Effect of Glutathione on Tyrosinase and the Significance of the Dopa Reaction. ∗

Abstract

Bloch, 1 as a result of his discovery of the “dopa reaction” and because it had been impossible to demonstrate tyrosinase in the skin of any of the slightly pigmented vertebrates, postulated the existence of an enzyme that accelerated the oxidation of dihydrox-phenylalanine. He named it “dopa oxidase”, and claimed that this enzyme was responsible for melanin formation in human skin. Such an enzyme has not been isolated and its existence has been questioned many times. The controversy in regard to this has been discussed in comprehensive reviews. 2 , 3

Recently it has been shown 4 that the activity of the enzyme tyrosinase is regulated by the oxidation-reduction potential of substances added to the substrate. It was evident from this that both the enzyme tyrosinase and its substrate tyrosine could be present in a cell without melanin formation taking place if a sufficiently strong-inhibitor such as reduced glutathione was also present. With this in mind, an investigation was started to determine whether the enzyme responsible for the “dopa reaction” was a separate and specific dopa oxidase or tyrosinase associated with an inhibitor. To investigate this possibility chemically the rates of melanin formation were determined in the following series of reaction mixtures:

1. Buffered dopa alone.

2. Buffered dopa plus various reversibly oxidizable substances.

3. Buffered dopa plus tyrosinase.

4. Buffered dopa plus tyrosinase and glutathione.

5. Buffered tyrosine plus tyrosinase.

6. Buffered tyrosine plus tyrosinase and glutathione.

Series 1 was included to study the rate of non-enzymatic oxidation in air. Series 2 was included to see if any of the reversibly oxidizable substances would accelerate or inhibit oxidation in air. The last 4 series were included to determine and compare the effect of glutathione on the rate of enzymatic oxidation of dopa and tyrosine.