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
2
3
4

No
No
Yes
Yes

No
Yes
Yes
No

7802 266
23427 1046
50091 2198
50150 1746

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