148 H. J. CORPER AND H. C. SWEANY 



Series VII. Elastic and connective tissue digesting enzymes. 



In order to demonstrate the presence or absence of elastic or 

 connective tissue digesting enzymes in the tubercle bacilli, an 

 indirect method of attack had to be resorted to. In the first 

 place it was necessary to demonstrate just how much digestion 

 was a result of the action of the enzymes upon the proteins 

 already obtained from the bacilli and then using this as control, 

 to determine whether there was really any digestive action 

 upon the elastic or connective tissues. Since the amino acid a 

 nitrogen method of Harding and McLean was the most delicate 

 and accurate method available for the purpose it was used as 

 the index of the digestion taking place during the course of the 

 experiments. 



Experiment I. Elastic tissue digesting enzyme. To 10 cc. of 

 emulsified human tubercle bacilli was added 25 cc. sterile 0.9 

 per cent salt solution, 5 cc. chloroform, and 10 cc. toluene. This 

 was placed in the incubator at 37°C. for twenty-four hours when 

 it was centrifugated and 8 cc. of the supernatant autolysate was 

 drawn off and filtered through a sterile hard filter paper. The 

 filtrate was divided into three equal portions, one was heated 

 for thirty minutes in boiling water to destroy the enzymes and 

 the other two portions were kept unheated. All were now 

 diluted to 5 cc. and to the heated and one of the unheated por- 

 tions was added 0.5 gram of elastic tissue prepared from lamb 

 lung.^ A definite amount of the solution (0.5 cc. each time) 

 was withdrawn immediately, one day, three days, five days, 

 seven days, and ten days after incubation at 37°C. and used for 

 determining the amino acid a nitrogen content. Similarly 8 

 cc. of autolysate were withdrawn at four days and eight days 

 and tested as above for digestive action upon the elastic tissue. 

 The results of the analyses are given in the following table, the 



* The elastic tissue was prepared by finely grinding lamb's lung and treating 

 it at room temperature, shaking frequently, with a large amount of 5 per cent 

 potassium hydroxide and 10 per cent acetic acid alternately until the potassium 

 hydroxide and acetic acid solutions used remained absolutely colorless and clear 

 after twenty-four hours contact. The elastic tissue thus prepared was washed 

 with water until absolutely free from acetic acid and was kept for use in distilled 

 water with toluene and chloroform as preservatives. 



