CAROTIN, THE PRINCIPAL YELLOW PIGMENT OF MILK FAT 433 



it was often noticed that when the serum had stood for some time 

 in a closed bottle in contact with a little supernatant air, an orange- 

 yellow scum always came to the top. This was found to be a water 

 insoluble protein which would not give up any color to petroleum 

 ether when its aqueous suspension was shaken with that solvent, but 

 when an equal volume of absolute alcohol was added and the shaking 

 with petroleum ether repeated, the latter solvent rose to the top as a 

 beautiful yellow layer. The pigment thus extracted gave a red-orange 

 carbon bisulphide solution showing the three carotin absorption bands. 



In order to show more conclusively the character of the protein 

 with which the serum carotin is evidently combined, the coagulation 

 temperature of the protein was determined. For this purpose 150 c.c. 

 of serum was diluted with an equal volume of a saturated solution of 

 ammonium sulphate. After filtering off the precipitated globulins, por- 

 tions of the globulin-free filtrate were submitted to fractional coagu- 

 lation. It was found that on carefully elevating the temperature to 

 80 C. and holding it at that temperature for a short time, the filtrate 

 from the coagulated albumins still yielded a large amount of carotin on 

 addition of alcohol and shaking with petroleum ether. On the other 

 hand the coagulated albumins yielded a comparatively small amount 

 of carotin. A similar result was obtained at temperatures of 81, 82, 

 83, 84, 85, and 85.5 C., although the amount of carotin in the 

 filtrate rapidly decreased with the increase in coagulation temperature. 

 At 86 C., however, the pigmented protein had completely coagulated, 

 and the filtrate yielded no carotin on treatment with alcohol and 

 petroleum ether. The coagulation temperature limits of the pigment 

 carrying protein therefore lie between 80 and 86 C., when the pro- 

 tein is in half saturated ammonium sulphate solution. There is no 

 marked coagulation at the lower temperature, but it is completely 

 coagulated at the upper temperature. 



The coagulation temperature of the protein which carries the 

 carotin in the blood was studied further with an aqueous solution of 

 the protein obtained in a manner similar to the one used in obtaining 

 the protein for the study previously reported. Briefly, an equal volume 

 of saturated (NH 4 ) 2 SO 4 solution was added to 200 cubic centimeters 

 of blood, rich in carotin, from Holstein Cow No. 221. The globulins 

 were filtered off and the golden-yellow filtrate heated carefully in a 

 water bath to a temperature of 79 C. The coagulated proteins were 

 filtered off. The yellow filtrate was saturated with (NH 4 ) 2 SO 4 

 in substance and let stand several hours. The golden-yellow pre- 

 cipitate was filtered off on a Buchner funnel. After allowing to suck 



