COPPER, ENZYMES, AND FERTILIZATION. 85 



I. Copper in the Egg Pigment. 



Since copper is an integral part of so many organic coloring 

 matters, it was natural to examine first of all the pigment of the 

 egg. This material, as is well known, is widely distributed in 

 granular form in the cytoplasm. As the granules are most nu- 

 merous in tangential sections, the pigment bodies are evidently 

 concentrated near the surface. This also seems to be true, to some 

 extent, immediately about the nucleus. 



It is entirely justifiable to identify these granules as the source 

 of the coloring matter secreted by the eggs. I consequently prer 

 cipitated the pigment from normal egg secretion by the chloroform 

 method described in an earlier paper (21 3 ). After incineration an 

 alkaline solution of the ash in twenty minutes gave a relatively 

 heavy, though incomplete, deposition of metallic copper on alumi- 

 num. The deposit was dissolved and gave a deep yellow color on 

 addition of potassium ethyl xanthate. 



Direct proof of copper in the pigment bodies in situ is also pos- 

 sible. Tangential sections which contain more of these granules 

 than others are the very ones that stain most heavily with hsema- 

 toxylin. This we should expect in the presence of copper, though 

 the test is not dependable if iron is also present. I therefore 

 adopted a procedure employed in the microchemical analysis of 

 minerals. The method depends on the formation of a triple nitrite 

 of potassium, copper, and lead ('94). 



To sections on the slide I added a trace of sodium acetate and a 

 somewhat larger quantity of potassium nitrite. The whole was 

 then acidulated with acetic acid. Last of all, a few grains of lead 

 acetate. The presence of copper, as little as 0.05 microgram, 1 if 

 crystals are desired, completes the conditions necessary for the 

 formation of the triple salt, K 2 CuPb(NO 2 ) , which is jet black. 



In this test, which differentiates between iron and copper, the 

 sections most darkened are the very kind most affected by hema- 

 toxylin. There is a marked graying of the cell contents to which 

 a partial reduction of the pigment bodies contributes. The gen- 

 eral effect is due to a decided increase in cytoplasmic granulation — 

 the granules being excessively minute and, like the microsomes of 



1 1 microgram (/xgr.) = i/ioooth milligram. 



