Joseph A. C o w a n s and Sidney A. Katz' Rutgers University Comden, N e w Jersey
I
I
Enzyme Activity and hadivation A laboratory experiment
Interest in instrumental measurements has resulted in the development of a potentiometric method for assaying urease (1). A new experiment utilizes this technique to demonstrate the liberation of ammonia by the action of urease on urea and the inhibition of this action by copper(I1) ions. A cationic sensitive glass electrode which develops a potential proportional to the logarithm of the ammonium ion concentration (2, 3) measures the ammonium ions liberated in this reaction. The potential measurements are made with a pH meter, and the meter output is recorded on a strip chart. Enzyme activity is calculated from the amount of ammonia liberated in 5 minutes by 1 ml of urease solution. In the presence of copper(I1) ions the action of the enzyme is inhibited, and, consequently, less ammonia is liberated. To correlate the potential of the cationic sensitive glass electrode with the ammoninm ion concentration of the reaction system, a calibration curve was necessary. A series of solutions from 1 X 10-' to 1 X lo-' M in ammonium ion maintained a t pH 7.0 by a 1 X lo-' M THAM (trishydroxymethyl amino methane) buffer was prepared. The test solutions were maintained a t 25.0°C by immersion in a thermostated water bath. Potentiometric measurements were made on the series using a Beckman 39137 cationic sensitive glass electrode and a 39170 fiber junction calomel reference electrode in conjunction with a Corning model 12 pH meter. Meter output was recorded on a LabLine Graphicorder 10. A plot of the potential against the logarithm of the ammonium ion concentration gave a straight line of the form: E
-
54.8 log [NH,+]
+ 229.4
Several aliquots, 1.00,5.00,and 10.0 ml, of urease solution were brought to a volume of exactly 60 ml with THAM buffer, immersed in the water bath and assayed for activity. The electrodes were immersed in the test solution, and exactly 10 ml of 0.500 M urea solution were added. Potentiometric measurements were made. The amount of ammonia liberated in 5 minutes was calculated with the above equation from the potential recorded 5 minutes after the addition of the urea solution. The activity of the enzyme was expressed in terms of the number of millimoles of ammonia formed in 5 minutes by 1 ml of urease solution. The results of six assays gave a mean activity of 4.55 X + 0.21 X 10-%millimoles NH3/ml. Author to whom correspondence should be directed. Partial finmeid support from the Rutgers University Rsearch Council is gratefully acknowledged.
The inhibition of the action of urease by copper(I1) ions was studied by mixing 10-ml aliquots of urease solution with exactly 50 ml of TEAM buffer containing known amounts of copper(I1) ion and then assaying by the above procedure. Typical data are presented in Table 1. Table 1.
Effect of Copper(l1) on Urease Activity
Activity* retained (%)
[cu(II)]
1.47 X lo-' 7.36 X lo-' 1.47 X lo-'
22.4;21.5;20.9' 15.3,14.4,15.4 9.2,10.3,11.4,6.1
" 100Yo = 0.0455 millimoles NHI liberated in 5 min/ml urease.
To insure that the decrease in ammonia liberation in the presence of copper(I1) ions was due to the inhibition of the enzyme rather than the formation of the tetraammine copper(I1) ion, the potentials of 3 X M ammoninm ion solutions containing varying amounts of copper(I1) ions were measured. The data in Table 2 show that copper(I1) ion does not interfere with the potentiometric determination of ammonia a t pH 7.0 in 1 X lo-' THAM buffera t 25'C. Table 2.
Effect of Copper(l1) Ion on the Determination of Ammonia a t DH 7.0 in THAM Buffer a t 25"
[CU(II)~ OW 1.03 X lo-' 5.15 X lo-' 1.03 X [NH,+I in all solutions
[NH.+] found 3.13 X lo-' 3.03 X 2.97 X lo-* 3.18 X lo-' =
Ammonia recovered (%) 98.2 96.2 93.2 99.9
3.19 X lo-=.
Data presented in Table 1 show that the activity of urease decreases with increasing copper(I1) ion concentration. This observation is in accord with the current understanding of the inhibition effects of metallic ions on enzyme systems (4),and this inhibition of urease has previously been reported by Bahabur and Chandra (6). This experiment, however, is of value in demonstrating the enzyme reaction in the laboratory. Once the student is convinced that he is seeing the liberation of ammonia by the action of urease on urea, the kinetics of the reaction will be of interest. The effects of copper (11) ions on urease activity introduce the concept of active sites. Although this experiment requires the use of a pH meter and recorder, it is superior to the Volume 42 Number 10, October 1965
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conventional =say method which involves quenching the reaction and determining the amount of ammonia liberated by a colorimetric technique (6). This experiment has the advantage of providing a direct and continual measure of the liberation of ammonia by the action of urease on urea under a wide variety of experimental conditions, and is of value in introducing the student to chemical instrumentation for biochemical investigations.
554 / Journal of Chemical Education
Literature Cited (1) Knm, S. A.,Anal. C h m . , 36,2500 (1964). (2) bz, 8. A,, AND RECHNITZ, G. A,, Z. A ~ ~Chem., L . 196, 248 (1963). (3) ~ C E N I T Z G. , A,, J. CHEM.EDUC.,41,385 (1964). (4) DIXON,MALCOLM, AND WEBB, EDWIN,C., "Enzymes," Academic Press, New York, 1964, pp. 345-6. CHANDRA, ",, Proe. Nat. Aead.Sd.,India, ( 5 ) BAHasnR,K,, Sect. A 32 Pt. 1, 72 (1962). (6) SUMNER, J., AND HAND,D., J . Bid. Chem., 76, 149 (1928).
