Histidine ammonia-lyase enzyme electrode for determination of L

Anal. Chem. 1980, 52, 1684-1690

1684

LITERATURE CITED (1) Kobos, R . K.; Rice, D. J.; Flourney, D. S. Anal. Chem. 1979, 5 1 ,

1122-1 125.

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 4, 2015 | http://pubs.acs.org Publication Date: September 1, 1980 | doi: 10.1021/ac50061a035

(2) Jensen, M. A.; Rechnitz, G. A. Anal. Chim. Acta 1978, 701, 125-130. (3) Riechel, T. L.; Rechnitz. G. A. J . Membr. Sci. 1978, 4 , 243-250. (4) Rechnitz, G.A,; Riechel, T. L.; Kobos, R. K.; Meyerhoff, M. E. Science 1978, 199,440-441. (5) Kobos, R. K.; Rechnitz, G. A. Anal. Lett. 1977, 10, 751-758. (6) Rechnitz, G. A.; Kobos, R. K.; Riechel, S.J.; Gebauer, C. R. Anal. Chim. Acta 1977, 94, 357-365. 171 Lessie. T. G.: Neidhardt. F. C. J , Bacteriol. 1967. 93. 1800-1810. (Si Tabor,".; Hayaishi, 0. j . B o / .Chem. 1952, 194,'171-175. ( 9 ) Tabor, H.; Mehler, A. H. Methods fnzymol. 1955, 2,228-233. (IO) Tabor, H. I n "Amino Acid Metabolism"; McElroy, W. D., Glass, H. B., Eds.; Johns Hopkins Press: Baltimore, Md., 1955;pp 373-390. (11) Rechler, M. M. J . Biol. Chem. 1969, 244, 551-559. (12) Mascini, M.; Guiibault, G. G. Anal. Chem. 1977, 49, 795-798. (13) White, W. C.;Guilbault, G. G. Anal. Chem. 1978, 50, 1481-1486.

(14) Taniguchi. S.;Sato, R.; Egani, F. I n "Inorganic Nitrate Metabolism";

McElroy, W. D., Glass, B.. Eds.; Johns Hopkins Press: Baltimore, Md., 1956;p 98. (15) Frankfater, A,; Fridovich, I. Biochim. Siophys. Acta 1970, 206,

457-472. (16) Walters, R. R . ; Johnson, P. A,; Buck, R. P. Anal. Chem.. following paper in this issue.

RECEIVED for review April 4, 1980. Accepted June 9, 1980. This work was s u m o r t e d bv National Science Foundation Grant No. CHE-7i-20491. One of the authors (R.R.W.) acknowledges the John Motley Morehead Foundation for financial support. This work was presented in part a t the 156th Meeting of the Electrochemical Society, Los Angeles, Calif., October 14-19, 1979.

Histidine Ammonia-Lyase Enzyme Electrode for Determination of L- Hist idine Rodney R. Walters,' Patricia A. Johnson, and Richard P. Buck* Kenan Laboratories of Chemistry, University of North Carolina, Chapel Hill, North Carolina 275 14

A potentiometric sensor for L-histidine has been made by immobilizing histidine ammonia-lyase on an ammonia gassensing electrode. The enzyme was purified from Pseudomonas sp. The effect on the electrode response of thiol and metal ion activation of the enzyme was examined. The response of the electrode was linear between 3 X and lo-' M histidine with a slope of 5 4 mV/decade. The sensor showed no decllne In response over one week of use. The response time was 3-8 min. The electrode responded only to histidine and ammonia. The effect of inhibitors of the enzyme reaction on the electrode response was found to be diminished by using hlgh enzyme activities on the electrode. A method for preventing leakage of the Inner filling solution of the ammonia electrode was also presented.

Enzyme electrodes have been developed for several amino acids ( I , 2). An electrode for L-histidine based on the decarboxylase enzyme and a C 0 2 sensor has been reported ( 3 ) , b u t details have not appeared in the literature. Histidine ammonia-lyase (E.C. 4.3.1.3.), which catalyzes the formation of urocanic acid and ammonia from histidine, should be very suitable for use with a n ammonia gas-sensing electrode as a histidine sensor. T h e relatively high optimum p H of this enzyme, approximately p H 9.2 ( 4 ) , should allow the sensor t o be optimal for both the enzyme and gas sensor. Lack of a commercially available source of histidine ammonia-lyase of sufficient activity may have discouraged previous work on this electrode. Several purification procedures have been published, however, in which enzyme of high activity a n d purity was obtained from bacteria (4-8). In this paper the enzyme has been purified from the same bacteria used with the histidine bacterial electrode (9). T h e use of histidine ammonia-lyase presents difficulties. T h e enzyme contains four readily oxidized sulfhydryl groups ( 7 ) and two metal binding sites ( I O ) . T h e activity and kinetic properties of the enzyme depend on both the oxidation state Present address: Department of Chemistry, Iowa State University, Ames, Iowa 50011. 0003-2700/80/0352-1684$01 .OO/O

of the enzyme and the metal activator used (10). In this paper, experiments have been conducted using several different systems for activating the enzyme electrode. T h e analytical characteristics of this electrode are compared with the previously reported bacterial electrode.

EXPERIMENTAL Apparatus. An Orion Model 601A pH meter and Beckman recorder were used for all potential measurements. Solutions were thermostated at 25.0 f 0.1 "C prior to and during measurement with a Beckman thermocirculator and low temperature accessory. An Orion Model 95-10 ammonia gas-sensing electrode was used in construction of the enzyme electrode. The gas-permeable membrane was replaced with a 0.2-pm pore size polytetrafluoroethylene membrane (type FG, Millipore Corp., Bedford, Mass.). Reagents. Bovine serum albumin (BSA), glutaraldehyde (25%), L-histidine hydrochloride monohydrate, and DEAE cellulose (medium mesh) were obtained from Sigma Chemical Co., St. Louis, Mo. Glutathione (reduced) was obtained from Calbiochem-Behring Corp., La Jolla, Calif. Protamine sulfate was obtained from ICN Pharmaceuticals, Inc., Cleveland, Ohio. All other chemicals were reagent grade. Procedure. Enzyme Preparation. Forty liters of Pseudomonas sp. ATCC 11299b (American Type Culture Collection, Rockville, Md.) were grown as described previously (9). The cells were harvested by continuous flow centrifugation, washed with a 0.25% NaC1-0.25% KCl solution, and stored at -10 "C. The enzyme purification was carried out at C-4 "C by a method similar t o the methods of Rechler ( 5 ) and Givot et al. (6). The cells were thawed and suspended in a volume of cold 0.05 M potassium phosphate buffer, pH 7.5, equivalent t o 4 X the weight of the cells and then homogenized in a blender for 30 s. The cells were disrupted in a French press by two passages through the press at 6.2 X 10'-7.6 X lo7 Pa. The cell debris was removed by centrifugation a t 27000g for 15 min; 2.3% protamine sulfate (0.16 mL/mg protein in the enzyme solution) was slowly added with stirring to the supernatant while maintaining the pH a t 7.3-7.5 with a dilute KOH solution. After 30 rnin of stirring, the precipitate was removed by centrifugation at 27 OOOg for 20 min. The supernatant was heated at 78-80 "C for 15 min and then cooled rapidly in an ice bath. The precipitate was removed as before. Ammonium sulfate was slowly added to the supernatant until 50% saturation (313 g/L) was reached while maintaining the pH at 7.3-7.5. After 30 min of equilibration, the precipitate was sepC 1980 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 52, NO. 11, SEPTEMBER 1980

_ _ _ _ _ _ _ ~

Table I. Purification of Histidine Ammonia-Lyase from 50 g of Packed Pseudomonas sp. Cells

step


crude extract protamine sulfate heat ammonium sulfate DEAF, cellulose ammonium sulfate

voltotal total specific ume, activity, protein, activity, mL U mg Uimg 190 219

203 12 500 4

470 430 340 310

130 67

3830 2340 500 85 21

21

0.12 0.18 0.68 3.6 6.3 3.2

arated as before. The precipitate was suspended in 10 mL of the phosphate buffer and dialyzed overnight against 10 L of the same buffer. After dialysis, the enzyme solution was removed from any undissolved precipitate and applied t o a 2.5 X 30 cm column of DEAF, celliilose which had equilibrated overnight with 0.05 M potassium phosphate huffer. pH 7.5. The protein was eluted using a gradient of 20 equal concentration steps (100 mL each) between 0.05 and 0.3 M potassium phosphate, pH 7.6, and a flow rate of 1.0 mL,/min. The enzyme eluted as a broad peak with retention volume 300 to 800 mL. The protein was precipitated by adding ammonium sulfate to 60% saturation and collected by centrifugation at 16 000g for 30 min. The precipitate was dissolved in 4 mL 0.003 M potassium phosphate, pH 7.5, and dialyzed overnight against 10 L of the same buffer. The concentrated enzyme was divided into 400-pL aliquots and stored at -10"C until used. A l.U-mL diquot was lyophilized and stored a t -10 O C . Rnz>,nze Assay. Histidine ammonia-lyase was assayed by the met,hod of Hassall et al. ( 1 1 ) . One milliliter of 0.15 M 2-amino2-tnethyl-1,3-propanediol, pH 9.2; 0.5 mL 0.03 M glutathione; and 0.5 tn1, enzyme solution (diluted if necessary to contain approximate!y 0.03 unit of activity) were mixed and incubated a t 26.0 "C for 15 min. One milliliter of 0.1 M L-histidine, pH 9.2, was added to initiate the reaction. The formation of urocanate was followed by the absorbance increase at 277 nm ( t = 18800). One unit of activity (U)is defined as that amount of enzyme which catalyzes the formation of 1 pmol urocanate/min at 25 "C. ['rotpin Mcasitrement. Protein was estimated by the method of Lowry et al. (12). EIectrode Preparation. Before assembling the ammonia electrode, the inside of the bottom cap, the lower threads of the outer body, O-rings, and the spacer were all greased with Apiezon L iJames G. Riddle Co., Plymouth Meeting, Pa.) to prevent leakage of the inner filling solution. Electrodes were prepared either by placing up to 10 pL of enzyme solution between the gas-permeable membrane and a circular dialysis membrane (Spectra/Por 2, Fisher Scientific Co., Raleigh, N.C.) or by chemically immobilizing the enzyme with glutaraldehyde. In the latter case, 10 pL enzyme solution, 20 pL 15% BSA. and 2 p L 12.5% glutaraldehyde were quickly mixed on the gas-permeable membrane and then air-dried for 25 min before placing in buffer solution. Most electrodes were activated before use each day by soaking for 1 h a t 25 "C in a 6.7 mM glutathione solution (pH approximately 8.8) freshly made by adding the glutathione to 0.1 M Tris buffer, pH 9.2. The electrodes were run in 10.0-mL solutions of histidine in 0.1 M Tris, pH 9.2, to which 10 pL 0.3 M glutathione (freshly made about twice a week and stored at 4 "C) and 5 pL 0.04 M citric acid-0.02 M MnCl, solution were added just before measurement. The electrodes were stored overnight at 4 "C in the 6.7 mM glutathione solution made earlier that day.

RESULTS A N D D I S C U S S I O N E n z y m e P u r i f i c a t i o n . T h e results of the purification, shown in Table I, are similar to those of Rechler (5) in both specific activity and overall yield. Additional purification steps were not undertaken since the enzyme is reported to be stable after the DEAE cellulose chromatography step ( 5 ) and further purification produces little or no increase in the specific activity (5, 6). Enzyme stored a t -10 'C lost approximately half of its activity over a 4-month period. Repeated freezing and thawing

1685

further reduced the activity. As a result, the units of enzyme activity placed on the electrode were only approximate values. T h e lyophilized enzyme appeared t o be stable indefinitely. E l e c t r o d e C o n s t r u c t i o n . T h e Orion ammonia electrode occasionally leaked inner filling solution around the gaspermeable membrane. Over-tightening of the bottom cap often caused the membrane t o wrinkle up. Greasing the components of the electrode with Apiezon L totally eliminated both problems. T h e bottom cap could be gently tightened, b u t yet electrodes soaked in the same solution for weeks showed no sign of leakage of the inner filling solution. After initiating this procedure, none of the electrodes since assembled have ever leaked. In addition, the O-rings have never needed to be replaced. T h e presence of the Apiezon L had no adverse effect on t h e electrode response. We highly recommend this procedure to others using this type of electrode. E l e c t r o d e Optimization. Choosing the optimum conditions for the enzyme electrode required careful consideration of the properties of the enzyme itself. T h e purification procedure converted histidine ammonia-lyase into the oxidized form. Reduction of the enzyme sulfhydryl groups by glutathione, P-mercaptoethanol, or dithiothreitol, in optimum concentration, converts the enzyme t o the more active reduced form (5). However, after activation with thiols, the enzyme must always be kept in a thiol solution or an irreversible loss of activity results (8). Both the oxidized and reduced forms of the enzyme bind t o several divalent cations (10). At a concentration of M, Cd2+and, t o a lesser degree, Mn"+ stimulate the activity of the oxidized enzyme (10). T h e reduced enzyme binds metals more strongly, requiring only M Mn" for activation (10). Assays of our purified enzyme, in agreement with previous literature results, indicated that when glutathione was deleted from the assay mixture, the activity 'was 75% lower ( I O ) , and when Mn2+ was added, the activity increased by 30% (5). It therefore seemed desirable t o add both glutathione and Mn2+ to the solutions analyzed with the enzyme electrode. However, preliminary experiments showed that, a t p H 9.2, t h e glutathione was rapidly oxidized and the Mn2+ rapidly oxidized and precipitated. This latter effect also caused a spurious response by the ammonia electrode. Addition of citric acid a t a concentration twice that of the Mn2+was observed to slow the precipitation enough to prevent interference in the electrode measurements. Low levels of citrate are reported not to inhibit the enzyme (8). Based on the above information, three systems for activating the enzyme electrode were chosen for further study in an effort to maximize the analytical characteristics of the electrode while minimizing any inconvenience or problems associated with the activation. T h e first utilized M CdC12, which could be included in the buffer solutions without precipitation occurring. The second system used 2: x M citric acid and M MnC12,a concentrated mixture of the two being added to the buffer solutions immediately before measurement. T h e final system contained 3 X M glutathione, 2 x M citric acid, and M MnCl,, and again these were added just before measurement (see experimental section). This latter system also required an initial reduction of the enzyme. Glutathione, a t a n optimum concentration of 6.7 mM, is reported t o activate histidine ammonia-lyase within 15 min a t 25 "C (5). At 25 "C, maximum activation of the enzyme electrode occurred in t o 1 h. T h e responses remained more reproducible if the electrode was activated daily, so the electrodes were generally activated for 1 to 1'/, h each day. Each of these systems was studied over a range of p H s around the optimum p H of the enzyme, which is generally

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ANALYTICAL CHEMISTRY, VOL. 5 2 , NO. 1 1 , SEPTEMBER 1980


Figure

CdC12; 3

120 100

-

W60 -

h

p o

v

40

-

20

7.7 8.2 8.7 9.2 9.7 10.2 7.7 8.2 8.7 9.2 97 10.2 77 82 8.7 9.2 9.7 10.2

PH Effect of pH on t h e electrode response. 0.05 M Tris ( 0 )and AMPD (M) buffers were used containing (a) M CdCI,; (b) 2 X M citric acid, M MnCI,; (c) 2 X M citric acid, M MnCI,, 3 X M glutathione. The electrodes were measured on Day 2 first in lo-' M histidine (lower curves) and then in M histidine (middle curves) and finally in buffer (upper curves). Precipitation of Mn2+ interfered with some of the measurements; the dashed lines show potentials based on the initial response of t h e electrode in those cases Figure 2.

reported to be p H 9.0 (5)to p H 9.2 (4). The enzyme is active over a broad pH range, with half-maximal activity reported at p H 8.1 and 9.9 (5). Of the many buffers available in this p H range, most are unsuitable. Pyrophosphate and glycine inhibit the enzyme, probably by chelation of the metal ions (13)or, in the case of glycine, by competitive inhibition (6). Ethylenediamine is also a n inhibitor (14), while diethanolamine ( 5 , 1 5 )was observed to interfere slightly with the ammonia sensor at high concentrations. Sodium carbonate caused rapid precipitation of both of the metal ions tested and thus was unsuitable. The two buffers finally chosen were Tris (7) and 2-amino-2-methyl-l,3-propanediol (AMPD) ( 1 1 ) . These buffers were used to cover the p H range 7.7 to 10.2. Each of the activation systems was tested in two ways using an enzyme electrode containing 10 FL (0.08 U) of purified histidine ammonia-lyase solution. Calibration curves for the electrode were run on three successive days in 0.05 M Tris buffer, p H 9.2, and evaluated for slope, linear range, and reproducibility (Figure 1). Each electrode was also tested on M and 10 * M L-histidine a t 0.5 pH Day 2 in buffer and unit intervals between p H 7.7 and 10.2 using 0.05 M Tris and AMPD buffers (Figure 2). Figures l a and l b compare responses for the Cd2+-and Mn2+-activated forms of the oxidized enzyme, respectively. Both electrodes were linear down to lo4 M histidine, and both showed a decline in t h e response slope on Day 2. T h e responses of each electrode improved on Day 3, apparently as

a result of the exposure to the more extreme p H solutions on Day 2. The Cd2+-activatedelectrode had a slightly larger slope (55 mV/decade average for Days 1 and 3 vs. 50 mV/decade for the Mn2+-activated electrode) but showed much more scatter of points around t h e least-squares fitted lines. This scatter was reproducible and slightly super-Nernstian between 3 x lo-* M and M histidine. I t has been reported that the oxidized form of histidine ammonia-lyase exhibits substrate activation in the presence of lo4 M Mn2+or Cd2+,with the effect most severe with Cd2+and observable between lo4 M and 4 X M histidine (10) (higher histidine concentrations were not tested). The effect of the nonlinear kinetics of the Cd2+-activatedelectrode on the response curve made it the least suitable of the systems tested, although it was the most convenient. M Mn2+activation, Figure T h e reduced enzyme with IC, showed the best overall reproducibility with an average slope of 53 mV/decade and linearity to M histidine or less. The reduced enzyme is reported to show linear kinetics M, therefore when the Mn2+ concentration is over 3 X this electrode did not show super-Nernstian behavior over any part of the calibration curve. T h e effect of the p H is shown in Figure 2. T h e largest response, as determined by the potential difference between the buffer and or lo-' M histidine, was observed between pH 9.2 and 9.7 for all three systems. This result was expected since the enzyme activity exhibits a broad maximum around

ANALYTICAL CHEMISTRY, VOL. 52,

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Table 11. Effect of High pH on Electrode Response E , mV 10-3 M

PH


9.2 9.7 10.2 9.2

buffer

his

Tris

68 61 68 76

AMPD AMPD

Tris

io-* M his

16 11 11 26

AE, mV 52

140

-

120

-

100

-

50 57 50

p H 9.2, while the ammonia sensor is most sensitive a t high p H where ammonia is the dominant species. The pK, of NH4+ is 9.25, so very little added sensitivity could be obtained by increasing the p H above about 9.7 because the sensitivity of the sensor began to level off while the enzyme activity began to decrease. Other problems began to appear at the higher pHs. At p H 10.2, the precipitation of M Mn2+occurred rapidly and interfered with the measurement (Figure 2b). This problem was not observed for the 10-j M Mn2+solutions used with the reduced form of the enzyme. A different problem arose a t high p H for this system, however. At p H 9.7 and 10.2, the potential differences between the and lo-*M histidine solutions were 64 and 69 mV, respectively (Figure 2c). These super-Nernstian responses were artifacts resulting from measuring the lo-* M histidine solutions a t all pHs prior to M histidine solutions. T o demonstrate this, measuring the the alternate measurement method was used, i.e., the electrode was run a t p H 9.2 first in M and lo-* M histidine and then similarly a t p H 9.7 and 10.2 and again at pH 9.2. Table I1 shows that the responses were not super-Nernstian and that exposure to the higher p H buffers caused a decline in the response of the electrode (Le., a shift to higher potentials). This effect was probably a result of an increased rate of oxidation of the enzyme a t high pH. The super-Nernstian response of Figure 2c was thus a result of decreased enzyme activity a t M histidine caused by exposure to high p H while measuring the M histidine solutions. These results indicate that high pH will have an adverse effect on t h e reproducibility of the electrode responses. Therefore p H 9.2 was chosen for further work rather than pH 9.7 where the response span was slightly larger. At this pH, AMPD was the best buffer in terms of buffering ability, but Tris was used because of its ready availability. Figure 2 shows that the responses in both buffers are nearly identical. Figures 1 and 2 show that the reduced enzyme with Mn2+activation gave the best response in terms of reproducibility, linearity, a n d overall range of the three systems tested. This system will be used in all other experiments. T h e effect of buffer concentration was tested using 0.05, 0.1, and 0.2 M Tris buffers (Figure 3). The within-day reproducibility of the electrode, as indicated by the responses in 0.05 M buffer measured a t the beginning and end of the day, was excellent between and lo-' M histidine. Increasing the buffer concentration shifted the calibration curves slightly as the activities of the solution species changed, but the slope remained nearly constant. The linear range no longer extended to lo-* M histidine, but this may have been the result of the better buffering ability preventing a slight enhancement of the response in 0.05 M Tris due to an ammonia-caused p H shift. T h e lack of significant changes in the response curve with buffer concentration indicated that the enzyme activity was not affected by the Tris buffers and therefore the Tris was not inhibiting the enzyme (12). A Tris concentration of 0.1 M was chosen for use in the remaining experiments. This buffer concentration also prevented a small change in p H of the samples upon addition of the acidic glutathione solution. Electrodes were next tested containing 2 FL (0.015 U) and 10 FL (0.08U) of the enzyme solution. A highly concentrated

-80-

>

-E w60-

LO

-

20

-

O t

5

L

3

2

-Log [ L - H I S ] Figure 3. Effect of the concentration of Tris buffer, pH 9.2, on the electrode response. The responses were measured in the following order: (0)0.05 M, ( 0 )0.1 M,(D) 0.2 M, and (0)0.05 M Tris. All the slopes were between 5 2 and 54 mV/decade

enzyme solution was made by dissolving a lyophilized enzyme sample in water in l/lo the original volume; 5 WL(0.8 U) of this was use4 in a third electrode. The electrode containing 0.015 U of activity showed a linear response down to M histidine and a short response time but had response slopes of only 45 mV/decade on Day 1 to 38 mV/decade on Day 4. Use of 0.08 U of activity resulted in an electrode containing approximately the optimal enzyme activity, i.e., producing the maximum electrode response (16). The linear range extended down to 3 X M histidine with slopes of 52 to 53 mV/ decade. This electrode showed a slight decline in the linear range from day to day. An excess (of enzyme, as in the electrode with 0.8 U of activity, did not improve the sensitivity any further but did slightly improve the response times, slopes, and reproducibility. The characteristics of this electrode will be discussed more fully in a later section. Electrodes prepared by immobilizing the enzyme beneath a dialysis membrane were compared to electrodes made by chemically cross-linking a BSA-enzyme mixture on the ammonia sensor. In the latter case, a variety of electrodes were made by varying the amount of glutaraldehyde and BSA used. The system which gave the largest response is similar to that used in a methionine electrode ( 1 7 ) . This system (see the experimental section) was used to make an electrode containing 0.8 U of enzyme activity and compared to a dialysis membrane type electrode containing the same amount of enzyme (Figure 4). The response times of the dialysis membrane electrode varied from 3-8 min compared to 8-12 min for the cross-linked electrode. The potentials reached were nearly identical in both cases, but the cross-linked electrode showed a small decline in response (1-2 mV/day at histidine concentrations of lo-* M) over an eight-day period of daily testing. The cross-linked electrode also required 30-40 min of soaking in buffer to reach the base-line potential after exposure to lo-' M histidine, compared to 20-30 min for the dialysis membrane electrode. The slow response of the cross-linked electrode was due a t least in part to the thickness of the protein layer which, when

1688

ANALYTICAL CHEMISTRY, VOL. 52, NO. 1 1 , SEPTEMBER 1980

ILO

-

120

-

100

-

>

80-

E

I

w I. 0

' 0 0

-

LO

-

20

-

I

I . 5

60

5

10


Time (minl Figure 4.

Time responses of electrodes containing enzyme immobilized

by a dialysis membrane (a) and by glutaraldehyde cross-linking with BSA (b). T h e histidine concentrations are, from top to bottom, lo-' M, 3 X M, M, 3 X M, M, 3 X M, M

swelled, was 500-800 pm thick compared to about 100 pm for the dialysis membrane and enzyme layer. The actual activity of enzyme in the cross-linked electrode may also have been lower due to denaturation of the enzyme during cross-linking or loss of enzyme from the layer while soaking. Response times comparable to the dialysis membrane electrode have been obtained with other cross-linked electrodes (17, 18), so it is possible that this electrode could be improved by varying the amounts of BSA and glutaraldehyde to provide a thinner immobilized enzyme layer which also retains a high enzyme activity. However, our results indicate that, unless one is using a very labile enzyme that can be stabilized by immobilization (19),it is better to use a dialysis membrane and avoid the timeand enzyme-consuming process of optimizing the cross-linking procedure. A dialysis membrane was used in all the experiments reported here. The effect of temperature on the enzyme electrode is shown in Figure 5. Increasing the temperature from 25 to 30 "C had little effect on the response curve, but response times did decrease slightly. A further increase to 35 "C caused a large decrease in the response. The response did not recover fully upon a return to 25 "C. All experiments were thus run at 25 O C to avoid any denaturation of the enzyme. Analytical Characteristics. The stability of an enzyme electrode can be characterized by both its storage stability and operational stability (18). T h e former was determined by measuring the response curves for an electrode containing 0.08 U of activity on Day 1 and Day 28. In the intervening time, the electrode was stored at 4 "C in 6.7 mM glutathione. Figure 6 shows that the electrode maintained a linear response down M histidine and a response slope of 52 mV/decade, to 3 X although the response curves did shift about 12 mV in potential. The operational stability was examined by determining the calibration curve of a n electrode containing 0.8 U of activity at least once each day for 8 days. Figure 7 shows the responses at each histidine concentration. At 3 x lo-' M histidine and above, the responses were nearly constant from day to day with no decline near the end of the 8-day period. On Day 4, the response slope appeared to decrease slightly, but this may have been due to incomplete activation of the enzyme that day. Electrodes containing this much enzyme activity would probably maintain excellent responses for a period of weeks t o months since electrodes containing as much activity showed only slight day-to-day declines in the responses. T h e average calibration curve obtained over the 8-day period is shown in Figure 8. T h e response is linear from 3

O t

5

L

3

2

-Log [ L - H ~ s ] Effect of temperature on the enzyme electrode: (0) 25 C. ' C , (A)35 O C

Figure 5.

(m) 30

5

L

3

2

-Log [ L - H ~ s I Figure 6. Storage stability of t h e enzyme electrode: ( 0 )Day 1 , (m) Day 28. The electrode was stored during the intervening time at 4 OC in 6.7 mM glutathione X 10-5to lo-' M histidine with a slope of 54 mV/decade. The standard deviation of the points averaged less than f 2 mV over this time period. Within a day, the responses typically changed no more than fl mV over the linear range of the electrode. The calibration curve for NH&l in the same buffer is also shown in Figure 8. This response is shifted about 55 mV from the histidine response, indicating that of the histidine present in the bulk solution, only is converted into measurable ammonia because of diffusional or kinetic considerations. This

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Table 111. Change in Potential of the Enzyme Electrode upon Addition of Possible Negative Interferences to a l o - ' M Histidine Solution. Two Electrodes Containing the Indicated Enzyme Activities Were Tested interferences, M all L-tryptophan glucose


urea L -lysine L-arginine urocanic acid elvcine

1

2

3

4

5

6

7

8

Day Figure 7. Operational stability of the enzyme electrode. The electrode contained 0.8 U of enzyme activity and was activated each day for 65-90 min in 6.7 mM glutathione

5

L

- L o g JIL-HISIo r

3

2

[NH;])

Figure 8. Standard calibration curves for the electrode in histidine ( 0 ) and ammonia The standard deviations of the histidine responses 1 over an eight-day time period are shown. The slope was 54 mV/decade for histidine and 58 mV/decade for ammonia

(m).

*

prevents the enzyme electrode from having the lower detection limits one might have expected at this high pH. Enzyme electrode interferences can be of two types: positive interferences (chemicals to which the electrode responds) and negative interferences (chemicals which inhibit the enzyme and prevent a response to the analyte) ( 1 6 ) . The latter interference has seldom been considered in previous enzyme electrode studies even though most enzymes are specific

AE,

0.08 u 0 -2 0 + '2

+4 +6 + 16

mV 0.8

u

-1 -2 -2 0 +1

+4 +12

enough to catalyze only one reaction but may be inhibited by a variety of substances which faintly resemble the substrate or product or which denature the enzyme. Solutions of lo-' M L-tryptophan, L-arginine, L-lysine, glycine, urea, urocanic acid, and D-glucose containing 10-5M L-histidine (added to keep the electrode a t a constant potential) were tested as possible positive interferences. As expected, the enzyme electrode did not respond to any of the compounds. Histidine ammonia-lyase has not been reported to have any other substrates except the synthetic analogues 2- and 4-fluoro-~-histidine(20). Among the compounds tested and found not to react were D-histidine, histamine, 7 other imidazole compounds, L-phenylalanine, L-leucine, L-isoleucine, L-aspartic acid, L-alanine, and L-tyrosine ( 4 , 13). Ammonia would, of course, be a major interference. It is possible that some amino acids or other compounds might interfere since the enzyme of the DEAE cellulose chromatography step is not electrophoretically pure ( 5 ) and thus could contain other deaminases. Should such interferences occur, the enzyme could be further purified to homogeneity using published procedures ( 5 , 6). Possible negative interferences were tested by measuring lo-' M solutions of the compounds tested above in the presence of lo-* M L-histidine using electrodes containing 0.08 and 0.8 U of activity. The shifts in potential from the potential measured in lo-' M histidine alone are shown in Table 111. A shift of more than 2 mV in the positive direction (i.e., toward decreased ammonia concentrations) was considered experimentally significant. Arginine and urocanic acid were minor negative interferences and glycine was a more significant interference of the electrode containing 0.08 U of activity. The electrode with a higher enzyme activity showed decreased interference by these compounds; in fact, arginine no longer interfered a t all. These results can be interpreted by assuming that the enzyme electrode response is nearly under diffusion control, as is the case for an electrode with a very high enzyme activity (21). The electrode response is controlled by the diffusion rates of substrate and product into and out of the immobilized enzyme layer. The response is therefore independent of the enzyme activity, and thus a moderate inhibition of the enzyme will not affect the response. The observed results indicate that the above enzyme electrodes are approaching this ideal behavior. Weak inhibitors, such as urocanic acid (61, have only a slight effect on the response, while a stronger competitive inhibitor, glycine ( 6 ) ,produces only a moderate decline in the electrode response, and this decline diminishes as the enzyme activity in the electrode increases. We conclude from this result that using an excess of enzyme activity in the electrode not only improves the electrode lifetime ( 1 6 ) but also decreases interferences by compounds which inhibit the enzyme. A few other compounds are also known to inhibit histidine ammonia-lyase and thus may be negative interferences of the enzyme electrode. These include EDTA and other Mn'+

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ANALYTICAL CHEMISTRY, VOL. 52, NO. 11, SEPTEMBER 1980

Table IV. Comparison of Histidine Enzyme and Bacterid Electrode Characteristics enzyme electrode response slope linear range response time lifetime interferences

bacterial electrode

52-54 mV/decade 3 x 10-5 to 1 0 - 2 M 3-8 min

more than 8 days ammonia + a few minor negative interferences excellent thaw enzyme and assemble requires daily activation and the addition of a thiol and Mn2+t o each sample

day-to-day reproducibility initial electrode preparation requirements for operation

47-56 mVIdecade 10-4 to 3 x 10-3 M 7-12 min 3 weeks

ammonia + several moderate positive interferences good grow and harvest bacteria and assemble requires only an initial heat treatment to reduce ammonia levels

Table V. Concentrations of Histidine ( 2 3 ) ,Ammonia ( 3 0 ) ,and Urea ( 3 1 ) in Blood Serum


histidine concentration 2x

normal M to 1.7 x

M

elevated above 1.9 x

M

8x -

chelating agents, L-cysteine, and high concentrations of the thiols used for activating the enzyme (22). C o m p a r i s o n of E n z y m e a n d B a c t e r i a l Electrodes for Histidine. Table IV summarizes some of the properties of the histidine bacterial electrode (9) and the enzyme electrode. Both electrodes have a similar sensitivity to histidine, but the enzyme electrode is clearly superior in terms of most analytical properties. T h e purified enzyme required about a week to prepare, but enough enzyme was obtained to make about 70 of t h e higher activity electrodes. With the lifetime of the enzyme electrode, the enzyme supply would be sufficient to last one to several years (the activity would probably be lost during storage faster than the enzyme could be used in electrodes). T h e bacterial electrode required no large inputs of time but did require a small amount of time each day for culturing the bacteria plus the time needed for harvesting the cells for each electrode. In the long term, the enzyme electrode probably would require a smaller input of time than the bacterial electrode. T h e bacterial electrode is the more durable. I t is not harmed by p H or temperature changes, whereas the enzyme electrode requires some care to prevent a loss of activity. I n a given analytical situation, the interferences of each electrode would probably be the decisive factor in choosing which electrode to use. One possible application of a histidine electrode would be in t h e diagnosis of a rare hereditary disorder, histidinemia (23). Methods of diagnosing this condition include measuring histidine by the Pauly diazotization reaction (24),the Knoop reaction with bromine ( 2 4 ) ,a snake venom L-amino acid oxidase method (25),a histidine ammonia-lyase method (13),condensation with o-phthalaldehyde (26),paper chromatography ( 2 4 ) , and amino acid analysis (27). T h e absence of urocanic acid can be measured by paper chromatography (28),and the presence of imidazole pyruvic acid, an alternative degradative product of histidine (23), can be measured by the ferric chloride test (29). All of these methods are either slow or lacking in specificity. Table V lists the typical values in serum for histidine, ammonia (the major interference of both electrodes), and urea (a major interference of the bacterial electrode). Using the maximum normal values for urea and ammonia and the calibration curves for the bacterial electrode in histidine, urea, and ammonia ( 9 ) ,it can be shown that the urea would cause an interference equivalent to nearly M histidine. Hence the bacterial electrode would not be suitable for measuring histidine in serum. With the enzyme electrode, the same approach yields an interference by ammonia equivalent to M histidine. This electrode would marginally about 1.3 x be able to detect elevated histidine levels. Neither electrode

ammonia concn M to 2.4 X

M

3.7 x

urea concn M to 6.7 X

M

would be able to measure normal histidine levels because of the interferences. If some of the interferences could be masked or removed, both electrodes would be useful for measuring histidine levels in the upper normal and elevated range. ACKNOWLEDGMENT T h e authors thank L. L. Spremulli and her students for their assistance in the enzyme purification.

LITERATURE CITED Guiibauit, G. G.; Sadar, M. H. Acc. Chem. Res. 1979, 72. 344-350. Cammann, K. Fresenius' Z.Anal. Chem. 1977, 287, 1-9. White, W. C. Ph.D. Dissertation, The University of New Orleans, New Orleans, La., 1977. Peterkofsky, A. J . Biol. Chem. 1962, 237, 787-795. Rechler, M. M. J . Biol. Chem. 1969, 244, 551-559. Givot, I.L.; Smith, T. A,; Abeles. R . H. J , Biol. Chem. 1969, 244, 6341-6353. Klee, C. B. J . Biol. Chem. 1970, 245, 3143-3152. Frankfater, A.; Fridovich, I . Biochlm. Biophys. Acta 1970, 206, 457-472. WaRers, R. R.; Moriarty. B. E.; Buck, R. P. Anal. Chem., preceding paper in this issue. Klee. C. B. J . Biol. Chem. 1972, 247, 1398-1406. Hassall, H.; Lunn, P. J.; Ryall-Wilson, J. Anal. Biochem. 1970, 3 5 , 326-334. Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275. Hassall, H . Anal. Biochem. 1970, 3 5 , 335-345. Tabor, H.; Mehler, A. H. Methods Enzymol. 1955, 2 , 228-233. Magasinik, B.; Kaminskas, E.; Kimhi, Y. Methods Enzymol. 1971, 778, 47-50. Guilbault, G. G. Compr. Anal. Chem. 1977, 8 . 1-70. Fung, K. W.; Kwn. S. S.; Sung, H. Y.; Guilbauk. G. G. Anal. Chem. 1979, 51, 2319-2324. White, W. C.; Guilbault, G. G. Anal. Chem. 1978, 5 0 , 1481-1486. Klibanov, A. M. Anal. Biochem. 1979, 9 3 , 1-25. Klee, C. B.; Kirk, K. L.; Cohen, L. A,; McPhie, P. J . Biol. Chem. 1975, 250, 5033-5040. Carr, P. W. Anal. Chem. 1977, 49, 799-802. Rechler, M. M.; Tabor, H. Methods Enzymol. 1971, 778, 63-69. LaDu, B. N. I n "The Metabolic Basis of Inherited Disease", Stanbury, J. B., Wyngaarden, J. B., Fredrickson, D. S . , Eds.; McGraw-Hill: New York, 1978; pp 317-327. Greenstein, J. P.; Wink, M. "Chemistry of the Amino Acids", Vol. 3, John Wiley & Sons: New York, 1961; Chapter 27. Baldridge, R. C.; Greenberg, N. J . Lab. Ciin. Med. 1963, 67, 700-707. Ambrose, J. A.; Crimm, A.; Burton, J.; Paullin, K.; Ross, C. Clin. Chem. 1969, 75,361-366. Ghadimi, H.; Partington, M. W.; Hunter A. Pediatrics 1962, 2 9 , 714-728. Levy, H. L.; Baden, H. P.; Shih, V. E. J . Pediah. 1969, 75, 1056-1058. Gerritsen, T.; Niederwieser, A. In "Clinical Biochemistry". Vol. 2, Curtius, H. C., Roth, M., Eds.; Walter de Gruyter: Berlin, 1974; p 1073. Ceriotti, G. In "Clinical Biochemistry", Vol. 2, Curtius, H. C., Roth, M.. Eds.; Walter de Gruyter: Berlin, 1974; p 1124. Toro, G.; Ackermann, P. G. "Practical Clinical Chemistry"; Little, Brown: Boston, 1975; p 148.

RECEIVED for review April 4, 1980. Accepted June 9, 1980. This work was supported by National Science Foundation Grant No. CHE-77-20491. One of the authors (R.R.W.) acknowledges the John Motley Morehead Foundation for financial support.

Histidine ammonia-lyase enzyme electrode for determination of L

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