160 Microscopic Histochemistry 



chemical and physical agents and is present in a number of 

 structures, especially in myeloid leucocytes, and (2) an ab- 

 solutely specific dopa oxidase which does not act on any 

 other substrate, is easily inactivated by chemical and phys- 

 ical agents, and is present only in cells concerned with the 

 elaboration of melanin (basal layer of epidermis, chromato- 

 phores, cells of melanoma, etc.). Although Bloch's theory 

 was criticized by several authors ( Heudorf er,^^ Przibram^^ ) , 

 it appears that the virtual specificity of the enzyme of mel- 

 anin-producing cells in vertebrate species is a well-estab- 

 lished fact. The only compound, besides dopa, to be attacked 

 by this enzyme is oxytyramin ( Mulzer and Schmalf uss ) .^^ In 

 insects and other species, other melanizing enzyme systems 

 may be present ( Hasebroek ) ,^^ and even vertebrates possess 

 other enzymes which will convert tyrosin and related com- 

 pounds to melanin. The pathway of melanin formation by 

 both types of enzymes, is essentially the same and leads 

 through derivatives of indole^^ (ring-closure of the side 

 chain ) . To rule out nonspecific phenol oxidases, it is advis- 

 able to incubate control sections with a different substrate 

 (e.g., naphthol or tyrosin). 

 Method {Laidlaw and Bhckhergs^^ modification) 



Use frozen sections of fresh material or of tissue fixed for 

 only a few hours in 5 per cent formalin. Longer fixation may 

 cause partial inactivation of the enzyme. Rinse sections very 

 briefly in distilled water and transfer them into a 0.1 per cent 

 solution of dihydroxyphenylalanin (the commercial sub- 

 stance is a mixture of the stereo-isomers ) , buffered with a 

 phosphate buffer to pH 7.3-7.5, in an open dish for 4-5 



57. Heudorfer, K.: Miinchen. med. Wchnschr., 68:266, 1921; Heudorfer, 

 K.: Arch. f. Dermat. u. Syph., 134:339, 1921. 



58. Przibram, H.: Arch. f. Entwcklngsmech. d. Organ., 48:140, 1921. 



59. Mulzer, P., and Schmalfuss, H.: Med. Klin, 27:1099, 1931. 



60. Hasebroek, K.: Fermentforsch., 5:1, 1922. 



61. Duliere, W. L., and Raper, H. S.: Biochem. J., 24:239, 1930; Mason, 

 H. S.: J. Biol. Chem., 168:433, 1947. 



62. Laidlaw, G. F., and Blackberg, S. N.: Am. J. Path., 8:491, 1932. 



