AMIDE, PEPTIDE, AND NITRATE NITROGEN 289 



PROCEDURE 



1. To the soln. remaining after all ammonia has been distilled 

 out, in the determination of amide nitrogen, add 4 mg. Devarda 

 alloy. Tilt the vessel so that the alloy is separated from the liquid. 



2. Then introduce 0.1 ml. 25% sodium hydroxide, and allow the 

 ammonia formed to diffuse into a fresh drop of standard acid. 1-2 

 hr. is sufficient for the reduction of the nitrate, and the distillation 

 of the ammonia will be complete within this period if the diffusion 

 cell of the authors is employed. 



3. Determine the amount of ammonia which was absorbed. 



4. Employ two sets of controls, one in which the sodium 

 hydroxide is added without the alloy, and a second in which the 

 alloy is added to a control mixture containing the reagents for the 

 amide determination, but no sample. Substract the sum of these 

 two controls from the titer obtained in step 3. 



Peptide Nitrogen 



SPECIAL REAGENTS (in addition to those required for measurement of 

 amino acid and ammonia) 



Peptidase Preparation (ammonia free). Seed skim milk contain- 

 ing 5% dextrose with spores of Aspergillus wentii and incubate 

 for 6-8 days at 30°. Culture the mold in 1 1. Erlenmeyer flasks 

 with a 2 cm. depth of medium. A satisfactory growth is obtained 

 when the heavy pad of mold has begun to crack and draw away 

 from the wall of the flask. Remove the pads; weigh, and freeze 

 for several hr. to inactivate any arginase which may be present. 

 Grind the pads in a mortar with sand and add enough water to 

 give a suspension containing about 10% dry matter. (Determine 

 the moisture on a small sample of the pad.) Bring the pH to 7 to 

 7.5 and cover the suspension with toluene. After standing 4-6 hr. 

 at room temperature, filter through sailcloth with the aid of a 

 vacuum. Add an equal vol. acetone to the filtrate, and dissolve 

 the precipitate which forms in one fourth the original vol. water. 

 Centrifuge, and shake the supernatant with permutite to remove 

 ammonia. Store this enzyme soln. under toluene in a refrigerator. 

 The activity will be maintained for months even though the soln. 

 will darken on standing. 



PROCEDURE 



1. Add an equal vol. of a protein-free extract (trichloroacetic 



