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Tyrosinase and polyphenoloxidase. The role of metallic ions in melanogenesis
17o
BIOCI-IIMICA ET BIOPI-I¥SICA ACTA
VOL. 9 (1952)
TYROSINASE AND POLYPHENOLOXIDASE. THE ROLE OF METALLIC IONS IN MELANOGENESIS by D E N I S IiEIITI~SZ Research Department o/ the Istituto Regina Elena per Io Studio e la Cura dei Tumori, Roma (Italy)
The mechanism by which tyrosinase acts on monohydric phenols has been under discussion for a long time 1-a. ONSLOWAND ROBINSON6 were the first to propose that the monophenolase action is not due to the enzyme molecule itself but to the o-quinones produced by the enzymic oxidation of o-dihydric phenols. After the adverse criticism of PUGH7 the same view was reaffirmed by KEILIN AND MANN8 and by CALIFANO AND KERT£S#. More recently WARBIJRGl° states also that probably in the case of monohydric phenol oxidation o-diphenols are first formed by a slow reaction, and these then act catalytically by virtue of the change o-diphenol ¢ o-quinone. This is the same catalytic change which, in presence of traces of catechol, enables the enzyme to oxidize hydroquinone, ascorbic acid and reduced coenzymes I and II 1°. However, most authors, following NELSON AND DAWSON ~, believe that the oxidation of the monohydric phenols is a function of the enzyme molecule itself and that the enzyme is primed whilst oxidizing an dihydric phenol and so acquires the capacity to act on monohydrie phenols, a-~ALLETTEAND DAWSONn, to explain the loss of monophenolase activity during the purification process, suppose that different centres of the same protein molecule are responsible for the monohydric phenol and dihydric phenol oxidation. The diminution of the activity on monohydric phenols (in their terminology the cresolase activity of tyrosinase) and the augmentation of the activity on o-dihydfic phenols (in their terminology the catecholase activity of tyrosinase) would be the result of the greater fragility and loss of part of the molecule responsible for the cresolase activity, or of the unmasking of new active centres responsible for the catecholase activity during the purification process. Evidence reported in this paper not only fails to support the above views, but shows that the cresolase or monophenolase activity of the enzyme belongs to free, and not protein-bound, metallic ions which hasten the spontaneous reaction 9 between o-quinones and monohydric phenols. In agreement between earlier theoretical 1~ and experimentaP~,14 work the oxidation of the monohydric phenols appears to be represented correctly by the following reactions (I)
(II)
o-dihydric phenol ~ o-quinone ~ ulterior products ~ melanin o-quinone + monohydric phenol + H~O = 2 o-dihydric phenol
of which only the first of (I) is enzymatic. l?eferenees p. x79.
VOL. 9 (1952)
I7~:
TYROSINASE AND POLYPHENOLOXIDASE METHODS
Enzyme preparation. Different p r e p a r a t i o n s of p o l y p h e n o l o x i d a s e ape-enzyme, f r o m p o t a t o peelings e x t r a c t e d w i t h HCN, were described earlierlS, TM. I n this w o r k a stable p o w d e r was used which is similar to one described recently 10, b u t of a more simple preparation. 13oo g of p o t a t o Feelings are e x t r a c t e d w i t h 700 ml of e x t r a c t i o n liquid (0.05 m o l a r K C N neutralized a n d buffered to p H 7)- The e x t r a c t is centrifuged for a few m i n u t e s to eliminate the s t a r c h a n d t h e n b r o u g h t u p to 3 0 % c o n c e n t r a t i o n in acetone a n d centrifuged again. The precipitate is discarded. The supern a t a n t is b r o u g h t up to 6 0 % c o n c e n t r a t i o n in acetone and centrifuged. The precipitate is resuspended in 18o ml of e x t r a c t i o n liquid. The t r o u b l e suspension o b t a i n e d is b r o u g h t up again to 6 o O concent r a t i o n in acetone, filtered on a B u c h n e r funnel a n d the precipitate is w a s h e d 5 t i m e s w i t h acetone. I t is dessicated first ill an air current, later in a dessicator u n d e r v a c u u m . I t can be pulverized after 24 h o u r s ; the p o w d e r is stable (at least 12 m o n t h s at + 2 ° C) and it is n o t hygroscopic. D u r i n g the whole procedure the e x t r a c t i o n liquid is m a i n t a i n e d at o °, acetone at - - 2 0 ° C; all operations are made in a cold r o o m a t + 2 ° C (for f u r t h e r details see ref. 16). I m m e d i a t e l y before use, the p o w d e r is s u s p e n d e d a t a ratio of 50 m g / m l in Pyrex-redistilled w a t e r a n d the liquid is s h a r p l y centrifuged for 30 m i n u t e s to eliminate the a b u n d a n t insoluble m a t t e r s . The clear solution obtained is practically inactive w i t h o u t added copper on D o p a ; its c o n t e n t in non-dialysable s u b s t a n c e s is 16 m g / m l and in copper 0 . o 2 % . I ml of the solution contains, if copper is added, 60 e n z y m e u n i t s (Fig. I) as defined b y LERNER et al. 1~. Aetivity determination. The a c t i v i t y of the e n z y m e was determined m a n o m e t r i c a l l y by m e a s u r i n g the o x y g e n u p t a k e in the W a r b u r g a p p a r a t u s a t 38o C. S u b s t r a t e s (in a m o u n t s s h o w n in the legends of the figures) were added f r o m the side a r m s after 15 m i n u t e s of t e m p e r a t u r e equilibration, at o time. The reaction m i x t u r e s still contained o.25 ml of the e n z y m e solution, 1.25 ml of o.I molar p h o s p h a t e or p h o s p h a t e - c i t r a t e buffer at p H 6.8, the a m o u n t m a r k e d on the figures respectively of copper and of o t h e r metals and Pyrex-redistilled w a t e r to make the total volume up to 2. 5 ml. The central well of the flasks contained o.1 ml of N a O H 15 %. All solutions were p r e p a r e d with Pyrex-redistilled w a t e r ; r e a g e n t s were chosen and glass-ware w a s h e d metal-free. The i m p o r t a n c e of these p r e c a u t i o n s was emphasised by •ARRON et a/A s, b y \¥ARBURG 1°, and recently b y the a u t h o r 16. Copper determination. Copper was d e t e r m i n e d w i t h s o d i u m d i e t h y l d i t h i o c a r b a m a t e according to I~EILIN AND ~ A N N 8. Colorimetric readings were made w i t h a Coleman s p e c t r o p h o t o m e t e r , at 44 ° m,u. 2,0 RESULTS
In[luence o/metals on Dopa oxidation. Influence of copper is reported in Fig. I. It is evident that the enzyme is already fully active in presence of 0.00005 millimols (0.08%) Cu and that presence or absence of excess copper is indifferent. The insignificantly lower activities of the enzyme measured with the highest amounts of copper are artefact and due to the fact that the enzyme is slightly and slowly inactivated in presence of a great excess of copper. This is shown by the two lower curves (A and B) of the Fig. I, where the Warburg flasks containing o.oooi millimols and o.oo16 millimols of Cu were reopened, the side arm cavities washed (with the aid of a pipette) 7 times with Pyrexredistilled water and then filled with the same amount of Dopa solution as previously. The new substrate is added to the old test solution after 7 minutes of temperature equilibration. As the whole procedure did not take more than 15 minutes, the potato enzyme was held 12o minutes at 3 8o C in Re/erences p. z79.
~' l
[ ° ° ° ° ° 5 m M°ls cu÷÷ o.oooi I °'°°°2 .... .
f 7'I t00
I ~ I , - ~ IO
~
I0.0004 o
~0.0008
.
.
.
"
"
. . . .
"t
10.00~6
B q
,"0.0
30
60
.
.
.
.
. . . .
50
0
90
~20
~50 ~80 Ninufes
Fig. I. The effect of v a r y i n g a m o u n t s of copper on the enzymic oxidation of I mg Dopa. For curves A a n d B see text. All flasks contain the s a m e a m o u n t of a p e - e n z y m e corresponding (in presence of copper) to 15 e n z y m e units.
172
D. KERTtSZ
VOL. 9 (1952)
c o n t a c t w i t h D o p a a n d Cu w i t h a l m o s t c o n t i n u o u s a g i t a t i o n before t h e second o time. T h e e n z y m e which a c t e d in presence of o . o o o l millimol Cu is (calculating t h e g r e a t e r dilution) o n l y 12% i n a c t i v a t e d ; t h e e n z y m e which a c t e d in presence of o.oo16 millitools of Cu is a b o u t 55 % i n a c t i v a t e d d u r i n g t h i s time. As it was f o u n d b y KOBOWlTZTM, c o p p e r is s t r i c t l y specific a n d c a n n o t be r e p l a c e d b y a l l y o t h e r m e t a l . T e s t s were m a d e on: c o b a l t (as c o b a l t o u s chloride), v a n a d i u m (as d i v a n a d y l t e t r a c h l o r i d e a n d v a n a d y l sulphate), nickel (as sulphate), zinc (as chloride), iron (as ferric chloride), c h r o m i u m (as p o t a s s i u m c h r o m i c sulphate), m a g n e s i u m (as sulphate) a n d m a n g a n e s e (as m a n g a n o u s sulphate). Cobalt in V i t a m i n BI~ is also comp l e t e l y i n a c t i v e , as is iron in C y t o c h r o m e C ( e x p e r i m e n t s n o t r e p o r t e d ) . i
•
0.0016 Millimol, Cu ÷÷ 0.0008 ,,
+
0.0004
0
250
~L
ro.ooo2
t olO.OOOl
200-
':9
., ,,
LO.O0005 " *
0.0
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,,
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~
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m M
Cu
o
0.01
,
',
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0.005
. . . .
o
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. . . .
+
0.0008
,,
200
--.,x'}~i"6x~i'~'g'-'+
150
100
56
0
.
Ig,i 30
100
,
90
I~
i. . ~ /-I" I
50
ic 60
'AY I I t-
~
150 180
ro, ooo2 .ram
f"
Minutes Fig. 2. The effect of varying amounts of copper on the enzymic oxidation of 0.90 mg tyrosine + o.i mg Dopa. Curve C represents maximum autoxidation, in presence of o.ooi6 millimol copper and in absence of apo-enzyme. All other flasks contain the same amount of apo-enzyme as in Fig. I.
30
60
I --
• {o, ooo, •
120
II
.'<"
90
10,00005 0,0
cu
. . . .
"
i - L_--¢ 120 150 180
~:
210
Minutes
Fig. 3. The effect of varying amounts of copper on the enzymic oxidation of 0.90 mg tyrosine + o.o 5 mg Dopa in o.o 5 molar citrate-phosphate buffer. All flasks contain the same amount of apo-enzyme as in Fig. I.
Influence o] copper on tyrosine oxidation. C o m p l e t e l y different is t h e action of c o p p e r on t y r o s i n e o x i d a t i o n . T h e e n z y m e has t h e s a m e a c t i v i t y in presence of o.oooo5, o . o o o i a n d o.o002 millimols of copper, h o w e v e r (Fig. 2), f u r t h e r a u g m e n t a t i o n of this a m o u n t of c o p p e r d e t e r m i n e s f u r t h e r a u g m e n t a t i o n of t h e a c t i v i t y on tyrosine. Using c i t r a t e p h o s p h a t e buffer of MclLwAIN i n s t e a d of SORENSE~'S p h o s p h a t e s p e r m i t s a g r e a t e r v a r i a t i o n of c o p p e r c o n c e n t r a t i o n a n d t h u s an e n l a r g e m e n t of t h e p r e v i o u s findings (Fig. 3), b u t , as was f o u n d b y MASON AND WRIGHT 1° for D o p a o x i d a t i o n a t p H 6.6, t h e e n z y m e is slightly less a c t i v e in presence of citrate. T h e lower a c t i v i t y of t h e e n z y m e in this e x p e r i m e n t is p a r t l y due also to t h e lower c o n c e n t r a t i o n of D o p a . Re/erences p. x79.
VOL. 9 (I952)
T Y R O S I N A SAND E POLYPHENOLOXIDASE
173
Influence o/other metals on tyrosine oxidation. The non-specificity of copper is shown in Fig. 4. Here, after h a v i n g added 0.00005 millimols of copper, equimolecular a m o u n t s of cobalt, vanadium, nickel, zinc, iron, chromium, magnesium and manganese (as before) were added. Cobalt, v a n a d i u m and nickel accelerate significantly the activity, b u t copper is about 2.5 times as active as the other three. Presence or absence of zinc, iron, chromium, magnesium and manganese is indifferent. Cobalt in Vitamin Bz2 is inactive or at least is not more active t h a n inorganic cobalt; Cytochrome C is slightly inhibitory (experiments not reported). ,
0.00,25 mM
0 []
o sl
+
im
~I
Cu + 0 . 0 0 0 0 5 rn FI Ni + " CO + ', V
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al
ie + ZA +
,,
M ~ "#"
,=1
h'll~ + Cr +
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CU
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%ol
CU
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0.0"I mM V + 0.00005 eJ 4. wJ 0.007 J, ,,/,. #l 0,0025 0.0001 0.00005 mM Cu
rnM
CU
tm al
~,,.S5 0 -
li
,/
Z, /o7 / o,
& / i a" f6
0
30
50
90 $20 Minutes
Fig. 4. The effect of equimolecular amounts of copper, nickel, cobalt, vanadium, iron, zinc, magnesium, manganese and chromium on the enzymic oxidation of o.9 mg tyrosine + o.o5 mg Dopa. All flasks contain the same amount of apoenzyme as in Fig. z.
0
V 30
60
aO
$20
Minu#es
Fig. 5. The effect of varying amounts of vanadium on the enzymic oxidation of o. 9 mg tyrosine + o.o5 mg Dopa. All flasks contain the same amount of apoenzyme as in Fig. I.
Fig. 5 shows t h a t an a u g m e n t a t i o n of v a n a d i u m concentration determines a corresponding a u g m e n t a t i o n of activity on tyrosine. Demonstration that copper accelerating tyrosine oxidation is /ree and non proteinbound. I n the lowest of the curves reported in the Fig. 2 and 3 it was shown t h a t the apo-enzyme has the same activity on tyrosine in presence of 0.o0005 millimols, o.oooz a n d 0.0002 millimols of copper and t h a t the activity begins to increase only when this a m o u n t is surpassed. I t is highly improbable t h a t in all the following trials, especially with the highest concentration used, copper should remain always b o u n d to protein. However, it can be easily demonstrated, b y two different methods, t h a t even the lowest a m o u n t of copper, which gives a measurable increase of the activity on tyrosine, is already in excess and is not protein-bound. Re]erences p. 179.
I74
D. KERT~SZ
VOL. 9 (I952)
One of t h e d e m o n s t r a t i o n s is b a s e d on t h e fact, f o u n d b y KUBOWlTZ19, t h a t c o p p e r b o u n d to p o l y p h e n o l o x i d a s e p r o t e i n does n o t dialyse (if n o t a g a i n s t Iso HCN). I n Fig. 6 i t is shown h o w dialysis a g a i n s t P y r e x r e d i s t i l l e d w a t e r influences t h e a c t i v i t y of t h e e n z y m e to which 0.0008 millimols c o p p e r were a d d e d . D i a l y s i s ffoo does n o t m o d i f y t h e a c t i v i t y on D o p a , where only t h e p r o t e i n - b o u n d specific c o p p e r is acting, b u t reduces t h e a c t i v i t y on t y r o s i n e a t t h e s a m e level as i t w o u l d h a v e w i t h o u t excess of copper. A n o t h e r , y e t m o r e categoric proof can be pres e n t e d b y v i r t u e of t h e c o m p l e x i n t e r r e l a t i o n s existing b e t w e e n ascorbic acid, catechol, c o p p e r a n d t h e apo0 30 60 90 q20 q50 M/nutes enzyme. KUBOWITZ 10 a n d KEILIN AND 1V[ANN8 h a v e Fig, 6. The effect of dialysis on the d i s c o v e r e d t h a t c o p p e r b o u n d to c a t a l y t i c a l l y a c t i v e enzymic oxidation of 0. 5 mg Dopa positions of t h e p o l y p h e n o l o x i d a s e p r o t e i n becomes (Curves A and B) and on theenzymic oxidation of 0.9 mg tyrosine s t r i c t l y specific for o - d i h y d r i c phenols a n d it loses its + 0.o 5 mg Dopa (Curves C and D). original c a p a c i t y to oxidize d i r e c t l y ascorbic acidlS; it After having added 0.0008 millican do so only in t h e presence of a c a t a l y t i c a m o u n t mols of copper, A and C are dialysed (see text), B and D not. All of catechol, b y v i r t u e of t h e change o - d i h y d r i c phenol flasks contain the same amount of ~ - o - q u i n o n e . I t was r e c e n t l y shown 16 t h a t c o p p e r apo-enzyme as in Fig, x. b o u n d to p r o t e i n s of t h e e n z y m e s o l u t i o n is i n c a p a b l e of t h e d i r e c t c a t a l y s i s of ascorbic acid o x i d a } I i I I tion also w h e n i t occupies positions which are 0.25mi Enzyme + O.O004mM Cu c a t a l y t i c a l l y inactive, a n d t h a t c o p p e r has a __ Enzyme+O.O004mM Cu ~25C c e r t a i n preference for t h e c a t a l y t i c a l l y a c t i v e '~-q ml Enzyme + 0.0001 mM Cu positions of t h e a p o - e n z y m e molecule. If c o p p e r is n o t in excess a n d it is all p r o t e i n 200 b o u n d , in presence of t r a c e s of cateehol, an a u g m e n t a t i o n of t h e a m o u n t of a p o - e n z y m e d e t e r m i n e s a p r o p o r t i o n a l a u g m e n t a t i o n of 150 t h e ascorbic acid o x i d a t i o n , which follows exclusively t h e i n d i r e c t m e c h a n i s m of KuBoWITZ19. If, however, c o p p e r is in excess over ~0c t h e protein, t h e n i n d i r e c t a n d direct o x i d a tion proceeds c o n t e m p o r a n e o u s l y , a n d an a u g m e n t a t i o n of t h e a m o u n t of a p o - e n z y m e 50 n o t only does n o t d e t e r m i n e an a u g m e n t a tion, b u t a c t u a l l y reduces t h e r a t e of ascorbic acid o x i d a t i o n , b e c a u s e it reduces t h e q u a n t i t y of free c o p p e r ions p r e s e n t 16. I n t h e ex0 10 20 30 40 50 60 90 q20 150 Minutes p e r i m e n t r e p o r t e d in Fig. 7, in presence of Fig. 7. The effect of varying apo-enzyme con- o . o o o i millimols of c o p p e r a n d of o.oooI milcentrations in presence of an excess (o.0o04 limols of c a t e c h o l a fourfold a u g m e n t a t i o n of millimols) and in presence of an amount not in excess (o.oooi millimols) of copper on the t h e a m o u n t of a p o - e n z y m e d e t e r m i n e s a proenzymic oxidation of 3.51 mg ascorbie acid p o r t i o n a l a u g m e n t a t i o n of t h e o x i d a t i o n ; on + o.oi mg catechol, i ml enzyme = 6o ent h e c o n t r a r y , in presence of o.ooo 4 millimols zyme units.
l' *ml
l
Re/erences p. z79.
VOL. 9 (I952)
TYROSINASE AND POLYPHENOLOXIDASE
I75
of copper and o.oooi millimols of catechol, the originally high rate of ascorbic acid oxidation is in fact reduced by a fourfold augmentation of the apo-enzyme concentration. DISCUSSION
ONSLOW AND ROBINSON6 suggested that o-quinones react directly with monohydric phenols because they found that by repeated adsorptions and elutions they could reduce the activity of the potato-enzyme on tyrosine but not on catechol, and that the lost activity on tyrosine could be restored by adding traces of catechol. We arrived at the same conclusion 9 because we found that the enzymic oxidation of tyrosine, after the induction period, proceeds at a constant linear rate which is independent of the quantity of the o-dihydric phenol added to shorten the induction period, and that in the absence of enzyme o-benzoquinone can oxidise tyrosine to an o-dihydric phenol. The constant rate of the monohydric phenol oxidation was also found by BEHM AND NELSON21; and LER•ER et al. 17 in the first of a series of valuable papers have not only rediscovered the same particularities of the monohydroxyphenol oxidation, but have also shown t h a t the induction period is proportional to the negative logarithm of the amount of o-dihydroxyphenol added, as it was predicted theoretically ~2. In criticising the direct o-quinone theory, NELSON AND DAWSON5 state: " U n d o u b t edly, the strongest argument against the view that products of catechol oxidation are alone responsible for the initial oxidation of monohydric phenols is the fact that the ratio of catecholase activity to monophenol action of the enzyme can be widely varied during the preparation of tyrosinase". As PUGH7 rejected ONSLOW AND ROBINSON'S views principally because she could n o t vary the relative activity of the enzyme on catechol and on p-cresol, it is disconcerting to see the same argument used as the strongest one in an exactly contrary sense. Whatever is the value of this argument, the fact remains that the relative activity of the enzyme could be changed previously only in the sense of a loss of the monophenolase activity; a change more reasonable to explain on the basis of the o-quinone theory s, 10 than admitting a specific monophenolase activity of the enzyme itself, which leads one necessarily to attribute quite new and unprecedented properties to the tyrosinase molecule n. However, a real difficulty which indeed can not be explained by the simple o-quinone theory is represented b y the different behaviour of enzymes of animal and vegetal origin. SetSia 9 and melanoma 17 enzymes oxidise Dopa very rapidly and tyrosine, if less rapidly, still at a considerable speed, whilst enzymes of vegetal origin 8,19 oxidise catechol and Dopa very rapidly, but their action on tyrosine is relatively moderate. So animal enzymes appear to function more as a tyrosinase and vegetal enzymes more as a catecholase or polyphenoloxidase; in the terminology of NELSON AND DAWSON5 the first present a low catecholase/cresolase ratio, the second a high one. This different specificity of animal enzymes makes it necessary to postulate the presence of an additional factor or factors, which can accelerate the action of vegetal enzymes on monohydric phenols, especially on tyrosine. I t is shown in this paper t h a t at least four metals, copper, cobalt, vanadium and nickel can accelerate the action of the potato enzyme on a monohydric phenol and can raise it to a higher level than it was in the original extract. Iron, zinc, magnesium, manganese and chromium were tried and found completely inactive, but an eventual References p. z79.
176
D. KERT~SZ
VOL. 9 (1952)
activity of some other metal, owing to the little specificity of the reaction, cannot be excluded a priori. The action of the four active metals is completely different in Dopa and in tyrosine oxidation. Copper is highly specific in Dopa, as it is in o-dihydric phenol oxidation in generaP, 19 and cannot be replaced b y any other metal as the prosthetic group of the enzyme; if the enzyme is once saturated b y the relatively small amount of copper required, it is fully active and this activity cannot be augmented by an excess of the same or of any other metal. As the active copper is completely protein-bound and all other metal is inactive, dialysis against Pyrex-redistilled water does not have any influence on the activity on Dopa (Fig. 6). ~ I Presence of copper in the small quantity sufficient to saturate the active positions of the enzyme molecule is also indis- ._~ = pensable in tyrosine oxidation, but with this amount of copper ~20~ alone the activity is only moderate. In agreement with the fact _~ +,discovered by KUBOWITZ19 that polyphenoloxidase protein can .'~'~ t2 bind more copper than it uses as prosthetic group and t h a t the ~ ISO o surplus copper is fixed on catalytically inactive positions ~ 1 ("falsche Stellungen") of the molecule, the activity on tyrosine o remains moderate until the inactive places also of the enzyme ~ ¢00 - q o molecule and of the impurities (which of course have only inactive places) are occupied (Fig. 2 and 3). The activity begins ~to increase only when this amount of copper is once surpassed SO and where there are free and non-protein-bound copper ions in fl [ solution. As excess copper dialyses easily, activity on tyrosine is reduced to the minimum level by dialysis against Pyrex-I I-3 t-5 redistilled water (Fig. 6); moreover, it catalyses directly asLogarithmof millimols corbic acid oxidation (Fig. 7), which can not be done b y activelymetal a d d e d or inactively-bound copper ~6. A definite relationship exists Fig. 8. Relationship between amount of metall between copper concentration and rate of tyrosine oxidation; added and enzymic tyroif the time required for the oxidation of 5 ° % tyrosine is plotted sine oxidation. Curve I : against the logarithm of the amount of copper added, a linear Copper in phosphate buffer (from the expericurve is obtained which presents a sharp break at the value ment reported in Fig. 2). where there are no free ions in solution, t h a t is where all copper Curve 2 : Copper in is bound. Below this value, as stated before, the time of half citrate-phosphate buffer (from the experiment oxidation remains constant (Fig. 8). reported in Fig. 3)The specificity of excess copper in tyrosine oxidation is Curve 3: Vanadium (from the experiment limited to the fact that it is more active than cobalt, vanadium reported in Fig. 5). or nickel (Fig. 4). T h a t the same quantitative relationship holds also for the other active metals as for copper is shown b y curve 3 of Fig. 8, where half oxidation time is plotted against the logarithm of vanadium concentration. The point and mechanism of intervention of metals results clearly from a comparison of their action on Dopa and on tyrosine. This comparison cannot be made on catechol and p-cresol; the difficulties of using catechol as substrate are well knownS, 19,z~ and p-cresol, besides being highly unphysiological, is too autoxidable to serve as a useful substrate to distinguish between monohydric phenol and o-dihydric phenol oxidation. Although the ulterior products of Dopa and of tyrosine oxidation (exactly as those of catechol and of p-cresol oxidation 23, ~4, 25) are unknown, it was shown b y RAPER'S funRe#fences p. **79.
VOL. 9 (1952)
TYROSINASE AND POLYPHENOLOXIDASE
177
damental researches that the first product of the tyrosine oxidation is Dopa and the second is its o-quinone 1, ,6, ~ and there can be no doubt that in the two over-all reactions : (I) Tyrosine -~ Dopa ~ o-quinone -+ ulterior products ~ melanin (II) Dopa ~ o-quinone --> ulterior products ~ melanin all the ulterior products, the nature and importance of which is yet under discussion 15, 2s, are identical. As the over-all tyrosine oxidation (I) is much slower than over-all Dopa oxidation (II), the reaction tyrosine ~ Dopa is rate-determining in tyrosine oxidation and, the two reactions being in all other respects identical, all agents which accelerate tyrosine but not Dopa oxidation necessarily intervene in the reaction tyrosine ~ Dopa. The so-called tyrosinase appears as a very complicated system, composed of: I. an enzyme with copper as prosthetic group, specific for o-dihydric phenols' 2. an o-dihydric phenol; 3. certain free metallic ions. The complexity of this system is increased b y the interdependence of its components inasmuch as the quantity of o-dihydric phenol (or respectively of o-quinone) present depends on the amount of the enzyme 12 and on both the quality and quantity of the metallic ions present. Yet more complications arise from the fact that only free metallic ions increase the originally low rate of tyrosine oxidation and in consequence all constituents which can bind metal ions, especially proteins inert on o-dihydric phenol oxidation, will influence the activity on monohydric phenols, as will the metallic ions always present in common laboratory distilled water. In this context it is useful to point out the decisive importance of the use of Pyrex-redistilled water: if the enzyme were prepared in common distilled water and dialysed afterwards against the same, its activity on tyrosine would not change and it would appear to belong to the protein molecule. The error introduced b y the use of common distilled water is greater in the case of pure or of dilute enzyme solutions16; moreover it is constant in a given L a b o r a t o r y and so escapes detection. Although names are not of very great importance, the name "tyrosinase" is probably the most inappropriate among the numerous designations used for the enzyme studied in this paper. Admitting the direct action of this enzyme on monohydric phenols and calling it tyrosinase, we already possessed a most particular enzyme, which I. if dilute, could act on other substances but not on its own substrate 29 9 ; 2. had a much greater action on a number of other substances than on tyrosineS, s, lo; 3. could oxidise other substances immediately, but tyrosine only after an induction period 1-5; 4. could act at distance, without immediate contact with its substrate TM,14; 5. ought to possess quite unique properties for an enzyme molecule ~1. The results reported in this paper are in agreement with the views that the oxidation of tyrosine is a non-enzymatic reaction between o-quinone and tyrosine and render the name tyrosinase still more inappropriate: this enzyme corresponds to the system polyphenoloxidase + o-dihydric phenol (or o-quinone) + metallic ions, which latter function probably as electron transmitters and hasten the non-enzymic reaction between tyrosine and o-quinone. The author cannot see any justification for the conservation of the term "tyrosinase" except to keep the name under which the discovery and the fundamental researches of B. BERTRAND and of H. S. RAPER were made.
Re/erences p. I79.
I7S
D. I~RT~SZ
VOL. 9 (I952)
SUMMARY
A s t u d y was m a d e to explain t h e different behaviour of animal a n d of vegetal polyphenoloxidases on monohydric- a n d dihydric-phenols. I. It was found t h a t t h e first phase of the tyrosine oxidation (the t r a n s f o r m a t i o n of tyrosine in Dopa) is considerably accelerated b y excess of copper, by cobalt, by v a n a d i u m and by nickel, whilst iron, zinc, c h r o m i u m , m a g n e s i u m a n d m a n g a n e s e are inactive. 2. Excess of copper, cobalt, v a n a d i u m a n d nickel h a v e no action on t h e second, enzymic phase of tyrosine oxidation (the t r a n s f o r m a t i o n of Dopa in its o-quinone) where only copper bound to polyphenoloxidase protein is active. 3. It was demonstrated, b y two i n d e p e n d e n t methods, t h a t the acceleration of tyrosine oxidation is determined b y free a n d non-protein-bound metal ions. 4. a n d t h a t there is a q u a n t i t a t i v e relationship between metal concentration and velocity of tyrosine oxidation. 5. Tyrosinase is identified with a complex s y s t e m composed of an e n z y m e specific for o-dihydric phenols, of an o-dihydric phenol (or o-quinone) a n d of free metallic ions. The role of the latter is to accelerate the spontaneous reaction between o-quinone and tyrosine. 6. The appropriateness of the n a m e " t y r o s i n a s e " is discussed.
L a pr6sente 6rude a 6t6 entreprise afin d'essayer d'expliquer le c o m p o r t e m e n t diff6rent des polyph6noloxydases animales et v6g6tales vis-a-vis des ph6nols mono- et divalents. I. Nous avons trouv6 que la premiere phase de l ' o x y d a t i o n de la tyrosine (transformation de la tyrosine en dopa) est acc616r6e consid6rablement par u n exc~s de cuivre, par le cobalt, le v a n a d i u m et le nickel, t a n d i s que le fer, le zinc, le chrome, le m a g n 6 s i u m et le m a n g a n e s e sont inactifs. 2. U n exc~s de cuivre, de cobalt, de v a n a d i u m ot~ de nickel n'exerce aucune action sur la seconde phase e n z y m a t i q u e de l ' o x y d a t i o n de la tyrosine (transformation de dopa en son o-quinone) oil seul le cuivre li6 g une prot6ine polyphenoloxydasique est actif. 3. Nous avons d6montr6 p a r d e u x m6thodes i n d @ e n d a n t e s que l'acc616ration de l ' o x y d a t i o n de la tyrosine est d6termin6e par des ions metalliques fibres et non li6s A une prot6ine et 4. qu'il existe une relation q u a n t i t a t i v e entre la concentration du m6tal et la vitesse de Foxydation de la tyrosine. 5. La tyrosinase a 6t6 identifi6e comme 6tant u n syst~me complexe compos6 d ' u n enzyme sp6cifique pour les ph6nols c o n t e n a n t 2 - O H en position o-, d ' u n tel ph6nol divalent (ou d ' u n e o-quinone) et d'ions m6talliques libres. Le r61e de ces derniers consiste ~ acc616rer la r6action spontan6e entre l'o-quinone et la tyrosine. 6. La justesse du nora "'tyrosinase'" est discut6e.
ZUSAMMENFASSUNG Es wurde ein Versuch gemacht, u m das verschiedene Verhalten yon tierischen u n d pflanzlichen Polyphenol-Oxydasen gegen einwertige u n d zweiwertige Phenole zu erklgren. i. Es wurde festgestellt, dass die erste Phase der O x y d a t i o n y o n Tyrosin (die Verwandlung yon Tyrosin in Dopa) durch einen Uberschnss yon Kupfer, durch Kobalt, durch V a n a d i u m n n d durch Nickel sehr beschleunigt wird, w~hrend Eisen, Zink, Chrom, Magnesium u n d Mangan i n a k t i v sind. 2. Ein ~Jberschuss von Kupfer, Kobalt, V a n a d i u m u n d Nickel iibt keine W i r k u n g auf die zweite, enzymatische Phase der T y r o s i n o x y d a t i o n (Verwandlung y o n Dopa in sein o-Chinon) aus; bier ist n u r das an Polyphenol-Oxydase-Protein gebundenes K u p f e r wirksam. 3. Es wurde m i t Hilfe zweier unabhiingiger Methoden bewiesen, dass die Beschleunigung der Tyrosin-Oxydation durch freie u n d nicht proteingebundene Metallionen bewirkt wird, u n d 4- dass zwischen der Metallkonzentration u n d der Geschwindigkeit der Tyrosin-Oxydation ein q u a n t i t a t i v e s Verh~ltnis besteht. 5. Tyrosinase wurde als ein komplexes System, bestehend aus einem fiir Phenole m i t 2 0 H Gruppen in o-Stellung spezifischen E n z y m , Bus einem solehen zweiwertigen Phenol (oder o-Chinon) u n d Bus freien Metallionen, identifiziert. Die Rolle der letzteren b e s t e h t darin, dass sie die spontane Reaktion zwischen o-Chinon u n d Tyrosin besch!eunigen. 6. Die Angemessenheit des N a m e n s " T y r o s i n a s e " wird diskutiert.
Re#rences p. z79.
VOL. 9 (1952)
TYROSINASE AND POLYPHENOLOXIDASE
179
REFERENCES 1 H. S. RAPER, Ergeb. Enzym[orsch., I (1932) 270. 2 H. S. RAPER, in BAMANN-MYRB.~CK: Methoden der Ferment/orschung, Leipzig (1941) 2476. 3 C. OPPENHEIMER, Die Fermente u. i. Wirkungen, S u p p l e m e n t , T h e H a g u e (1939) 16o8. 4 W. FRANKE, in NORD-WEIDENHAGEN: Handbuch der Enzymologie, Leipzig (194 o) 731. 5 j . M. NELSON AND C. R. DAWSON, Advances in Enzymol., 4 (1944) 99. 6 M. W. ONSLOW AND M. E. ROBINSON, Biochem. J., 22 (1928) x327. C. E. M. PUSH, Biochem. J., 24 (193o) 1442. s D. KEILIN AND T. MANN, Proc. Roy. Soc., B, 125 (1938) 187. 9 L. CALIFANO AND D. KI~RTESZ, Nature, 142 (1938) lO36; Enzymol., 6 (1939) 233. 10 O. WARBURG, Heavy Metal Prosthetic Groups, O x f o r d (1949) 173. 11 M. F. MALLETTE AND C. R. DAWSON, Arch. Biochem., 23 (1949) 29. 12 D. KERT~SZ, Enzymol., 12 (1948) 254 ; 13 (1949) 182. 13 D. IKERT~SZ, Compt. rend. soc. biol., 143 (1949) 1469; Nature, 165 (195o) 523. 14 D. I~ERT~SZ AND P. CASELLI, Bull. soc. chim. biol., 32 (195 o) 583. 15 D. KERT~SZ, Bull. soc. chim. biol., 32 (195 O) 587 • 16 D. KERT~SZ, Bull. soc. chim. biol., 33 (1951) 14oo. 17 A. B. LERNER, T. B. FITZPATRICK, E. CALKINS, AND W . H. SUMMERSON, J. Biol. Chem., 178 (1948) 185. 18 E. S. G. BARRON, R. H. DE MEIO, AND F. I~LEMPERER, J. Biol. Chem., 116 (i936) 626. 19 V. KUBOWITZ, Biochem. Z., 298 (1938) 32. 2o I-I. S. MASON AND C. J. WRIGHT, J. Biol. Chem., I8o (1949) 235. 3x R. C. BEI~M AND J. M. NELSON, q u o t e d as in press, ref. 5, page 138. 33 loc. cir. 12, page 26o. 3 3 H. JACKSON, Biochem. J., 33 (1939) 1452. 3t H. S. MASON, L. SCHWARTZ, AND D. C. PETERSON, J. Am. Chem. Soc., 67 (1945) 1233. 35 E. A. H. ROBERTS AND D. J. W o o D , Nature, 165 (I95O) 32. ~s H. S. RAPER, Biochem. J., 2o (1926) 735. 37 C, E. M. PUSH AND H. S. RAPER, Biochem. J., 2I (I927) 137o. ~s D. IZERT~SZ, Nature, 168 (1951) 69729 C. E. M. PUSH, Biochem. J., 27 (1933) 478. Received
12
November
I2th,
I951
BIOCI-IIMICA ET BIOPI-I¥SICA ACTA
VOL. 9 (1952)
TYROSINASE AND POLYPHENOLOXIDASE. THE ROLE OF METALLIC IONS IN MELANOGENESIS by D E N I S IiEIITI~SZ Research Department o/ the Istituto Regina Elena per Io Studio e la Cura dei Tumori, Roma (Italy)
The mechanism by which tyrosinase acts on monohydric phenols has been under discussion for a long time 1-a. ONSLOWAND ROBINSON6 were the first to propose that the monophenolase action is not due to the enzyme molecule itself but to the o-quinones produced by the enzymic oxidation of o-dihydric phenols. After the adverse criticism of PUGH7 the same view was reaffirmed by KEILIN AND MANN8 and by CALIFANO AND KERT£S#. More recently WARBIJRGl° states also that probably in the case of monohydric phenol oxidation o-diphenols are first formed by a slow reaction, and these then act catalytically by virtue of the change o-diphenol ¢ o-quinone. This is the same catalytic change which, in presence of traces of catechol, enables the enzyme to oxidize hydroquinone, ascorbic acid and reduced coenzymes I and II 1°. However, most authors, following NELSON AND DAWSON ~, believe that the oxidation of the monohydric phenols is a function of the enzyme molecule itself and that the enzyme is primed whilst oxidizing an dihydric phenol and so acquires the capacity to act on monohydrie phenols, a-~ALLETTEAND DAWSONn, to explain the loss of monophenolase activity during the purification process, suppose that different centres of the same protein molecule are responsible for the monohydric phenol and dihydric phenol oxidation. The diminution of the activity on monohydric phenols (in their terminology the cresolase activity of tyrosinase) and the augmentation of the activity on o-dihydfic phenols (in their terminology the catecholase activity of tyrosinase) would be the result of the greater fragility and loss of part of the molecule responsible for the cresolase activity, or of the unmasking of new active centres responsible for the catecholase activity during the purification process. Evidence reported in this paper not only fails to support the above views, but shows that the cresolase or monophenolase activity of the enzyme belongs to free, and not protein-bound, metallic ions which hasten the spontaneous reaction 9 between o-quinones and monohydric phenols. In agreement between earlier theoretical 1~ and experimentaP~,14 work the oxidation of the monohydric phenols appears to be represented correctly by the following reactions (I)
(II)
o-dihydric phenol ~ o-quinone ~ ulterior products ~ melanin o-quinone + monohydric phenol + H~O = 2 o-dihydric phenol
of which only the first of (I) is enzymatic. l?eferenees p. x79.
VOL. 9 (1952)
I7~:
TYROSINASE AND POLYPHENOLOXIDASE METHODS
Enzyme preparation. Different p r e p a r a t i o n s of p o l y p h e n o l o x i d a s e ape-enzyme, f r o m p o t a t o peelings e x t r a c t e d w i t h HCN, were described earlierlS, TM. I n this w o r k a stable p o w d e r was used which is similar to one described recently 10, b u t of a more simple preparation. 13oo g of p o t a t o Feelings are e x t r a c t e d w i t h 700 ml of e x t r a c t i o n liquid (0.05 m o l a r K C N neutralized a n d buffered to p H 7)- The e x t r a c t is centrifuged for a few m i n u t e s to eliminate the s t a r c h a n d t h e n b r o u g h t u p to 3 0 % c o n c e n t r a t i o n in acetone a n d centrifuged again. The precipitate is discarded. The supern a t a n t is b r o u g h t up to 6 0 % c o n c e n t r a t i o n in acetone and centrifuged. The precipitate is resuspended in 18o ml of e x t r a c t i o n liquid. The t r o u b l e suspension o b t a i n e d is b r o u g h t up again to 6 o O concent r a t i o n in acetone, filtered on a B u c h n e r funnel a n d the precipitate is w a s h e d 5 t i m e s w i t h acetone. I t is dessicated first ill an air current, later in a dessicator u n d e r v a c u u m . I t can be pulverized after 24 h o u r s ; the p o w d e r is stable (at least 12 m o n t h s at + 2 ° C) and it is n o t hygroscopic. D u r i n g the whole procedure the e x t r a c t i o n liquid is m a i n t a i n e d at o °, acetone at - - 2 0 ° C; all operations are made in a cold r o o m a t + 2 ° C (for f u r t h e r details see ref. 16). I m m e d i a t e l y before use, the p o w d e r is s u s p e n d e d a t a ratio of 50 m g / m l in Pyrex-redistilled w a t e r a n d the liquid is s h a r p l y centrifuged for 30 m i n u t e s to eliminate the a b u n d a n t insoluble m a t t e r s . The clear solution obtained is practically inactive w i t h o u t added copper on D o p a ; its c o n t e n t in non-dialysable s u b s t a n c e s is 16 m g / m l and in copper 0 . o 2 % . I ml of the solution contains, if copper is added, 60 e n z y m e u n i t s (Fig. I) as defined b y LERNER et al. 1~. Aetivity determination. The a c t i v i t y of the e n z y m e was determined m a n o m e t r i c a l l y by m e a s u r i n g the o x y g e n u p t a k e in the W a r b u r g a p p a r a t u s a t 38o C. S u b s t r a t e s (in a m o u n t s s h o w n in the legends of the figures) were added f r o m the side a r m s after 15 m i n u t e s of t e m p e r a t u r e equilibration, at o time. The reaction m i x t u r e s still contained o.25 ml of the e n z y m e solution, 1.25 ml of o.I molar p h o s p h a t e or p h o s p h a t e - c i t r a t e buffer at p H 6.8, the a m o u n t m a r k e d on the figures respectively of copper and of o t h e r metals and Pyrex-redistilled w a t e r to make the total volume up to 2. 5 ml. The central well of the flasks contained o.1 ml of N a O H 15 %. All solutions were p r e p a r e d with Pyrex-redistilled w a t e r ; r e a g e n t s were chosen and glass-ware w a s h e d metal-free. The i m p o r t a n c e of these p r e c a u t i o n s was emphasised by •ARRON et a/A s, b y \¥ARBURG 1°, and recently b y the a u t h o r 16. Copper determination. Copper was d e t e r m i n e d w i t h s o d i u m d i e t h y l d i t h i o c a r b a m a t e according to I~EILIN AND ~ A N N 8. Colorimetric readings were made w i t h a Coleman s p e c t r o p h o t o m e t e r , at 44 ° m,u. 2,0 RESULTS
In[luence o/metals on Dopa oxidation. Influence of copper is reported in Fig. I. It is evident that the enzyme is already fully active in presence of 0.00005 millimols (0.08%) Cu and that presence or absence of excess copper is indifferent. The insignificantly lower activities of the enzyme measured with the highest amounts of copper are artefact and due to the fact that the enzyme is slightly and slowly inactivated in presence of a great excess of copper. This is shown by the two lower curves (A and B) of the Fig. I, where the Warburg flasks containing o.oooi millimols and o.oo16 millimols of Cu were reopened, the side arm cavities washed (with the aid of a pipette) 7 times with Pyrexredistilled water and then filled with the same amount of Dopa solution as previously. The new substrate is added to the old test solution after 7 minutes of temperature equilibration. As the whole procedure did not take more than 15 minutes, the potato enzyme was held 12o minutes at 3 8o C in Re/erences p. z79.
~' l
[ ° ° ° ° ° 5 m M°ls cu÷÷ o.oooi I °'°°°2 .... .
f 7'I t00
I ~ I , - ~ IO
~
I0.0004 o
~0.0008
.
.
.
"
"
. . . .
"t
10.00~6
B q
,"0.0
30
60
.
.
.
.
. . . .
50
0
90
~20
~50 ~80 Ninufes
Fig. I. The effect of v a r y i n g a m o u n t s of copper on the enzymic oxidation of I mg Dopa. For curves A a n d B see text. All flasks contain the s a m e a m o u n t of a p e - e n z y m e corresponding (in presence of copper) to 15 e n z y m e units.
172
D. KERTtSZ
VOL. 9 (1952)
c o n t a c t w i t h D o p a a n d Cu w i t h a l m o s t c o n t i n u o u s a g i t a t i o n before t h e second o time. T h e e n z y m e which a c t e d in presence of o . o o o l millimol Cu is (calculating t h e g r e a t e r dilution) o n l y 12% i n a c t i v a t e d ; t h e e n z y m e which a c t e d in presence of o.oo16 millitools of Cu is a b o u t 55 % i n a c t i v a t e d d u r i n g t h i s time. As it was f o u n d b y KOBOWlTZTM, c o p p e r is s t r i c t l y specific a n d c a n n o t be r e p l a c e d b y a l l y o t h e r m e t a l . T e s t s were m a d e on: c o b a l t (as c o b a l t o u s chloride), v a n a d i u m (as d i v a n a d y l t e t r a c h l o r i d e a n d v a n a d y l sulphate), nickel (as sulphate), zinc (as chloride), iron (as ferric chloride), c h r o m i u m (as p o t a s s i u m c h r o m i c sulphate), m a g n e s i u m (as sulphate) a n d m a n g a n e s e (as m a n g a n o u s sulphate). Cobalt in V i t a m i n BI~ is also comp l e t e l y i n a c t i v e , as is iron in C y t o c h r o m e C ( e x p e r i m e n t s n o t r e p o r t e d ) . i
•
0.0016 Millimol, Cu ÷÷ 0.0008 ,,
+
0.0004
0
250
~L
ro.ooo2
t olO.OOOl
200-
':9
., ,,
LO.O0005 " *
0.0
250
,,
"
J~-
-.--'o
I
~
F
*
0.02
m M
Cu
o
0.01
,
',
z~
0.005
. . . .
o
0.0032
. . . .
+
0.0008
,,
200
--.,x'}~i"6x~i'~'g'-'+
150
100
56
0
.
Ig,i 30
100
,
90
I~
i. . ~ /-I" I
50
ic 60
'AY I I t-
~
150 180
ro, ooo2 .ram
f"
Minutes Fig. 2. The effect of varying amounts of copper on the enzymic oxidation of 0.90 mg tyrosine + o.i mg Dopa. Curve C represents maximum autoxidation, in presence of o.ooi6 millimol copper and in absence of apo-enzyme. All other flasks contain the same amount of apo-enzyme as in Fig. I.
30
60
I --
• {o, ooo, •
120
II
.'<"
90
10,00005 0,0
cu
. . . .
"
i - L_--¢ 120 150 180
~:
210
Minutes
Fig. 3. The effect of varying amounts of copper on the enzymic oxidation of 0.90 mg tyrosine + o.o 5 mg Dopa in o.o 5 molar citrate-phosphate buffer. All flasks contain the same amount of apo-enzyme as in Fig. I.
Influence o] copper on tyrosine oxidation. C o m p l e t e l y different is t h e action of c o p p e r on t y r o s i n e o x i d a t i o n . T h e e n z y m e has t h e s a m e a c t i v i t y in presence of o.oooo5, o . o o o i a n d o.o002 millimols of copper, h o w e v e r (Fig. 2), f u r t h e r a u g m e n t a t i o n of this a m o u n t of c o p p e r d e t e r m i n e s f u r t h e r a u g m e n t a t i o n of t h e a c t i v i t y on tyrosine. Using c i t r a t e p h o s p h a t e buffer of MclLwAIN i n s t e a d of SORENSE~'S p h o s p h a t e s p e r m i t s a g r e a t e r v a r i a t i o n of c o p p e r c o n c e n t r a t i o n a n d t h u s an e n l a r g e m e n t of t h e p r e v i o u s findings (Fig. 3), b u t , as was f o u n d b y MASON AND WRIGHT 1° for D o p a o x i d a t i o n a t p H 6.6, t h e e n z y m e is slightly less a c t i v e in presence of citrate. T h e lower a c t i v i t y of t h e e n z y m e in this e x p e r i m e n t is p a r t l y due also to t h e lower c o n c e n t r a t i o n of D o p a . Re/erences p. x79.
VOL. 9 (I952)
T Y R O S I N A SAND E POLYPHENOLOXIDASE
173
Influence o/other metals on tyrosine oxidation. The non-specificity of copper is shown in Fig. 4. Here, after h a v i n g added 0.00005 millimols of copper, equimolecular a m o u n t s of cobalt, vanadium, nickel, zinc, iron, chromium, magnesium and manganese (as before) were added. Cobalt, v a n a d i u m and nickel accelerate significantly the activity, b u t copper is about 2.5 times as active as the other three. Presence or absence of zinc, iron, chromium, magnesium and manganese is indifferent. Cobalt in Vitamin Bz2 is inactive or at least is not more active t h a n inorganic cobalt; Cytochrome C is slightly inhibitory (experiments not reported). ,
0.00,25 mM
0 []
o sl
+
im
~I
Cu + 0 . 0 0 0 0 5 rn FI Ni + " CO + ', V
"
"l-
al
ie + ZA +
,,
M ~ "#"
,=1
h'll~ + Cr +
"
~J
iJ
CU
0.00005
%ol
CU
* o '-' + ,',
0.0"I mM V + 0.00005 eJ 4. wJ 0.007 J, ,,/,. #l 0,0025 0.0001 0.00005 mM Cu
rnM
CU
tm al
~,,.S5 0 -
li
,/
Z, /o7 / o,
& / i a" f6
0
30
50
90 $20 Minutes
Fig. 4. The effect of equimolecular amounts of copper, nickel, cobalt, vanadium, iron, zinc, magnesium, manganese and chromium on the enzymic oxidation of o.9 mg tyrosine + o.o5 mg Dopa. All flasks contain the same amount of apoenzyme as in Fig. z.
0
V 30
60
aO
$20
Minu#es
Fig. 5. The effect of varying amounts of vanadium on the enzymic oxidation of o. 9 mg tyrosine + o.o5 mg Dopa. All flasks contain the same amount of apoenzyme as in Fig. I.
Fig. 5 shows t h a t an a u g m e n t a t i o n of v a n a d i u m concentration determines a corresponding a u g m e n t a t i o n of activity on tyrosine. Demonstration that copper accelerating tyrosine oxidation is /ree and non proteinbound. I n the lowest of the curves reported in the Fig. 2 and 3 it was shown t h a t the apo-enzyme has the same activity on tyrosine in presence of 0.o0005 millimols, o.oooz a n d 0.0002 millimols of copper and t h a t the activity begins to increase only when this a m o u n t is surpassed. I t is highly improbable t h a t in all the following trials, especially with the highest concentration used, copper should remain always b o u n d to protein. However, it can be easily demonstrated, b y two different methods, t h a t even the lowest a m o u n t of copper, which gives a measurable increase of the activity on tyrosine, is already in excess and is not protein-bound. Re]erences p. 179.
I74
D. KERT~SZ
VOL. 9 (I952)
One of t h e d e m o n s t r a t i o n s is b a s e d on t h e fact, f o u n d b y KUBOWlTZ19, t h a t c o p p e r b o u n d to p o l y p h e n o l o x i d a s e p r o t e i n does n o t dialyse (if n o t a g a i n s t Iso HCN). I n Fig. 6 i t is shown h o w dialysis a g a i n s t P y r e x r e d i s t i l l e d w a t e r influences t h e a c t i v i t y of t h e e n z y m e to which 0.0008 millimols c o p p e r were a d d e d . D i a l y s i s ffoo does n o t m o d i f y t h e a c t i v i t y on D o p a , where only t h e p r o t e i n - b o u n d specific c o p p e r is acting, b u t reduces t h e a c t i v i t y on t y r o s i n e a t t h e s a m e level as i t w o u l d h a v e w i t h o u t excess of copper. A n o t h e r , y e t m o r e categoric proof can be pres e n t e d b y v i r t u e of t h e c o m p l e x i n t e r r e l a t i o n s existing b e t w e e n ascorbic acid, catechol, c o p p e r a n d t h e apo0 30 60 90 q20 q50 M/nutes enzyme. KUBOWITZ 10 a n d KEILIN AND 1V[ANN8 h a v e Fig, 6. The effect of dialysis on the d i s c o v e r e d t h a t c o p p e r b o u n d to c a t a l y t i c a l l y a c t i v e enzymic oxidation of 0. 5 mg Dopa positions of t h e p o l y p h e n o l o x i d a s e p r o t e i n becomes (Curves A and B) and on theenzymic oxidation of 0.9 mg tyrosine s t r i c t l y specific for o - d i h y d r i c phenols a n d it loses its + 0.o 5 mg Dopa (Curves C and D). original c a p a c i t y to oxidize d i r e c t l y ascorbic acidlS; it After having added 0.0008 millican do so only in t h e presence of a c a t a l y t i c a m o u n t mols of copper, A and C are dialysed (see text), B and D not. All of catechol, b y v i r t u e of t h e change o - d i h y d r i c phenol flasks contain the same amount of ~ - o - q u i n o n e . I t was r e c e n t l y shown 16 t h a t c o p p e r apo-enzyme as in Fig, x. b o u n d to p r o t e i n s of t h e e n z y m e s o l u t i o n is i n c a p a b l e of t h e d i r e c t c a t a l y s i s of ascorbic acid o x i d a } I i I I tion also w h e n i t occupies positions which are 0.25mi Enzyme + O.O004mM Cu c a t a l y t i c a l l y inactive, a n d t h a t c o p p e r has a __ Enzyme+O.O004mM Cu ~25C c e r t a i n preference for t h e c a t a l y t i c a l l y a c t i v e '~-q ml Enzyme + 0.0001 mM Cu positions of t h e a p o - e n z y m e molecule. If c o p p e r is n o t in excess a n d it is all p r o t e i n 200 b o u n d , in presence of t r a c e s of cateehol, an a u g m e n t a t i o n of t h e a m o u n t of a p o - e n z y m e d e t e r m i n e s a p r o p o r t i o n a l a u g m e n t a t i o n of 150 t h e ascorbic acid o x i d a t i o n , which follows exclusively t h e i n d i r e c t m e c h a n i s m of KuBoWITZ19. If, however, c o p p e r is in excess over ~0c t h e protein, t h e n i n d i r e c t a n d direct o x i d a tion proceeds c o n t e m p o r a n e o u s l y , a n d an a u g m e n t a t i o n of t h e a m o u n t of a p o - e n z y m e 50 n o t only does n o t d e t e r m i n e an a u g m e n t a tion, b u t a c t u a l l y reduces t h e r a t e of ascorbic acid o x i d a t i o n , b e c a u s e it reduces t h e q u a n t i t y of free c o p p e r ions p r e s e n t 16. I n t h e ex0 10 20 30 40 50 60 90 q20 150 Minutes p e r i m e n t r e p o r t e d in Fig. 7, in presence of Fig. 7. The effect of varying apo-enzyme con- o . o o o i millimols of c o p p e r a n d of o.oooI milcentrations in presence of an excess (o.0o04 limols of c a t e c h o l a fourfold a u g m e n t a t i o n of millimols) and in presence of an amount not in excess (o.oooi millimols) of copper on the t h e a m o u n t of a p o - e n z y m e d e t e r m i n e s a proenzymic oxidation of 3.51 mg ascorbie acid p o r t i o n a l a u g m e n t a t i o n of t h e o x i d a t i o n ; on + o.oi mg catechol, i ml enzyme = 6o ent h e c o n t r a r y , in presence of o.ooo 4 millimols zyme units.
l' *ml
l
Re/erences p. z79.
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of copper and o.oooi millimols of catechol, the originally high rate of ascorbic acid oxidation is in fact reduced by a fourfold augmentation of the apo-enzyme concentration. DISCUSSION
ONSLOW AND ROBINSON6 suggested that o-quinones react directly with monohydric phenols because they found that by repeated adsorptions and elutions they could reduce the activity of the potato-enzyme on tyrosine but not on catechol, and that the lost activity on tyrosine could be restored by adding traces of catechol. We arrived at the same conclusion 9 because we found that the enzymic oxidation of tyrosine, after the induction period, proceeds at a constant linear rate which is independent of the quantity of the o-dihydric phenol added to shorten the induction period, and that in the absence of enzyme o-benzoquinone can oxidise tyrosine to an o-dihydric phenol. The constant rate of the monohydric phenol oxidation was also found by BEHM AND NELSON21; and LER•ER et al. 17 in the first of a series of valuable papers have not only rediscovered the same particularities of the monohydroxyphenol oxidation, but have also shown t h a t the induction period is proportional to the negative logarithm of the amount of o-dihydroxyphenol added, as it was predicted theoretically ~2. In criticising the direct o-quinone theory, NELSON AND DAWSON5 state: " U n d o u b t edly, the strongest argument against the view that products of catechol oxidation are alone responsible for the initial oxidation of monohydric phenols is the fact that the ratio of catecholase activity to monophenol action of the enzyme can be widely varied during the preparation of tyrosinase". As PUGH7 rejected ONSLOW AND ROBINSON'S views principally because she could n o t vary the relative activity of the enzyme on catechol and on p-cresol, it is disconcerting to see the same argument used as the strongest one in an exactly contrary sense. Whatever is the value of this argument, the fact remains that the relative activity of the enzyme could be changed previously only in the sense of a loss of the monophenolase activity; a change more reasonable to explain on the basis of the o-quinone theory s, 10 than admitting a specific monophenolase activity of the enzyme itself, which leads one necessarily to attribute quite new and unprecedented properties to the tyrosinase molecule n. However, a real difficulty which indeed can not be explained by the simple o-quinone theory is represented b y the different behaviour of enzymes of animal and vegetal origin. SetSia 9 and melanoma 17 enzymes oxidise Dopa very rapidly and tyrosine, if less rapidly, still at a considerable speed, whilst enzymes of vegetal origin 8,19 oxidise catechol and Dopa very rapidly, but their action on tyrosine is relatively moderate. So animal enzymes appear to function more as a tyrosinase and vegetal enzymes more as a catecholase or polyphenoloxidase; in the terminology of NELSON AND DAWSON5 the first present a low catecholase/cresolase ratio, the second a high one. This different specificity of animal enzymes makes it necessary to postulate the presence of an additional factor or factors, which can accelerate the action of vegetal enzymes on monohydric phenols, especially on tyrosine. I t is shown in this paper t h a t at least four metals, copper, cobalt, vanadium and nickel can accelerate the action of the potato enzyme on a monohydric phenol and can raise it to a higher level than it was in the original extract. Iron, zinc, magnesium, manganese and chromium were tried and found completely inactive, but an eventual References p. z79.
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activity of some other metal, owing to the little specificity of the reaction, cannot be excluded a priori. The action of the four active metals is completely different in Dopa and in tyrosine oxidation. Copper is highly specific in Dopa, as it is in o-dihydric phenol oxidation in generaP, 19 and cannot be replaced b y any other metal as the prosthetic group of the enzyme; if the enzyme is once saturated b y the relatively small amount of copper required, it is fully active and this activity cannot be augmented by an excess of the same or of any other metal. As the active copper is completely protein-bound and all other metal is inactive, dialysis against Pyrex-redistilled water does not have any influence on the activity on Dopa (Fig. 6). ~ I Presence of copper in the small quantity sufficient to saturate the active positions of the enzyme molecule is also indis- ._~ = pensable in tyrosine oxidation, but with this amount of copper ~20~ alone the activity is only moderate. In agreement with the fact _~ +,discovered by KUBOWITZ19 that polyphenoloxidase protein can .'~'~ t2 bind more copper than it uses as prosthetic group and t h a t the ~ ISO o surplus copper is fixed on catalytically inactive positions ~ 1 ("falsche Stellungen") of the molecule, the activity on tyrosine o remains moderate until the inactive places also of the enzyme ~ ¢00 - q o molecule and of the impurities (which of course have only inactive places) are occupied (Fig. 2 and 3). The activity begins ~to increase only when this amount of copper is once surpassed SO and where there are free and non-protein-bound copper ions in fl [ solution. As excess copper dialyses easily, activity on tyrosine is reduced to the minimum level by dialysis against Pyrex-I I-3 t-5 redistilled water (Fig. 6); moreover, it catalyses directly asLogarithmof millimols corbic acid oxidation (Fig. 7), which can not be done b y activelymetal a d d e d or inactively-bound copper ~6. A definite relationship exists Fig. 8. Relationship between amount of metall between copper concentration and rate of tyrosine oxidation; added and enzymic tyroif the time required for the oxidation of 5 ° % tyrosine is plotted sine oxidation. Curve I : against the logarithm of the amount of copper added, a linear Copper in phosphate buffer (from the expericurve is obtained which presents a sharp break at the value ment reported in Fig. 2). where there are no free ions in solution, t h a t is where all copper Curve 2 : Copper in is bound. Below this value, as stated before, the time of half citrate-phosphate buffer (from the experiment oxidation remains constant (Fig. 8). reported in Fig. 3)The specificity of excess copper in tyrosine oxidation is Curve 3: Vanadium (from the experiment limited to the fact that it is more active than cobalt, vanadium reported in Fig. 5). or nickel (Fig. 4). T h a t the same quantitative relationship holds also for the other active metals as for copper is shown b y curve 3 of Fig. 8, where half oxidation time is plotted against the logarithm of vanadium concentration. The point and mechanism of intervention of metals results clearly from a comparison of their action on Dopa and on tyrosine. This comparison cannot be made on catechol and p-cresol; the difficulties of using catechol as substrate are well knownS, 19,z~ and p-cresol, besides being highly unphysiological, is too autoxidable to serve as a useful substrate to distinguish between monohydric phenol and o-dihydric phenol oxidation. Although the ulterior products of Dopa and of tyrosine oxidation (exactly as those of catechol and of p-cresol oxidation 23, ~4, 25) are unknown, it was shown b y RAPER'S funRe#fences p. **79.
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damental researches that the first product of the tyrosine oxidation is Dopa and the second is its o-quinone 1, ,6, ~ and there can be no doubt that in the two over-all reactions : (I) Tyrosine -~ Dopa ~ o-quinone -+ ulterior products ~ melanin (II) Dopa ~ o-quinone --> ulterior products ~ melanin all the ulterior products, the nature and importance of which is yet under discussion 15, 2s, are identical. As the over-all tyrosine oxidation (I) is much slower than over-all Dopa oxidation (II), the reaction tyrosine ~ Dopa is rate-determining in tyrosine oxidation and, the two reactions being in all other respects identical, all agents which accelerate tyrosine but not Dopa oxidation necessarily intervene in the reaction tyrosine ~ Dopa. The so-called tyrosinase appears as a very complicated system, composed of: I. an enzyme with copper as prosthetic group, specific for o-dihydric phenols' 2. an o-dihydric phenol; 3. certain free metallic ions. The complexity of this system is increased b y the interdependence of its components inasmuch as the quantity of o-dihydric phenol (or respectively of o-quinone) present depends on the amount of the enzyme 12 and on both the quality and quantity of the metallic ions present. Yet more complications arise from the fact that only free metallic ions increase the originally low rate of tyrosine oxidation and in consequence all constituents which can bind metal ions, especially proteins inert on o-dihydric phenol oxidation, will influence the activity on monohydric phenols, as will the metallic ions always present in common laboratory distilled water. In this context it is useful to point out the decisive importance of the use of Pyrex-redistilled water: if the enzyme were prepared in common distilled water and dialysed afterwards against the same, its activity on tyrosine would not change and it would appear to belong to the protein molecule. The error introduced b y the use of common distilled water is greater in the case of pure or of dilute enzyme solutions16; moreover it is constant in a given L a b o r a t o r y and so escapes detection. Although names are not of very great importance, the name "tyrosinase" is probably the most inappropriate among the numerous designations used for the enzyme studied in this paper. Admitting the direct action of this enzyme on monohydric phenols and calling it tyrosinase, we already possessed a most particular enzyme, which I. if dilute, could act on other substances but not on its own substrate 29 9 ; 2. had a much greater action on a number of other substances than on tyrosineS, s, lo; 3. could oxidise other substances immediately, but tyrosine only after an induction period 1-5; 4. could act at distance, without immediate contact with its substrate TM,14; 5. ought to possess quite unique properties for an enzyme molecule ~1. The results reported in this paper are in agreement with the views that the oxidation of tyrosine is a non-enzymatic reaction between o-quinone and tyrosine and render the name tyrosinase still more inappropriate: this enzyme corresponds to the system polyphenoloxidase + o-dihydric phenol (or o-quinone) + metallic ions, which latter function probably as electron transmitters and hasten the non-enzymic reaction between tyrosine and o-quinone. The author cannot see any justification for the conservation of the term "tyrosinase" except to keep the name under which the discovery and the fundamental researches of B. BERTRAND and of H. S. RAPER were made.
Re/erences p. I79.
I7S
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SUMMARY
A s t u d y was m a d e to explain t h e different behaviour of animal a n d of vegetal polyphenoloxidases on monohydric- a n d dihydric-phenols. I. It was found t h a t t h e first phase of the tyrosine oxidation (the t r a n s f o r m a t i o n of tyrosine in Dopa) is considerably accelerated b y excess of copper, by cobalt, by v a n a d i u m and by nickel, whilst iron, zinc, c h r o m i u m , m a g n e s i u m a n d m a n g a n e s e are inactive. 2. Excess of copper, cobalt, v a n a d i u m a n d nickel h a v e no action on t h e second, enzymic phase of tyrosine oxidation (the t r a n s f o r m a t i o n of Dopa in its o-quinone) where only copper bound to polyphenoloxidase protein is active. 3. It was demonstrated, b y two i n d e p e n d e n t methods, t h a t the acceleration of tyrosine oxidation is determined b y free a n d non-protein-bound metal ions. 4. a n d t h a t there is a q u a n t i t a t i v e relationship between metal concentration and velocity of tyrosine oxidation. 5. Tyrosinase is identified with a complex s y s t e m composed of an e n z y m e specific for o-dihydric phenols, of an o-dihydric phenol (or o-quinone) a n d of free metallic ions. The role of the latter is to accelerate the spontaneous reaction between o-quinone and tyrosine. 6. The appropriateness of the n a m e " t y r o s i n a s e " is discussed.
L a pr6sente 6rude a 6t6 entreprise afin d'essayer d'expliquer le c o m p o r t e m e n t diff6rent des polyph6noloxydases animales et v6g6tales vis-a-vis des ph6nols mono- et divalents. I. Nous avons trouv6 que la premiere phase de l ' o x y d a t i o n de la tyrosine (transformation de la tyrosine en dopa) est acc616r6e consid6rablement par u n exc~s de cuivre, par le cobalt, le v a n a d i u m et le nickel, t a n d i s que le fer, le zinc, le chrome, le m a g n 6 s i u m et le m a n g a n e s e sont inactifs. 2. U n exc~s de cuivre, de cobalt, de v a n a d i u m ot~ de nickel n'exerce aucune action sur la seconde phase e n z y m a t i q u e de l ' o x y d a t i o n de la tyrosine (transformation de dopa en son o-quinone) oil seul le cuivre li6 g une prot6ine polyphenoloxydasique est actif. 3. Nous avons d6montr6 p a r d e u x m6thodes i n d @ e n d a n t e s que l'acc616ration de l ' o x y d a t i o n de la tyrosine est d6termin6e par des ions metalliques fibres et non li6s A une prot6ine et 4. qu'il existe une relation q u a n t i t a t i v e entre la concentration du m6tal et la vitesse de Foxydation de la tyrosine. 5. La tyrosinase a 6t6 identifi6e comme 6tant u n syst~me complexe compos6 d ' u n enzyme sp6cifique pour les ph6nols c o n t e n a n t 2 - O H en position o-, d ' u n tel ph6nol divalent (ou d ' u n e o-quinone) et d'ions m6talliques libres. Le r61e de ces derniers consiste ~ acc616rer la r6action spontan6e entre l'o-quinone et la tyrosine. 6. La justesse du nora "'tyrosinase'" est discut6e.
ZUSAMMENFASSUNG Es wurde ein Versuch gemacht, u m das verschiedene Verhalten yon tierischen u n d pflanzlichen Polyphenol-Oxydasen gegen einwertige u n d zweiwertige Phenole zu erklgren. i. Es wurde festgestellt, dass die erste Phase der O x y d a t i o n y o n Tyrosin (die Verwandlung yon Tyrosin in Dopa) durch einen Uberschnss yon Kupfer, durch Kobalt, durch V a n a d i u m n n d durch Nickel sehr beschleunigt wird, w~hrend Eisen, Zink, Chrom, Magnesium u n d Mangan i n a k t i v sind. 2. Ein ~Jberschuss von Kupfer, Kobalt, V a n a d i u m u n d Nickel iibt keine W i r k u n g auf die zweite, enzymatische Phase der T y r o s i n o x y d a t i o n (Verwandlung y o n Dopa in sein o-Chinon) aus; bier ist n u r das an Polyphenol-Oxydase-Protein gebundenes K u p f e r wirksam. 3. Es wurde m i t Hilfe zweier unabhiingiger Methoden bewiesen, dass die Beschleunigung der Tyrosin-Oxydation durch freie u n d nicht proteingebundene Metallionen bewirkt wird, u n d 4- dass zwischen der Metallkonzentration u n d der Geschwindigkeit der Tyrosin-Oxydation ein q u a n t i t a t i v e s Verh~ltnis besteht. 5. Tyrosinase wurde als ein komplexes System, bestehend aus einem fiir Phenole m i t 2 0 H Gruppen in o-Stellung spezifischen E n z y m , Bus einem solehen zweiwertigen Phenol (oder o-Chinon) u n d Bus freien Metallionen, identifiziert. Die Rolle der letzteren b e s t e h t darin, dass sie die spontane Reaktion zwischen o-Chinon u n d Tyrosin besch!eunigen. 6. Die Angemessenheit des N a m e n s " T y r o s i n a s e " wird diskutiert.
Re#rences p. z79.
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REFERENCES 1 H. S. RAPER, Ergeb. Enzym[orsch., I (1932) 270. 2 H. S. RAPER, in BAMANN-MYRB.~CK: Methoden der Ferment/orschung, Leipzig (1941) 2476. 3 C. OPPENHEIMER, Die Fermente u. i. Wirkungen, S u p p l e m e n t , T h e H a g u e (1939) 16o8. 4 W. FRANKE, in NORD-WEIDENHAGEN: Handbuch der Enzymologie, Leipzig (194 o) 731. 5 j . M. NELSON AND C. R. DAWSON, Advances in Enzymol., 4 (1944) 99. 6 M. W. ONSLOW AND M. E. ROBINSON, Biochem. J., 22 (1928) x327. C. E. M. PUSH, Biochem. J., 24 (193o) 1442. s D. KEILIN AND T. MANN, Proc. Roy. Soc., B, 125 (1938) 187. 9 L. CALIFANO AND D. KI~RTESZ, Nature, 142 (1938) lO36; Enzymol., 6 (1939) 233. 10 O. WARBURG, Heavy Metal Prosthetic Groups, O x f o r d (1949) 173. 11 M. F. MALLETTE AND C. R. DAWSON, Arch. Biochem., 23 (1949) 29. 12 D. KERT~SZ, Enzymol., 12 (1948) 254 ; 13 (1949) 182. 13 D. IKERT~SZ, Compt. rend. soc. biol., 143 (1949) 1469; Nature, 165 (195o) 523. 14 D. I~ERT~SZ AND P. CASELLI, Bull. soc. chim. biol., 32 (195 o) 583. 15 D. KERT~SZ, Bull. soc. chim. biol., 32 (195 O) 587 • 16 D. KERT~SZ, Bull. soc. chim. biol., 33 (1951) 14oo. 17 A. B. LERNER, T. B. FITZPATRICK, E. CALKINS, AND W . H. SUMMERSON, J. Biol. Chem., 178 (1948) 185. 18 E. S. G. BARRON, R. H. DE MEIO, AND F. I~LEMPERER, J. Biol. Chem., 116 (i936) 626. 19 V. KUBOWITZ, Biochem. Z., 298 (1938) 32. 2o I-I. S. MASON AND C. J. WRIGHT, J. Biol. Chem., I8o (1949) 235. 3x R. C. BEI~M AND J. M. NELSON, q u o t e d as in press, ref. 5, page 138. 33 loc. cir. 12, page 26o. 3 3 H. JACKSON, Biochem. J., 33 (1939) 1452. 3t H. S. MASON, L. SCHWARTZ, AND D. C. PETERSON, J. Am. Chem. Soc., 67 (1945) 1233. 35 E. A. H. ROBERTS AND D. J. W o o D , Nature, 165 (I95O) 32. ~s H. S. RAPER, Biochem. J., 2o (1926) 735. 37 C, E. M. PUSH AND H. S. RAPER, Biochem. J., 2I (I927) 137o. ~s D. IZERT~SZ, Nature, 168 (1951) 69729 C. E. M. PUSH, Biochem. J., 27 (1933) 478. Received
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November
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I951