Chemistry of Coat-Colour in Animals. 



41 



half its weight of chloroform water and sufficient kieselguhr to make a good 

 paste, and the whole mixture was thoroughly ground up for some time. 

 The resulting black mass was finally transferred to a cheese cloth and the 

 ferment fluid expressed by means of a hydraulic or efficient screw press. 

 After being filtered through a soft paper, this fluid is white with a slight 

 opalescence, provided the removal of the capillaries has been complete. 

 The spectrum of oxyhemoglobin cannot be detected, and the fluid does not 

 show the presence of a trace of iron by the Prussian blue test. The extracts 

 of the skins of all the other rabbits were prepared in exactly the same way, 

 care being taken in the case of the English rabbits to cut away the black 

 portions of the skin. A series of tubes was next prepared in the following 

 manner : 2 c.c. of the ferment fluid were placed in each of a number of narrow 

 test-tubes, together with three drops of an approximately 2-per-mille suspen- 

 sion of tyrosine in water, or other chromogen, and OT c.c. of a 0"05-per-cent. 

 solution of hydrogen peroxide.* This quantity of hydrogen peroxide cannot 

 greatly be exceeded, since it has been shown by Bachf that larger amounts 

 seriously inhibit the reaction. When an extract was to be tested for the 

 presence of an inhibitor, it was added to the ferment fluid in the exact 

 amount subsequently stated. Chloroform was employed as a preservative, 

 since toluol, which was used by Miss Durham, if added in excess, rises to the 

 surface and prevents the free access of oxygen. By means of very dilute 

 sodium carbonate the contents of the tubes were carefully rendered faintly 

 alkaline to litmus. The tubes were then plugged with cotton wool and 

 incubated at a temperature of 87° C. As the reaction was found to have 

 taken place in 12 hours the tubes were always examined after that time. 

 An additional reason for doing this was because in some cases, when the 

 oxidation had been slight, it was noticed that the colour had a tendency to 

 fade after a longer period. Some idea of the amount of the pigment 

 produced may be obtained from the following experiment : — 



Ten cubic centimetres of the ferment fluid were incubated for 24 hours 

 with an excess of tyrosine and 0'5 c.c, of a 0-05-per-cent. solution of hydrogen 

 peroxide. This was acidified with 1 c.c. of 10-per-cent. sulphuric acid, and 

 made up to 50 c.c. with distilled water. This solution was then titrated 

 with potassium permanganate. It required 9-85 c.c, of a - 2-per-cent.j 

 solution to remove entirely the colour due to the melanin produced. 



* For these experiments Merck's " perhydrol " was used. 



+ Bach, ' Ber. der Deutsch. Chem. Gesell.,' vol. 41, p. 216 (1908). 



| According to Bach's method of estimating the amount of melanin, a 0-002-per-cent. 

 solution of potassium permanganate was found sufficient. In the above experiment, 

 however, this strength proved to be totally inadequate. 



