ESMOND R. LONG 102 1 



for varying periods and in different types of apparatus. Precipitation was carried out 

 by acetic acid at the iso-electric point of the bulk of the colloid present, and by all of 

 the protein precipitants commonly used, ammonium sulphate and trichloracetic acid 

 proving the most valuable. In addition to this, cleavage of the protein material pres- 

 ent was effected by proteolytic enzymes and acid hydrolysis, in an effort to deter- 

 mine any parallelism between tuberculin activity and the size of protein molecules 

 present. The results of this investigation are briefly summarized in a later article,' 

 from which I quote with slight change: 



When tubercle bacilli are grown on a non-protein medium of known chemical composi- 

 tion, tuberculin activity develops coincidently with the appearance of protein in the medium, 

 the protein being derived presumably by extraction or autolysis of tubercle bacilli. If tuber- 

 culin prepared on this medium is subjected to dialysis, as a general rule the active substance, 

 together with the protein, remains in the dialysing sac, all of the original constituents 

 of the medium, as well as the substances responsible for the characteristic odour and most of 

 the colour, diffusing out. Occasionally traces of substance of protein nature also appear to 

 diffuse, and when this occurs tuberculin activity appears in the diffusate. 



The addition of acetic and other acids precipitates from tuberculin prepared on this me- 

 dium a protein which has moderate tuberculin activity. But precipitation of the protein of 

 the solution is never complete by this method, and, moreover, the protein-containing filtrate 

 from the precipitate retains tuberculin activity. Full saturation with ammonium sulphate, 

 however, precipitates at the same time all of the protein and all of the active substance. 



The ammonium sulphate precipitate contains both coagulable and non-coagulable pro- 

 tein and also proteose. When the protein is destroyed by pepsin with little destruction of the 

 proteose, activity is destroyed. When the proteose alone is disintegrated by the action of 

 trypsin in neutral solution or by erepsin, the activity is not affected. 



The active principle of tuberculin withstands without loss of activity four hours' heating 

 at 120° C. and 14-lb. pressure in the presence of N/io hydrochloric acid. Up to this point 

 the protein of the solution remains in such state that it can be precipitated by trichloracetic 

 acid. The precipitate is active. Strengths of acid of N/6 and above hydrolyse the protein so 

 that it no longer precipitates with trichloracetic acid, and at the same time tuberculin activity 

 disappears (see Figs. 2 and 3). 



The activity of tuberculin thus definitely appears associated with a protein substance. 

 It has not been found possible, by any of a variety of means tried, to separate from this sub- 

 stance any non-protein fraction with tuberculin activity, and therefore it seems doubtful 

 that the close association of activity with protein is merely one of physical absorption. The 

 possibility remains open that in the protein molecule itself activity may be a function of a 

 certain group. The apparent persistence of activity on mild hydrolysis with beginning pro- 

 tein disintegration would support this view. The fact that with further hydrolysis of protein 

 activity disappears, makes it unlikely, however, that an active group can be split off, free 

 from the rest of the protein. 



An advance of the first importance which may lead to solution of the whole prob- 

 lem has been made by Dr. Seibert^ in the crystallization of the water-soluble protein 

 found in the tuberculin preparations we have used. Fractional coagulation and other 

 methods of concentrating the activity of the water-soluble tuberculin protein failing, 



' Long, E. R., and Seibert, F. B.: Tubercle, 8, iii. 1926. 



^ Seibert, F. B.: Science, 63, 619. 1926; Tr. 22d Ann. Meeting,, Nat. Tuherc. Assoc, p. 274. 1926; 

 Tr.2jd Ann. Meeting, Nat. Tuberc. Assoc, p. 245. 1927; Science, 66, 433. 1927. 



