374 Determination of Tryptophan [April 



used the mercury stilfate-sulfiiric acid method^ in work on casein, 

 this treatment has always been reported as giving figures somewhat 

 lower than actual valnes. Tryptophan diminishes in quantity after 

 a time, and may disappear, during the progress of tryptic digestion. 

 In this laboratory we have found that the mercury sulfate-sulfuric 

 acid method of Hopkins and Cole does not completely precipitate 

 the tryptophan present in the digestion mixture. After precipitating 

 the tryptophan-mercury-sulfate product from a casein digestion 

 mixture, fikering, neutrahzing with calcium hydroxide and remov- 

 ing the calcium sulfate and insoluble calcium salts, we obtained a 

 filtrate that gave a characteristic tryptophan test with glyoxylic and 

 sulfuric acids. Obviously the Hopkins-Cole method did not com- 

 pletely precipitate tryptophan. Because of the smallness of the 

 amounts of tryptophan usually derived from proteins, this method is 

 necessarily dependent on the use of relatively large quantities of 

 protein. The tedious nature of the methods for the purification of 

 large amounts of proteins led us to endeavor to devise an accurate 

 process involving the use of small amounts of protein. * 



The Solution of the problem seemed to depend on perfecting a 

 method for the quantitative determination of small amounts of 

 indol. We desired to use i^-napthoquinone mono-sodium sulfonate, 

 such as Herter* employed in his work on indol, but could not find 

 it on the market. We prepared a substance that reacted with indol, 

 giving a deep violet colored Solution such as Herter obtained, but the 

 substance formed by the combination of indol with our supposed 

 ;8-napthoquinone mono-sodium sulfonate was not soluble in Chloro- 

 form. Herter used Chloroform to extract the indol-containing Com- 

 pound. Further use of our reagent was abandoned. It is probable 

 that we had an isomer of Herter's reagent differing mainly from his 

 in its reaction with Chloroform. We prepared our reagent by 

 cautiously oxidizing " Eikonogen," the Photographie developer, by 

 means of concentrated nitric acid. The oxidation was quite satis- 

 factory but the_ substance obtained was evidently an isomer, bearing 

 the sulfonic acid radical on a benzene nucleus other than the one 

 holding the quinone linkages. 



* Hopkins and Cole: Jour. of PhysioL, 1901-2, xxvii, p. 418; Ibid.j 1903, 

 xxix, p. 451. (Mercury sulfate-sulfuric acid method; Isolation of tryptophan.) 



* Herter and Foster: Jour. of Bio!. Chent., 1905-6, i, p. 257; Ibid., 1906-7, ii, 

 p. 267. (ß-napthoquinone reaction with indol.) 



