COPPER, ENZYMES, AND FERTILIZATION. 89 



A. Lipolysin — I. 



The lipolysin first tested was prepared as follows : the eggs were 

 permitted to secrete as usual ; the agglutinin was salted out with 

 ammonium sulphate, which incidentally also precipitates much of the 

 pigment. After filtration, the lipolysin iteslf was thrown down by 

 means of barium, redissolved in dilute hydrochloric acid and precipi- 

 tated by acetone which unless in great excess holds the remaining 

 agglutinin in solution. The lipolysin so secured was a white powder 

 free from pigment, though in the course of time it turned slightly 

 purple. 



After incineration a solution of the ash gave a positive reaction 

 for copper with potassium ethyl xanthate. 



Lipolysin — 77. 



This precipitate had exactly the same history as the foregoing except 

 that charcoal was used in place of barium. Moreover, the powder did 

 not change color but remained snow white and therefore may have 

 been a somewhat purer product than lot I. .By the xanthate test, 

 lot II. also contained copper. 



B. Agglutinin — /. 



The agglutinin first examined came from Asterias egg-secretion which 

 had been freed from lipolysin by the barium method. The xanthate 

 test, carried out as before, gave a perceptible reaction for copper but 

 as the separation from the other constituents of the secretion was in- 

 complete, this result is inconclusive. 



Agglutinin — II. 



A second test was made, this time with Arbacia agglutinin which had 

 been merely salted out with ammonium sulphate and contained there- 

 fore considerable quantities of pigment. In this case the xanthate 

 test was markedly positive. 



A gglutinin — III. 



Arbacia agglutinin whose separation from pigment and other im- 

 purities was accomplished by chloroform, charcoal, and final differen- 

 tial precipitation from acetone. This material was negative for copper. 



In all I examined five different lots of agglutinin, and in every 

 case preparations whose purity I supposed to be high were the ones 

 that proved to be either completely negative for copper, doubtful, 

 or at most very slightly positive. It appears that copper is not an 

 essential constituent or even regular associate of pure agglutinin. 



These results, together with the triple-nitrite test and the con- 

 siderations that suggest a cortical concentration of the secretions, 

 justify my assumption that copper is present in the cortex of the 

 egg immediately beneath the vitelline membrane. This copper, 



