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

Abstract

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 obtained 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 foaming) is added, and the mixture allowed to stand ten minutes at room temperature (18° or over), three minutes at 40°, or two minutes at 50°. The ammonia is then drawn off by ten minutes' aeration (Folin's method) into 20 c.c. of N/50HCl. 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 K₂CO₃). 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.