Eur J. Biochcm.

f98,317-326 (1991)
FEBS 1991
001429569100342R

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase
Alison J. WINDER and Henry HARRIS
Sir William Dunn School of Pathology, University of Oxford, England

(Received September 11, 1990/February 3, 1991) - EJB YO 1089

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been
developed. The tyrosine hydroxylase assay uses ~-[carboxy-’~C]tyrosine as the substrate. I4CO2 is released
from the products of the hydroxylation and further metabolism of ~-[carboxy-’~C]tyrosine by incubation with
ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase
activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with
Besthorn’s hydrazone (3-methyl-2-benzothiazolinonehydrazone) to form a pink pigment with an absorbance
maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two
enzyme activities are described.

It is well established that tyrosinase from lower organisms released on the hydroxylation of tyrosine. A major defect is
catalyses the first two steps of melanin synthesis: the hydrox- that additional 3 H 2 0 is released during polymerisation of
ylation of tyrosine to dopa, and the oxidation of dopa to intermediates to form melanin. Tritium exchange between L-
dopaquinone [l]. The classical view is that this is also true [3,5-3H]tyrosine and water leads to high background values.
for the mammalian enzyme (21; it has been suggested that Hence, for adequate sensitivity, the assay requires a long incu-
mammalian tyrosinase may catalyse a third step in melanin bation period, which means that there may be considerable
synthesis: the oxidation of 5,6-dihydroxyindole [3, 41. There loss of the second tritium due to melanin formation.
have, however, been reports of the separation of melanogenic Further assays for the tyrosine hydroxylase activity of
tyrosine hydroxylase and dopa oxidase activities on purifi- tyrosinase have been described [7, 111, but these also have
cation of the mammalian enzyme [5, 61. To determine conclu- problems associated with the requirement for L-dopa as a
sively whether the tyrosine hydroxylase and dopa oxidase cofactor and its potential for further metabolism. The ‘14C02
activities of tyrosinase are inherent in one protein molecule, assay’ described here is based on the method of Okuno and
satisfactory assays for these two activities are required. There Fujisawa for assaying neuronal tyrosine hydroxylase [12], an
are many problems associated with the existing assays so the iron-based enzyme that has completely different cofactor re-
aim of this work was to develop improved assays for both quirements from copper-based tyrosinase, and is involved in
activities. the synthesis of catecholamine neurotransmitters and hor-
mones, not melanogenesis [13]. The substrate is L-[carboxy-
Tyrosine hydroxylase assay 14C]tyrosine which is hydroxylated to ~-[carboxy-’~C]dopa,
and the L-dopa is further metabolised during the course of an
The tyrosine hydroxylase activity of tyrosinase requires L- assay. By incubating the reaction mixture with ferricyanide, L-
dopa, the product of the reaction, as a cofactor. In the absence dopa and any of its metabolites further along the melanogenic
of cofactor, no activity [7] or very low activity [8] has been pathway are decarboxylated, while only a very small pro-
reported. In general, a cofactor is required at a low concen- portion of 14C02is released from the ~-[carboxy-~~C]tyrosine
tration relative to the substrate, but a relatively high concen- substrate. For every molecule of tyrosine hydroxylated, one
tration of L-dopa is required [9]. If tyrosinase does catalyse molecule of 14C02is released from products, and this can be
the first two steps of melanin synthesis, addition of L-dopa as assayed radiometrically. The problems associated with the
a cofactor also represents addition of a competitive substrate. requirement for L-dopa as a cofactor have been overcome to
Oxidation of L-dopa by dopa oxidase activity will deplete the a large extent in the assay by using D-dopa rather than L-dopa
concentration of cofactor. The requirement for L-dopa as a as a cofactor.
cofactor thus makes the tyrosine hydroxylase activity of tyro-
sinase difficult to assay.
The Pomerantz method [lo] is used most frequently to Dopa oxidase assay
assay the tyrosine hydroxylase activity of tyrosinase. It uses The most widely used in vitro assay for the dopa oxidase
~-[3,5-~H]tyrosine as the substrate and measures the 3 H 2 0 activity of tyrosinase is the dopachrome method [14]. L-Dopa
Correspondence to A. J. Winder, Sir William Dunn School of
is used as the substrate, and the formation of dopachrome,
Pathology, University of Oxford, South Parks Road, Oxford, OX1 which has an absorbance maximum at 475 nm, is measured
3RE, England spectrophotometrically. Autoxidation of dopaquinone is very
Abbreviation. MBTH, 3-methyl-2-benzothiazolinonehydrazone. rapid, and none of the intermediates between L-dopa and
Enzyme. Tyrosinase (EC 1.14.18.1). dopachrome shows absorbance in the visible spectrum, hence
318

MBTH Dopaquinone

coo-
t NH:

'0H

Dark pink pignient

Fig. 1. The reaction of dopuquinone with M B T H

the intermediates do not interfere in the assay. Since the assay spectrophotometer. It is assumed that the reaction of MBTH
does not measure the direct product of L-dopa oxidation, with dopaquinone is very rapid relative to the enzyme-
dopaquinone, it relies on the assumption that all the catalysed oxidation of L-dopa. Thus, the rate of production
dopaquinone is converted to dopachrome. This is probably of the pink pigment can be used as a quantitative measure of
not correct since reactive intermediates, such as dopaquinone enzyme activity.
and leukodopachrome, are trapped in the final melanin poly-
mer. A major disadvantage of this method is that dopachrome
is unstable in aqueous solution and is further oxidised even MATERIALS AND METHODS
while its accumulation is being monitored. This instability General methods
results in the assay being linear for only a short period (2-
3 min). The absorption coefficient of dopachrome ( E ~ , ~ Chemicals. ~-[carboxy-'~C]Tyrosine(52 Ci/mol) and L-
3700 M - ' cm-') is relatively low [I41 and the assay is there- 3,4-dihydro~y-phenyl-[l-'~C]alanine (~-[carboxy-'~C]dopa;
fore of only low sensitivity. Furthermore, the value of the 5.5 Ci/mol) were obtained from Amersham International
absorption coefficient was determined at pH 5.6, not the pH (Amersham, UK). Bovine serum albumin fraction V, catalase
of the assay, pH 6.8, since the decomposition of dopachrome (no. CIOO), D-dopa, L-dopa, Hepes, iodoacetamide, MBTH,
at the latter pH was too rapid for it to be determined accurately mushroom tyrosinase, l-phenyl-2-thiourea, potassium ferri-
~41. cyanide, sodium diethyldithiocarbamate, Triton X-100 and L-
Many other assays for dopa oxidase activity have been tyrosine were obtained from Sigma (Poole, UK). Collagen
described [15 -201 but all have inherent defects. The assay type I was from Boehringer (Lewes, UK). N,N'-Dimethyl-
described here exploits the fact that dopaquinone, the direct formamide and perchloric acid were from BDH (Poole, UK).
product of L-dopa oxidation, is highly reactive. Dopaquinone NaC1/Pi (137 mM NaCl, 2.7 mM KCI, 8.1 mM Na2HP04,
is trapped as a stable coloured product that cannot react 1.5 mM KH2P04, pH 7.3) was from Oxoid (Basingstoke,
further and its formation is monitored spectrophoto- UK). Trypsin was from Gibco (Paisley, UK). Liquid scin-
metrically. tillant Unisolve E was from Koch-Light Lab. Ltd (Haverhill,
An assay in which the quinone formed on the oxidation UK). All other reagents were of analytical grade.
of catechol is reacted with Besthorn's hydrazone (3-methyl- Cell culture. RVH 421 human malignant melanoma cells
2-benzothiazolinonehydrazone hydrochloride, or MBTH) to [21] were grown in Leibovitz L-15 medium (Gibco); HeLa
give a pink product has been described previously [15, 161. (human cervical carcinoma, ATCC no. CCL2) cells were cul-
Disadvantages of the method are that it is a stopped assay, it tured in minimum essential medium (Gibco);GM1604 human
has to be done at a low pH (4.2), and the pink pigment formed fibroblasts [22] and PG19 mouse melanoma cells [23] were
has to be extracted using an organic solvent. cultured in Dulbecco's modified Eagle's medium. For these
The new 'MBTH assay' uses MBTH to trap dopaquinone lines, media were supplemented with 10% (by vol.) foetal calf
formed on the oxidation of L-dopa. Presence of a low concen- serum (Imperial Laboratories, Andover, UK) and 2 mM L-
tration of N,N'-dimethylformamide in the assay mixture ren- glutamine (Gibco). PC12 rat phaeochromocytoma cells [24]
ders MBTH soluble, and the method can be used over a range were grown in collagen-coated tissue culture flasks (prepared
of pH values. MBTH reacts with dopaquinone by a Michael according to manufacturer's directions); the medium was
addition reaction as shown in Fig. 1; the formation of the RPMI 1640 supplemented with 5% (by vol.) foetal calf serum
dark pink product is monitored continuously using a (Imperial Laboratories), 10Yn (by vol.) horse serum (heat-
 319

inactivated at 56°C for 40 min; Gibco) and 2 mM L-gluta- UK) inside a 20-ml glass liquid scintillation vial (Canberra-
mine. HeLa, GM1604 and PG19 cell lines were passaged by Packard, Pangbourne, UK). The vial was sealed with a 16-
incubation (37 "C for 5 min) in NaCI/P,/EDTA/trypsin(NaCl/ mm subaseal stopper (Gallenkamp, Loughborough, UK) and
Pi containing 0.02%, mass/vol., EDTA and 0.125%, mass/ the plastic vial cap fitted over this had a 5-mm-diameter hole
vol., trypsin). RVH 421 and PC12 cells were incubated in bored in its centre. Surrounding the reaction tube was a filter
NaCI/P,/EDTA alone. paper strip (Whatman no. 1, 1.7 cm x 8.5 cm) which was
Protein determination. Protein concentrations were deter- soaked with 0.2 m10.5 M potassium hydroxide solution. The
mined using a protein assay kit (Bio-Rad, Heme1 Hempstead, standard assay mixture contained 0.1 ml assay buffer A (0.5 M
UK), based on the method of Bradford [25]. Bovine serum potassium phosphate pH 7.5), 0.1 m15 mM D-dopa in 2.9 mM
albumin was used as a reference standard. HC1, 0.1 ml ~-[carboxy-'~C]tyrosine (2.6 Ci/mol; 2.6 pCi/ml)
Spectrophotometric measurements. Spectra of samples and and 0.2 ml enzyme preparation. This gave final concentrations
absorbance values at selected wavelengths were determined of 100 mM potassium phosphate, 1 mM D-dopa and 0.2 mM
using a PU8800 spectrophotometer (Pye Unicam, Cambridge, L-tyrosine, and a final pH of 7.2. The vial was sealed and the
UK). reaction mixture was pre-incubated at 37°C for 5- 10 min
Statistical analysis. Mean values were compared using with shaking (60 strokes min- '). Tyrosine hydroxylase en-
Student's t-test. Errors are stated at the level of 95% confi- zyme preparation was injected through the subaseal stopper
dence. Best straight lines were determined by linear regression using a 250-pl-capacity HPLC syringe (Anachem, Luton, UK)
analysis, and regression goodness of fit was determined using and the mixture incubated at 37°C for 10 min with shaking
the F-test. Curves were drawn by fitting a polynomial re- (60 strokes min-I). The reaction was stopped by injecting
gression equation. 0.1 ml 6% (massjvol.) perchloric acid through the subaseal
stopper using a 1-ml disposable syringe (Becton-Dickinson,
Enzyme preparation Oxford, UK). Products of the hydroxylation of tyrosine were
RVH 421 melanoma cells were used as a source of both decarboxylated by injecting 0.2 ml 150 mM potassium ferri-
human melanogenic tyrosine hydroxylase and dopa oxidase. cyanide in 1 M potassium phosphate pH 7.3 and incubating
Cells were harvested as for passage, washed three times with at 60°C for 6 min with vigorous shaking (120 strokes min- ').
ice-cold NaCl/P,, then used to prepare enzyme. The methods This reaction was stopped rapidly by injecting 0.2 ml 12%
used to prepare both tyrosine hydroxylase and dopa oxidase (mass/vol.) perchloric acid. Vials were left overnight at room
samples differ slightly from the optimised procedure which temperature then the reaction tubes were removed from the
was only developed after the experiments reported in this vials and 12 ml liquid scintillant (Unisolve E) added to the
paper had been done [26]. Details of the optimised procedure filter papers. Radioactivity was determined in a Packard Tri-
are included below. Carb liquid scintillation spectrometer (model 544) and cpm
Tyrosine hydroxylase preparation. Cells were resuspended values were corrected for quenching by the method of channels
by vortexing in 4 vol. of tyrosine hydroxylase (TH) buffer ratio. During assays, vials were handled in a fume cupboard
(20 mM potassium phosphate pH 6.5, O S % , by vol., Triton to prevent possible release of I4CO2into the laboratory.
X-100, 5 mM iodoacetamide), and sonicated on ice for 10 s Decarboxylation controls. Decarboxylation control vials
(MSE ultrasonic disintegrator model 7100; setting 5 pm). Ex- were used to estimate the fraction of 14C that could be re-
tracts were centrifuged briefly in a microfuge (9000 g for I rnin covered from ~-[carhoxy-'~C]dopaas I4CO2; 50 p1 L-[car-
at 4' C) and the supernatant dialysed against 20 mM potas- h ~ x y - ' ~C ] d o p (0.018
a pCi; 2.6 Ci/mol) in TH buffer was
sium phosphate, pH 6.5 (1 1 changed four times over 24 h). added to a standard assay mixture containing unlabelled L-
Dialysed extracts were stored at - 20°C. Control extracts of tyrosine and no enzyme. The volume was made up to 0.5 ml
other cell types were prepared similarly. with TH buffer and the vial processed exactly as for a routine
Dopa oxidase preparation. Cells were resuspended by vor- assay. To calculate the fraction of 14C recovered as 14C02,
texing in 5 vol. dopa oxidase (DO) buffer (50 mM sodium the mean radioactivity (dpm) for decarboxylation control vials
phosphate pH 6.9) and frozen by placing in a -20°C deep was divided by the mean radioactivity for vials containing
freeze. On thawing, the suspension was sonicated on ice for 0.018 pCi ~-[carboxy-'~C]dopa and a filter paper strip soaked
30 s (setting 5 pm), then centrifuged in a microfuge (9000 g in 0.2 ml 0.5 M potassium hydroxide solution. The fraction
for 15 min at 4 T ) . The centrifugation step was repeated with of 14C recovered from ~-[carboxy-'~C]tyrosine as 14C02was
the resulting supernatant, and the final supernatant was used estimated similarly, using vials containing the amount of L-
in assays. Control extracts of other cell types were prepared [carbo~y-'~C]tyrosine routinely used in assays (0.26 pCi).
similarly.
Optimiscd enzyme preparation. Cells were resuspended by
Dopa oxidase assay
vortexing in 4 vol. enzyme (E) buffer (20 mM potassium phos-
phate pH 7.5, 0.5%, by vol., Triton X-loo), and sonicated on M B T H assay f o r dopa oxidase. In general, the reaction
ice for 30 s (setting 5 pm). Extracts were centrifuged in a mixture (total volume 1.0 ml) contained 490 pl assay buffer
microfuge (9000 g for 15 min at 4"C), the centrifugation step B (100 mM sodium phosphate pH 7.1, 4%, by vol., N,N'-
repeated, and the final supernatant dialysed against 20 mM dimethylformamide), 200 pl 5 mM L-dopa, 290 pl 20.7 mM
potassium phosphate pH 7.5 (1 1 for 1 h then 4 1 overnight) MBTH and 20 ~1 enzyme preparation. This gave final concen-
before freezing at - 20 "C. trations of 50mM sodium phosphate, 2% (by vol.) N , N -
Mushroom tyrosinase. Mushroom tyrosinase was reconsti- dimethylformamide, 1 mM L-dopa and 6 mM MBTH, and a
tuted in the buffer appropriate to the enzyme assays being final pH of 6.9. Reaction mixture, without enzyme, was made
done and diluted to the required activity. up in a 1.6-mi plastic cuvette (Sarstedt, Numbrecht, FRG)
and incubated at 37°C for 10 min. Reaction mixture tempera-
Tyrosine hydroxylase assay ture was checked using a thermistor thermometer (Camlab,
14CO, assayfor tyrosine hydroxylase. Each assay was done Cambridge, UK), dopa oxidase enzyme preparation added,
in a plastic test tube (40 mm x 11 mm; Sterilin, Hounslow, and the cuvette contents mixed by inversion. The initial rate
320
of absorbance increase at 505 nm was monitored spectro- pH 7.2 to pH 8.0 (Fig. 2A). Background did not vary with
photometrically, routinely over a period of 5 min. Assay mix- pH, providing that the pH of the ferricyanide solution was
ture temperature was rechecked at the end of the assay. adjusted to give the optimum pH for the decarboxylation step.
Dopachrome assay for dopa oxidase. The reaction mixture The lowest pH in the optimum range, pH 7.2, was used for
(total volume 1.O ml) contained 490 p1 assay buffer C (50 mM the assay because this minimises the spontaneous oxidation
sodium phosphate pH 6.9), 200 pl 5 mM L-dopa, 290 p1 dis- of dopa to melanin, the rate of which increases with increasing
tilled water and 20 pl enzyme preparation. This gave final PH .
concentrations of 50 mM sodium phosphate and 1 mM L- The initial rate (v) of enzyme-catalysed tyrosine hydrox-
dopa, and a final pH of 6.9. The same method was used as ylation increased with increasing temperature between 20 -
for the MBTH assay except that absorbance increase was 50"C, the rate of increase falling off slightly at temperatures
monitored at 475 nm. above 40 "C (Fig. 2 B). Background increased significantly
Pigment preparation. To prepare a sample of the pigment with increasing temperature over this range ( P < 0.02; data
formed in the MBTH assay, a standard MBTH assay mixture not shown). Physiological temperature, 37"C, was used for
was made up, dopa oxidase enzyme preparation added to give the assay.
a rate of absorbance increase dASo5of 0.05-0.10 min-' and There was substrate inhibition at concentrations of L-tyro-
the mixture incubated (37°C for 10 min). The reaction was sine above 0.1 mM, illustrated by the deviation from linearity
stopped by adding 130 pl 2.4% (massivol.) perchloric acid in in a Hanes plot (Fig. 2C). Routinely, 0.2 mM L-tyrosine was
assay buffer D (50 mM sodium phosphate pH 7.1, 2%, by used in the assay since enzyme activity is maximal at this
vol., NJ"'dimethy1formamide). The pH was adjusted to 6.9 concentration. A K, of 0.1 mM for L-tyrosine has been esti-
by adding 60 p1 assay buffer D and 140 pl 1 M potassium mated for a human melanoma cell extract using the 14C02
hydroxide in assay buffer D without dimethylformamide; then assay (derived from data shown in Fig. 2 C). Previously report-
the mixture was centrifuged in a microfuge (9000 g for 5 min ed values for mammalian enzymes range over 0.02-0.05 mM
at room temperature). The supernatant was used as a sample [27] to 0.7 mM [7].
of pigment. Sample volumes ranging over 20 - 200 p1 can be used satis-
Estimation of molar absorption coefficient of' the pigment. factorily in the assay system described. The assay can be used
Assays were done using optimised enzyme preparations under to measure the tyrosine hydroxylase activity of turbid or non-
standard conditions except that a low concentration of L-dopa turbid crude cell extracts and of purified enzyme.
(0.03-0.07 mM) was used. Absorbance was measured at 1-
min intervals over a 20-min period. Curves were fitted to the Cojactor requirement
results, and extrapolated to infinite time to determine the A cofactor is required for the tyrosine hydroxylase reac-
absorbance of the product formed on the complete oxidation
tion; in the absence of cofactor no activity could be detected
of L-dopa substrate. These values were used to estimate the (Tables 1 and 2). In the I4CO2 assay, the problems inherent
molar absorption coefficient ( E ~ of~ the
~ )pink pigment.
in using L-dopa as a cofactor have been largely overcome by
using D-dopa instead. The optimum concentration of L-dopa
RESULTS cofactor was 0.1 -0.2 mM (Fig. 3). D-Dopa stimulated tyro-
sine hydroxylase activity to the same extent at a concentration
Tyrosine hydroxylase assay of 1 mM, a 5-10-fold higher concentration (Fig. 3). At a
Stock solutions concentration of 2 mM, D-dopa stimulated tyrosine hy-
droxylase activity slightly more ( z25%). However, a concen-
A frequent problem with radiometric assays is the purity tration of 2 mM D-dopa was not used routinely because the
of the labelled substrate. The ~-[carboxy-'~C]tyrosine used in low solubility of dopa resulted in cofactor solution making up
the 14C02assay is available 97 - 99% pure, and no contami- a large proportion of the assay mixture and limited the amount
nation by dopa is detectable by thin-layer chromatography of sample that could be used. This decreased the sensitivity of
(data not shown). the assay with low activity samples.
Dopa and dopamine are autoxidised in the presence of In order to maintain the concentration of cofactor in assay
water, oxygen and light. To minimise problems due to this mixtures in which both tyrosine hydroxylase and dopa oxidase
instability, cofactor solutions were used within a maximum of activities are present, the cofactor must not be readily oxidised
4 h of their preparation, stored on ice and in the dark, and by dopa oxidase. The rates of oxidation of L-dopa and D-
made up in 2.9 mM HCI because low pH decreases the rate dopa, in the presence and absence of L-tyrosine, are compared
of autoxidation. ~-[carboxy-'~C]Dopa was stored at - 20 "C, in Table 1. Oxidation rates for the concentrations of com-
thawed just before use, then treated similarly to other dopa pounds used in tyrosine hydroxylase assays are included. In
solutions. the absence of tyrosine, 0.2 mM L-dopa was oxidised approxi-
mately ten times more rapidly than 1 mM D-dopa. Oxidation
Optimisation of assay conditions of 0.2 mM L-dopa was inhibited by 0.2 mM L-tyrosine, the
concentration of L-tyrosine used in the 14C02 assay. The
Phosphate and Hepes assay buffers give equivalent tyro-
increased rate of absorbance increase on the addition of
sine hydroxylase activities, but with both buffers a slightly
0.2 mM L-tyrosine to 0.2 mM D-dopa is thought to be due to
higher activity ( zSo/,)was obtained with potassium than with
oxidation of L-tyrosine. The much lower rate of oxidation of
sodium salts, although this is not significant ( P > 0.1; data
D-dopa compared with L-dopa makes this a preferable
not shown). Routinely, 100 mM potassium phosphate was
cofactor for the assay.
used as the assay buffer. Tyrosine hydroxylase activity was
significantly lower (z1So/) in 200 mM compared with
100 mM or 50 mM buffer (P< 0.02; data not shown). Optirnisation of decarhoxylation conditions
Tyrosine hydroxylase activity increased with increasing Decarboxylation conditions were adjusted to give mini-
pH from pH 6.4 to pH 7.2, then only decreased slightly from mum release of 14C02from ~-[carboxy-'~C]tyrosinesubstrate
 321

0.6

7. 0.2 c / I
0 . 0 ~ " " " "
20 30 40 50 60

PH Temperalux ("c)

C D
0.8
o'8 I
0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 1.2

[L-TY~I(mM) [ Enzyinc J (ai-bii mi-)( u ii i is)

Fig. 2. Optimisation of conditionsfor the 14C02assay. Assays were done using human tyrosine hydroxylase preparation at a fixed concentration,
except in (D). Conditions were standard and not varied except: (A) p H ; (B) temperature; (C) substrate (L-tyrosine) concentration. In (D) the
conccntration of enzyme was varied. Points represent the means of three (A, B) or four (C, D) rate determinations corrected for background.
Error bars (A, B, D) represent f SEM and errors not shown are smaller than the symbols. All errors are less than 10%. In (D) goodness of
fit of the straight line is significant at 0.1,% (F'1,4
= 2607)

8.0
Table 1. Comparison of the rates of oxidation of L-dopa and D-dopa
I MBTH assays were done under standard conditions with a fixed
concentration of human dopa oxidase preparation. Values are the
d
means of three rate determinations, corrected for the appropriate
*
blank rate, SEM (n.d., not determined)

Substrate Substrate lo3 x 1l

concn
no L-Tyr 0.2 mM L-Tyr

A 10.00
None
L-Dopa
D-Dopa
D-Dopa
-
0.2
0.2
1 .0
Of0
94+ 1
2*0
10f0
0+0
61 f 0
4f1
n.d.
[Cofactor] (mM)

Fig. 3. Cofactorsfor the tyrosine hydroxylase reaction. Comparison of

L-dopa (0)and D-dopa ( 0 )as cofactors. The amount of tyrosine
but maximum release of 14C02 from the products of its hy-
hydroxylated in a 10-min assay is shown. Assays were done with a
fixed concentration of human tyrosine hydroxylase preparation and droxylation and further metabolism. Conditions were
conditions were standard except that cofactor type and concentration optimised by decarboxylating known amounts of either L-
were varied. Each point represents the mean of three determinations, [carb~xy-'~C]tyrosine, or ~-[carboxy-'~C]dopain the presence
corrected for background, SEM * of unlabelled L-tyrosine. Incubation with 35 mM potassium
322
14 Table 2. Tyrosine hydroxyla~yeactivities of various samples
Assays were done under standard conditions with a fixed volume of
c 12 - sample prepared using the tyrosine hydroxylase method. Values are
i
v 10 -
the means of three rate determinations, corrected for background,
f SEM; BSA, bovine serum albumin
p
c a -
E
R 6 -
Sample Final protein
concn
v
c

mg ml-' pmol min

TH buffer 0.00 Of2
BSA 0.40 8f4
RVH 421 extract 0.20 303 f 1
0 5 10 15 20 25 30
RVH 421 extract 0.20 8 f 7"
PG19 extract 0.19 38f2
T i e (min) HeLa extract 0.71 - 4f2
Fig. 4. Linearity of the 14C02 ussuy with respect to time. Assays GM1604 extract 0.39 3*4
were done with a fixed concentration of human tyrosine hydroxylase PC12 extract 1.20 - 2f3
preparation under standard conditions. Each point rcpresents the
mean of four determinations, corrected for background, f SEM. a D-Dopa cofactor omitted
Errors not shown are smaller than the symbols; all errors are less than
10%. Goodness of fit of the straight line is significant at 0.1 YO(F1,4=
2239)
to use D-dopa as a substrate, and enzyme inactivation on
oxidation of dopa, make this enzyme difficult to assay accu-
ferricyanide at 6 0 T for 6 min proved optimal (data not rately by a stopped assay method. The 14C02assay can never-
shown). The action of ferricyanide was stopped immediately theless detect hydroxylation of L-tyrosine by the mushroom
after the incubation by addition of excess perchloric acid. This enzyme (data not shown).
is important to minimise release of 14C02 from labelled L-
tyrosine. Leaving the vials at room temperature overnight
after the decarboxylation step significantly increased the re- Specficity of the assay
covery of 14C02 from labelled L-dopa ( P < 0.001; data not
shown), but not from labelled L-tyrosine ( P > 0.1; data not When substrate or enzyme was omitted from the reaction
shown). Presence of human melanoma cell extract had no mixture, no tyrosine hydroxylase enzyme activity could be
significant effect on the amount of 14C02 recovered from detected (data not shown and Table 2). No activity could be
labelled L-dopa ( P > 0.1 ; data not shown), indicating that detected when dopa cofactor was omitted, confirming the
further metabolism of L-dopa does not affect recovery of specificity of the assay for tyrosinase (Table 2).
I4CO2. Under optimised conditions, approximately 0.3% of The tyrosine hydroxylase activity of tyrosinase was detect-
the I4C in ~-[carboxy-'~C]tyrosine was collected, compared ed only in cells expected to possess this activity. It was mea-
with 85 -90% of the 14Cin ~-[carboxy-'~C]dopa and products sured in human (RVH 421) and mouse (PG19) melanoma
of its oxidation. The 14C02 released from ~-[carboxy-'~C]- cell extracts, but not in HeLa cells, a non-melanoma human
tyrosine substrate in the absence of enzyme was subtracted as tumour cell line, GM1604 human fibroblasts, o r rat phaeo-
background and the slightly incomplete recovery of 14C02 chromocytoma (PC12) cells (Table 2). A bovine serum albu-
from products corrected for by running decarboxylation con- min solution of comparable protein concentration to enzyme
trols. Since background and percentage decarboxylation are preparations had no significant activity in the 14C02 assay
very reproducible, correction can be made by a standard ( P > 0.1 ; Table 2).
factor. Okun et al. have suggested that peroxidase is responsible
for the hydroxylation of tyrosine in melanin synthesis [28],
but this claim has since been refuted [29]. Controls were done
Validity of'the a s . s q to confirm that peroxidase is not involved. Addition of
The amount of L-tyrosine hydroxylated was generally lim- catalase, which inhibits peroxidase by decomposing hydrogen
ited to less than 5 % of the total substrate ( 5 nmol in a standard peroxide but does not inhibit tyrosine hydroxylase, had no
assay) to obtain an accurate estimate of initial rate. The assay significant effect on tyrosine hydroxylase activity ( P > 0.1 ;
is nevertheless linear with respect to time even when more than data not shown).
5% of the L-tyrosine was hydroxylated (Fig. 4). Routinely, an Enzyme inhibitors were used to test that the assay mea-
incubation period of 10 min was used. This gives adequate sures the activity of the tyrosine hydroxylase involved in mel-
sensitivity and accuracy: duplicate determinations varied by anin synthesis, and not the neuronal enzyme. Diethyldithio-
no more than 10% and usually by less than 5%. A short carbamate, a copper chelator which inhibits melanogenic tyro-
incubation time decreases depletion of cofactor and melanin sine hydroxylase, produced marked inhibition ( = 85%) of
formation. There was no increase in background with increas- enzyme activity at a concentration of 10 mM (data not
ing incubation time. N o lag period was observed under shown). In contrast, 2,2'-dipyridyl, which is an iron chelator
optimised conditions (Fig. 4), although a lag did occur when and inhibits neuronal tyrosine hydroxylase, showed only a
cofactor concentration was well below optimum. very slight inhibition of activity ( z 12%) at a concentration
When a human melanoma cell extract was assayed, the of 5 m M (P < 0.02; data not shown). This slight inhibition
14C02assay was linear with respect to enzyme concentration may be due to chelation of copper at the high concentration
(Fig. 2D). The assay thus provides a quantitative measure of of inhibitor used. The results confirm the specificity of the
the amount of enzyme. The ability of mushroom tyrosinase assay for the tyrosine hydroxylase activity of tyrosinase.
 323
1 .o

0.8

0.6
8
-2

'
P
0.4
f
0 2 4 6 8 10

0.2 [rnTHI (mM)

Fig. 6. Effect of M B T H concentration on initial rate of uhsorhance
increuse at 505 nm. Assays were done with a fixed concentration of
0.0 I I I I
human dopa oxidase preparation and conditions were standard except
350 400 450 500 550 600 650
that the concentration of MBTH was varied. Each point represents
the mean of three rate determinations, corrected for the appropriate
Wavelength (nm) blank rate, SEM. Errors not shown are smaller than the symbols;
all errors are less than 5%
Fig. 5. Ahsorhunce spectrum o j t h e pigment jormedin the M B T H u.s.suy

concentration, the dimethylformamide did not affect dopa

Sensitivity of the ussuy oxidase activity (data not shown).
The assay, as described, has a sensitivity of the order of
5 0 0 pmol substrate converted with a 95% confidence level of Optimisation of assay conditions
f 10% for the standard error of the mean (n = 3). Satisfactory
results can be obtained with incubation periods of as long as Assay buffer type and concentration have little effect on
1 h, corresponding to a turnover of approximately 10 pmol dopa oxidase activity. Use of sodium Hepes instead of sodium
min-'. phosphate had no significant effect ( P > 0.1 ; data not shown),
but use of potassium instead of sodium phosphate stimulated
enzyme activity by approximately 20% ( P < 0.001; data not
Dopa oxidase assay shown). Routinely, 5 0 m M sodium or potassium phosphate
Properties of the pigment was used as the assay buffer.
The concentration of MBTH used in the assay was 6 mM.
The pigment formed in the MBTH assay has an ab- At this concentration and p H 6.9, MBTH is soluble and satu-
sorbance maximum at 5 0 5 nm (Fig. 5). On exposure to 505- rating at the maximum rate at which the assay was used
nm light a t 37°C under standard assay conditions, the pigment (Fig. 6). MBTH did not inhibit dopa oxidase activity at the
was stable for at least 30 min (data not shown). It is therefore concentrations tested; if it had, the rate would be expected to
quite sufficiently stable to perform assays that routinely take decrease instead of reaching a plateau at concentrations of
only 5 min. The molar absorption coefficient of the pigment MBTH above saturation. When no MBTH was present, an
under standard assay conditions has been estimated: csos increase in absorbance was seen at 5 0 5 nm due to the forma-
29000 1000 M - ' cm-' (mean SEM, n = 4). Since thisde- tion of dopachrome which absorbs at this wavelength. At
termination is preliminary, enzyme activity in this report is concentrations of MBTH below saturation, the absorbance
expressed in terms of the rate of increase in absorbance at increase at 505 nm is the sum of the absorbance of the pink
5 0 5 nm. pigment and of dopachrome formed from dopaquinone that
is not trapped.
The optimum pH for the assay is p H 6.9 (Fig. 7A). The
Stock solutions
stability of dopaquinone, and hence the efficiency of quinone
To minimise problems due to the instability of L-dopa, trapping by MBTH, decreases with increasing pH. However,
stock solutions were used within a maximum of 4 h of their a concentration of 6 m M MBTH was saturating at pH values
preparation and stored on ice and in the dark. L-Dopa is not as high as p H 7.3 (data not shown). Therefore, the measured
readily soluble in aqueous solution, therefore the maximum p H optimum is thought to reflect enzyme activity. Enzyme
concentration of stock solution used was 7.5 mM, and rou- activity cannot be determined at higher pH values because
tinely the stock concentration was 5 mM. MBTH is not sufficiently soluble. There is no significant vari-
MBTH solutions were prepared freshly on the day of use, ation in the absorption spectrum of the pigment over the pH
but are stable at room temperature for at least 12 h. MBTH range tested (data not shown). The rate of L-dopa autoxida-
hydrochloride is an acidic salt (pH 3 -4 for a 20 m M solution tion increases with increasing pH, and this is reflected in a
at room temperature) and its solubility is highly dependent on significant increase in the blank rate: h i s o sof 0.001 min-'
pH and ionic strength. A relatively high concentration of at pH 5.8, increasing to 0.004 min-' at pH 7.3 ( P < 0.02; data
buffer, 5 0 mM phosphate, is required to overcome the acidity not shown).
of the salt and give the optimum assay pH. Adequate Physiological temperature, 3 7 T , was used for the
solubilisation of MBTH was achieved by including 2 % (by optimised assay. The initial rate of enzyme-catalysed dopa
vol.) N,N'-dimethylformamide in the assay mixture. At this oxidation increased with increasing temperature over the
324
A
70

.-C
v
E
>
30
X
3 20

5.5 6.0 6.5 7.0 7.5 8.0 8.5 20 30 40 50 60

pH Temperature ("C)

C D
12 , I 120

100 -

80 -

60 ~

0 2 4 6 a

[Enzyme] (arbiuary units)

Fig. 7. Optimisation of condition.s,for the M B T H assay. Assays were done using human dopa oxidase preparation at a fixed concentration,
except in (D). Conditions were standard and not varied except: (A) pH; (B) temperature; (C) substrate (L-dopa) concentration. In (D) the
concentration of enzyme was varied. Points represent the means of two (A, B) or three (C, D) rate determinations, corrected for the appropriate
blank rates. In (D) error bars represent +_ SEM. Errors not shown are smaller than the symbols and all errors are less than 3%. Goodness of
fit of the straight line is significant at 0.1% ( F l , b= 3751). In (A), no interpolation implicd

range 25 - 50 "C (Fig. 7 B). The rate of spontaneous L-dopa Validity of the ussay
oxidation also increased significantly with increasing tempera-
ture over this range: ~ l . of40.001
~ ~min-'
~ at 2 5 T , increasing
With 1 mM L-dopa as substrate, at rates of absorbance
increase d A 5 0 5 of the order of 0.1 min-', the assay is linear
to 0.007 min-' at 50°C (P < 0.001 ; data not shown).
In aqueous solution at neutral pH and 37 " C ,the maximum with respect to time for approximately 2 min. Generally,
samples were diluted to yield a maximum d A 5 0 5 of
solubility of L-dopa was approximately 8 mM. A concen-
0.06 min- ',giving a linear increase in absorbance for at least
tration of 1 mM L-dopa was used routinely in the assay so this
is not a saturating substrate concentration. This concentration 5 min (data not shown). The mean rate of absorbance increase
gave an acceptably low blank rate due to autoxidation (dA505 over the 5-min period is a measure of initial rate. Duplicate
0.002 -0.003 min-') and was readily achieved despite the low rate determinations varied by no more than l o % , and usually
solubility of L-dopa. The effect of substrate concentration on by less than 5%.
enzyme activity is shown by the Hanes plot in Fig. 7C. For a The MBTH assay was linear with respect to enzyme con-
human melanoma cell extract, a K, of 0.7 0.2 mM for L- centration when a human melanoma cell extract was assayed
dopa (mean SEM, n = 3) has been estimated using the (Fig. 7 D). Rate determinations thus provide a quantitative
MBTH assay. This is in good agreement with previous deter- measure of the amount of enzyme. A similar result was
minations for other mammalian enzymes made using the obtained when mushroom tyrosinase was used as a source of
dopachrome assay, for example 0.6 mM [7] and 0.5 mM [30]. dopa oxidase activity (data not shown).
A volume of 20 p1 enzyme preparation was used routinely
in assays. This can be increased slightly to enhance sensitivity, Specificity of the assay
the concentrations and volumes of L-dopa and/or MBTH
solutions being adjusted appropriately. The assay is suitable N o dopa oxidase activity could be detected when substrate
for measuring dopa oxidase activity of non-turbid crude cell was omitted from the reaction mixture (data not shown).
extracts and of purified enzyme. When substrate was included, but enzyme buffer substituted
 325
Table 3. Dopu oxiduse uctivities of' various samples approximately seven times that measured by the dopachrome
Assays were done under standard conditions with a fixed volume of method (data not shown). The MBTH assay thus appears to
sample prepared using the dopa oxidase method. Values are the means be about seven times more sensitive. The molar absorption
of three rate determinations, not corrected for the blank rate, k SEM; coefficient of the pink pigment is seven to eight times greater
BSA, bovine serum albumin
than that of dopachrome, which implies that the pink pigment
Sample Final protein 103 and dopachrome are formed at the same rate.
concn

mg ml-' min-' DISCUSSION

DO buffer 0.00 2.5 f 0.3
BSA 0.06 2.1 k 0 . 1
The new techniques described for assaying the tyrosine
BSA 0.12 1.6 f 0.0 hydroxylase and dopa oxidase activities of tyrosinase have
BSA 0.18 1.5k0.1 distinct advantages over traditional methods.
RVH 421 extract 0.12 58.5 f 2.5
PG19 extract 0.15 8.7 k 0.3
HeLa extract 0.19 1 .0 f 0.3 Tyrosine lzydroxylase assuy
GM1604 extract 0.02 2.0 f 0.0 The 14C02assay for tyrosine hydroxylase activity is highly
PC12 extract 0.11 0.6 0.3
specific: for every molecule of tyrosine hydroxylated, one
molecule of 14C02 is released from products on their
decarboxylation. In contrast, the Pomerantz method is not
for enzyme, an increase in absorbance was seen due to spon- entirely specific. It measures the formation of 3 H 2 0 on the
taneous oxidation of L-dopa (Table 3). Solutions of bovine hydroxylation of tyrosine, but additional 3 H 2 0is released on
serum albumin, giving a final protein concentration in the further metabolism and this leads to inaccuracy.
assay mixture of greater than 0.1 mg ml-', significantly de- A maximum 5% conversion of substrate is allowed in the
creased the rate of spontaneous L-dopa oxidation to less than I4CO2 assay to ensure that there is no significant decrease
that obtained with enzyme buffer ( P < 0.001 ; Table 3). This in rate due to substrate depletion. The incubation period is
possibly reflects the quenching of free radicals involved in minimised to decrease autoxidation and enzymic oxidation of
spontaneous L-dopa oxidation by solutions of relatively high D-dopa cofactor, and to prevent significant melanin forma-
protein concentration. Therefore, with cell extracts of final tion, since labelled substrate and intermediates may be trapped
protein concentration greater than or equal to 0.1 mg ml-', in the polymer. The trapping of label in melanin is unlikely to
bovine serum albumin controls are used to correct for the be a serious problem. Hearing et al. have reported that melanin
blank rate. formed from ~-[carhoxy-'~C)tyrosine by mammalian tyrosin-
The MBTH assay detects dopa oxidase activity only in ase contains only approximately 1% of the I4C initially pre-
cells expected to possess this activity. Dopa oxidase activity sent in the L-tyrosine substrate, the remainder of the label
was detectable in both human melanoma (RVH 421), and being released as I4CO2 further along the pathway [31].
mouse melanoma (PG19) cell extracts. No such activity was The use of D-dopa rather than L-dopa as a cofactor has
found in extracts of HeLa cells, GM1604 human fibroblasts notable advantages. At the concentrations at which D-dopa
or rat phaeochromocytoma (PC12) cells (Table 3). and L-dopa are used as cofactors, D-dopa is oxidised ten
There is evidence that dopa can be converted to times less rapidly than L-dopa by the dopa oxidase activity
dopachrome in the presence of peroxidase and hydrogen per- of tyrosinase. Thus, the optimum concentration of D-dopa
oxide [29]. To test that the dopa oxidase activity measured in cofactor is depleted much less rapidly than that of L-dopa.
crude cell extracts by the MBTH assay is not due to peroxi- The lack of correlation between rates of oxidation of L-dopa
dase, assays were done in the presence of catalase, which and D-dopa, and their effectiveness as cofactors for the tyro-
inhibits peroxidase but not dopa oxidase. Catalase had no sinase hydroxylase reaction, may be related to the mechanism
significant effect ( P > 0.1; data not shown) on the dopa oxi- of enzyme catalysis. Detailed mechanistic studies have only
dase activity of a human melanoma cell extract, showing that been done for tyrosinase from lower organisms [32,33]. There-
this activity is not due to peroxidase. fore, at present, any explanation in mechanistic terms assumes
Tyrosinase inhibitors were also used to verify that the that one enzyme catalyses both tyrosine hydroxylation and
MBTH assay measures the dopa oxidase activity of tyrosinase. dopa oxidation in human cells, and that its catalytic mechan-
Diethyldithiocarbamate at a concentration of 10 mM, and ism is the same as that of the tyrosinase from lower forms.
phenylthiourea at a concentration of 0.7 mM, completely in- Another explanation for the lack of correlation could be that
hibited the dopa oxidase activity of a human melanoma cell dopa acts as an allosteric activator of mammalian tyrosinase,
extract (data not shown). This confirms the specificity of the as has been suggested previously [9].
assay for the dopa oxidase activity of tyrosinase. Over a wide range of concentrations of L-[carboxy-
''C]dopa, only 85-90% of the I4C can be recovered as
I4CO2. This may be due to impurity of the L-[carboxy-
Comparison with the dopachrome assay ''C]dopa standard (available approximately 96% pure), oxi-
The dopachrome assay is linear with respect to time for dation of ~-[carboxy-'~C]dopa to products from which the
only 2-3 min under conditions in which the MBTH assay 14C cannot be released as I4CO2, or to I4CO2 remaining in
shows a linear relationship with respect to time for at least aqueous solution. The slightly incomplete recovery of 14C
5 min (data not shown). The decrease in rate seen with the can be corrected for by running decarboxylation controls in
dopachrome assay reflects the instability of dopachrome. parallel with each set of assays. Alternatively, since recovery
When a human melanoma cell extract was assayed for dopa is very reproducible, a standard correction factor can be used.
oxidase activity by the dopachrome and MBTH methods, the The sensitivity of the 14C02assay is comparable to that
rate of absorbance increase measured by the MBTH assay was of the optimised Pomerantz microassay [34], being of the order
326
of 500 pmol substrate converted. Routinely, the incubation REFERENCES
period required for the 14C02assay is 10 min whereas a 3-h
incubation period is used for the Pomerantz microassay. It is 1. Lerch, K. (1987) Meth0d.Y Enzymol. 142, 165 - 169.
feasible that a microassay procedure could be developed for 2. Lerner, A. B., Fitzpatrick, T. B., Calkins, E. & Summerson, W.
the 14C02assay. H. (1949) J . B i d . Chem. 178, 185-195.
No melanogenic tyrosine hydroxylase activity can be de- 3. Hearing, V. J., Korner, A . M. & Pawelek, J. M. (1982) 1. Invest.
Dermatol. 79, 16 - 18.
tected in a PC12 cell extract, these cells having been reported 4. Korner, A. & Pawelek, J . (1982) Science217, 1163-1165.
to possess neuronal tyrosine hydroxylase activity [24]. The fact 5. Hogeboom, G. H. & Adams, M. H. (1942) J . Biol. Chem. 145,
that the assay measures the activity of the tyrosine hydroxylase 273 - 279.
involved in melanin synthesis, and not the neuronal enzyme, 6. Pomerantz, S. H. & Li, J. P.-C. (1974) Nature 252, 241 -243.
is confirmed by inhibitor studies and its requirement for a 7. Laskin, J. D.&Piccinini, L. A. (1986) J . Biol. Chem.261.16626-
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Dopa oxidase assay
10. Pomerantz, S. H. (1964) Biochem. Biophys. Res. Commun. 16,
The MBTH assay for dopa oxidase activity has a much 188-194.
more sound theoretical basis than the traditional dopachrome 11. Husain, I., Vijayan, E., Ramaiah, A,, Pasricha, J. S. & Madan,
assay. It measures the formation of the direct product of N. C. (1982) J . Invest. Dermatol. 78, 243-252.
dopa oxidation, trapping it as a stable compound. In contrast, 12. Okuno, S. & Fujisawa, H. (1983) Anal. Biochem. 129,405-411.
dopachrome is not the direct product and is not stable. The 13. Tank, A. W. & Weiner, N. (1987) Methods Enzymol. 142,71-82.
MBTH assay is approximately seven times more sensitive than 14. Mason, H. S. (1948) J . Biol. Chem. 172, 83-99.
15. Mazzocco, F. & Pifferi, P. G. (1976) Anal. Biochem. 72, 643-
the dopachrome assay in terms of rate of absorbance increase.
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This is apparently because the molar absorption coefficient of 16. Pifferi, P. G. & Baldassari, L. (1973) Anal. Biochem. 52, 325-
the pink pigment is seven to eight times greater than that of 335.
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Human melanoma cell extracts used in optimisation of Dermutol. 86, 570- 572.
dopa oxidase and tyrosine hydroxylase assays were prepared 20. Vachtenheim, J. Duchofi, J . & MatouS, B. (1985) Anal. Biochem.
differently, but an optimised method for preparing both activi- 146,405-41 0.
ties has now been devised [26]. Both enzyme activities can 21. McCormick, D., Wallace, I., Kirk, J., Dinsmore, S . & Allen, I.
also be detected in a mouse melanoma cell extract and a (1983) Br. J . Exp. Pathol. 64, 103-115.
commercial mushroom tyrosinase preparation. It is important 22. Der, C. J. & Stanbridge, E. J. (1981) Cell 26,429-438.
to dialyse melanoma cell extracts for use in tyrosine hy- 23. Jonasson, J., Povey, S. & Harris, H. (1977) J . Cell Sci. 24, 217-
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24. Greene, L. A. & Tischler, A. S. (1976) Proc. Nail Acad. Sci. USA
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Applications of’the assays 27. Jara, J. R., Solano, F. & Lozano, J. A. (1988) Pigment Cell Res.
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The assays described here promise to be useful tools in the 28. Okun, M. R., Donnellan, B., Patel, R. P. & Edelstein, L. M.
further characterisation of the tyrosine hydroxylase and dopa (1973) J . Invest. Dermutol. 61, 60-66.
oxidase activities of tyrosinase. In the light of the possibility 29. Smith, P. I. & Swan, G . A. (1976) Biochem. J . 153,403-408.
that these two activities may not be inherent in one enzyme 30. Pomerantz, S. H. (1963) J . Biol. Chem. 238,2351 -2357.
molecule, their slightly different patterns of behaviour with 31. Hearing, V. J., Ekel, T. M., Montague, P. M. & Nicholson, J. M.
respect to pH and enzyme inhibitors are of interest. The (1980) Biochim. Biophys. Acta 611, 251 -268.
finding that diethyldithiocarbamate is a much more effective 32. Cabanes, J., Garcia-Canovas, F., Lozano, J. A. & Garcia-
inhibitor of dopa oxidase than tyrosine hydroxylase activity Carmona, F. (1987) Biochim. Biophys. Acta 923, 187-195.
has been reported previously [8]. The assays should prove to 33. Lerch, K. (1981) in Metal ions in biological systems, vol. 13:
be useful in defining the functions of the various cDNA clones Copperproteins (Siegel, H., ed.) pp. 143 - 186, Marcel Dekker,
New York.
encoding mammalian tyrosinase and tyrosinase-related pro-
34. Townsend, D., Guillery, P. & King, R. A. (1984) Anal. Biochem.
teins that have been reported recently [35 - 381. They also have 139, 345 - 352.
potential for use in the study of melanin-related disorders such 35. Kwon, B. S., Haq, A. K., Pomerantz, S. H. & Halaban, R. (1987)
as albinism, vitiligo and melanoma. Proc. Nail Acad. Sci. U S A 84, 7473 - 7477.
36. Muller, G., Ruppert, S., Schmid, E. & Schutz, G. (1988) E M B O
This work was supported by the Cancer Research Campaign. J . 7,2723 -2730.
A.J.-W. was in rcceipt of a Medical Research Council research 37. Ruppert, S., Muller, G., Kwon, B. & Schutz, G. (1988) EMBO J .
studentship and a scholarship from St Hugh’s College, Oxford, and 7,2715-2722.
the Dee Corporation. We would like to thank Wendy Brownsill for
38. Shibahara, S . , Tomita, Y., Sakakura, T., Nager, C., Chaudhuri,
technical assistance and Phil Winder for help with preparation of the
B. & Muller, R. (1986) Nucleic Acids Res. 14, 2413-2427.
manuscript. Thanks are also due to Dr Mike Bramwell and Dr V. J.
Hearing for helpful discussions.