56 



Scientific Proceedings (56). 



33 (850) 



The preparation of soy bean urease in solid form and its use in 

 urea determination. 



By Donald D. Van Slyke and Glenn E. Cullen. 



[From the Rockefeller Institute for Medical Research, New York.] 



In the course of work in which we have been utilizing the soy 

 bean urease, recently introduced by Marshall into analytical 

 chemistry, we have found it advantageous to prepare and keep 

 the enzyme in solid form. The fine-ground beans are covered 

 with 5 parts of water and allowed to stand an hour with occasional 

 shaking. The extract is then pressed through cheese cloth, and 

 either filtered or centrifuged. The enzyme in solid form is ob- 

 tained from the extract by either: (1) Precipitation, by pouring 

 the extract into at least 10 volumes of acetone; (2) concentration 

 of the extract to dryness at room temperature at a pressure less 

 than 1 mm. The dry powder obtained by either method can be 

 dissolved in a few seconds in 10 parts of water, and the solution 

 obtained is so active that it permits very rapid analyses. Urine 

 (human) is diluted tenfold. Three c.c. (= 0.3 c.c. urine) are 

 mixed in a 100 c.c. test tube with 2 c.c. of an 8 per cent, urease 

 solution. A drop of caprylic alcohol (to prevent subsequent foam- 

 ing) is added, and the mixture allowed to stand ten minutes at 

 room temperature (18 0 or over), three minutes at 40 0 , or two 

 minutes at 50 0 . The ammonia is then drawn off by ten minutes' 

 aeration (Folin's method) into 20 c.c. of N 50HCI. The stoppers 

 are placed in the tubes as soon as the urease is added, and the 

 aeration run a half minute before opening the tube to add the 

 alkali (4 grams solid K 2 C0 3 ). Blood: 5 c.c. of freshly drawn blood 

 are mixed with 1 c.c. of 5 per cent, potassium citrate, 1 c.c. of 

 8 per cent, urease, and 4 drops of caprylic alcohol. Remainder as 

 with urines. If Folin's colorimetric method for determining the 

 ammonia is used, 1 c.c. of blood suffices. Aqueous tissue extracts 

 are brought to a volume of 0.5 to 1 c.c. per gram of tissue, and 5 

 c.c. portions are treated as described for blood, except that only I 

 drop of caprylic alcohol is needed. In case the extracts have been 

 acidified with acetic acid to coagulate proteins, 1 c.c. of 15 per 



