ENZYMES OF THE TUBERCLE BACILLUS 143 



Summary. Tubercle bacilli possess a pepsin-like enzyme 

 which, though feeble in action, is capable of liberating amino 

 acid a nitrogen from the bacillary bodies in the presence of 

 0.2 per cent hydrochloric acid. 



Series III. Erepsin-like enzymes 



Attempts were made to determine the presence of erepsin- 

 like enzymes by using casein in acid solution and testing nephe- 

 lometrically, as recommended by Kober, by using both the 

 bacillary emulsion and autolysate therefrom, but both attempts 

 failed on account of turbidities produced in the solutions by the 

 bacillary emulsion and autolysate even before adding the pre- 

 cipitant. It was finally decided to test for erepsin-like enzymes 

 by preparing a peptone from Witte's peptone which was com- 

 pletely precipitated by ten volumes of methyl alcohol so that 

 no perceptible trace was dissolved therefrom by means of methyl 

 alcohol, and using as criterion the splitting of this peptone in 

 acid solution, testing for such splitting by means of Harding 

 and MacLean's quantitative amino acid a nitrogen test. 



Four cubic centimeters of tubercle bacilli were diluted with 

 sterile physiological salt solution to 10 cc, 2 cc. toluene and 1 

 cc. chloroform were added, and the mixture, after shaking thor- 

 oughly, was placed in the incubator at 37°C. for twenty-four 

 hours, after which the centrifugated supernatant liquid was 

 filtered through a sterile hard filter paper and divided into two 

 equal portions. One portion (5 cc.) was heated thirty minutes 

 at 100°C., while the other portion (5 cc.j remained unheated. 

 To each was added sufficient sterile physiological salt solution 

 to make 10 cc, sufficient hydrochloric acid to make 0.2 per cent, 

 2 cc. toluene, 1 cc. chloroform and 1 cc. 0.25 per cent pure 

 peptone solution (completely precipitable by ten volumes of 

 methyl alcohol). From each of these tubes a control of 0.5 cc. 

 was withdrawn and to this ten volumes of methyl alcohol was 

 added, the whole centrifugated at 3000 revolutions until per- 

 fectly clear and the supernatant clear methyl alcohol analyzed 

 quantitatively for amino acid content. At definite intervals 



