Inhibitors of Tyrosinase

ABSTRACT It has been reported that if Drosophila larvae are ground to a fine paste with sand, the homogenate shows little tyrosinase activity, but that if the larvae are allowed to blacken in chloroform vapour before grinding, activity is increased. This has been interpreted as showing the effect of an intracellular inhibitor, set free by rupturing the cells, but destroyed by chloroform. This inhibitor has been identified by previous authors as a dehydrogenase. It is here suggested that the lack of activity of Drosophila extracts prepared with sand is due to destruction of tyrosinase as it oxidizes naturally occurring aromatic substrates. It is shown that tyrosinase is destroyed by oxidizing the aromatic substrate present in the cuticle of Calliphora larvae, or by very low concentrations of homocatechol. The aromatic substrate of Calliphora larvae is concentrated in the cuticle, and would be set free by fine grinding. Drosophila or Calliphora larvae yield a more active extract when ground with sand than when simply crushed, provided that they are tested soon after grinding. The tyrosinase activity of such extracts is not increased by chloroform or methanol. The compound between o-quinones and amino-acids is capable of oxidizing ascorbic acid or excess amino-acid without the aid of an enzyme, and of simultaneously reducing methylene blue. This reaction, rather than the activity of dehydrogenases, is probably responsible for most of the ability of damaged insect tissue to bleach methylene blue. The blood of insects normally contains dissolved oxygen in equilibrium with the air. The reaction involved in the blackening of insect blood may consume all the dissolved oxygen. Previous observations on fluctuations in the oxidation-reduction potential of the blood of Calliphora larvae with age are probably due to changes in the rate at which oxygen is consumed by the blood after it is shed. There does not therefore appear to be any valid evidence that tyrosinase is inhibited in vivo by the action of dehydrogenases. The absence of tyrosinase activity in undamaged tissue is probably due to the structure of the cytoplasm, which keeps enzyme and substrate apart. Instances of the inhibition of tyrosinase reported in Crustacea and Echino-dermata seem to be susceptible of the same explanation as in insects. The supposed inhibitory effect of sulphydryl groups reported for vertebrate melanophores is shown to be due to the combination of sulphydryl groups with o-quinones, which prevents the formation of melanins.