Table 11. Determination of Small Amounts of Bromide in Chloride, [CI-] = 6.6 X [Br-] X 106 M
Taken
Found
3.30
3.75 5.70 9.5 14.5
6.60 10.0 13.3
Error
$13.6 -13.6 -5.0
+11.2
C
I
19
0.09
0.19
0.09
0.19
0.09
0.19
0.09
,
0.19
009
I
E, VOLTS vs SCE
Figure 3. Synergistic effect of chloride on the electrodeposition of bromide [CI-] = 6.6 X 10-4M;the bromide concentration for A, B, C, D, and E is 0, 3.3 X 6.6 X 1.3 X and 1.66 X 10-5M,respectively: pre-electrolysis potential, 0.22 V vs. SCE: scan rate, 2 mV/sec; electrode area, 2.22 f 0.07 mm*; electrolysis time, 2.0 min
ditions is'more than 100 mV more negative than this. Depending on the conditions ( i e . , pre-electrolysis potential and chloride concentration), the sensitivity of this version of cathodic stripping analysis for bromide determinations is a t least three times greater than for the simultaneous halogen determination procedure described above. This increase in sensitivity is due to the synergistic effect of chloride on the electrodeposition of bromide. The standard addition method is best suited for bromide determinations of this kind since the reproducibility of the stripping curves from sample to sample is not sufficiently good to allow the use of calibration curves. Table II gives the results of the application of this method to the determination of bromide in synthetic samples. The error of the method is a little over 10%. It should be pointed out that since this method depends very much on the concentration of chloride, determination of bromide in solutions of differing chloride concentration results in dissolution curves of unequal peak currents for the same bromide concentration. Interference by diverse ions in cathodic stripping analy-
sis of halides are caused by species capable of forming insoluble mercury salts. Metallic ions such as copper, lead, and anions such as phosphate have no effect on the deter-, mination of bromide and/or chloride. Sulfide can seriously interfere with the determination, causing severe distortion of the stripping curve. Iodide can also interfere with the determination; however, this interference depends primarily on the halide composition of the sample. In the case of samples rich in chloride, iodide has a negative effect on the chloride peak which is indicative of destruction of the mercurous chloride deposit (14). When the bromide is the predominant species of the sample, iodides cause a displacement reaction similar to the one observed with bromide-chloride binary mixtures (14), and the procedures described above should also be applicable to the determination of trace iodides and bromides in various mixtures. To date, only measurements of bromide and chloride have been performed by this method. However, similar measurements are possible for iodide and bromide mixtures. Additionally, it should be possible to extend this method to certain mixtures of all three halides.
ACKNOWLEDGMENT We are indebted to James Fennessy for his assistance during the course of this work. Received for review December 7, 1973. Accepted March 18, 1974. This work was supported through a grant to the Atmospheric Analysis Laboratory by the Arizona Mining Association.
Immobilized Enzyme Electrode for the .Determinationof Arginase H. Edward Booker and John L. Haslam D e p a r t m e n t of Chemistry, University of Kansas, L a w r e n c e , Kan. 66044
A dual and sequential enzyme catalyzed reaction is utilized for the determination of arginase, an important metabolic enzyme. The progress of the enzymic reaction is monitored by a cation selective electrode responsive to NH4+, a product of the reaction. Experiments indicate a precision of about 3% with a detection range of about 1.6 to 16 units of arginase an'd an average time requirement of less than 10 minutes per analysis. The change in potential with time is measured, and the amount of arginase 1054
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, N O . 8, J U L Y 1974
is determined from a previously prepared calibration curve. The utility of this procedure is exemplified by its use to study several parameters which affect the catalytic efficiency of arginase. The results of studies on the effect of pH, temperature, substrate concentration, and immobilized enzyme concentration using this new potentiometric method for arginase are presented. The possibility of extended application is likewise discussed.
Updike and Hicks ( I ) presented the first description of an enzyme electrode, although the use of enzymes as a functional part of an electrochemical device was probably first reported in 1962 ( 2 ) . Specific potentiometric sensors have since been modified and used by Guilbault for several substrates and enzymes by immobilizing appropriate enzyme arginase. The enzyme arginase (L-arginine ureohydrolase E.C. 3.5.3.1) was discovered in 1901 by Kossel determining amygdalin, in conjunction with a cyanide-selective membrane electrode. The work reported here makes use of the seemingly parallel developments in ion-selective electrode technology and enzyme immobilization technology to uniquely devise a potentiometric procedure for the determination of the enzyme arginase. The enzyme arginase (L-arginine ureohydrolase E.C. 3.5.3.1) was discovered in 1901 by Kossel and Dakin (10) and is found in both animals and plants. While the mammalian liver is the primary source of arginase, i t has also been identified in lesser extent in several human tissues including breast (11) kidney (12), testes, salivary glands, epidermis, and erythrocytes (13). Arginase catalyzes the terminal reaction in the familiar Krebs and Henseleit ( 1 4 ) cycle, converting L -arginine to ornithine and urea. For many years, a significant handicap to the study of the properties of arginase has been the lack of a rapid and completely reliable method of assay for this important metabolic enzyme. Methods of determining arginase activity have included measurement of a decrease in arginine concentration (15-19), an increase in ornithine concentration (20), or an increase in urea concentration and subsequent decomposition of the urea with jack-bean urease followed by manometric (21-24) determination of the COz formed or colorimetric (25-27) or titrimetric (28) determination of ammonia. The more recent methods for determining arginase are cumbersome in that they require either formation and isoS. Updike and G. Hicks, Nature (London), 214, 986 (1967) L. C Clark, J r . , and C. Lyons, Ann. N . Y . Acad. Sci., 102, 29 (1962). G . G . Guilbault and J G. Montalvo. Jr.. Anal. Lett.. 2, 283 (1969) G . G. Guilbault and A . G . Montalvo, Jr.. J. Amer. Chem. SOC., 92, 2533 (1970). G . G . Guilbault and E. Hrabankova. Anai. Lett., 3, 53 (1970) G . G . Guilbault and E. Hrabankova. Anai. Chem.. 42, 1779 (1970) G . G. Guilbault and E. Hrabankova, Anai. Chim. Acta, 56, 285 (1971 ) . G. A Rechnitz and R . Llenado, Anai. Chem., 43, 283 (1971). R A. Llenado and G . A. Rechnitz, Anal. Chem., 43, 1457 (1971) A. Kossel and H. D . Dakin, 2. Physioi. Chem.. 41, 321 (1904). E Baldwin, "Dynamic Aspects of Biochemistry," Cambridge University Press, Cambridge, 1953, pp 317-320. J. Cabello, "La Arginasa Hepatica," Universidad de Chile, Santiago de Chile, 1955, p 49. J. H . Reynolds, J . Follette, and W. N . Valentine, J. Lab. Clin. Med., 50, 78 (1957). A. A. Krebs and K . Henseleit, 2. Physioi. Chem., 210, 33 (1932) S. Sakaguchi, J . Biochem.. 37, 231 (1950). D D . Gilboe and J . N . Williams, Proc. SOC. Exp. Bjoi. Med., 91, 537 (1956) R. L. Ward and Paul A Srere, Anai. Biochem.. 18, 102 (1967) D . D . Gilboe and J . N . Williams, Proc. SOC.Exp. Bioi. Med.. 91. 535 (1956) W F. Loeb and R. A. Stuhlrnan, Clin. Chem., 1 5 , 162 (1969). L R Linderstrorn, L. Weil, and H. Holter, C. R. Trav. Lab.. Carlsberg, Ser. Chim., 21, 7 (1935). L. Weil and M A. Russell, J. Bioi. Chem., 106, 505 (1934) A Hunter and J 6 . Pettigrew, Enzymoiogia. 1 , 341 (1936) D . D . Van Slyke and R . M . Archibald, J. Bioi. Chem., 165, 293 (1946) R . M . Archibald. in "Methods in Enzymology," Vol. I l l , S P. Colowick and N . 0. Kaplan, Ed., Academic Press, New York, N.Y.. 1955, p 1044. A Hunter and J A. Dauphinee, Proc. Roy. SOC. (London), Ser. B, 97, 209 (1924). C. J . Gentzkow. J. Bioi. Chem., 143, 531 (1942) A . Hunter and C. E. Downs, J. Biol. Chem.. 155, 173 (1944) A Hunter and J. A. Dauphinee, J. Bioi. Chem., 85, 627 (1929)
lation of a derivative of urea (29, 30) or time consumed in color development (31). Some of the methods currently in use either lack sensitivity and are too excessively complex for routine use (32-34) or require elaborate, expensive equipment and specialized personnel (35-37). It is for these reasons that a study was undertaken to apply a potentiometric technique using the immobilized urease electrode as the sensing device for the determination of arginase. This procedure is based on a dual arginase-urease system in which NH4f is generated by a simple two step reaction. These reactions are summarized as follows in which L-arginine is first hydrolyzed by arginase
L-arginine
H
0~c-c-(cH,),-NH, I
HO'
I
NH, L-ornithine
+
c=o \ SH,
(1)
urea
to produce L-ornithine plus urea. The urea is subsequently hydrolyzed in the presence of the immobilized urease electrode to produce ammonium ion. This permits continuous, automatic recording of the progress of the enzymatic reaction by monitoring production of the ammonium ion. This procedure represents the first and only example of the use of an electrochemical or potentiometric technique for the assay of arginase. During the course of this work, a paper was published by Rechnitz and Neubecker (38) in which a dual enzyme catalyzed reaction was used in conjunction with an ammonium ion selective membrane electrode to measure arginine concentrations. Another dual enzyme system was reported earlier (22) for the determination of arginine from carbon dioxide production. The work reported here uses a similar approach, but is concerned with the determination of arginase rather than arginine.
EXPERIMENTAL Apparatus. A
B e c k m a n c a t i o n selective electrode w a s s u i t a b l y m o d i f i e d f o r use as a n u r e a responsive electrode by p l a c i n g a thin film o f urease, i m m o b i l i z e d in p o l y a c r y l a m i d e gel, over i t s sensing bulb. T h e p o t e n t i a l w a s m o n i t o r e d us. a n SCE p l a c e d in a 0.1M T r i s / T r i s HC1 s a l t b r i d g e in c o n j u n c t i o n with a B e c k m a n e x p a n d o m a t i c pH m e t e r a n d a V a r i a n s t r i p c h a r t recorder. A B e c k m a n DU w a s u s e d for a l l s p e c t r o p h o t o m e t r i c m e a s u r e m e n t s a n d a F o r m a thermostated water b a t h was used t o control t h e temperature. (29) D. M Greenberg. in "Methods in Enzymology," Vol. 1 1 , S. P. Colowick and N. 0. Kaplan. Ed., New York, N . Y . , 1955, p 368. (30) P. S. Satoh and Y. ito, Anal. Biochem.. 23, 219 (1968). (31) J. W Geyer and D . Dabich,Anai. Biochem., 39, 412 (1971). (32) R . T. Manning and S Grisola, Proc. SOC. Exp. Bioi. Med., 95, 225 (1957). (33) G. M . Ugarte, M . E. Pino, P. Peirano, and E. Marusic, J. Lab. Ciin. Med., 55, 522 (1960). (34) C. E. Cornelius and R . Freedland, Cornell Vet., 52, 344 (1962) (35) W. H. Marsh, B . Fingerhut, and H. Miller, Clin. Chem., 11, 624 (1965). (36) S. Kaihara and H. Wagner, J r . , Anal. Biochem., 24, 515 (1968) (37) P. Righetti, L. D e Luca. and G. Wolfe, Anal. Biochem., 22. 225 (1968). (38) G. A Rechnitz and T. A. Neubecker, Anal. Lett., 5 , 653 (1972). A N A L Y T I C A L C H E M I S T R Y , V O L . 46,
NO. 8,
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Urease electrode response curves for arginase
(Ai 15 75
units, (6)11 82 units, fCI 7 88 units fDI
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( A ) Urease electrode response toward urea (8)Cation electrode resoonse toward NH4+
Chemicals and Reagents. The arginase (L-arginineureohydrolase, EC 3.5.3.1), 21 units/mg, urease (urea amidohydrolase, E.C. 3.5.1.5), 2.5 units/mg, L-arginine, diacetylmonoxime, and tris(hydroxymethy1)aminomethane (Tris) were purchased from Sigma Chemical Company. The thiosemicarbazide was purchased from Fairmount Chemical Company. The polymer and cross-linking reagents used in the preparation of enzyme gels for the construction of the enzyme electrode were obtained from Eastman Organic Chemicals. All other chemicals were analytical reagent grade. Procedure. The requisite experimental conditions for the potentiometric method for arginase were ascertained by the commonly used spectrophotometric procedure in which the urea resulting from arginine hydrolysis is determined directly from its reaction with diacetylmonoxime to form a colored complex (39) which is assayable at XJ30: As applied here, this procedure showed that a minimum period of one-half hour was required for optimum arginase activity when the enzyme is activated with manganese (1.42 mg/ml) in 0.1M Tris buffer, pH 7.0 and stabilized by 0.02M glycine at 25 "C (40).The arginine concentration was 0.06M for all studies. These substances were found to have a negligible effect upon the urease electrode when used at the prescribed concentrations. Preparation and Response of the Urease Electrode. The urease electrode was constructed after the method of Guilbault (3). The performance of the electrodes prepared by this procedure was checked by measuring the potential of known urea concentrations prepared in 0.1M Tris buffer at pH 7.0. Figure 1 is typical of the Nernstian response exhibited by the urease electrode toward urea (curve A ) and for the unmodified cation electrode toward NH4+ (curve B ) . The absolute potential which one measures with the enzyme electrode is dependent upon the concentration of urease utilized during the immobilization. For electrodes used in this study, the urease concentration was controlled at 410 mg per electrode preparation. Such an electrode is linear over the range from 5.10- to 5.10- 5Murea with a 50-mV Nernstian-like slope. Potentiometric Measurement. For a typical experiment, 24 ml of the assay reagent was placed into the thermostated reaction vessel and mixing was initiated by a magnetic stirbar. The calomel-immobilized urease electrode pair was then removed from the O . M , pH 7.0 storage buffer and immersed into the assay buffer. The 0.1M Tris/HCl salt bridge used in conjunction with the SCE minimized any effects from leakage of potassium ion from the KC1 filling solution. With the recorder adjusted to the most sensitive (1 mV full scale) setting, the background potential was recorded with stirring until a steady-state value was obtained. For consistency, the background potential was generally recorded for about 3 minutes. The effects of any urease impurities are includ(39) B Mellerup,Clin Chem 13, 900 (1967) (40) H Edward Booker Ph D Thesis, University of Kan , 1973
Kansas Lawrence
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giving a plot of millivolts as a function of time. A family of curves is obtained in this manner by varying the concentration of arginase. Prior to measuring a solution of different concentration. the electrodes are washed in 0.1M Tris hffer of the same pH as the solution being measured until a low value of the potential is observed. The urease electrode is stored in 0.1M Tris of the appropriate pH between measurements. Standard calibration curves were constructed by one of the following methods. Method I: The Initial Slope Method. A tangent is drawn to the kinetic curve at the initial stage of the reaction. A calibration curve is then constructed by plotting the slope, A E / A T , as a function of enzyme units. Method II: Lapsed Time-Slope Method. A second method eliminates the initial part of the curve in determining the rate of potential change. In this method, tangents are drawn t o the progress curves at 30 seconds. Method III: Timed Potential Method. For the final method presented here, the potential was obtained after a constant time and plotted as a function of enzyme concentration. All calibration curves were constructed based upon units determined spectrophotometrically at pH 9.5. To obtain the exact units produced at pH 7.5, these units should be divided by 11.4.
RESULTS AND DISCUSSION Response Curves. Representative curves for arginase concentrations from 1.58-15.75 units per 25 milliliters are shown in Figure 2. The potential which is observed is proportional to the urea concentration and, therefore, to the concentration of arginase. This potential is, of course, governed by the Nernst equation, which for 25 "C is shown below:
Eobsd = E"'
+ 0.0591 log (urea)
13)
This equation may be differentiated with respect to time to yield Equation 4. dEobsd -
E
dt
1 d(urea) 0.0391 ___ ~(urea) df
When conditions are such that the reciprocal of urea concentration is changing relatively more slowly with time than the time differential of (urea), the rate of change in the potential, dE,,,,/dt, is then directly proportional to the rate of change in urea concentration with time. If this equation holds for the early part of the reaction, a calibration curve may be constructed by drawing a tangent to the enzymic progress curves and plotting the slope (AE/ as a function of enzyme concentration. Proof of the validity of the above equation using the
1 9 1
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Figure 3. Arginase calibration curves, initial slope method ( A ) The immobilized urease electrode contained 410 m g urease/cm3 gel. Circles and triangles represent the data for different identically prepared electrodes ( B ) The immobilized urease electrode contained 205 mg urease/cm3 gel
Beckman cation electrode has been reported. The proof resided in the observation that calibration plots of the change in potential with time were linear with change in concentration for both substrate and enzyme for several systems studied (41). Further verification is provided by this work. Curves prepared by the initial slope procedure are shown in Figure 3 for two electrodes prepared identically and simultaneously and for another electrode utilizing a different amount of urease. Each data point represents the average of triplicate runs. The data represented by curve A are for two electrodes containing 410 milligrams of urease per milliliter of polymer gel. The two electrodes show good agreement and the linear region for these two electrodes is from 1.58 to 39.38 units of arginase. The average deviation for all points in this range of arginase concentration is 10.126 mV/min, giving a precision of about 3%. Data for curve B were obtained with an electrode containing 205 mg urease per cm3 gel. A comparison of curves A and B indicates that a greater linear region is obtained for the electrode containing the lesser amount of urease. However, a greater error (kO.91 mV/min) is obtained using this electrode. In addition, the sensitivity is approximately a factor of two greater (1.7) for the electrode with a larger amount of immobilized urease. The results of assays performed in the same manner, but in absence of substrate, indicated that approximately 20-24% of the initial reaction was not due to the enzymatic reaction but is produced by arginase itself or extraneous monovalent cations. Suitable ion exchange and/or dialysis procedures may be helpful in reducing this potential change. This suggests, however, that the reproducibility of curves A and B will depend upon sample history as well as electrode history. While this poses a limitation for the use of the initial slope procedure for the assay of arginase from undetermined sources, it does not distract from its use for samples whose history is known. Furthermore, if purer enzyme were used in construction of the calibration curve, contributions to the initial slope would possibly be negligible allowing the use of one calibration curve for identically prepared electrodes. An assay conducted in the absence of substrate indicated that 96% of the steady-state potential is obtained after 15 seconds and 98% a t 30 seconds. Curve A of Figure 4 represents data obtained by drawing tangents to the enzy(41) G . G. Guilbault, R K . Smith, and J. G . Montalvo. Jr., Anal. Chem., 41, 600 (19691.
Arginase, units
Figure 4. Arginase calibration curves, lapsed. time slope method and timed-potential method ( A ) Data points were obtained by constructing, tangent's to the curve after the initial 30 seconds. (S) Data points were obtained by takin$ the potential after a constant time of 30 seconds.
matic progress curves a t 30 seconds after addition of arginase. Most of the contaminants which produce an electrode response in the arginase sample will appear during this 30-second t i d e period. Construction of the calibration curve by this procedure resulted in a considerable diminishing of the sensitivity and applicable range compared to the initial slope method. This procedure does have the advantage, however, of being applicable to almost any sample regardless of its history and can thus be used for purity determinations as in preparative enzyme studies. This is true because only the chemical reaction will be measured a t 30 seconds or longer. Each data point in Figure 4 for curve A represents the average of triplicate analyses for each of two identically prepared urease electrodes containing 410 mg of urease per cm3 of gel. The average deviation of points in the linear region of the curve is k0.05 mV/min, which corresponds to 0.3 unit of arginase or about a 2% error. A typical calibration curve obtained by Method 111 is shown by B of Figure 4. A constant time of 30 seconds was chosen for reading the potential, although any time greater than this during which the reaction is still linear would also be suitable. This method, by relying on an absolute potential, has the same disadvantages as Method I and, therefore, is not recommended as a general procedure for preparing calibration curves. The procedure can be used, for example, to follow changes in enzyme activity due to denaturation when working with samples having the same history. The average deviation for all data points on curve B is f0.19 mV/min, which corresponds to 0.2 unit of arginase. Because of its independence of sample history and comparatively good precision, Method I1 is the only method recommended for preparation of calibration curves for the assay of arginase by this potentiometric technique. This procedure was used in all subsequent studies which were performed to demonstrate the utility of the potentiometric method for arginase determination. As with any enzyme assay, a knowledge of the kinetic parameters governing the enzymes' activity was of interest. Results of experiments on the effect of pH, temperature, and substrate concentration as determined by this procedure are discussed below. The experimentally determined pH dependence of the arginase-catalyzed reaction is shown in Figure 5 for a 0.83-mg sample of arginase prepared in 0.1M Tris of the ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 8, J U L Y 1974
1057
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.
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-
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0
10
20 I
30 I
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50 1
60 I
,M x
(ARGININE)
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I
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Figure 7. Substrate concentration dependence of the arginase catalyzed reaction 7.0
7.5
8.0
8.5
9.0
Held constant was 1.24 m g of arginase per 25 ml total reaction volume at p H 7.5 and 25 "C
9.5
P"
Figure 5. Effect of pH upon the potentiometric arginase determination. Measurements were made at 25 O C in 0 . l M Tris buffer and a substrate concentration of 0.06M at 25.0 mi. The salt bridge solution was replaced with 0.1M Tris of the appropriate pH for each study
30
t
-1
I
t
25
41
30
35
40
45
50
Temp e r a t u r a ,t
Figure 6. Effect of temperature upon the potentiometric assay of arginase Data taken are for 0.083 mg arginase per 25 rnl with an arginine concentration of 0.06M. All studies were performed in 0.1MTris buffer at pH 7.5
Consideration must also be given to the pH optimum of the immobilized urease. An optimum pH of 6-7 has been reported by Fishbein et al. (46) and Gorin et al. (47), respectively, for urease. The stability and activity of urease drops off rapidly at pH values much greater than this. This factor may be contributing to the observed drop in rate above pH 9.0 in Figure 5. For these reasons, a compromise pH of 7.5 was chosen for all subsequent studies. The results of studies on the effect of temperature on the potentiometric method for arginase are shown in Figure 6. Proper interpretations of these data must take into consideration the temperature effect on the immobilized urease used to measure the arginase reaction. The optimum temperature for urease with respect to both stability and activity is 25 "C. Arginase, on the other hand, is reported to be very stable with respect to heat (29) and the temperature optimum is reported to be 50 "C (48).Figure 6 shows an increase in the rate of reaction with an increase in temperature over the range 25-40 "C. The maximum rate is obtained a t a temperature of 40 "C. The 10 "C lower temperature optimum observed and the rapid drop above 40 "C may be attributed to the denaturation of urease. This inactivation appears to be reversible in that about 94% of the expected potential was obtained when an 8.33 x 10-2M solution of urea was measured with the electrode following the temperature study. However, in order to achieve maximum stability and performance of the urease electrode, a temperature of 25 "C was used for all other studies. The concentration of substrate is also an important factor affecting the rate of a given enzymatic reaction. The correlation between enzyme activity and substrate concentration is given by the Michaelis-Menten (49) equation shown below. In this equation, V, is the initial rate of reaction, Vm,, is the maximum attainable rate for a given set of conditions, K , is the Michaelis constant and [SI is the substrate concentration.
appropriate pH buffer. The maximum rate occurred a t a pH of 9.0 using our potentiometric procedure. This pHactivity profile has the same general shape as that reported for cobalt(I1)- or nickel(I1)-activated arginase (42), while the optimum p H for manganese(Jl)-activated arginase is reported by Bach and Killip (43) as 9.2 and Roholt and Greenberg (44) as 10.2 with most standard assay procedures being performed at a pH of 9.5 (45). Unfortunately, at this pH most of the NH3 produced in the hydrolysis of urea will remain in this form and only a small fraction will be converted to NH4+ ion, which is the form necessary to give a response at the ammonium ion electrode.
Km values relate to substrate concentration and are useful in predicting the range a t which enzyme-catalyzed
(42) D. M. Greenberg, in "The Enzymes." Voi. I , Part I I , J. 6 . Sumner and K . Murback, Ed., Academic Press. New York, N.Y., 1951, p 893. (43) S. J. Bach and J. D. Killip, Biochem. J., 77. 7P (1960) (44) 0.A. Roholt, J r . , and D. M. Greenberg, Arch. Biochem. Biophys.. 62, 454 (1956). (45) "Worthington Enzyme Manual," Worthington Biochemical Corporation, Freehold, N.J , 1972, p 149.
(46) W. N. Fishbein, T. S. Winter, and J. D. Davidson, J. Bioi. Chem., 240, 2402 (1965). (47) G . Gorin, E. Fuchs, L. G. Butler, S. L . Chapin, and R . T Hersh, Biochem.. 1, 911 (1962). (48) D M . Greenberg and Mostafa S. Moharned, Arch. Biochem., 8, 365 (1945). (49) L. Michaelis and M. L. Menten, Biochem. Z.,49, 333 (1913).
1058
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ANALYTICAL CHEMISTRY, VOL. 46, NO. 8, JULY 1974
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-
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1
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reactions are analytically practical (50, 51). The effect of substrate concentration on the potentiometric arginase assay is shown in Figure 7. Enzyme conbentration was held constant a t 1.24 mg while the substrate concentration was varied from 0 to 70mM. The initial rate increases with arginine concentration until the enzyme is saturated and additional substrate causes no further increase in the rate. A Lineweaver-Burk (52) plot of the reciprocal of the initial rate us. the reciprocal substrate concentration is shown in Figure 8. The linear plot gives a slope equal to K,/V,,, and an intercept equal to 1/Vmax. The Michaelis constant calculated from the slope and intercept is 1.26 x 10-3M and is in very good agreement with a value of 1.5 x 10-3M reported by Greenberg (44) for which the determination was made by a spectrophotometric procedure. It should be possible to extend this potentiometric procedure to the analysis of arginase in serum samples, thus providing a convenient means for diagnosis of liver disorders. This possibility exists because the serum of normal persons (39) and persons with other diseases but no liver disease show no significant arginase activity (33). On the other hand, patients with cirrhosis of the liver or hepatitis show serum arginase levels ranging from about 7-16 pmoles urea per ml of serum (33).This range can be easily adjusted to fall within the region for application of this technique. It is of interest that arginase has greater hepatocellular specificity in mammals than the transaminases and dehydrogenases which are commonly measured as a means of diagnosing hepatic diseases (33). In addition, these enzymes are so widely distributed that injury to other organs such as the heart, muscles, or pancreas may also increase their serum activity. Arginase, on the other hand, is present largely only in the liver and increased serum levels are therefore of value in signaling specific liver disorders. Of the ions present in serum, sodium and potassium will be the most serious interferences in a procedure which uses a cation-specific electrode. Consequently, experiments were performed to determine the tolerance level of sodium on the assay procedure. Sodium ions (prepared in O.lM, pH 7.5 Tris buffer) were added to enzyme solutions (1.62 mg/ml) such that the ion's concentration as NaCl varied from 3.5 x to 3.5 x 10-lM. The subsequent assay was conducted in the usual manner-ie., 1.0 ml of the enzyme solution was injected into 24.0 ml of the assay buffer reagent after recording the background potential and the enzymatic response curve was recorded. (50) G. G. Guilbault. Anal. Chem., 42, 334R (1970). (51) G. G. Guilbault. in "Enzymatic Methods of Analysis." Pergamon Press, New York, N Y . , 1970 (52) H . Lineweaver and D J. Burk, J. Arner. Chem. SOC.,56, 658 (1934).
Figure 9.
Effect of sodium ions upon the potentiometric arginase
assay Measurements were made for 1.62 mg/ml arginase in 0.1M Tris buffer, pH 7.5 at 25 "C
Tangents were drawn to the kinetic curves after subtracting the initial 30 seconds. The results of these experiments are presented in Figure 9. The dashed line represents the potential expected for unadulterated arginase. There appears to be no effect upon the response for concentrations of sodium ion less than 10- 4M. The sensitivity of detection a t 3.5 X 10- *M sodium is about 82% of maxima and decreases by about 17% per decade increase in salt concentrations up to 3.5 x 10-IM. The enzymatic reaction becomes obliterated at higher salt.concentrations under conditions where ammonium ion production is small. The average salt content of serum is approximately ?/z this value (53) while the average content of urea in serum is 2-7 pmoles per ml ( 5 4 ) . If necessary, these levels can be diminished or eliminated by a combination of dialysis and/or ion exchange procedures, thus making the method directly applicable to serum samples.
CONCLUSIONS Compared with the spectrophotometric procedure generally used for the assay of arginase, this potentiometric technique offers a great advantage in terms of the time required per assay. However, in the spectrophotometric procedure, the enzymatic reaction can proceed for longer times and, therefore, smaller amounts of enzyme are required; thus the spectrophotometric procedure can be made more sensitive than the potentiometric method. This comparatively lower sensitivity of detection is the main drawback to the potentiometric procedure. The potentiometric procedure, on the other hand, has the advantages of not requiring blanks, thereby conserving the enzyme and reagents, and of requiring fewer reagents, thus being less cumbersome. Another advantage offered by the potentiometric method is the capability of using the immobilized urease electrode repeatedly and continuously for several analyses. A still further advantage of the potentiometric method is its ease of adaptability to automation, thus allowing for continuous assays. The equipment required is also simple and inexpensive and may be found in most clinical and analytical laboratories.
( 5 3 ) "Handbook of Clinical Laboratory Data," 2nd ed.. The Chemical Rubber Co.. Cleveland, Ohio, 1968. (54) E. Bernt and H U. Bergmeyer, in "Methods of Enzymatic Analysis," H. U. Bergmeyer, Ed., Academic Press, New York, N . Y . , 1963, p 405. A N A L Y T I C A L C H E M I S T R Y , V O L . 46, N O . 8, J U L Y 1974
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ACKNOWLEDGMENT The authors wish to thank Ralph N , Adams for many helpful discussions and suggestions during the course of this work.
Received for review June 8, 1973. Accepted March 5, 1974. Acknowledgment is made to the University of Kansas’ General Research Fund for partial support of this research.
Absolute Determination of Atmospheric Halocarbons by Gas Phase Coulometry Daniel Lillian and Hanwant Bir Singh Department of Environmental Sciences, Rutgers University-The
State University of New Jersey, New Bruns wick, N.J. 08903
Gas phase coulometry was evaluated and used for the measurement of ambient halocarbons since the method permits operation at maximum sensitivity and is absolute. Ionization efficiencies were determined for 14 compounds to range from less than 0 to 90%. Reciprocal efficiencies were verified to increase linearly with flow rate as predicted by a stirred tank reactor model. At intermediate efficiencies (40 to 60%), coulometric measurements of CC13F and CC14 exhibited a constant error of about 25% over a representative concentration range. At efficiencies greater than 90%, the per cent error in coulometric analysis was less than 5% compared to primary standards. Compounds identified and measured coulometrically in the New Brunswick, N.J., area were CCI3F, CHd, CH3-CC13, CC14, CHCI=CCIz, and CCIZ=CCIZ.
Under optimum conditions of near 100% ionization in an electron capture (EC) detector, strongly electron-absorbing compounds produce a response of such great sensitivity that femtogram gram) analysis has been suggested ( I ) . In an ongoing study of the formation and decay of atmospheric halocarbons, present in concentrations less than (v/v), a coulometric gas chromatographic method of analysis based on gas phase electron absorption has been evaluated and used for measuring the ambient concentrations of several such compounds. Developed by Lovelock ( 2 ) , this method is based on a 1:l equivalency a t 100% ionization between the number of solute molecules in a carrier stream to the number of electrons absorbed by them in the EC detector. Accordingly, the solute concentration can be calculated directly from the number of electrons absorbed. At ionization efficiency of less than 10070, the use of two identical detectors in series enables one to determine the fractional ionizations in the EC detector and thereby maintain the absolute nature of analysis by correcting for the unionized molecules. When operated coulometrically at 100% ionization efficiency, an EC detector is a t its maximum sensitivity since all solute molecules are “counted.” Since the method is absolute, sources of mixing and contamination errors inherent in the preparation of extremely dilute calibration mixtures are precluded. An additional advantage of this mode of operation, particularly in air chemistry studies, is the ability to determine the cbncentration of an unknown compound and possibly deduce its identity from spatial (1) J. E. Lovelock, in “Gas Chrornatatography 1968.” C. L. A. Harbourn, Ed., Elsevier, Amsterdam, 1969. (2) J . E. Lovelock, R . J . Maggs, and E. R . Adlard, Ana/. Chem., 43, 1962 (1971).
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and temporal distributions of concentrations. Furthermore, rate of thermal electron attachment is an excellent aid for confirmation of identity when used in conjunction with retention data. The compounds selected initially for an evaluation of coulometric application were: CC14, SFs, CC13F, CBrFzCBrF2, C C I F C C ~ ~ , CHJI, CC12F2, CHC13, CH3-CC13, CC12F-CClF2, CHCl=CC12, trans-CHCl=CHCl, CH2= CC12, and CHzC12.
THEORY Consider two identical EC detectors in series and let their signals be X I and X2 in coulombs due to an injection of a plug of W moles of compound AB. If p is the fractional ionization of AB, at high ionization efficiencies and low solute concentrations, one can write:
Xi
Xj
= PQ
(1)
- PQ)
= P(Q
p = 1 - - x2
(2)
(3)
XI
where, from Faraday’s law, CJ is equal to 96,500 W . The gram moles of solute W in the EC detector are, therefore, from Equations 1 and 3
W =
XI 96,500(1
-
2)
(4)
For an injection of V ml of sample a t t “C, the volumetric mixing ration CAB is: CAB
=
8.5 X
X
v(1-
(273
+ t)(X,)
(5)
$2)
With a recorder, the signals and x2are simply the respective chromatogram areas in coulombs. Ionization efficiencies can theoretically be predicted by considering the EC detector as a stirred tank reactor in which molecules undergo pseudo-first-order reactions with electrons present in large excess. For a carrier gas passing through the detector at constant flow rate Fo simple mass balance gives:
where K is the first-order rate constant and V d is the detector volume. Verification of the model offers the possib i l i t ~of determining optimum parameters for coulometric analysis.
