in the number of reactive sites or the dissociation of a urease-inhibitor complex with the inhibitor assumed to be of natural origin. With the recognition that potassium ion can function as an inhibitor and phosphate ion as an activator of urease it appears that neither of the above explanations are correct and that the dilution effect is simply the consequence of a change in the relative concentrations of enzyme, activator and inhibitor.18s19
ExperimentalReagents.-The stock 0.1, 0.2, 0.3, 0.4 and 0.5 $1 buffer solutions were prepared from reagent grade dipotassium hydrogen phosphate and potassium dihydrogen phosphate and from the corresponding sodium salts. In every case irrespective of the concentration of the buffer, the $H of the solution after final dilution was 7.0 =t 0.02 a t 25'. A 1.0 M stock solution was prepared daily from urea which had been recrystallized from ethanol. The crystalline urease was prepared from Arlington jackbean meal by the method of DouncelBO all operations subsequent to the initial extraction being conducted at 5'. The thrice recrystallized urease obtained from 400 g. of meal was dissolved in 5 ml. of water 1% saturated with hydrogen sulfide at 0' and this stock solution stored a t 5'. The water used for the dilution of the enzyme stock solution was also 1% saturated with hydrogen sulfide a t 0'. The water used for all solutions was redistilled from an all-glass apparatus.Procedure.-In general the procedure used w a 5 a modification of that described by Van Slyke and Cullen9 in which the aeration step was eliminated and the ammonia determined by the method of Conway.*l In practice 2.0ml. aliquots of one of the above buffer solutions were placed in eight 5.0-ml. volumetric flasks, 1.0 ml. of 0.016, 0.020, 0.028, 0.032, 0.040, 0.060, 0.10 and 0.20 M urea solution added to successive flasks and the latter placed in a bath at 25 * 0.02'. After thermal equilibrium was obtained 0.78 (18) 0. H. Straus and A. (21) E. J Conway, "Micro-diffusion Analysis and Volumetric Error," D Van Nostrand Co , New York, M. Y , 1940, p 73ml. of a diluted enzyme solution was added to each of the above solutions and the mixtures vigorously stirred with a rod kept in each flask. After 3 minutes 0.5 ml. of 2.0 N sulfuric acid was added to each flask, the solution again stirred, the flasks withdrawn from the bath, the stirring rods washed and the volume of solution in each flask made up to 5.0 ml. The diluted enzyme solutions were prepared so as to contain approximately 1 microgram of protein nitrogen per ml. of solution. These solutions which were 0.01 M in the appropriate buffer were allowed to stand for 5 hours a t 25' prior to use. For the determination of liberated ammonia 1.0-ml. aliquots of approximately 0.005 N hydrochloric acid containing Tashiro indicator*' was placed in the central chamber of a Conway dish, a 1.0-ml. aliquot of one of the above 5.0 ml. solutions placed in the outer chamber, the lid, lubricated with glycerol containing sodium hydroxide, placed in position so as to permit the rapid intr...