43 MISSOURI AGRICULTURAL EXP. STA. RESEARCH BULLETIN NO. 12 



ether, even after making strongly alkaline with potassium hydroxide. 

 Amyl alcohol extracted the pigment. 



7. Experiment 6 repeated on 200 c.c. of the same serum gave the 

 same result. The entire pigment in the golden-yellow nitrate was 

 coagulated by boiling. The coagulum was not soluble in water. 



8. Fifty c.c. of serum (Cow No. 2) was diluted to 350 c.c. with 

 water, a pinch of salt added and the solution heated on the steam 

 bath, with stirring until cloudiness appeared. On adding a few drops 

 of glacial acetic acid, a sharp coagulation took place. On filtering, 

 the filtrate was bright yellow in color. On saturation of the filtrate 

 with (NH 4 ) 2 SO 4 in substance, a comparatively small amount of deep 

 yellow precipitate was thrown down leaving a colorless supernatant 

 solution. The yellow precipitate, which was contaminated with a little 

 (NH 4 ) 2 SO 4 , was readily soluble in water, giving a perfectly clear 

 yellow solution from which the yellow color was again entirely thrown 

 down on saturation with (NH 4 ) 2 SO 4 in substance, or on the addition 

 of mercuric nitrate. The latter precipitate when still moist would not 

 give up its color to petroleum ether until first moistened with absolute 

 alcohol. The bright yellow pigment now found in the petroleum ether 

 gave a red-orange CS 2 solution which showed the three carotin 

 absorption bands. 



9. Two 350 c.c. portions of serum (Jersey Cow No. 2) were 

 treated respectively as follows : 



Portion A was treated with (NH 4 ) 2 SO 4 in substance to one half 

 saturation, according to the formula 



VC 2 

 X= 



18.158 .54 C 2 



Where V original volume of protein solution. 

 C = desired saturation as grams in 10 c.c. 

 X = grams to be added to give the required 

 saturation. 



The globulins which precipitated carried down some of the pig- 

 ment, but on dissolving them in 150 c.c. of warm water containing some 

 (NH 4 ) 2 SO 4 , and adding (NH 4 ) 2 SO 4 to half saturation, they were 

 thrown down practically colorless. The yellow filtrate from this pre- 

 cipitation was added to the other globulin-free filtrate and the com- 

 bined solutions diluted to 1500 c.c. with distilled water. This solution 

 was now raised to a temperature of 75 C in a water bath. 15 c.c. of 

 i% per cent acetic acid added and the temperature raised to 80 C, 

 when a sharp coagulation occurred. The solution was filtered, giving a 



