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Partial purification and characterization of the soluble polyphenol oxidases from suspension cultures of Papaver somniferum
Plant Science Letters, 34 (1984) 315--322
315
Elsevier Scientific Publishers Ireland Ltd.
PARTIAL PURIFICATION AND CHARACTERIZATION OF THE SOLUBLE POLYPHENOL OXIDASES FROM SUSPENSION CULTURES OF P A P A V E R S O M N I F E R U M
AN-FEI HSU, EDWIN B. KALANand DONALD D. BILLS Eastern Regional Research Center*, 600 E. Mermaid Lane, Philadelphia, PA 19118 (U.S.A.) (Received September 14th, 1983)
(Revision received December 13th, 1983) (Accepted December 13th, 1983)
SUMMARY
Suspension cultures of Papaver s o m n i f e r u m contained at least four heterogenous forms of polyphenol oxidase (PPO) by polyacrylamide gel electrophoresis (PAGE). Two were purified approx. 100-fold by ammonium sulfate precipitation, ultracentrifugation, carboxymethylcellulose chromatography and Sephacryl S-200 chromatography; they had the same Kin-value (5 × 10 -3 M) for catechol, similar heat stability and both were active toward o~liphenols but not toward monophenols, and were inhibited similarly in vitro by sodium cyanide,/~-mercaptoethanol, sodium sulfite, sodium ascotbate and L-cysteine. However, diethyldithiocarbamate greatly inhibited one purified form compared to the other. K e y words: Papaver s o m n i f e r u m -- Callus -- Polyphenol oxidase
INTRODUC~ON
PPO (EC 1.14.18.1) has been implicated in the biosynthesis of opium alkaloids in the opium poppy, P. s o m n i f e r u m [1], apparently in the pathway from tyrosine to the opium alkaloids [.2--4]. PPO from acetone powder of the whole plant has been isolated and partially purified [5], although its
*Agricultural Research Service, U.S. Department of Agriculture. Abbreviations: DIECA, diethyldithiocarbamate; DOPA, dihydroxyphenylalanine; PAGE, polyacrylamide gel electrophoresis;PPO, polyphenol oxidase; PVP, polyvinylpyrrolidone. 0304-4211/84/$03.00
© 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
316
direct involvement in the biosynthesis of morphine alkaloids has not been demonstrated. Although crude extracts from P. somniferum were shown to convert (-+)-[3-3H] reticuline to [3H] salutaridine in the presence of hydrogen peroxide [ 6 ] , peroxidase isolated from seedlings of P. somniferum was ineffective in catalyzing oxidation of reticuline [ 7]. However, horseradish peroxidase and a crude p o p p y enzyme fraction were able to transform morphine alkaloids into N-oxides and morphine to p s e u d o m o r p h i n e [8]. Since the involvement of PPO or peroxidase in the oxidation step of morphine biosynthesis is not clear, the results of this s t u d y should contribute to a better understanding of the nature of PPO occurring in opium p o p p y and its relationship to phenanthrene alkaloid biosynthesis is needed. MATERIALS AND METHODS
Suspension cultures of P. somniferum were induced from seedling callus as previously reported [ 9]. Sephacryl S-200" was purchased from Pharmacia C o m p a n y and carboxymethylcellulose was obtained from Whatman Company. Acrylamide (N, hr-methylene bisacrylamide) was purchased from Eastman Kodak Company.
Isolation and purification o f PPO Suspension cells obtained by filtration were homogenized in a motordriven glass-Teflon Potter-Elvehjem homogenizer with 3 vols. of sodium phosphate buffer (pH 6.8, 5 mM) containing 20 mM sodium ascorbate and 0.2 M sucrose. The homogenate was passed through four layers of cheese cloth and centrifuged at 3000 X g for 10 min. Solid ammonium sulfate was added to the supernatant to 85% saturation, and the mixture was centrifuged at 48 000 X g for 20 min. The pellet was redissolved in homogenization buffer (minus sodium ascorbate) to a protein concentration of 10 mg/ml. Dialysis was performed overnight with 0.01 M sodium phosphate buffer (pH 6.8) containing 0.2 M sucrose. The dialysate was centrifuged at 100 000 X g for 1 h and the supernatant was used as the crude soluble PPO. PPO assay procedure Enzyme assays were performed in 1.0 ml vol. containing 10 mM catechol in 0.1 M sodium phosphate buffer (pH 6.8 and saturated with O2) at r o o m temperature. An appropriate a m o u n t of enzyme was added and activity was monitored at 430 nm with a Beckman Model 22 recording spectrophotometer. Under these conditions, linearity was maintained for 5 min with one unit of PPO-activity defined as a change in absorbance of 0.001/min at 430 nm at 25°C. * R e f e r e n c e to b r a n d or firm n a m e d o e s n o t c o n s t i t u t e e n d o r s e m e n t by t h e U.S. Departm e n t o f A g r i c u l t u r e over o t h e r s o f a similar n a t u r e n o t m e n t i o n e d .
317
Ion exchange chromatography Activated preswollen carboxylmethylceUulose was packed into a 2.5 × 20cm column and equilibrated at 4°C with 0.01 M sodium phosphate buffer (pH 6.8) containing 0.2 M sucrose. The sample was applied and followed with three void volumes of the buffer. A sodium chloride gradient was applied in a stepwise fashion with concentrations of 10, 60, 100 and 200 mM sodium chloride in 200 ml of buffer. Three millimeter fractions were collected and 200-pl aliquots were used to determine PPO-activity. Protein concentration was determined b y the L o w r y procedure [10].
Sephacryl S-200 chromatography Each fraction containing PPO-activity from CM~ellulose column was separately pooled, concentrated to 1 ml in an Amicon apparatus and then loaded on a Sephacryl S-200 column (1 × 60 cm), equilibrated with 0.05 M sodium phosphate buffer (pH 6.8) containing 0.2 M sucrose. The eluent was continuously monitored at 215 nm and PPO-activity was assayed as described before.
PAGE Discontinuous PAGE was carried o u t on 7.5% acrylamide gels as described previously [ 11]. During electrophoresis, the temperature was maintained at 0 -2°C by circulating ice-water through the electrophoretic cells. PPO-activity in the gels was d e t e c t e d b y immersing the gel for 30 min in 20 ml of t 0 mM catechol (0.1 M of sodium phosphate buffer, pH 6.8) containing 0.5% p-phenylenediamine as described b y Benjamin and Montgomery [12]. PPO mobilities was expressed as a ratio of the mobility of bromophenol blue. RESULTS
Four distinct heterogenous forms of PPO were separated by CM-cellulose chromatography followed b y increasing ionic strength from 10 mM to 200 mM of sodium chloride (Fig. 1). No further PPOs were eluted from the column with salt concentrations above 200 mM. Fractions obtained by CM-ceUulose were separately pooled and concentrated to a small volume with an Amicon ultrafiltration apparatus and subjected t o PAGE. Staining with p-phenylenediamine, peak I of Fig. 1 contained t w o PPO bands (Rf 0.30 and 0.24), peak II contained only one PPO (Rf 0.24, designed as PPO-A), peak III contained one PPO (Rf 0.16, designed as PPO-B) and peak IV contained a mixture of PPO (Rf 0.16 and 0.10). A t t e m p t s to separate t w o PPOs in peak I and IV b y either cation or anion exchange resins with continuous or step gradients were n o t successful. Further purification of PPO-A and B was achieved b y chromatography on a Sephacryl S-200 column which separated PPO-activity from the major prorein peak. This step resulted in the greatest purification.
318 IOmM NoCI
&
60ram
IOOmM
200ram
i
&
&
2.0 O.b
i 1.5
& o.6 E
E
c I0 0.4!
0.5
0.2 ,~ --I
0
" 0
x°
/
I ~ m ~'~
I 50
. o ~ o J ) V ' ~
I00
Iv - -
150
l 200
~
..o o...e_o_e_4
250
FRACTION NUMBER
Fig. 1. CM-ceilulose c h r o m a t o g r a p h y o f s o l u b l e p o l y p h e n o l oxidase. T h e c r u d e p r e p a r a t i o n o f s o l u b l e P P O was l o a d e d o n t h e CM-cellulose c o l u m n . T h e s e p a r a t i o n o f p r o t e i n s was d e s c r i b e d u n d e r Materials a n d M e t h o d s . P r o t e i n p e a k (o o ) was d e t e r m i n e d b y Al00 run, a n d P P O - a c t i v i t y ( A , , 0 n m , • • ) was d e t e c t e d as d e s c r i b e d in t h e t e x t .
Table I provides a summary of the purification steps for PPO. Ammonium sulfate precipitation and ultracentrifugation produced little purification of the isoenzymes. Only 60% of the total activity was recovered in the soluble fraction (100 000 × g supematant), while the remainder (40%) was detected TABLE I P U R I F I C A T I O N O F PPO F R O M S U S P E N S I O N C U L T U R E S O F P. S O M N I F E R U M Purification step
Total act. (units) a
Total protein (rag) b
1. C r u d e PPO extract 2. A m m o n i u m s u l f a t e precipitate 3. S o l u b l e f r a c t i o n 4. CM-celluiose Peak I Peak II P e a k III Peak IV 5. S e p h a c r y l S - 2 0 0 PPO-A PPO-B
286
283
1.01
100
1
440
230
1.91
150
2
330
100
3.30
115
3
135 120 70 12
40 15 7 2
3.38 8 10 6
47 41 24 4
3 8 10 6
0.62 0.35
92 97
58 34
Spec. act. (units/rag)
a u n i t s : a b s o r b a n c e c h a n g e o f 0 . 0 0 1 at 4 3 0 n m p e r m i n . b p r o t e i n d e t e r m i n e d b y L o w r y assay. CBased o n t h e c o m p a r i s o n o f t o t a l a c t i v i t y t o c r u d e P P O e x t r a c t . dBased o n a m m o n i u m s u l f a t e p r e c i p i t a t e fraction.
Yield c (%)
20(13) d 12(08) d
Purification fold
92 96
319
in precipitates obtained by low speed centrifugation. A greater degree of purification was obtained by chromatography on Sephacryl S-200 (Table I) which yielded roughly 92- and 96-fold of purification based on the specific activity of the crude PPO. Effect o f catechol concentration on PPO activity The rate of formation of quinone products by PPO-A increased with increasing concentration of catechol up to 20 mM with linearity according to classical kinetics (Fig. 2). A double reciprocal plot yielded a straight line with a K m of 5 X 10 -3 M for catechol. PPO-B had the same K m as PPO-A when catechol was used as a substrate. Inhibitor studies All compounds used in this study inhibited PPO-activity (Table II), and the extent of inhibition depended on the concentration of the inhibitor. Sodium cyanide and ~-mercaptoethanol were most effective for both PPOA and PPO-B, while diethyldithiocarbamate (DIECA) had different inhibition effects on PPO-A and PPO-B. At a concentration of I raM, DIECA almost completely inhibited PPO-B activity, while it inhibited less than 40% of PPO-A activity. With the same concentration of inhibitors, the order of J
1.2
0.8
E
E ~
0.4
f
01,
013 I/IS] (mM) -I
l
I0
I
20
I
30
I
40
[S] CATECHOL (raM)
Fig. 2. E f f e c t o f c a t e c h o l c o n c e n t r a t i o n o n P P O - A activity. C o n s t a n t a m o u n t o f PPO-A (100 ug) was i n c u b a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f c a t e c h o l . T h e values o f Vmax and K m w e r e d e t e r m i n e d b y a least-squares fit o f t h e d a t a o n a d o u b l e reciprocal plot. T h e p r o d u c t c o n c e n t r a t i o n was c a l c u l a t e d b a s e d o n log e(M- ' c m - 1)= 3.324.
320 T A B L E II E F F E C T O F PPO I N H I B I T O R S O F T H E P P O - A A N D B Inhibitor
Inhibitor conc. ( m M )
Percent inhibition
PPO-A Control a
PPO-B
0
0
0
Diethyldit hiocarbamate
10 1
100 34
100 95
Na-Ascorbate
10 1
100 20
100 8
L-Cysteine
10 1
100 18
100 49
Na-Sulfite
10 1
100 24
100 40
#-Mercaptoethanol
10 1
100 61
100 68
Na-Cyanide
10 1
100 59
100 83
a E n z y m e a c t i v i t y was d e t e r m i n e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s , a n d t h e control e x p e r i m e n t was t h e i n c u b a t i o n o f e n z y m e a n d s u b s t r a t e w i t h o u t t h e a d d i t i o n o f inhibitors.
effectiveness o f the inhibitors for PPO-A was: ~-mercaptoethanol - sodium cyanide > DIECA > sodium sulfite ~ sodium ascorbate and L-cysteine. T he degree o f effectiveness on PPO-B was DIECA > sodium cyanide >/3-mercapt o e t h a n o l > a-cysteine ~ sodium sulfite > sodium ascorbate. TABLE III SUBSTRATE SPECIFICITY OF PPO-A AND B FROMSUSPENSION CULTURES OF P. SOMNIFER UM Substrate
Catechol P-Cresol Tyrosine Pyrogallol Caffeic acid C h l o r o g e n i c acid
PPO a c t i v i t y a (percentage) PPO-A
PPO-B
100 b 3 0 61 11 18
100 b 0 3 40 14 7
a E n z y m e a c t i v i t y was d e t e r m i n e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . bWhe h i g h e s t e n z y m e a c t i v i t y was assigned a value o f 100%.
321
Substrate specificity PPO-A and B preparation from culture cells of P. somniferum had strong activity toward o
322
PPO contains Cu ~ [26], and cyanide and DIECA are Cu2+-chelating agents [1]. With m u s h r o o m catechol oxidase, however, cyanide was competitive to molecular oxygen, a participant in the reaction [16]. The inhibition o f PPO by --SH c o m p o u n d s and other reducing agents is well d o c u m e n t e d [17]. Some --SH c o m p o u n d s appear to react with the enzyme's Cu ~, b u t reducing agents can affect the enzyme reactions in several ways [1]. For example, reducing agents may act on the quinoid products of PPO-activity and convert them non-enzymatically back to the phenolic precursors, thus decreasing the a m o u n t of p r o d u c t and increasing the a m o u n t of substrate w i t h o u t involvement with the enzyme. Ascorbic acid is aa reducing agent, b u t our preliminary results suggest that it functions at least in part as a competitive inhibitor of PPO from P. somniferum. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
A.M. Mayer and E. Harel, Phytochemistry, 18 (1979) 193. M.F. Roberts, Phytochemistry, 10 (1971) 3021. M.F. Roberts, J. Pharm. Pharmacol., 25 (1973) 115. M.F. Roberts, Phytochemistry, 13 (1974) 119. S.S. Asghar and V. Siddiqi, Enzymologia, 39 (1969) 289. C.C. Hodges and H. Rapoport, Biochemistry, 21 (1982) 3729. T. Robinson and W. Nagel, Phytochemistry, 21 (1982) 535. D. Va$fljfabvi and M. Petz-Stifter, Phytochemistry, 21 (1982) 1533. A.-F. Hsu, J. of Natl. Products, 44 (1981) 408. O.H. Lowry, N.J. Rosebraugh, A.L. Farr and tLJ. Randall, J. Biol. Chem., 193 (1951) 265. B.J. Davis, AnrL N.Y. Acad. Sci., 121 (1964) 404. N.D. Benjamin and M.W. Montgomery, J. F o o d Sci., 38 (1973) 799. M. Santo, Phytochemistry, 21 (1982) 1229. C.T. Zenin and Y.K. Park, J. of F o o d Sci., 43 (1978) 646. R.L. Jolley, Jr., L.I-L Evans, N. Makino and H.S. Mason, J. Biol. Chem., 249 (1974) 335. H.W. Duckworth and J.E. Coleman, J. Biol. Chem., 245 (1970) 1613. J.K. Palmer and J.B. Roberts, Science, 157 (1967) 200.
315
Elsevier Scientific Publishers Ireland Ltd.
PARTIAL PURIFICATION AND CHARACTERIZATION OF THE SOLUBLE POLYPHENOL OXIDASES FROM SUSPENSION CULTURES OF P A P A V E R S O M N I F E R U M
AN-FEI HSU, EDWIN B. KALANand DONALD D. BILLS Eastern Regional Research Center*, 600 E. Mermaid Lane, Philadelphia, PA 19118 (U.S.A.) (Received September 14th, 1983)
(Revision received December 13th, 1983) (Accepted December 13th, 1983)
SUMMARY
Suspension cultures of Papaver s o m n i f e r u m contained at least four heterogenous forms of polyphenol oxidase (PPO) by polyacrylamide gel electrophoresis (PAGE). Two were purified approx. 100-fold by ammonium sulfate precipitation, ultracentrifugation, carboxymethylcellulose chromatography and Sephacryl S-200 chromatography; they had the same Kin-value (5 × 10 -3 M) for catechol, similar heat stability and both were active toward o~liphenols but not toward monophenols, and were inhibited similarly in vitro by sodium cyanide,/~-mercaptoethanol, sodium sulfite, sodium ascotbate and L-cysteine. However, diethyldithiocarbamate greatly inhibited one purified form compared to the other. K e y words: Papaver s o m n i f e r u m -- Callus -- Polyphenol oxidase
INTRODUC~ON
PPO (EC 1.14.18.1) has been implicated in the biosynthesis of opium alkaloids in the opium poppy, P. s o m n i f e r u m [1], apparently in the pathway from tyrosine to the opium alkaloids [.2--4]. PPO from acetone powder of the whole plant has been isolated and partially purified [5], although its
*Agricultural Research Service, U.S. Department of Agriculture. Abbreviations: DIECA, diethyldithiocarbamate; DOPA, dihydroxyphenylalanine; PAGE, polyacrylamide gel electrophoresis;PPO, polyphenol oxidase; PVP, polyvinylpyrrolidone. 0304-4211/84/$03.00
© 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
316
direct involvement in the biosynthesis of morphine alkaloids has not been demonstrated. Although crude extracts from P. somniferum were shown to convert (-+)-[3-3H] reticuline to [3H] salutaridine in the presence of hydrogen peroxide [ 6 ] , peroxidase isolated from seedlings of P. somniferum was ineffective in catalyzing oxidation of reticuline [ 7]. However, horseradish peroxidase and a crude p o p p y enzyme fraction were able to transform morphine alkaloids into N-oxides and morphine to p s e u d o m o r p h i n e [8]. Since the involvement of PPO or peroxidase in the oxidation step of morphine biosynthesis is not clear, the results of this s t u d y should contribute to a better understanding of the nature of PPO occurring in opium p o p p y and its relationship to phenanthrene alkaloid biosynthesis is needed. MATERIALS AND METHODS
Suspension cultures of P. somniferum were induced from seedling callus as previously reported [ 9]. Sephacryl S-200" was purchased from Pharmacia C o m p a n y and carboxymethylcellulose was obtained from Whatman Company. Acrylamide (N, hr-methylene bisacrylamide) was purchased from Eastman Kodak Company.
Isolation and purification o f PPO Suspension cells obtained by filtration were homogenized in a motordriven glass-Teflon Potter-Elvehjem homogenizer with 3 vols. of sodium phosphate buffer (pH 6.8, 5 mM) containing 20 mM sodium ascorbate and 0.2 M sucrose. The homogenate was passed through four layers of cheese cloth and centrifuged at 3000 X g for 10 min. Solid ammonium sulfate was added to the supernatant to 85% saturation, and the mixture was centrifuged at 48 000 X g for 20 min. The pellet was redissolved in homogenization buffer (minus sodium ascorbate) to a protein concentration of 10 mg/ml. Dialysis was performed overnight with 0.01 M sodium phosphate buffer (pH 6.8) containing 0.2 M sucrose. The dialysate was centrifuged at 100 000 X g for 1 h and the supernatant was used as the crude soluble PPO. PPO assay procedure Enzyme assays were performed in 1.0 ml vol. containing 10 mM catechol in 0.1 M sodium phosphate buffer (pH 6.8 and saturated with O2) at r o o m temperature. An appropriate a m o u n t of enzyme was added and activity was monitored at 430 nm with a Beckman Model 22 recording spectrophotometer. Under these conditions, linearity was maintained for 5 min with one unit of PPO-activity defined as a change in absorbance of 0.001/min at 430 nm at 25°C. * R e f e r e n c e to b r a n d or firm n a m e d o e s n o t c o n s t i t u t e e n d o r s e m e n t by t h e U.S. Departm e n t o f A g r i c u l t u r e over o t h e r s o f a similar n a t u r e n o t m e n t i o n e d .
317
Ion exchange chromatography Activated preswollen carboxylmethylceUulose was packed into a 2.5 × 20cm column and equilibrated at 4°C with 0.01 M sodium phosphate buffer (pH 6.8) containing 0.2 M sucrose. The sample was applied and followed with three void volumes of the buffer. A sodium chloride gradient was applied in a stepwise fashion with concentrations of 10, 60, 100 and 200 mM sodium chloride in 200 ml of buffer. Three millimeter fractions were collected and 200-pl aliquots were used to determine PPO-activity. Protein concentration was determined b y the L o w r y procedure [10].
Sephacryl S-200 chromatography Each fraction containing PPO-activity from CM~ellulose column was separately pooled, concentrated to 1 ml in an Amicon apparatus and then loaded on a Sephacryl S-200 column (1 × 60 cm), equilibrated with 0.05 M sodium phosphate buffer (pH 6.8) containing 0.2 M sucrose. The eluent was continuously monitored at 215 nm and PPO-activity was assayed as described before.
PAGE Discontinuous PAGE was carried o u t on 7.5% acrylamide gels as described previously [ 11]. During electrophoresis, the temperature was maintained at 0 -2°C by circulating ice-water through the electrophoretic cells. PPO-activity in the gels was d e t e c t e d b y immersing the gel for 30 min in 20 ml of t 0 mM catechol (0.1 M of sodium phosphate buffer, pH 6.8) containing 0.5% p-phenylenediamine as described b y Benjamin and Montgomery [12]. PPO mobilities was expressed as a ratio of the mobility of bromophenol blue. RESULTS
Four distinct heterogenous forms of PPO were separated by CM-cellulose chromatography followed b y increasing ionic strength from 10 mM to 200 mM of sodium chloride (Fig. 1). No further PPOs were eluted from the column with salt concentrations above 200 mM. Fractions obtained by CM-ceUulose were separately pooled and concentrated to a small volume with an Amicon ultrafiltration apparatus and subjected t o PAGE. Staining with p-phenylenediamine, peak I of Fig. 1 contained t w o PPO bands (Rf 0.30 and 0.24), peak II contained only one PPO (Rf 0.24, designed as PPO-A), peak III contained one PPO (Rf 0.16, designed as PPO-B) and peak IV contained a mixture of PPO (Rf 0.16 and 0.10). A t t e m p t s to separate t w o PPOs in peak I and IV b y either cation or anion exchange resins with continuous or step gradients were n o t successful. Further purification of PPO-A and B was achieved b y chromatography on a Sephacryl S-200 column which separated PPO-activity from the major prorein peak. This step resulted in the greatest purification.
318 IOmM NoCI
&
60ram
IOOmM
200ram
i
&
&
2.0 O.b
i 1.5
& o.6 E
E
c I0 0.4!
0.5
0.2 ,~ --I
0
" 0
x°
/
I ~ m ~'~
I 50
. o ~ o J ) V ' ~
I00
Iv - -
150
l 200
~
..o o...e_o_e_4
250
FRACTION NUMBER
Fig. 1. CM-ceilulose c h r o m a t o g r a p h y o f s o l u b l e p o l y p h e n o l oxidase. T h e c r u d e p r e p a r a t i o n o f s o l u b l e P P O was l o a d e d o n t h e CM-cellulose c o l u m n . T h e s e p a r a t i o n o f p r o t e i n s was d e s c r i b e d u n d e r Materials a n d M e t h o d s . P r o t e i n p e a k (o o ) was d e t e r m i n e d b y Al00 run, a n d P P O - a c t i v i t y ( A , , 0 n m , • • ) was d e t e c t e d as d e s c r i b e d in t h e t e x t .
Table I provides a summary of the purification steps for PPO. Ammonium sulfate precipitation and ultracentrifugation produced little purification of the isoenzymes. Only 60% of the total activity was recovered in the soluble fraction (100 000 × g supematant), while the remainder (40%) was detected TABLE I P U R I F I C A T I O N O F PPO F R O M S U S P E N S I O N C U L T U R E S O F P. S O M N I F E R U M Purification step
Total act. (units) a
Total protein (rag) b
1. C r u d e PPO extract 2. A m m o n i u m s u l f a t e precipitate 3. S o l u b l e f r a c t i o n 4. CM-celluiose Peak I Peak II P e a k III Peak IV 5. S e p h a c r y l S - 2 0 0 PPO-A PPO-B
286
283
1.01
100
1
440
230
1.91
150
2
330
100
3.30
115
3
135 120 70 12
40 15 7 2
3.38 8 10 6
47 41 24 4
3 8 10 6
0.62 0.35
92 97
58 34
Spec. act. (units/rag)
a u n i t s : a b s o r b a n c e c h a n g e o f 0 . 0 0 1 at 4 3 0 n m p e r m i n . b p r o t e i n d e t e r m i n e d b y L o w r y assay. CBased o n t h e c o m p a r i s o n o f t o t a l a c t i v i t y t o c r u d e P P O e x t r a c t . dBased o n a m m o n i u m s u l f a t e p r e c i p i t a t e fraction.
Yield c (%)
20(13) d 12(08) d
Purification fold
92 96
319
in precipitates obtained by low speed centrifugation. A greater degree of purification was obtained by chromatography on Sephacryl S-200 (Table I) which yielded roughly 92- and 96-fold of purification based on the specific activity of the crude PPO. Effect o f catechol concentration on PPO activity The rate of formation of quinone products by PPO-A increased with increasing concentration of catechol up to 20 mM with linearity according to classical kinetics (Fig. 2). A double reciprocal plot yielded a straight line with a K m of 5 X 10 -3 M for catechol. PPO-B had the same K m as PPO-A when catechol was used as a substrate. Inhibitor studies All compounds used in this study inhibited PPO-activity (Table II), and the extent of inhibition depended on the concentration of the inhibitor. Sodium cyanide and ~-mercaptoethanol were most effective for both PPOA and PPO-B, while diethyldithiocarbamate (DIECA) had different inhibition effects on PPO-A and PPO-B. At a concentration of I raM, DIECA almost completely inhibited PPO-B activity, while it inhibited less than 40% of PPO-A activity. With the same concentration of inhibitors, the order of J
1.2
0.8
E
E ~
0.4
f
01,
013 I/IS] (mM) -I
l
I0
I
20
I
30
I
40
[S] CATECHOL (raM)
Fig. 2. E f f e c t o f c a t e c h o l c o n c e n t r a t i o n o n P P O - A activity. C o n s t a n t a m o u n t o f PPO-A (100 ug) was i n c u b a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f c a t e c h o l . T h e values o f Vmax and K m w e r e d e t e r m i n e d b y a least-squares fit o f t h e d a t a o n a d o u b l e reciprocal plot. T h e p r o d u c t c o n c e n t r a t i o n was c a l c u l a t e d b a s e d o n log e(M- ' c m - 1)= 3.324.
320 T A B L E II E F F E C T O F PPO I N H I B I T O R S O F T H E P P O - A A N D B Inhibitor
Inhibitor conc. ( m M )
Percent inhibition
PPO-A Control a
PPO-B
0
0
0
Diethyldit hiocarbamate
10 1
100 34
100 95
Na-Ascorbate
10 1
100 20
100 8
L-Cysteine
10 1
100 18
100 49
Na-Sulfite
10 1
100 24
100 40
#-Mercaptoethanol
10 1
100 61
100 68
Na-Cyanide
10 1
100 59
100 83
a E n z y m e a c t i v i t y was d e t e r m i n e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s , a n d t h e control e x p e r i m e n t was t h e i n c u b a t i o n o f e n z y m e a n d s u b s t r a t e w i t h o u t t h e a d d i t i o n o f inhibitors.
effectiveness o f the inhibitors for PPO-A was: ~-mercaptoethanol - sodium cyanide > DIECA > sodium sulfite ~ sodium ascorbate and L-cysteine. T he degree o f effectiveness on PPO-B was DIECA > sodium cyanide >/3-mercapt o e t h a n o l > a-cysteine ~ sodium sulfite > sodium ascorbate. TABLE III SUBSTRATE SPECIFICITY OF PPO-A AND B FROMSUSPENSION CULTURES OF P. SOMNIFER UM Substrate
Catechol P-Cresol Tyrosine Pyrogallol Caffeic acid C h l o r o g e n i c acid
PPO a c t i v i t y a (percentage) PPO-A
PPO-B
100 b 3 0 61 11 18
100 b 0 3 40 14 7
a E n z y m e a c t i v i t y was d e t e r m i n e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . bWhe h i g h e s t e n z y m e a c t i v i t y was assigned a value o f 100%.
321
Substrate specificity PPO-A and B preparation from culture cells of P. somniferum had strong activity toward o
322
PPO contains Cu ~ [26], and cyanide and DIECA are Cu2+-chelating agents [1]. With m u s h r o o m catechol oxidase, however, cyanide was competitive to molecular oxygen, a participant in the reaction [16]. The inhibition o f PPO by --SH c o m p o u n d s and other reducing agents is well d o c u m e n t e d [17]. Some --SH c o m p o u n d s appear to react with the enzyme's Cu ~, b u t reducing agents can affect the enzyme reactions in several ways [1]. For example, reducing agents may act on the quinoid products of PPO-activity and convert them non-enzymatically back to the phenolic precursors, thus decreasing the a m o u n t of p r o d u c t and increasing the a m o u n t of substrate w i t h o u t involvement with the enzyme. Ascorbic acid is aa reducing agent, b u t our preliminary results suggest that it functions at least in part as a competitive inhibitor of PPO from P. somniferum. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
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