COPPER, ENZYMES, AND FERTILIZATION. 83 



It is sufficient to say that whenever I was able to avoid interfer- 

 ence, due to small amounts of iron, I found positive indications 

 for traces of copper. Material was, therefore, prepared for the 

 more careful analyses made this winter at Amherst. 



Immature eggs were taken directly from the ovaries ; mature 

 eggs were shed spontaneously under conditions which I have de- 

 scribed elsewhere (22 1 ) and which precluded any contact whatever 

 with sea-water, fresh water, dermal secretions, appendages, or 

 detritus of any kind. The eggs were tested with sperm and found 

 to be normal. 



Two c.c. of mature eggs dissolved in concentrated nitric acid 

 constitute sample A. This was subsequently diluted to 50 c.c. with 

 water doubly distilled from glass. Fractions of this material were 

 used either directly or, after evaporation to dryness and incinera- 

 tion in glazed china crucibles. The ash was redissolved in sul- 

 phuric acid, usually neutralized with ammonia, and the volume re- 

 stored to that of the original fraction. In all these operations 

 great care is essential. All reagents were tested repeatedly and 

 found negative for copper. 



1. Copper as Cupric Hydroxide — Cu(OH) 2 . 



1 c.c. of A evaporated to dryness and incinerated. Ash dissolved 

 in H0SO4. Excess NH 4 0H should give the blue of cupric hydroxide. 

 Result : positive but exceedingly faint. 



2. Copper as Cupric Cyanide — Cu(CN) 2 . 



To 5 c c. of A, added concentrated KCN solution. Should give yel- 

 low precipitate of Cu(CN), 2 . The precipitate should dissolve in H 2 S0 4 

 and this solution should give test for Cu(OH) 2 . Result: positive 

 and distinct throughout. 



3. Copper as Cupric Ferrocyanide — Cu 2 Fe(CN) 6 . 



Ash from 10 c.c. of A redissolved and neutralized as before. To 

 avoid interference by iron, the reagent, a 1 :3s solution of potassium 

 ferrocyanide, K 4 Fe (CN) C in distilled water, was carefully run over 

 the surface of the test solution as in the ring reaction for proteins with 

 nitric acid. In the present case also a ring is formed; this quickly 

 thickens — the upper layers being composed of the blue ferrocyanide of 

 iron ; the lower of brown cupric ferrocyanide. 



4. Copper as Cupric Xanthate — Cu(C 3 H-S 2 0) 2 . 



Redissolved ash from A, in the presence of potassium ethyl xanthate 

 should give a yellow color due to the formation of copper xanthate 



