446 BROOKS. 



The black coloring matter is deposited in much the same way as resin, 

 namely in the heartwood, in insect stings and wounds. In other words, 

 the deposition of the coloring matter, as in the case of the resins in gen- 

 eral, occurs simultaneously with the death of the cells. The leaves of 

 herbarium specimens of many of the Ebenacece turn inky black after a 

 short time. Black ebony is also found occasionally in the heartwood of 

 3 r oung twigs. Therefore, it is very evident that the conditions of the color 

 formation are not those usually prevailing when humus substances are 

 formed. This color is exceedingly stable and although alcoholic potash 

 extracts a small amount of brown substance which is precipitated by 

 acids in amorphous flocks, it is certain that the black substance itself 

 has not the properties usually ascribed to humus. 



The explanation of the manner of formation of the coloring matter - was 

 suggested by the fact that fresh leaves of the species Diospyros nitida 

 Men', (cunalon, Tagalog,) become black on drying, or blacken in an hour 

 or two when bruised or crushed and exposed to the air. I have been 

 able to show that this result is brought about by the action of an oxidizing 

 enzyme on an insoluble chromogenic substance in the leaves. The same 

 enzyme and chromogen occur in the wood and the enzyme was detected 

 in the sapwood. Its ability to cause the formation of a black, insoluble 

 substance resembles that of tyrosinase and laccase. However, it probably 

 is not identical with either of these oxidases. 



The behavior of the enzyme was studied by making an aqueous ex- 

 tract of the macerated, fresh, green leaves, and heating it as well as 

 the bruised leaves to 100° for a few minutes, after which treatment they 

 did not blacken on exposure to the air. Therefore the enzyme is de- 

 stroyed by heat. Dilute hydrochloric acid also renders it inactive. Sam- 

 ples of the original leaf pulp or of the turbid extract when exposed to 

 the air turn very black in about one hour. A portion set aside in a 

 test tube blackened at the surface, the color gradually extending down 

 into the solution, whereas a sample kept in an atmosphere of carbon- 

 dioxide showed almost no color change after twenty-four hours. 



The extract does not turn guaiacum to a blue color directly, but 

 develops the tint immediately on the addition of a few drops of hydrogen 

 peroxide. A blue color is quickly produced on shaking with a naph- 

 thol and p-phenylenediamine (the Ebhmann-Spitzer reagent). 



It is highly probable that this oxidase also exists in other species of the 

 Ebenacece. Thus Bacon 28 found that extracts of the fruit of Diospyros canomoi 

 DC. blackened very quickly on exposure to the air. The extract appeared to 

 contain an acid of the tannic acid series. The black color produced by Diospyros 

 leaves is much used by the Moros in the southern Philippine islands; it is quite 

 fast to washing and to light. Textiles dyed with this black and the dark red of 

 mangkudu are highly prized by Americans and Europeans. 



"-"This Journal (1906), 1, 1029. 



