io6 The Iodine Complex of Thyreoglobulin 



readily soluble laevorotatory form. This was then iodized according to 

 the Pauly method. One determination on the snow-white product gave 63 

 per cent iodine (theoretical 65 per cent). The slightly lower value may 

 be due to an admixture of a small amount of di-iodohistidine anhydride. 



Iodized tryptophane. Tryptophane was prepared from commercial casein 

 by the Hopkins-Cole method. 21 Several attempts were made to iodize 

 the pure crystals by the method of Neuberg, 22 but in no case was a substance 

 obtained containing more than 6.3 per cent iodine. The preparation finally 

 made for physiological testing was obtained by dissolving one milligram 

 molecule of tryptophane in 4 cc. of | NaOH, cooling by immersing in ice 

 water and, while keeping cool and stirring well, adding drop by drop 6 cc. 

 of aqueous N iodine solution. The mixture was allowed to stand at ice box 

 temperature for twenty-four hours, then filtered off. The precipitate was 

 well washed with cold water and dried over sulphuric acid in a vacuum desic- 

 cator. The product obtained is light brown in color, readily soluble in alka- 

 lies, reprecipitated on acidifying and liberates a very small amount of iodine 

 to chloroform on shaking therewith. Duplicate determinations on this 

 gave 41.5 and 41.9 per cent iodine (the theoretical for mono- and di-iodo- 

 tryptophane are 38.4 per cent and 55.7 per cent respectively). 



B. Methods. 



Determination of iodine. The Hunter 23 method with slight modifications 

 was employed. The material to be analyzed, taken in quantities of 0.05- 

 2 grams was mixed with 15 grams of fusion mixture and covered with 10 

 grams of fusion mixture as suggested by Hunter. To conduct the fusion the 

 Roger's ring burner was found to be much more satisfactory in ensuring a 

 uniform rapid heating without overheating. With the size of the flame once 

 determined one "finds ten minutes to be ample time to give a satisfactory, 

 easily removable fusion. In the treatment with alkaline hypochlorite it 

 was considered best to warm to 40C. for ten minutes. In acidifying it is 

 very important to make sufficiently acid and then always to the same degree. 

 Sulphuric acid of 25 per cent strength was used here and since the same 

 amounts of fusion mixtures and hypochlorite were used in each case the 

 acidity was well controlled by always adding the same amount of acid. 

 In removing the excess of chlorine gentle boiling was continued for forty 

 minutes after the negative test of the vapors by starch iodine paper. In 

 this way the blank test on the reagents never was more than 0.1 cc. of a 

 ^ Na2S 2 O3.5H 2 O solution. 



Physiological testing by the Hunt method. The method employed was that 

 of feeding the same quantity of iodine, in the different combinations, to 

 white mice in such a manner as to make as certain as possible the entire 

 consumption of the material fed. In order to do this each mouse was first 



21 Journ. of PhysioL, xxvii, p. 418, 1901. 



22 Biochem. Zeitschr., vi, p. 276, 1907. 



23 This Journal, vii, p. 321, 1910. 



