Diverse Ions. The effect of many diverse ions was studied using the procedure described in the experimental section for the analysis of 4 ppm of bismuth. An error of less than 2.5 was considered negligible. The results of this study are summarized in Table 111. Iron(II1) and mercury(I1) are reduced by the ascorbic acid and can be tolerated only in small amounts. Iron(I1) slowly reduces the heteropoly acid to heteropoly blue, but if the samples are measured very soon after preparation, small amounts of iron(I1) can be tolerated. Apparently the reduction by iron is considerably slower than that by ascorbic acid and has only a small effect on the rate. Fluoride interferes seriously, probably by complexing with the Mo(V1). Chloride is the only other important anion interference but no explanation for its interference can presently be offered.
Advantages of the Rate Method. The reaction rate method has many advantages over conventional spectrophotometric methods using similar chemical systems. In the rate method it is not necessary to make absorbance measurements at a very carefully selected time in order to avoid or compensate for blank absorption. The presence of other materials that absorb at the measurement wavelength or that impart a turbidity to the solution will not normally interfere in the reaction rate method. These advantages coupled with the excellent precision and good sensitivity obtainable should make this a very useful method. RECEIVED for review December 23,1968. Accepted January 30, 1969.
Use of Ion Selective Electrodes in Enzymic Analysis. Cation Electrodes for Deaminase Enzyme Systems G. G. Guilbault, R. K. Smith, and J. G. Montalvo, Jr. Chemistry Department, Louisiana State Unicersity in
New Orleans, New Orleans, La. 70122
The response, selectivity, and use of the 3 9 0 4 7 and 39137 Beckman cationic electrodes for the determination of N H 4+ activity are reported. Both electrodes were found to respond to NH4: in the concentration range 10-1 to lO-5M with a deviation of f 0.5 mV in the 10-1 to 1O-M region and =t20 mV at 10-4 to lo-5M region. The response characteristics of the micro 3 9 0 4 7 electrode were found to be Ag+ > H+ > K+ > NH4+> Na+ > Li+>>Mg*, Ca2+. Those of the 39137 electrode were Ag+ > K+ > H+ > NH4+? Na+ > Li+ > Mgz+, Ca2+. At a pH of 7 the only serious interferences in the assay of [NH 4+] are Ag+ and K+. Both electrodes were used for the assay of deaminase enzyme systems: u rea-u rea se, g luta m ine-g Iu ta m in a se, as pa ragi neasparaginase, D- and L-amino acids-amino acid oxidases. All substrates and enzymes could be assayed with a precision and accuracy of about 2.5%.
CATION-SENSITIVE glass electrodes ( I , 2) are finding widespread use in analytical chemistry. Excellent reviews on ion selective electrodes and their applications have been prepared by Rechnitz (3, 4). Beckman ( 5 ) markets a cationic electrode (39137, formerly designated 78137) that has a reported selectivity H+ > Ag+ > K*, NH4+ > Na’ > Li+ >> Mgz+, Ca2+. This electrode has been used for the determination of sodium ions in acidic silica sol systems (6), for estimating exchangeable potassium in soils ( 7 ) , to study the weak complex formed by sodium ion and malic acid (S), and for the direct determination of alkali (1) G. Eisenman, D. 0. Rudin, and J. U. Casby, Science, 126, 831 (1957). (2) G. Eisenman, Biophys. J., 2, 259 (1962). (3) G. A. Rechnitz, Chem. Eng. News, June 12, 1967, p 146. (4) G. A. Rechnitz, in “Glass Electrodes for Hydrogen and Other Cations,” G. Eisenman, Ed., Marcel Dekker, Inc., New York, 1967. (5) Beckman Instrument Co., Bulletin 7017-a, Beckman Instrument Co., Fullerton, Calif. (6) T. A. Taulli, ANAL.CHEM.,32, 186 (1960). (7) M. M. Mortland, Quarterly Bulletin, Michigan Agricultural Sta., 43, (3), 491 (1961). (8) G. A. Rechnitz, T a h t a , 11, 617 (1964). 600
ANALYTICAL CHEMISTRY
metal ions by a tetraphenylboron titration (9, IO). Rechnitz (11) has studied the response of the cation-sensitive electrode to alkali metal ions in nonaqueous solution, and Jacobson (12)
has developed a method for the continuous assay of potassium in the presence of ammonia with the Beckman cation electrode. Katz and Rechnitz have used the cation-sensitive glass electrode (39137) for the determination of urea (13) and urease (14) via complete conversion of urea to NH 4+ by enzyme-catalyzed hydrolysis. The potential of the solution after hydrolysis is proportional to the amount of NH4+produced and hence to the concentration of urea or urease. Katz (15) applied this method to study the effect of various ions on the urease catalyzed hydrolysis or urea. Although considerable attention has been paid to use of the Beckman cation selective electrode for determination of various cations, a complete study of the selectivity of the electrode towards NH4+ has not been made (16-18). In this paper the results of a thorough investigation of the response, selectivity and use of the cation electrode for determination of NH4+ activity are reported. The application of this electrode to a study of the kinetics of deaminase enzyme systems (urease, asparaginase, glutaminase, amino acid oxidase and amine oxidases) is likewise reported. EXPERIMENTAL
Apparatus. All potentiometric measurements were made with a Beckman research model pH meter. EMF cs. time curves for the dynamic response measurements and enzyme (9) G. A. Rechnitz, S. B. Zamochnick, and S. A. Katz, ANAL. CHEM.,35, 1322 (1963). (10) J. E. McClure and G. A. Rechnitz, ibid., 38, 136 (1966). (11) G. A. Rechnitz and S. B. Zamochnick, Talanta, 11,979 (1964). (12) H. Jacobson, ANAL.CHEM.,38, 1951 (1966). (13) S. A. Kat2 and G. A. Rechnitz, Z. Anal. Chem., 196,248 (1963). (14) S. A. Katz, Anal. Chem., 36, 2500 (1964). (15) S. A. Katz and J. A. Cowans, Biochem. Biophys. Acta, 107, 605 (1965). (16) L. Budd, J. Elecrroanal. Chem., 5, 35 (1963). (17) K. Cammann, Z. Anal. Chem., 216,287 (1966). (18) G. A. Rechnitz and G. Kugler, ibid., 210, 174 (1965).
reactions were obtained by displaying the output signal of the pH meter on a Brown potentiometric recorder. The cation electrodes used in this study were the 39047 micro glass electrode and two 39137 large glass electrodes, one obtained in May of 1968, the second in May, 1967. All electrodes were from Beckman Instrument Co. A conventional Beckman saturated calomel electrode was used in all studies. In those studies involving Ag+ ion a salt bridge filled with tris-HNO3 buffer was used to prevent contamination of the SCE. All measurements were carried out in a thermostated cell at 0.1 "C. 25 Chemicals. Stock solutions of all ions were prepared by dissolving the reagent grade chemicals (Fisher) in tris (hydroxymethyl amino-methane) buffer, 0.1M, pH 7.0. The tris was used both to maintain a constant pH and a constant ionic strength. A series of standard solutions were prepared by suitable dilution of the stock solutions, keeping the ionic strength constant at 0.1M by the addition of appropriate volumes of tris. Enzymes. Stock 1 mg/ml solutions of all enzymes were prepared in triply distilled water. The activity of all enzymes was assayed by standard procedures and was found to be: urease (jack bean, Sigma, Type IV, 2.50 unit per mg), glutaminase (E. Coli, Sigma, 0.5 unit per mg-one unit is that amount which effects the deamination of 1 pmole of glutaasparaginase (E. Coli, mine per minute at pH 4.9 and 37 "C), Sigma, 20 units per mg-1 unit will liberate 1 pmole of L-amino ammonium nitrogen per minute at pH 8.6 and 37 "C), acid oxidase (Crotolus Adamanteus venom, Sigma, Type I, activity 0.3 units/mg), D-amino acid oxidase (hog kidney, Sigma, activity 0.059 units/mg). Substrates. Stock 0.1M solutions of all substrates: urea, asparagine, glutamine, D- and L-tyrosine, proline, and leucine (Sigma) were prepared in 0.1M tris buffer, pH 7.0. Dilutions were made to the optimum substrate concentrations as described below. Procedure. POTENTIOMETRIC METHODS. Potentiometric measurements for the construction of calibration curves and the study of the effect of interfering ions were carried out in the conventional manner. Into the electrochemical cell (50 ml capacity) were placed 25 ml of solution, a cation electrode, and a reference electrode. All solutions were magnetically stirred [bar made of Teflon (Dupont)]. All electrode potentials were rapidly attained, and generally the equilibrium potentials were read within 2.5 minutes after immersion of the electrodes into the test solution. The cation electrode was soaked in triply distilled water between measurements. Electrode response curves were obtained by addition of concentrated solutions of the ions to either triply distilled water or to some concentration of ion tested. The resulting emf cs. time curves were smooth and reproducible after an initial mixing period at constant pH and ion strength. Determination of Urease, Glutaminase, Asparaginase, Dand L-Amino Acid Oxidase. To 24 ml of tris buffer, pH 7.0, is added 1 ml of a solution of the enzyme to be analyzed (urease, glutaminase, asparaginase, D- or L-amino acid oxidase). The 39137 cation selective electrode and calomel electrode are immersed in the solution and the potential is automatically recorded. The potential of the system should correspond to a low level of NH4+,as determined from a calibration curve. A large positive potential indicates the presence of extraneous alkali metals (K+, Na+, etc.) which will interfere, If such a potential is observed, add 1 gram of a cation exchange resin (Dowex 50 or equivalent) and rapidly stir the solution for 5 min. Filter off the resulting solution and add 1 ml of the appropriate substrate solution (urea, glutamine, asparagine, D-proline or L-tyrosine) such that the overall substrate concentration is 10-2M. The change in potential with time, due to production of NH4+,is automatically recorded. The enzyme present can be calculated from a plot of AE/min cs. enzyme concentration.
-2
-3
-4
-4.3
log [":],M Figure 1. Calibration plots of potential (vs. SCE) YS. log NH4+ for three cationic specific electrodes p = 0.1M A - Beckman 39137 electrode (1968) Slope = 52.5 1.5 mV B - Beckman 39137 electrode (1967) Slope = 53.0 k 1.5 mV C - Beckman 39047 electrode Slope = 53.5 k 1.0 mV
Determination of Urea, Glutamine, Asparagine, D- or LAmino Acid. The procedure as described above is followed, except that a 1-mg/ml solution of the appropriate enzyme (40-mg/ml solution of D-amino acid oxidase) is added. At zero time 1 ml of a solution of the substrate to be analyzed (urea, glutamine, asparagine, D- or L-amino acid) is added. Again the change in potential with time, AE/min, is recorded, and the concentration of substrate present is calculated from a calibration plot of AE/min cs. concentration. RESULTS AND DISCUSSION
Response of Electrodes to Ammonium Ion. The Beckma 39047 and 39137 cation electrodes have sensing tips comprised of specially formulated glass (the 39137 electrode is identified with the composition 2 7 z NazO, 4 % A1203and 6 9 z S O n ) which measures the activity of monovalent cations in a manner analogous to pH determination with a glass electrode. The glass electrode tip is a membrane which is sensitive to ionic activity differences. A standard solution within the electrode has a fixed ionic activity, and the unknown solution with which the tip of the electrode comes in contact has an activity, with respect to the ion sensed, which may be either the same or different from that of the internal solution. This activity difference between the internal standard solution and the test solution is indicated as a voltage. The voltage change due to VOL. 41, NO. 4, APRIL 1969
601
Table I. Selectivity Ratios for Various Cations Relative to NH4+ T = 2 5 i 0.10 "C. All potentials were determined in 0.1M tris buffer, pH 7.0, except H+which was determined in triply distilled water KM+/NH4+ Cation 39137 (1967) 39137 (1968) 39047 0.402 0.246 Ag+ a H+ 0.40 0.468 0.404 K+ 0.43 0.416 0.427 Na+ 3.16 4.03 3.94 Li+ 56.5 83.5 95.6 Mg2+ 3.1 x 104 2.6 x 104 1,230 Wis-HNO, salt bridge used to prevent fouling of SCE Tris-HNO, buffer used as supporting electrolyte and solvent
-%
activity differences in test solutions should obey the Nernst equation : 0.0591 EobB= E"' log qC+)(at 25 "C) (1)
I-
+7
A
r 90
60
w 30 v)
z w ~
I-
0
Because the cationic electrodes (39047 and 39137) respond to NH4+,a plot of E cs. log of NH4+concentration should be a straight line with a slope of 59.1 mVat 25 "C. A plot for three such electrodes is shown in Figure 1. Lines A and B are the responses of two 39137 electrodes, one recently purchased (line A) and one a year old (line B). Line C is for the 39047 micro cationic electrode. All electrodes respond to (NH4+) over the concentration range 10-1 to 10-jM. The voltages of the two 39137 electrodes are close to one another, whereas the potential of the 39047 micro electrode is considerably less positive. Evidently, the filling solution of the micro electrode (yellow for the 39047 and colorless for the 39137) and/or the internal reference differs appreciably from that of the macro electrode. Otherwise, Curve C in Figure 1 should agree with Curves A and B. Another 39047 electrode recently obtained from Beckman had a colorless filling solution and give voltages for line C very close to A and B. Both of the 39137 electrodes give excellent reproducible voltages from day to day at concentrations of lO-4M and above (f0.5 mV average). At a concentration of 10-5 to lO-4M higher deviations result (f20 mV). Slightly higher deviations are observed with the 39047 electrode. None of the electrodes give a completely Nernstian response to [NHd-], having slopes of about 53 mV/ decade change in concentration. The hydrolysis of NH4+ ion in solution was considered in calculations of the (NH4+)activity reported in Figure 1 : NH4+
+ H20 e NHI + H30+
=
5.5 X lo-"'.
(2) At a pH of 7 the ratio of (NH4+)/(NH3)is about 180; hence almost all of the NH4+is in the cationic form. Dynamic Response Characteristics. The response characteristics of the ammonium-ion sensitive electrode (39137) were evaluated by exposing the electrode to a rapid change in ammonium ion concentration and recording the resulting emf us. time function. Some typical response curves obtained ( p = 0.1M) are reproduced in Figure 2 . The electrode was soaked in distilled water, then exposed to 10-1 (A), 10-2 (B), 10-3 (C), 10-4 (D), and 5 X lO-5M (E) concentrations of NH4+. Essentially identical curves were obtained upon going from more concentrated to less concentrated solutions. All curves are smooth and of identical shape. The expected emf values were obtained in all cases. The response rates are all very fast, the response half-time, t;, obtained being less than 1 second. The high response rate of this electrode indicates that continuous monitoring of ammonium ion activity in aqueous solutions is possible. 602
ANALYTICAL CHEMISTRY
Kh
Q
-30
D -60
E -90
0
I
I
5
IO
TIME (MIN.) Figure 2. Dynamic response curves of the 39137 cationic electrode to various concentrations of NH4+ u , = 0.1M A - 0 to 1W'M B - 0 to 10-*M c - o to 10-JM D - 0 to 1 e 4 M E - 0 to 5 X lO-'M The pretreatment of the electrode had a pronounced effect on the electrode response. The electrode exhibited a faster, better response if soaked for several days before use in triply distilled water. More drift was observed if the electrode is soaked in ("49 solutions before use, rather than in water. The best potential readings were obtained after about 2.5 minutes, although readings can be taken after about 30 seconds. Effect of Cations on Electrode Response. The response of the Beckman 39137 electrode is reported to be H+ > Ag+ > K+, NH4+ > Na+ > Li+. In order to establish the selectivity of the cationic electrodes, the selectivity ratios, K, defined by Eisenman's simplified Equation 1 : Mz+
=
0.1
E(Ml+ = 0
7
--E(::
I 1.J
=
RT In K . ~ ~ + / . ~ ~ + . (3)
7
were determined for 3 electrodes with respect to NH4+. These ratios are reported in Table I. These results indicate that the selectivity of the cationic electrode varies from electrode to electrode, and may be different from that claimed by the manufacturer. The selectivity ratios of the large cationic electrode, recently obtained [39137 (1968)J are vastly different from those of the small (39047) electrode and even from the large cationic electrode obtained a year ago [39137 (1967)J. The order of selectivity of the small electrode found agrees with that claimed except that the electrode responds better to NH4+than to Na+: Ag+ > H+ > K+ > NH4+ >
-50’ 2.0
3.0
4.0
5 .O
I
I
I
I
to
3.0
4.0
5.0
P Figure 3. Calibration plot of potential (vs. SCE) vs. p [Ag+l at various NH4+concentrations 0.1M A-lO-ZM NH,+ B - 1 0 - 3 ~ NH,,+ c -1 0 - 4 ~ NH~+ D .- 1 0 - 5 ~ N H ~ + / . I=
Na+ > Li+ >> Mgz+. The order of the large electrode is different from that claimed: Ag+ > KT > H+ > NH4+ > Na+ > Li+ >> Mgz+. These observations are consistent with the finding by Eisenman (19, 20) that two different batches of Beckman 78137 electrodes were found to differ both in respect to their electrode properties and the characteristics of ionic diffusion. This indicated that the two batches, although supposed to be identical, must have differed slightly in actual composition (NazO/A1203ratio) and degree of hydration. It should be pointed out that these selectivity ratio differences obtained here with the various electrodes would include the effects of cesium and rubidium ions. Tests of the effect of these cations (Ag+, K+, Na+ and Li+) on the cationic electrode were made over the concentration range of NH4+ ion of 10-2M to 10-jM. The concentration range of the cations was varied from 10-2 to 10-jM. The ionic strength was kept constant at 0.1M and the pH at 7 with tris buffer. Some typical response curves describing the results of this study are given in Figures 3-6 for Ag-, K+, Na+ and Li+-NH4+ solutions. The potential response of the electrode is plotted cs. p ( M + )at various (NH4+) concentrations. The horizontal dashed lines indicate the response expected if (19) G . E. Eisenman, in “The Electrochemistry of Cation-Sensitive Glass Electrodes,” from Advances in Analytical Chemistry and Instrumenration, C. N. Reilley, Ed., Vol. 4, 1965, p 213. (20) “Glass Electrodes for Hydrogen and Other Cations,” G . Eisenman, Ed., Marcel Dekker, Inc., New York, 1967, p 137.
k+l
Figure 4. Calibration plot of potential (vs. SCE) vs. p [K+]at various NH4+ concentrations p = 0.1M A - 10-*M NH4+ B - 10-JM m4+ c - 1 0 - 4 ~ NH,+ D - 1 0 - 5 ~ NH,+
(M+) had no effect on the electrode response. Thus in an analysis of (NH4+) in concentrations above 10-4 either by direct potentiometry or in an enzymic analysis, Li+ would not be a serious interference at concentrations below 5 X 10-3114 and sodium below 5 x 1 0 - 4 ~ .Experimental findings showed that H+ does not interfere below lO-4M(pH 4); above IO-SM (pH 8) interference due to conversion of NH4+ to NH3 is important, K+ and Ag+ are serious interferences and must be removed. Mgz+, Ca2+, and other divalent ions have very little effect. Plots for Mgz+ similar to those in Figures 3-6 yielded calibration plots that agreed with the calibration plot due to NH4+ given above within experimental error. Assay of Deaminase Enzymes. Because of its rapid response to (NH4+), its approximately Nernstian response and good selectivity, the cationic electrode (39137 and 39047) should be an ideal tool for following the activity of deaminase enzymes: Substrate
Deaminase
> NH4+
Katz and Rechnitz (13,14) have studied the use of the 39137 electrode for assay of urea and urease. The potentials recorded after enzymic hydrolysis were proportional to the concentration of urea and urease. In this study we attempted to develop continuous automatic assay procedures for the urease, glutaminase, asparaginase, amino acid oxidase, glutamate dehydrogenase and amine oxidase enzyme systems, for the specific assay of both enzyme and substrate. VOL. 41, NO. 4, APRIL 1969
603
I I)(
-d
-t
IOC
w 100 0
u
vj
v)
B
ui
vi >
w
0 v
Y
Y
v)
3
50
0
L -I
=! I 0
-50
2
4
3
P
5
[Nil
I
2
p =
A
NH4+
c - 1 0 - 4 ~ NH,+ D -1 0 - 5 ~ NH~+
0.1M
- 10-'M
c-io-4~ o - 5 ~
D -i
Urea Table 11. Substrates and Enzymes Determined Electrochemically with the ("49 Sensitive Electrode
Range
Re1 error %
0.5-100 pg/ml 0.0010-0.04 units 0.5-10 Mg/ml 0.0010-0.40 units 0.5-10.0 pg/ml 0.0010-0.40 units 1-40 fig/ml 1-40 pg/ml 10-100 pg/ml 10-100 pg/ml 0.010-0.2 units 0.05-1 .O units
2.0
Substance
I
I
NH4+
B - 1 0 - 3 ~ NH,+
B - 1 0 - 3 ~ NH,+
Urea Urease Glutamine Glutaminase Asparagine Asparaginase L-Tyrosine L-Leucine D-Tyrosine D-Methionine L-Amino acid oxidase D-Amino acid oxidase
I
4
Figure 6. Calibration plot of potential (vs. SCE) vs. p &i+] at various NH4+concentrations
0.1M
A - 10-'M
I
3
P[Li+l
Figure 5. Calibration plot of potential (vs. SCE) vs. p ma+] at various NH4+concentrations p =
-50
Glutamine Asparagine
> NH4+
Glutaminase
> NH4+ > NH4+ > NH4+ > NH4+
Oxidase
Amino acid
5.0 5.0
Amine
3.0 3.0
Glutamic acid
2.5 3.5
Urease
Asparaginase
2.5
2.0 2.5 3.5 4.0
NH,+ NH,+
Oxidase Dehydrogenase
->
NH4+
Initially, before initiation of th= enzymic reaction, a zero potential should be recorded, because the enzyme and substrate are molecular, and not ionic. Then upon enzymic reaction a potential would be observed due to production of NH4+: E = E"' 0.0591 log ("a+) (4)
+
Table 111. Effect of Various Ions on the Urea-Urease Reaction
Urea
=
0.01M
Urease = 0.04 pg/ml
Ion
Conc., M
% Inhibition
Ag+
10-3 10-4 10-5 10-6 10-5 10-6 10-2
100 93.5
Hg*+ Kf
10-3
604
ANALYTICAL CHEMISTRY
63.9
17.5 75 43 82.5 43
Differentiation of this equation with respt -t to time yields:
dE-- 0.0591 dr
1 d(NH4+) (NH4+) dr
~
~
(5)
Assuming that 1/(NH4+) is changing relatively more slowly with time than the time differential of ("49 in the initial stage of reaction, the rate of change in the potential, dE/dt, is then directly proportional to the rate of change in the ("49 concentration with time. Thus, instead of a point by point relation of the potential reading to the log of the (NHd+) concentration for calculation of the progress of the enzymic
Table IV. Effect of pH on the Rate of Hydrolysis of Urea by Urease (Urease) = 0.04 pg/ml
PH 7.0
7.5 8.0
8.5 9.0 9.5
a0
Urea = 0.01M AmV/min 500 500 450 400 375 350
70 60
50
40
30 20 I,
w7
0 30 60amv 90
150
TIME
210
270
(sec.)
Figure 7. Response curves of potential (vs. SCE) vs. time for the urease catalyzed hydrolysis of urea Urea
= O.OlM, pH = 7.0, Tris Buffer, 0.1M A - 0.04 units urease B - 0.016 units urease C - 0.008 units urease D - 0.004 units urease E - 0.002 units urease
reaction, we need only take the initial rate of reaction by drawing a tangent to the E cs. time curves (illustrated in Figure 7 for urea-urease). The validity of the above assumptions and procedure has been proved by Guilbault et al. for enzyme reactions using a platinum indicator electrode (21-24). The same reasoning should hold for the ("49 selective cationic electrode, and proof of the validity of this method lies in the observation that calibration plots of the change in potential with time, AElAt, are linear with change in concentration of both enzyme and substrate in all systems studied. Some results obtained in the use of this method for the analysis of various enzymes and substrates are indicated in Table 11. The urease, glutaminase, asparaginase and D- and (21) G. G. Guilbault,D. N. Kramer, and P. Cannon, ANAL.CHEM., 34, 842 (1962). (22) Zbid.,36, 606 (1964). (23) G. G. Guilbault,D. N. Kramer, and P. Cannon,Anal. Biochem., 5, 208 (1963). (24) G. G. Guilbault, D. N. Kramer, and P. Goldberg, J . Phys. Chem., 67, 1747 (1963).
L-amino acid oxidase systems worked well. Attempts to get the glutamic acid dehydrogenase system to work failed, probably because of the presence of ions in the enzyme. This was indicated by the large potentials observed before initiation of the enzymic reaction and the serious drift problems encountered. All attempts to remove the ionic interferences failed. Similar problems were sometimes encountered in assay of the other enzyme systems, but such problems were generally eliminated by pretreatment of the enzyme and/or substrate with an ion exchange resin. A potential problem can be easily spotted by a pretesting of the potential of the enzyme and substrate with the cationic electrode. If the potential is not indicative of a low level of NH4+, the presence of ionic contaminants is indicated. The only serious interferences at pH 7 are K+ and Ag+ which exhibit a better response to the cationic electrode than NH4+. This effect is demonstrated in Table I11 which shows the effect of these two ions on the urea-urease system as the apparent per cent inhibition. In the case of Ag+ the effect is evidently not only a response of the electrode to Ag+ but also an inhibition of the enzyme. Hgz+, Pb2+, and Cu2+ are inhibitors of urease. The effect of pH on the assay of the urea-urease system is shown in Table IV. A maximum rate of change in potential with time was observed at a pH of 7-7.5. This same pH optimum was observed for all the enzyme systems studied. At higher pH's ( > 7.5) increasing amounts of NH4+ are present in the form of NH3 to which the electrode is not responsive. Methods for the assay of the monoamine and diamine oxidase systems were not initially successful, due primarily to too low an activity of the enzyme (producing too little NH4+ to be measured). Slight rates were observed. Both these enzymes are currently being purified, and will be retested at a later date. A comparison of the sensitivity of the electrochemical procedure described herein to fluorescence methods (25) indicates the electrochemical methods to be about two orders of magnitude less sensitive for the assay of the amino acids and the amino acid oxidases (25). The methods should be quite useful for the assay of the other enzyme systems that liberate NH4+, where sensitive fluorometric methods are not available.
RECEIVED for review October 24, 1968. Accepted December 22, 1968. (25) G. G. Guilbault and J. Hieserman, Anal. Biochem., 26,1(1968).
VOL. 41, NO. 4, APRIL 1969
605
