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 mono-
hydric 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 mono-
phenolase 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 activ-
ity, 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-qui-
nones and monohydric phenols. In agreement between earlier theoretical 1~ and experi-
mentaP~,14 work the oxidation of the monohydric phenols appears to be represented
correctly by the following reactions
(I) o-dihydric phenol ~ o-quinone ~ ulterior products ~ melanin
(II) 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) TYROSINASE
AND POLYPHENOLOXIDASE I7~:

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 super-
n 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 concen-
t 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 ~' l [ ° ° ° ° ° 5 m M°ls cu÷÷
or absence of excess copper is indifferent. The in- o.oooi . . . .

significantly lower activities of the enzyme meas- f I °'°°°2 ....

7'I I0.0004 " "
ured with the highest amounts of copper are arte- I ~ I ~0.0008 . . . .
t00 , - ~ o
fact and due to the fact that the enzyme is slightly "t
10.00~6 . . . .
IO
and slowly inactivated in presence of a great excess ~ B q ,"0.0 . . . .
of copper. This is shown by the two lower curves
(A and B) of the Fig. I, where the Warburg flasks 50

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 Pyrex- 0 30 60 90 ~20 ~50 ~80
Ninufes
redistilled water and then filled with the same
amount of Dopa solution as previously. The new 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
substrate is added to the old test solution after 7 I mg Dopa. For curves A a n d B see
minutes of temperature equilibration. As the whole 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
procedure did not take more than 15 minutes, the (in presence of copper) to 15 e n z y m e
potato enzyme was held 12o minutes at 3 8o C in units.
Re/erences p. z79.
 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 milli-
tools 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 com-
p 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 I ~ F

* 0.02 m M Cu
o 0.01 , ',
0 0.0016 Millimol, Cu ÷÷
z~ 0.005 . . . .
• 0.0008 ,, 250
250 o 0.0032 . . . .
+ 0.0004 ,, ':9
ro.ooo2 .,
+ 0.0008 ,,

~L t olO.OOOl ,, 200
200- LO.O0005 " J~- -.--'o
* 0.0 " --.,x'}~i"6x~i'~'g'-'+

150

100 . ,
100 'AY I I t- I~
.'<"
I --
II
i. . ~ ro, ooo2 .ram cu
56 50 /-I" I • {o, ooo, . . . .

Ig,i ic ~ f"
•
10,00005
0,0
" ~:

i - L_--¢
0 30 60 90 120 150 180 30 60 90 120 150 180 210
Minutes Minutes
Fig. 2. The effect of varying amounts Fig. 3. The effect of varying amounts of
of copper on the enzymic oxidation of copper on the enzymic oxidation of 0.90 mg
0.90 mg tyrosine + o.i mg Dopa. Curve tyrosine + o.o 5 mg Dopa in o.o 5 molar
C represents maximum autoxidation, citrate-phosphate buffer. All flasks contain
in presence of o.ooi6 millimol copper the same amount of apo-enzyme as in Fig. I.
and in absence of apo-enzyme. All
other 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 Cu + 0 . 0 0 0 0 5 rn FI CU
0 o Ni + "
[] sl CO + ',
+ im
V "l- al
ie + ,, * 0.0"I mM V + 0.00005 rnM CU
ZA + o 0.007 eJ 4. wJ tm
~I "
~J M ~ "#" ,=1
'-' 0,0025 J, ,,/,. #l al
h'll~ + + 0.0001
iJ
Cr + " ,', 0.00005 mM Cu
0.00005 CU

~,,.S5 0 -
li ,/

%ol Z,
/o7
/ o,
&
/ i a"
f6

0 30 50 90 $20 0
V 30 60 aO $20
Minutes
Minu#es
Fig. 4. The effect of equimolecular Fig. 5. The effect of varying amounts of
amounts of copper, nickel, cobalt, vana- vanadium on the enzymic oxidation of
dium, iron, zinc, magnesium, manganese o. 9 mg tyrosine + o.o5 mg Dopa. All
and chromium on the enzymic oxidation flasks contain the same amount of apo-
of o.9 mg tyrosine + o.o5 mg Dopa. All enzyme as in Fig. I.
flasks contain the same amount of apo-
enzyme as in Fig. z.

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 cor-

responding 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 protein-
bound. 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
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 - Iso
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 pre-
s 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 apo- 0 30 60 90 q20 q50
enzyme. KUBOWITZ 10 a n d KEILIN AND 1V[ANN8 h a v e M/nutes

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 Fig, 6. The effect of dialysis on the

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 theen-
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 zymic oxidation of 0.9 mg tyrosine
original c a p a c i t y to oxidize d i r e c t l y ascorbic acidlS; it + 0.o 5 mg Dopa (Curves C and D).
After having added 0.0008 milli-
can 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 dialys-
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 ed (see text), B and D not. All
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

~25C
l' *ml
__ 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
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

l
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 KuBo-
WITZ19. 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 -
0 10 20 30 40 50 60 90 q20 150 t y of free c o p p e r ions p r e s e n t 16. I n t h e ex-
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 mil-
centrations in presence of an excess (o.0o04
millimols) and in presence of an amount not 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
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 pro-
enzymic 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 en-
zyme units. t h e c o n t r a r y , in presence of o.ooo 4 millimols
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 concen-
tration.

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-dihy-
droxyphenol 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 strong-
est 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 unprecedent-
ed properties to the tyrosinase molecule n.
However, a real difficulty which indeed can not be explained by the simple o-qui-
none 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 cate-
cholase 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 re-
quired, 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 satu-
rate 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 as- Logarithmof millimols
corbic acid oxidation (Fig. 7), which can not be done b y actively- metal a d d e d
or inactively-bound copper ~6. A definite relationship exists Fig. 8. Relationship be-
tween amount of metall
between copper concentration and rate of tyrosine oxidation; added and enzymic tyro-
if 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 experi-
curve 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 fun-
Re#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 con-
text 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 proba-
bly 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 induc-
tion 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 oxi-
dation 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 polyphenol-

oxidases 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 Foxy-
dation 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

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Received November I2th, I951

12