildman (27) reported the inhibition of spinach leaf polyphenol oxidase by pnitrophenol. Robb et al. (22) observed that p-nitrophenol and sodium fluoride inhibited the tyrosinase from broad bean 1eaveE (Vicia fuba L) noncompetitively. Fluoride is known to complex with copper (22). Sodium fluoride and different copper-complexing agents had no significant effect on
0SII)ATION
OF CATECHOL
the Tecoma leaf enzyme even at low pH values. Thiourea caused no reduction in the activity of the Tecoma leaf enzyme whereas it had been reported as an inhibitor of tea polyphenol oxidase (28) and mammalian tyrosinasc (24). It is interesting to note the insensitivity of the purified Tecoma leaf enzyme to carbon monoxide while t’he sensitivity of the tyrosinase to carbon monoxide is very significant (29). The observation that copper could not be det,ected in the purified Tecoma leaf enzyme preparations affords further evidence to deduce that the Tecoma leaf enzyme is not a copper protein like the tyrosinase. The molecular weight of the purified Tecoma leaf enzyme is estimated approximately as 200,000. An approximate molecular weight of 100,000 has been reported by Kubowitz (30) for the phenolase from white potatoes (Xolanum tuberosum). The polyphenol oxidase of common mushroom (Psalliota campestris) had an approximate molecular weight of 133,000 f 10,000. Another significant molecular property of the tyrosinase, in contrast to the Tecoma leaf enzyme, is their heterogeneity. The multiple molecular forms of mushroom tyrosinase were separated by Bouchilloux et al. (31). The four molecular forms of the tyrosinase, in this case, differed from each other in their substrate specificity. Robb et al. (32) achieved the separation of the different molecular forms of the tyrosinase from broad bean leaves (V&a faba L) by starch gel electrophoresis and it was found that the different forms did not differ decisively in their substrate specificity (22). On the contrary, the purified DDQ-forming enzyme from Tecoma leaves gives only one band on disc electrophoresis at different pH values. The cellular distribution of the DDQforming enzyme from Tecoma leaves seems to be important in view of the multiplicity of catechol oxidases in tissues of higher plants (33). The DDQ-forming enzyme is predominantly localized in the soluble fraction of the cell. Moreover, it exhibits only a single pH optimum. Hare1 et al. (34) have
IN
PLANTS
III
2.55
shown t’hat in apples catcchol oxidases are present in several subcellular fractions. These authors (33) subsequently showed t,he multiplicity of phenol oxidases in the same subcellular fraction. The purified Tecoma leaf enzyme loses its activity under mild denaturing condit’ions. When the Tecoma leaf enzyme is exposed to acid and alkaline pH, there is a diminution of its activity. Kenten (16) has described the tyrosinase from T’icia fhba leaves as a latent, enzyme which gains activation on treatment with acid and alkali. Robb et al. (35) concur with the idea of Kenten (16) that t’he exposure of the broad bean tprosinase, for a limited time, to pH 3.0~3..5 or 10-11.5, leads to the activation of the enzyme. They found that t’he activity of this enzyme was promoted by mild denaturing conditions. In contrast to the broad bean tyrosinase, treatment of the Tecoma leaf enzyme with urea leads to a progressive reduction in enzyme activity. The purified Tecoma leaf enzyme does not show any peroxidase activity. The enzyme is not inhibited by added catalase and hydrogen peroxide does not seem to be a product of the enzymatic reaction. It has been pointed out that the DDQforming enzyme from Tecoma leaves is similar to t’he spinach leaf enzyme (a) in its specificity for catechol. The lack of sensitivity of the spinach leaf enzyme t’o diethyldithiocarbamate was another distinctive feature of this enzyme. The Tecowza leaf enzyme shares this property. Many other copper-chelating agents have been tested with the purified Tecoma leaf enzrme and none of these was found to inhibit the enzyme. Moreover, the purity of the Tecoma leaf enzyme has been checked. It appears that the DDQ-forming enzyme is a distinctive one for the metabolism of catechol in Tecoma leaves. An interesting aspect of the spinach leaf enzyme is that it is stimulated by FAD (2) though an absolute requirement for it was not observed. The reaction was markedly inhibited by atebrine. Atebrine was, however, without effect 011the Tecorna leaf enzyme and it was not activated by FAD to any significant degree. The uv and visible
256
KANDASWAMI
FIGS. 4 and 5. Mechanism
AND
for the formation
spectrum of the purified enzyme did not indicate the presence of any flavin moiety in the enzyme. The Tecoma leaf enzyme is optimally active at pH 7.1, whereas the spinach leaf enzyme (2) exhibits an optimum pH of 7.4. The Tecoma leaf enzyme is much more susceptible to Hg2+ than the spinach leaf enzyme. The extreme susceptibility of the purified Tecoma leaf enzyme to reducing agents is a noteworthy feature. It will be instructive to review the available information concerning the mechanisms that may operate for the formation of carbon-oxygen-linked oxidation products of phenolic compounds, like DDQ. Forsyth and colleagues (36) suggested that the enzymatic oxidation of catechol leads to the formation of o-benzoquinone in presence of mushroom polyphenol oxidase. Further oxidation of benzoquinone derivatives is believed to result in coupling by both carbon-carbon and carbon-oxygen bonds. Brown (15) believes that oxidation products such as DDQ arise by the coupling of aryloxy radicals. According to him (15) the enzymatic oxidation of catechol leads to the formation of aryloxy radicals which then form different products depending on the mode of coupling. DDQ might arise by the oxygen-para coupling of aryloxy radicals (Fig. 4). Thomson (37) while discussing the formation of larger monomeric aromatic sub-
VAIDYANATHAN
of diphenylene
dioxide
2,3-quinone.
FIG. 5
stances found in nature, draws attention to the oxidative dimerization of simple phenolic precursors. In describing the mechanism of oxidative processes, he emphasizes the importance of oxidation by one-electron abstracting agents. Oxidation by oneelectron abstracting agents give rise to mesomeric radicals (ArO.) which may dimerize or react with other radicals forming new C-C, C-O, or O-O bonds. He suggests that enzymatic oxidation may take the same course. The mechanism for the formation of DDQ as suggested by Thomson (37) is delineated in Fig. 5. In this scheme 2,3’ ,4’trihydroxydiphenylether has been implicated as an intermediate. At this juncture, it would be premature to speculate on the possible mode of formation of DDQ in presence of the Tecoma leaf enzyme. At t’he same time we are tempted to think that the strong inhibition of the enzyme by cysteamine might indicate the involvement of free radicals in the formation of DDQ from catechol.
O~XIDATION
OF CATECHOL
ACKNOWLEDGMENT The financial Atomic Energy, acknowledged.
18. NAIR,
support from the Department of Government of India is gratefully REFERENCES
1. KAND~~~AMI,
C.,
AND VAIDYANATHAN,
IN
C. S.
(1972) Biochem. J. 128, 30 P. L. C. (1964) Arch. 2. N~IR, P. M., AND VINING, Biochem. Biophys. 106, 422. 3. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 4. ANDREWS, P. (1964) Biochem. J. 91,222. 5. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404. 6. NXR, P. M., AND VAIDYANATHAN, C. S. (1964) Anal. Biochem. 7, 315. 7. ARNOW, L. E. (1953) J. Biol. Gem. 118,531. 8. BOSCON, L., PO~RIE, W. D., AND FENNENIO, 0. (1962) J. Food Sci. 27,574. 9. SISLER, E. C., AND EVANS, H. J. (1958) Plant Physiol. 33, 255. 10. PATIL, S. S., AND ZUCKER, M. (1965). J. Biol. Chem. 240,393s. 11. SANDELL, E. B. (1944) Calorimetric Estimation of Traces of Metals, Inter-science, New York. 12. ST~IRK, G. R., AND L)AWSON, C. R. (1958) AnaZ. Chem. 30,91. 13. VAUGHAN, P. F. T., AND BUTT, V. S. (1970) Biochem. J. 119, 89. 14. N~K.~No, M., TSUTSUNI, Y., AND DANOWSKI, T. S. (1968) Proc. Sot. Esp. Biol. Med. 129, 960. Coupling of 15. BROWN, B. R. (1967) in Oxidative Phenols. (Taylor, W. I., and Battersby, A. R., eds.), p, 167, Marcel Dekker, New York. 16. KENTEN, R. H. (1958) Biochem. J. 68,224. 17. SW.~IN, T., MAPSON, L. W., AND ROBB, D. A. (1966) Phytochemistry 6, 469.
PLANTS
III
P. R/I., ,iND VAIDy.4NdTKAN,
257 C. S. (1964)
Phytochemistry 3, 235. 19. SCOTT, A. I. (1967) in Oxidative Coupling of Phenols (Taylor, W. I., and Battersby, A R., eds.), p. 15, Marcel Dekker, New York. 20. PIATTELLI, M., FATTORUSSO, E., NICOL.LUS, R. A., AND ;LIASON, s. (1965) Tetrahedron 21, 3229. 21. ABUKHhRM.4, D. A., .tND WOOI.HOt-HE, l-1. 9. (1966) i\‘eu, Phytol. 66,477. 22. Ronn, D. A., hfAPSON, L. W., .4ND Pn-m, T. (1966) Phytochemistry 6, 665. 23. MALMSTROM, B. G., ‘LSD NIELANDS, J. B. (1964) Annu. Rev. Biochem. 33, 331. 24. POMERANTZ, S. (1963) J. Biol. Chem. 233,235l. 25. OMURA, T. (1961) J. Biochem. 60, 389. 26. JAMES, W. 0. (1953) Annu. Rev. Plant Ph,ysiol. 4, 59. S. G. (1946) Arch. 27. BONNER, J., AND WILDM.IN, Biochem. 10, 497. 28. GREGORT, R. P. F., .%IXD BEND.ILL, I). S. (1966) Biochem J. 101,569. 29. KEILIN, II. (1929) Proc. Roy. Sot. Ser. B 104, 206. 30. KUBOWITZ, F. (1938) Biochem. 2. 299, 32. 31. BOIXHILLOUX, S., MCMAHILL, P., IND M.UON, II. S. (1963) J. Biol. Chem. 238, 1699. 32. Ronn, D. A., MAPSON, L. W., .YND Smart, T. (1966) Phytochemistry 4, 731. 33. HAREL, E., MAYER, A. M., ASD SH~IS, Y. (1965) Phytochemistry 4, 783. 34. HAREL, E., MAYER, A. M., .~XD SH~IN, Y. (1964) Physiol. Plant. 17,921. 35. Ronn, D. A., M.~PSOX, L. W., AND &V.ZIN, T. (1964) Nature (London) 207, 503. 36. FORSYTH, W. C. G., QUESNEL, D. C., AND ROBERTS, J. B. (1960) Biochim. Biophys. Acta 37, 322. 37. THOMSON, R. H. (1964) in, Biochemistry of Phenolic Compounds (Harborne, .J. B., ed.) p. 1, Academic Press, New York.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Oxidation III. Purification
and Properties
247-257
166,
(1973)
of Catechol
in Plants
of the Diphenylene
Dioxide
2,3-Quinone-Forming
Enzyme System from Tecoma Leaves C. KANDASWAMI’ Department
of Biochemistry,
AND
Indian Received
C. S. VAIDYANATHAN
Fnstitute August
of Science, Bangalore-12,
India
15, 1972
In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricaleium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120.fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of t,he enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35’C. The K, for catechol was determined as 4 X lo-” M. The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activit.y. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show an\ peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.
The enzymatic conversion of catechol to diphenylene dioxide 2 ,3-quinone catalyzed by Tecoma leaf extracts was reported in an earlier communication (1). A partially purified enzyme system from Tecoma leaves was found to convert catechol to diphenylene dioxide 2,3-quinone (DDQ),2 stoichiometrically (Eq. 1). Interestingly, this preparation was found to be resistant to copperchelating agents. These results suggested 1 Senior Research Fellow of the Department of Atomic Energy, Government of India. 2 Abbreviation used: DDQ = Diphenylenedioxide 2,3-quinone.
the possibility of occurrence of a distinct enzyme, for the metabolism of catechol, in Tecoma leaves. Such an enzyme has been reported in spinach leaves by Nair and Vining (2). But nothing is known about the distribution of this enzyme in plants. It was worthwhile, therefore, to determine precisely the nature of the enzyme system which mediated the conversion of catechol to DDQ, in Tecoma leaves. To this end, further purification of the enzyme system from Tecoma leaves was made. The characterization of this enzyme system from Tecoma leaves is presented in this paper. 247
Copyright .411 rights
@ 1973 by Academic Press, of reproduction in nny form
Inc. reserved.
248
KANDASWAMI
AND
VAIDYANATHAN
OH
CATECHOL
DIPHENYLENE DIOXIOE-2,3 - qUlNONE
EQUATION
MATERIALS
AND
METHODS
Chemicals. Catechol was purchased from E. Merck Ag., Darmstadt, W. Germany. Protamine sulfate was obtained from Mann Research Laboratories, Inc., New York. Beef liver catalase, yeast alcohol dehydrogenase, bathocuproin sulfonate, NAD, NADP, NADH, ATP, FAD, FMN, GSH, etc. were procured from Sigma Chemical Co., St. Louis, MO. DEAE-Sephadex and Sephadex G-150 were obtained from Pharmacia, Uppsala, Sweden. Protein estimation. The protein content of the enzyme preparation was estimated by the method of Lowry et al. (3). Crystalline bovine serum albumin was used as standard. Determination of the approximate molecular weight of the enzyme. The method of Andrew (4) was used to assess the approximate molecular weight of the Tecoma leaf enzyme, Gel filtration was performed in a column (1.5 X GOcm) of Sephadex G-150, calibrated by using standard proteins. Disc electrophoresis. Polyacrylamide gel electrophoresis was done following the method of Davis (5) using Tris-glycine buffer, pH 8.3, and fl-alanine buffer, pH 4.3. Electrophoresis was carried out for 1 hr at room temperature with a current of 8 mA per tube. The gel was stained overnight with amido black and destained using 10% acetic acid. Enzyme assay. The enzyme activity was determined as follows. The reaction mixture consisting of 0.1 M sodium phosphate buffer, pH 7.1 (1.0 ml), 3 rmoles of catechol, enzyme solution (0.5 ml), and water in a total volume of 2 ml was incubated at 35°C for 15 min. The reaction was terminated by the addition of 0.2 ml of 0.5 N HCl and 3.0 ml of distilled chloroform were then added to the mixt,ure and the product was extracted into chloroform. DDQ formed was measured spectrophotometrically by measuring the absorbance at 410 nm. Catechol was estimated in the reaction mixtures following the calorimetric method of Nair and Vaidyanathan (6). A unit of activity is defined as the amount of enzyme which would catalyze the formation of 1 @mole of DDQ under the standard conditions of assay described. Estimation of dihydric phenols. The disappearance of o-dihydric phenols in the reaction mix-
tures was followed by the calorimetric method of Arnow (7). Assay of polyphenol oxidase. The assay of polyphenol oxidase was performed by the spectrophotometric method of Boston et al. (8), based on the increase in OD per minute at 470 nm, when the phenolic substrate is incubated with the enzyme preparation. The indirect spectrophotometric assay of Sisler and Evans (9) was also used to determine polyphenol oxidase activity. Preparation of potato phenolase. Purified preparations of pot,ato phenolase were obtained by adopting the purification procedure devised by Patil and Zucker (lo), up to the first DEAEcellulose column chromatographic stage. Estimation of copper in enzyme samples. For the estimation of copper in enzyme preparations, a calorimetric method of assay, using dithizone, as described by Sandell (11) was adopted. Estimation of microquantities of copper in the enzyme preparations was also done by the method of Stark and Dawson (12) as modified by Vaughan and Butt (13). Enzymatic determination of small quantities of Hg02. For the determination of small quantities of Hz02 produced, if any, during the conversion of catechol to DDQ, catalyzed by the Tecoma leaf enzyme, the micromethod described by Nakeno et al. (14) was used. Oxygen uptake. Oxygen uptake studies were carried out in Warburg flasks at 30°C as follows. The reaction mixture contained 1.5 ml of 0.1 M sodium phosphate buffer, pH 7.1, 4.5 pmoles of catechol, and 0.25 ml of water. The reaction was started by the addition of 0.75 ml of the Tecoma leaf enzyme from the side arm. Catechol disappearance in the reaction mixture was measured concurrently. RESULTS
Purification of the enzyme system from Tecoma leaves. All the operations were performed between stated otherwise.
0
and
5”C,
unless
Step 1, Preparation of crude extract. Fresh, leaves of Tecoma stans L, were
mature t,aken,
the
midribs
from
the
leaves
mere
OXIDATION
OF CATECHOL
detsc,hed and hvashed several times with glass-distilled water, and ground in a chilled mortar with acid-washed sand (9 g) and extracted with 175 ml of 0.01 M sodium phosphate buffer, pH .5.7. After squeezing through cheesecloth the greenish extract was cent’rifuged at 12,OOOgfor 15 mm. The clear supernatant fluid obtained was designated as the crude extract. Step 2,. T,reatnzent with protamine sulfate. Nine milliliters of 2% aq. protamine sulfate solution were added to the crude extract (180 ml) drop by drop wit,h gentle stirring. After 15 min the extract was centrifuged at 12,OOOg for 10 mm to get a clear supern&ant fraction. Step 3. Tricalcium phosphate gel fractionation. An S5-ml quantity of tricalcium phosphate gel (20 mg/ml) was centrifuged and to the residue obtained were added 188 ml of the supernatant fluid from Step 2 (pH 5.7) with stirring. After 20 min the mixture was centrifuged at 12,000g for 10 min. The gel supernatant fraction did not show any activity. The gel was washed twice with 40 ml of 0.02 RI sodium phosphate buffer (pH 6.0) and later the enzyme was eluted twice with 30 ml of 0.05 M sodium phosphate buffer, pH 7.0. The gel was removed by centrifugation and the eluate was processed further. Step 4. DEAE-Sephadex chromatography. DEAESephadex was equilibrated with 0.05 M sodium phosphate buffer and packed into a column (1.5 X 20 cm). The gel eluate obtained from Step 3 was concentrated to 8 ml by aquacide dialysis. The concentrate was loaded onto the column of DEAESephadex equilibrated with 0.05 M sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl. The column was initially washed with 40 ml of 0.05 M sodium phosphate buffer, pH 7.0. After this, the column was successively washed with 40-ml quantities of 0.05 M sodium phosphate buffer (pH 7.0) containing 0.15 and 0.2 nt NaCl, respectively. Finally the enzyme was eluted twice with 20-ml portions of 0.05 nc sodium phosphate buffer, pH 7.0, containing 0.25 M T‘JaCl. This eluate was designated as the purified enzyme. By following the purification procedure
IN PLANTS
245
III TABLE
I
SUMMARY OF PURIFICATION OF THE DDQ-FORMING ENZYME FROM Tecowaa LEAVES
Step Volume
I II III IV
180 188 60 40
Total activity (units) 105.30 157.10 70.22 62.86
Protein (w/ml)
4.50 2.20 0.44 0.10
Specific activity (units ‘mg protein)
-
0.13 0.38 2.66 15.72
outlined above, a 120.fold purified enzyme was obtained with nearly 60% recovery. A summary of the purification proccdurc is given in Table I. Electrophoresis of the puri$ed enzyme. The purified Tecoma leaf enzyme was analyzed by disc electrophoresis on polyacrylamide gel by the method of Davis (5). A prominent single band was observed in the gel which stained darkly. Only one prominent band was detected when electrophoresis was performed at pH 8.3 and pH 4.3 (fig. 1). Molecular weight of the Tecoma leaf DDQforming enzyme. The molecular weight. of the purified Tecoma leaf enzyme as determined by gel filtration of t#he protein on Sephadex G-150 and G-200 was approximately 200,000. E$ect oj pH on enzyme activity. Figure 2 shows the effect of pH on the purified Tecoma leaf enzyme. The optimum pH of the reaction was determined by using 0.1 M citrate-O.2 M sodium phosphate and 0.1 nr sodium phosphate buffers. Optimal act’ivity was observed around pH 7.1. There was no appreciable rate of activity at pH values above 7.8 and below 4.5. Eflect of substrate concentration. The optimal substrate concentration for the reaction was found to be 1.5 X 10d3 M. The ILL value for catechol was determined as 4 X 10e4 M from a Lineweaver-Burk plot. E$ect of enzyme concentration. The reaction rate was found to be linear up to a protein concentration of 50 pg. Effect of temperature. The optimum temperature for the enzyme activity was found to be 35°C. The activity fell off rapidly beyond 50°C. Time-course and stoichiometry of the
250
KANDASWAMI
AND
VAIDYANATHAN
presence of the purified Tecoma leaf enzyme is shown in Table III. The data indicated a requirement for 1.5 atoms of oxygen per mole of catechol oxidized when measurements were made after 5 min. Substrate spec$city. It was of interest to compare the substrate specificity of the purified Tecoma leaf enzyme with that of the plant polyphenol oxidases which oxidize a bewildering range of phenolic substrates (15). Various 0-dihydric phenols were tested as substrates for the purified Tecoma 0.60 I
FIG. 1. Polyacrylamide gel electrophoresis of the purified DDQ-forming enzyme from Tecoma leaves. Polyacrylamide gel electrophoresis of the purified protein was performed employing Trisglycine buffer, pH 8.3. Electrophoresis was carried out for 1 hr at room temperature with a current of 8 mA per tube. The gel was stained with amido black and destained using 10% acetic acid.
reaction. The effect of different periods of incubation on the formation of DDQ, catalyzed by the purified Tecoma leaf enzyme, showed a linear relationship between the enzyme activity and the incubation period up to 15 min. One mole of DDQ was formed for 2 moles of catechol metabolized, in presence of the purified Tecoma leaf enzyme. The stoichiometry of DDQ formation catalyzed by the purified Tecoma leaf enzyme is presented in Table II. Oxygen uptake. The conversion of catechol to DDQ, catalyzed by the purified Tecoma leaf enzyme, proceeded only under aerobic conditions. The oxygen consumption during the oxidation of catechol to DDQ, in
a 0;
6.2
6.6
7.0
7.4
7
PH
FIG. 2. Effect of pH on the purifigd Tecoma leaf enzyme. The reaction mixture consisbd of 1.0 ml of 0.1 M sodium phosphate buffer of different pH values, 3 pmoles of catechol, 50 rg of purified Tecoma leaf enzyme, and water to a total volume of 2 ml was incubated at 35°C for 15 min.
TABLE CONVERSION OF PURIFIED
II
CATECHOL
TO
Tecoma LEAF
DDQ
BY
THE
ENZYME
STOICHIOMETRY~
Time (min)
Catechol disappeared bole)
DDQ formed (wale)
5 10 15
0.31 0.63 0.89
0.15 0.30 0.42
(1 Standard assay conditions were used and the reaction was performed for various intervals of time.
OXIDATIOX TABLE
OF CATECHOL
III
OXYGEN CONSUMPTION DURING THE CONVERSION OF CATECHOL TO DDQ BY THE PURIFIED Tecoma LEAF ENZYME” Time (min)
Catechol oxidized Oxygen consumed (patorns) bole) ..~ .~~~
5 10 15
0.40 0.84 1.60
0.28 0.58 1.10
(LThe composition :he s:ame as described
of the reaction mixt,ure is for oxygen-uptake studies.
TABLE
IV
OXIDATION OF VARIOUS O-DIHYDRIC PHENOLS BY THE PURIFIED Tecoma LEAF ENZYME” Compound tested
Catechol DL-Dihydroxyphenylslanine Caffeic acid Chlorogenic acid 3,4-Dihydroxybenzoic acid 2,3-Dihydroxybenzoic acid Dihydrocaffeic acid Homoprotocatechuic acid
pmoles of compound disappeared 4.20 0.01 0 0.02 0.01 0.03 0 0
The composition of the reaction mixture was the same as described for the standard assay except that 5 X lo-’ M of each phenolic substrate xas utilized in t,he assay in each case.
leaf enzyme and the results are summarized in Table IV. ,4 perusal of the results presented in Table IV would show that the Tecoma leaf enzyme is highly specific for catechol. The ability of the purified Tecoma leaf enzyme to attack vie-trihydric phenols was studied. Pyrogallol and gallic acid did not serve as substrates for the purified Tecoma leaf enzyme. These substrates were tested by the indirect spectrophotometric method. The purified enzyme was feebly active with monohydric phenols like L-tyrosine and p-cresol. The unreactivity of the monohydric phenols was confirmed by oxygen uptake studies and by calorimetric assay met,hod. Obviously the Tecoma leaf enzyme did not possess hydroxylase activity toward monohydric phenols. The purified Tecoma leaf enzyme was not
IN
PLANTS
231
III
able to oxidize hydroquinone, t’hereby showing that the enzyme did not possess lactase activity (15). The Tecoma leaf enzyme failed to display any activity toward such flavonoids as phloridzin and quercetin. Comparison of the puriJied Tecoma leaf enzyme with potato polypkenol oxidase. The substrate specificity of the potato phenolase was examined in order to get a comparative picture with the Tecoma leaf enzyme. It is known that potato phenolase can attack many polyphenols in addition to monohydric phenols (15). It was earlier observed that the purified Tecoma leaf enzymes were incapable of at,tacking o-dihydric phenols like Dopa, caffeic acid, chlorogenic acid, protocatechuic acid etc. A partially purified preparation of pot’ato phenolase was obtained by following the method of Patil and Zucker (10). The different o-dihydric phenols were tested in presence of t’his preparation and the results are summarized in Table V. Chlorogenic acid was found Do be the most preferred substrate for the phenolase among the o-dihydric phenols Dested. The activity of the pot,ato phenolase towards di- and trihydric phenols and monophenols was tested by the indirect spectrophotometric met’hod (9). Potato phenolase was found to be very active with trihydric phenols like pyrogallol. E$ect of metal ions on the purified Tecoma leaj enzyme. The response of the Tecoma leaf TABLE
V
OXIDATION OF 0-DIHYDRIC PHENOLS BY THE PURIFIED POTATO PHENOLASE~ Substrate
Chlorogenic acid Catechol Caffeic acid DL-Dihydroxyphenylalanine 3,4-Dihydroxybenzoic a The reaction sodium phosphate of the enzyme, 1 tested, and water Incubations were The assay of the imetrically (Ref.
fimoles of substrate disappeared
acid
0.47 0.36 0.18 0.08 0.25
mixture consisted of 0.1 M buffer, pII 6.8 (1.0 ml), 0.5 ml Fmole of each substrate to be in a total volume of 2.0 ml. carried out at 30°C for 10 min. enzyme was performed color7).
252
KANDASWAMI
AND
enzyme to various metal ions was studied. Activity was not increased by supplementing the enzyme with any of the following salts (at lo4 M and 5 X lo4 M), CdS04, MgSCh , MnSOc , FeS04, and CuS04. Of the different metal ions tested HgCl, , FedSOd , and ZnS04 inhibited the reaction to 56,40, and 32% at 5 X lo4 M. E$ect of metal-chelating agents on the purified Tecoma leaf enzyme. The response of the Tecoma leaf enzyme to various metalchelating agents was studied and the results are shown in Table VI. Ferrous inhibitors such as o-phenanthroline and a-n’-dipyridyl did not inhibit the enzyme to any appreciable degree. The Tecoma leaf enzyme was found to be insensitive to diethyl dithiocarbamate and salicylaldoxime. The effect of different copper-chelating agents such as diethyldithiocarbamate, salicylaldoxime, potassium ethylxanthate, and bathocuproin sulfonate was tested at various concentrations (Table VII). They caused very little inhibition of the oxidation of catechol in presence of the purified Tecoma leaf enzyme. Estimation of copper in the purijed Tecoma leaf enzyme. T\Tocopper could be detected in the purified Tecoma leaf enzyme preparations by following different micromethods. TABLE
VI
EFFECT
OF METAL-CHELATING AGENTS ON THE PURIFIED Tecoma LEAF ENZYMES Final concenSupplement Catechol tration disappeared
(4 None (Y,or’-dipyridyl o-Phenanthroline S-Hydroxyquinoline Salicylaldoxime Diethyldithiocarbamate EDTA
10-d 5 x 10-d 10-d 5 x lo-4 lo-4 5 x lo-4 5 x 10-h 5 x 10-d 10-4
5 x
10-d
(Irmoles) 0.88 0.81 0.72 0.82 0.74 0.74 0.68 0.79 0.72 0.81 0.72
Each chelating agent was preincubated with the enzyme for 5 min prior to the addition of the substrate. Standard conditions of assay were employed.
VAIDYANATHAN TABLE
VII
EFFECT OF COPPER-CHELSTING AGENTS ON THE PURIFIED Tecoma LEAF ENZYME* Addition
Concentration (M)
None Salicylaldoxime
Diethyldithiocarbamate Bathocuproin sulfonate
Potassium xanthate
10-4 5 x 10-4 10-Z 10-d 5 x 10-d 10-a 10-4 5 x 10-4 lo-3
ethyl5 x
10-b 10-d 10-S
Catechol disappeared (pmoles) 0.87 0.81 0.78 0.75 0.78 0.71 0.69 0.85 0.79 0.77
0.79 0.72 0.70
a Each inhibitor was incubated with the enzyme for 5 min prior to the addition of the substrate. Standard conditions of assay were employed.
Effect of thiol inhibitors. The Tecoma leaf enzyme was found to be insensitive t’o thiol inhibitors like pHMB, iodoacetat’e, and N-ethylmaleimide. E$ect of reducing agents on the enzyme. An interesting property of the DDQ-forming enzyme, from Tecoma leaves, was its extreme susceptibility to reducing agents like GSH and cysteine. Cysteamine suppressed the reaction completely at 5 X lOA M.
E$ect of axide, sulfide, and other complexing agents on the purified Tecoma leaf enzyme. None of the copper-complexing agents such as cyanide, aside, sulfide, and citrate exhibited any significant effect on the purified Tecoma leaf enzyme. E$ect of dialysis of the puri$ed Tecoma leaf enzyme against copper-chelating agents. Extensive dialysis of the purified DDQforming enzyme from Tecoma leaves against copper-chelating agents and cyanide caused no reduction in the activity of the enzyme providing additional evidence t,hat the Tecoma leaf enzyme was not a copper protein. E$ect of cofactors on DDQ-forming enzyme from Tecoma leaves. The effect of various
OSIDSTION
OF CATECHOL
cofactors like l+lN, FAD, NAD, etc., on the purified Tecoma leaf enzyme was studied. NADI’ caused about 2S% stimulation of the enzvme activity. Other compounds testtld did not enhance the reaction rate appreciably. FAD and FRIN did not have any activating effect. The effect of atebrine 011 t.he purified Tecowa leaf enzyme was examined in order t’o know whet,her there was a flavin requirement, for the enzyme. Atcbrinc at, a cow centratJion of 5 X 10d4 31 had no effect on the oxidation of catcchol by the Teconza leaf enzyme. E.flect of carbon monoxide. The purified DDQ-forming enzyme from Tecoma leaves was insensitive to carbon monoxide. Efect of p-nitrophenol and sodium jluoride on the pu+ed Tecoma leaf enzyme. The activity of the Tecoma leaf enzyme n-as not affected by p-nitrophcnol or sodium fluoride. E#ect qf thiourea on the Tecoma leaf enzyme. The &ect of various concentrations of thiourea, on the Tecoma leaf enzyme was st’udicld. The results showed that t’hiourea did not cause any inhibition of the enzyme activity at a concent’ration of 10U3hi. Treatment of the Teconza leaf enzyme with denaturing agents. It has been reported that the tyrosinase from broad bean leaves is a latent, enzyme and that the act’ivity of the cbnzyme can be enhanced considerably by treatment with acid and alkali (16). The DDQ-forming enzyme from Tecoma lcaves was exposed for a limited time to pH 3.0-3.5 and 10-l 1. Such treatments did not activate the enzyme. On the other hand, prolonged t’reatment caused inhibition of the enzyme activit’y. The purified Tecoma leaf enzyme was found to low its activity under mild denaturing conditions in contrast to the broad bean tyrosinase (17). At a conce&ation of 6 YI urea half the enzyme activity was lost. The purified Tecoma leaf enzyme was t’reated with different concentrations of urea for varying intervals of time and the activity was assayed in each case. A IO-min treatment of the enzyme with S M urea resulted in nearly 60°jo loss in enzyme activity (Fig. 3). i3jeect of catalase on the Tecoma leaf enzyme. Beef liver catalase added to the
IN
01 0
PLANTS
I 5
25’ c
III
I I 10 15 Time (mins.)
I 20
FIG. 3. Effect of urea treatment on the purified DDQ-forming enzyme from Tecoma leaves. The purified Tecoma leaf enzyme was incubated with varying concentrations of urea for various intervals of time. The enzyme activity was assayed standard conditions described under under Materials and Methods.
reaction mixture (300 pg-1 mg) had nc inhibitory effect on the Tecoma leaf enzyme. It was observed that catalase did not alter the time-course of the DDQ-forming enzyme markedly. E’nxymatic determination of hydrogen peroxide. Attempts were made to estimate very small amounts of H202 produced, if any, during the conversion of catechol to DDQ by the purified Tecoma leaf enzyme. 50 Hz02 could be detected during the enzymat,ic conversion of catechol to DDQ. Intracellular distribution oj the Tecoma leaf enzyme. Table VIII shows the distribution of DDQ-forming enzyme in different subcellular fractions. Cellular fractionation of Tecoma leaves was carried out in saline medium by the procedure of Nair and Vaidyanathan (1s). The activity was mainly localized in the soluble fraction. DISCUSSION
A wide range of structurally diverse phenolic compounds found in higher plants and microorganisms-tannins, lignins, antibiotics, alkaloids, melanins-are recognized to be derived from the oxidative coupling of simple phenolic substances (15, 19). The formation of such natural products involve frequently carbon-carbon and carbon-oxygen linkages. The importance of the occur-
254
KANDASWAMI TABLE
AND
VIII
CELLULAR DISTRIBUTION OF THE DDQ-FORMING ENZYME FROM Tecoma LEAVES" Fraction
Chloroplast fraction Mitochondrial fraction Soluble fraction
Catechol oxidized (@moles) 0.12 0 0.88
a The assay conditions employed were standard except that the purified enzyme was replaced by 0 5 ml each of the above fractions in each case.
rence of the carbon-oxygen coupling during the formation of catechol melanins in higher plants and fungi has been emphasized (20). Nair and Vining (2) obtained a partially purified enzyme preparation from spinach leaves which converted catechol to DDQ. This preparation was not inhibited by diethyldithiocarbamate and was specific for catechol. The DDQ-forming enzyme isolated from Tecoma leaves was found to catalyze the stoichiometric conversion of catechol to DDQ. A perusal of the results shows that this enzyme is different from the polyphenol oxidases. At this stage it is necessary to interpret the results obtained with this enzyme system in the light of our present day knowledge concerning the specificity and catalytic activity of the phenol oxidases. A scrutiny of the substrate specificity of the purified DDQ-forming enzyme from Tecoma leaves would reveal the high specificity of the enzyme for catechol. This enzyme is not able to oxidize trihydric phenols and complex polyphenols like the flavonoids and thus differs markedly from the potato phenolase (21). The Tecoma leaf enzyme also lacks the hydroxylase activity towards monohydric phenols, which is a characteristic feature of the potato polyphenol oxidase (15). The tyrosinase isolated from the leaves of the broad bean (Vi&a faba L.) resembles the potato enzyme in its two basic catalytic activities, namely, the orthohydroxylation of monohydric phenols and the dehydrogenation of o-dihydric phenols (22). A significant feature in regard to the substrate pattern of the broad bean tyrosinase is that, substituted catechols containing a free carboxyl
VAIDYANATHAN
group, such as protocatechuic, caffeic, and chlorogenic acids, are not particularly good substrates, Dopa being a notable exception. The substrate specificity of the Tecoma leaf DDQ-forming enzyme seems to distinguish it from the broad bean tyrosinase. Another important aspect of the substrate specificity of the DDQ-forming enzyme from Tecoma leaves concerns its action toward hydroquinone. Hydroquinone is a preferred substrate for lactase whereas its oxidation is not catalyzed by tyrosinase (15) and this constitutes an important difference between tyrosinase and lactase (15). The DDQ-forming enzyme from Tecoma leaves is totally unreactive toward hydroquinone. It is evident, therefore, that the Tecoma leaf enzyme does not display lactase activity. The balance of evidence obtained on the DDQ-forming enzyme from Tecoma leaves indicates that it is not a copper protein. The purified Tecoma leaf enzyme was insensitive to a range of copper-chelating agents like salicylaldoxime, diethyldithiocarbamate, potassium ethylxanthate, and bathocuproin sulfonate. Moreover, the Tecoma leaf enzyme was not inactivated by dialysis against copper-chelating agents and cyanide. On the other hand, the dialysis of the mushroom tryosinase against cyanide (23) was sufficient to deprive the enzyme of practically all of its copper and its activity. Pomerantz (24) prepared the apoenzyme of tyrosinase from hamster melanoma by treatment with KCN or diethyldithiocarbamate. Omura (25) prepared the apoenzyme of Rhus vernicefera lactase by dialyzing the enzyme against cyanide or EDTA. Phenolic substances like p-nitrophenol and resorcinol form inactive complexes with copper proteins (26). These compounds had no effect on the Tecoma leaf enzyme. Bonner and Wildman (27) reported the inhibition of spinach leaf polyphenol oxidase by pnitrophenol. Robb et al. (22) observed that p-nitrophenol and sodium fluoride inhibited the tyrosinase from broad bean 1eaveE (Vicia fuba L) noncompetitively. Fluoride is known to complex with copper (22). Sodium fluoride and different copper-complexing agents had no significant effect on
0SII)ATION
OF CATECHOL
the Tecoma leaf enzyme even at low pH values. Thiourea caused no reduction in the activity of the Tecoma leaf enzyme whereas it had been reported as an inhibitor of tea polyphenol oxidase (28) and mammalian tyrosinasc (24). It is interesting to note the insensitivity of the purified Tecoma leaf enzyme to carbon monoxide while t’he sensitivity of the tyrosinase to carbon monoxide is very significant (29). The observation that copper could not be det,ected in the purified Tecoma leaf enzyme preparations affords further evidence to deduce that the Tecoma leaf enzyme is not a copper protein like the tyrosinase. The molecular weight of the purified Tecoma leaf enzyme is estimated approximately as 200,000. An approximate molecular weight of 100,000 has been reported by Kubowitz (30) for the phenolase from white potatoes (Xolanum tuberosum). The polyphenol oxidase of common mushroom (Psalliota campestris) had an approximate molecular weight of 133,000 f 10,000. Another significant molecular property of the tyrosinase, in contrast to the Tecoma leaf enzyme, is their heterogeneity. The multiple molecular forms of mushroom tyrosinase were separated by Bouchilloux et al. (31). The four molecular forms of the tyrosinase, in this case, differed from each other in their substrate specificity. Robb et al. (32) achieved the separation of the different molecular forms of the tyrosinase from broad bean leaves (V&a faba L) by starch gel electrophoresis and it was found that the different forms did not differ decisively in their substrate specificity (22). On the contrary, the purified DDQ-forming enzyme from Tecoma leaves gives only one band on disc electrophoresis at different pH values. The cellular distribution of the DDQforming enzyme from Tecoma leaves seems to be important in view of the multiplicity of catechol oxidases in tissues of higher plants (33). The DDQ-forming enzyme is predominantly localized in the soluble fraction of the cell. Moreover, it exhibits only a single pH optimum. Hare1 et al. (34) have
IN
PLANTS
III
2.55
shown t’hat in apples catcchol oxidases are present in several subcellular fractions. These authors (33) subsequently showed t,he multiplicity of phenol oxidases in the same subcellular fraction. The purified Tecoma leaf enzyme loses its activity under mild denaturing condit’ions. When the Tecoma leaf enzyme is exposed to acid and alkaline pH, there is a diminution of its activity. Kenten (16) has described the tyrosinase from T’icia fhba leaves as a latent, enzyme which gains activation on treatment with acid and alkali. Robb et al. (35) concur with the idea of Kenten (16) that t’he exposure of the broad bean tprosinase, for a limited time, to pH 3.0~3..5 or 10-11.5, leads to the activation of the enzyme. They found that t’he activity of this enzyme was promoted by mild denaturing conditions. In contrast to the broad bean tyrosinase, treatment of the Tecoma leaf enzyme with urea leads to a progressive reduction in enzyme activity. The purified Tecoma leaf enzyme does not show any peroxidase activity. The enzyme is not inhibited by added catalase and hydrogen peroxide does not seem to be a product of the enzymatic reaction. It has been pointed out that the DDQforming enzyme from Tecoma leaves is similar to t’he spinach leaf enzyme (a) in its specificity for catechol. The lack of sensitivity of the spinach leaf enzyme t’o diethyldithiocarbamate was another distinctive feature of this enzyme. The Tecowza leaf enzyme shares this property. Many other copper-chelating agents have been tested with the purified Tecoma leaf enzrme and none of these was found to inhibit the enzyme. Moreover, the purity of the Tecoma leaf enzyme has been checked. It appears that the DDQ-forming enzyme is a distinctive one for the metabolism of catechol in Tecoma leaves. An interesting aspect of the spinach leaf enzyme is that it is stimulated by FAD (2) though an absolute requirement for it was not observed. The reaction was markedly inhibited by atebrine. Atebrine was, however, without effect 011the Tecorna leaf enzyme and it was not activated by FAD to any significant degree. The uv and visible
256
KANDASWAMI
FIGS. 4 and 5. Mechanism
AND
for the formation
spectrum of the purified enzyme did not indicate the presence of any flavin moiety in the enzyme. The Tecoma leaf enzyme is optimally active at pH 7.1, whereas the spinach leaf enzyme (2) exhibits an optimum pH of 7.4. The Tecoma leaf enzyme is much more susceptible to Hg2+ than the spinach leaf enzyme. The extreme susceptibility of the purified Tecoma leaf enzyme to reducing agents is a noteworthy feature. It will be instructive to review the available information concerning the mechanisms that may operate for the formation of carbon-oxygen-linked oxidation products of phenolic compounds, like DDQ. Forsyth and colleagues (36) suggested that the enzymatic oxidation of catechol leads to the formation of o-benzoquinone in presence of mushroom polyphenol oxidase. Further oxidation of benzoquinone derivatives is believed to result in coupling by both carbon-carbon and carbon-oxygen bonds. Brown (15) believes that oxidation products such as DDQ arise by the coupling of aryloxy radicals. According to him (15) the enzymatic oxidation of catechol leads to the formation of aryloxy radicals which then form different products depending on the mode of coupling. DDQ might arise by the oxygen-para coupling of aryloxy radicals (Fig. 4). Thomson (37) while discussing the formation of larger monomeric aromatic sub-
VAIDYANATHAN
of diphenylene
dioxide
2,3-quinone.
FIG. 5
stances found in nature, draws attention to the oxidative dimerization of simple phenolic precursors. In describing the mechanism of oxidative processes, he emphasizes the importance of oxidation by one-electron abstracting agents. Oxidation by oneelectron abstracting agents give rise to mesomeric radicals (ArO.) which may dimerize or react with other radicals forming new C-C, C-O, or O-O bonds. He suggests that enzymatic oxidation may take the same course. The mechanism for the formation of DDQ as suggested by Thomson (37) is delineated in Fig. 5. In this scheme 2,3’ ,4’trihydroxydiphenylether has been implicated as an intermediate. At this juncture, it would be premature to speculate on the possible mode of formation of DDQ in presence of the Tecoma leaf enzyme. At t’he same time we are tempted to think that the strong inhibition of the enzyme by cysteamine might indicate the involvement of free radicals in the formation of DDQ from catechol.
O~XIDATION
OF CATECHOL
ACKNOWLEDGMENT The financial Atomic Energy, acknowledged.
18. NAIR,
support from the Department of Government of India is gratefully REFERENCES
1. KAND~~~AMI,
C.,
AND VAIDYANATHAN,
IN
C. S.
(1972) Biochem. J. 128, 30 P. L. C. (1964) Arch. 2. N~IR, P. M., AND VINING, Biochem. Biophys. 106, 422. 3. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 4. ANDREWS, P. (1964) Biochem. J. 91,222. 5. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404. 6. NXR, P. M., AND VAIDYANATHAN, C. S. (1964) Anal. Biochem. 7, 315. 7. ARNOW, L. E. (1953) J. Biol. Gem. 118,531. 8. BOSCON, L., PO~RIE, W. D., AND FENNENIO, 0. (1962) J. Food Sci. 27,574. 9. SISLER, E. C., AND EVANS, H. J. (1958) Plant Physiol. 33, 255. 10. PATIL, S. S., AND ZUCKER, M. (1965). J. Biol. Chem. 240,393s. 11. SANDELL, E. B. (1944) Calorimetric Estimation of Traces of Metals, Inter-science, New York. 12. ST~IRK, G. R., AND L)AWSON, C. R. (1958) AnaZ. Chem. 30,91. 13. VAUGHAN, P. F. T., AND BUTT, V. S. (1970) Biochem. J. 119, 89. 14. N~K.~No, M., TSUTSUNI, Y., AND DANOWSKI, T. S. (1968) Proc. Sot. Esp. Biol. Med. 129, 960. Coupling of 15. BROWN, B. R. (1967) in Oxidative Phenols. (Taylor, W. I., and Battersby, A. R., eds.), p, 167, Marcel Dekker, New York. 16. KENTEN, R. H. (1958) Biochem. J. 68,224. 17. SW.~IN, T., MAPSON, L. W., AND ROBB, D. A. (1966) Phytochemistry 6, 469.
PLANTS
III
P. R/I., ,iND VAIDy.4NdTKAN,
257 C. S. (1964)
Phytochemistry 3, 235. 19. SCOTT, A. I. (1967) in Oxidative Coupling of Phenols (Taylor, W. I., and Battersby, A R., eds.), p. 15, Marcel Dekker, New York. 20. PIATTELLI, M., FATTORUSSO, E., NICOL.LUS, R. A., AND ;LIASON, s. (1965) Tetrahedron 21, 3229. 21. ABUKHhRM.4, D. A., .tND WOOI.HOt-HE, l-1. 9. (1966) i\‘eu, Phytol. 66,477. 22. Ronn, D. A., hfAPSON, L. W., .4ND Pn-m, T. (1966) Phytochemistry 6, 665. 23. MALMSTROM, B. G., ‘LSD NIELANDS, J. B. (1964) Annu. Rev. Biochem. 33, 331. 24. POMERANTZ, S. (1963) J. Biol. Chem. 233,235l. 25. OMURA, T. (1961) J. Biochem. 60, 389. 26. JAMES, W. 0. (1953) Annu. Rev. Plant Ph,ysiol. 4, 59. S. G. (1946) Arch. 27. BONNER, J., AND WILDM.IN, Biochem. 10, 497. 28. GREGORT, R. P. F., .%IXD BEND.ILL, I). S. (1966) Biochem J. 101,569. 29. KEILIN, II. (1929) Proc. Roy. Sot. Ser. B 104, 206. 30. KUBOWITZ, F. (1938) Biochem. 2. 299, 32. 31. BOIXHILLOUX, S., MCMAHILL, P., IND M.UON, II. S. (1963) J. Biol. Chem. 238, 1699. 32. Ronn, D. A., MAPSON, L. W., .YND Smart, T. (1966) Phytochemistry 4, 731. 33. HAREL, E., MAYER, A. M., ASD SH~IS, Y. (1965) Phytochemistry 4, 783. 34. HAREL, E., MAYER, A. M., .~XD SH~IN, Y. (1964) Physiol. Plant. 17,921. 35. Ronn, D. A., M.~PSOX, L. W., AND &V.ZIN, T. (1964) Nature (London) 207, 503. 36. FORSYTH, W. C. G., QUESNEL, D. C., AND ROBERTS, J. B. (1960) Biochim. Biophys. Acta 37, 322. 37. THOMSON, R. H. (1964) in, Biochemistry of Phenolic Compounds (Harborne, .J. B., ed.) p. 1, Academic Press, New York.