Improvinq the melanin formation assay for mammalian assay tyrosinase
Because of the multi-functionality of tyrosinase (EC 1.14.18.1) in the melanin
biosynthetic pathway, there are several methods to measure its enzymatic activity. These
methods differ in the substrate used, the step of the pathway measured, and the analytical
technique used to follow the tyrosinase-catalyzed reaction. It is difficult to establish
which method is the best one to evaluate tyrosinase activity. However, in samples where
melanogenesis capability is the desired parameter to be determined, the melanin formation
assay is surely the more suitable one. This assay was introduced by Chen and Chavin (1965)
and further studied by Hearing and Ekel (1976) and ourselves (1988). Basically, the assay
consists in the determination of the insoluble 14C-labelled eumelanin formed
from L-(U-14C)-Tyrosine.
The major problem of this method is the low percentage of L-Tyrosine hydroxylated by
tyrosinase that is incorporated into the eumelanic polymer. Most of the substrate, once
oxidized in the tyrosine-catalyzed reactions, remains as soluble intermediates of the
pathway, and these intermediates are washed before their incorporation into the polymer.
Comparison between tyrosine hydroxylase and melanin formation assays, shows that the
percentage of L-tyrosine hydroxylated which actually is incorporated into the polymer
ranges from less than 10% in 1 hour of assay, to 60% in assays performed for long times of
incubation, 8 hours.
In our laboratory we have tested some modifications of the melanin formation assay in
order to improve it by increasing the percentage of tyrosine hydroxylated which reaches
the polymer using assays of 1 hour incubation time. These modifications enhance the
radioactivity incorporated up to 7 times and they could be very useful when samples
showing low tyrosinase activity are being dealt with.
Taking into account the methodology described by Hearing and Ekel (1976) or ourselves
(Jara et al, 1988), there are two easy ways to improve the sensitivity of the method :
1. once the time of incubation of the sample with the substrate has elapsed, the reaction
is stopped by addition of NaOH up to a 60 nM concentration. Then the reaction mixture is
left in such a basic medium for 5 minutes, to allow radioactive intermediates to
polimerize rapidly into eumelanin, since the rates of the chemical
steps in the Raper-Mason pathway increase at high pH. Higher concentrations of NaOH must
not be used, since some degree of solubilization of the polymer occurs. After this
treatment, samples are applied to Whatman 3MM filter paper discs and washed in 0.1 M HCL
and other solvents according to the published method (Hearing and Ekel, 1976).
2. Bearing in mind that divalent metal cations also increase the rate of the chemical
steps in the Raper-Mason pathway, we tried the addition to the reaction media of 1 mM of
several cations. Ni2+ proved to be the most efficient one, increasing the
radioactivity incorporated to the insoluble polymer up to 7 times in some samples. This
effect and the one produced by NaOH were not additive, as it can be seen in the Table,
mean and S.D. of five experiments performed using purified tyrosinase from Harding-Passey
melanosomes, and 1 hour incubation time.
Effect of 1 mM Ni2+ or basic treatment on melanin formation
Assay number |
Ni2+ |
NaOH |
neT cpm/h |
1 |
No |
No |
7802 ±
266 |
References
1. Chen YM, Chavin W (1965) : Anal Biochem 13:234-258
2. Hearing VJ, Ekel T (1976) : Biochem J 157:549-557
3. Jara JR, Solano F, Lozano JA (1988) : Pigment Cell Res. (in the press)
J.R. Jara, F. Solano and J.A. Lozano
Dept Biochemistry, Faculty of Medicine
University of Murcia, Spain