contained as much as 3 to 4% carbonate as determined by potentiometric pH titration. Where applications necessitated the removal of carbonate, the anion exchange procedure was satisfactory.
that the commercial resins used in this study were more stable then the resins prepared by Hale, Packham, and Pepper, or possibly that any alteration of the resin which does occur does so on first contact with the base, with no further changes occurring with successive treatments. This possibility was not investigated. Removal of Other Alkali Metals. I n the one base solution t h a t contained potassium ion in addition to sodium ion, the purification procedure completely removed the potassium as well as the sodium ions. S o other attempts were made t o test the procedure for the removal of other alkali metals, but earlier studies with lithium, sodium, and potassium, plus some studies including rubidium and cesium, indicated sodium is consistently the first of the alkali metals to breakthrough on tetramethylammonium ion forms of Dowex 50 W-X8 and 12 (14). Thus, it can be predicted that alkali metals other than sodium should be removed with an efficiency comparable to or even greater than the efficiency of removing sodium ion. Removal of Carbonate Ion. T h e absorption of C 0 2 from the atmosphere is a well known problem with strong bases, but a n anion exchange resin procedure can be used effectively to remove this impurity ( I S ) . Commercial bases supplied in plastic bottles
Preliminary Polarographic Studies.
Preliminary attempts were made t o use the above-purified TLIAH, TEAH, and TPXH solutions as supporting electrolytes for polarographic analysis. TEAH seemed to work well, in agreement with the findings of Zlotowski and Kolthoff (20), permitting about 1 p.p.m. sodium ion to be detected. TPAH allowed about 5 p.p.m. sodium ion to be detected. TMAH gave undesirably high background currents, but no further polarographic investigations were made. ACKNOWLEDGMENT
The help of Charles E. Forbes with some of the preliminary experiments is acknowledged. LITERATURE CITED
(1) BgFes, R. G., “Determination
of
pH, Wiley, New York, 1964. (2) Bjerrum, J., Schwarzenbach, G., Sillen, L. G., “Stability Constants, Part I, Organic Ligands,” The Chemical Society, London, 1957. (3) Blaedel, W. J., Olsen, E. D., BLIchanan, R. F., ANAL. CHEM.32, 1866 (1960). (4) Buser, W., Helv. Chim. Acta 34, 1635 (1951).
(5) Cundiff, R. H., AIarkunas, P. C., ANAL.CHEM.34, 584 (1962). (6) Hale, D. K., Packham, D. I., Pepper, K. W., J . Chem. SOC.1953, p. 844. (7) Hapeman, R. C., Distillation Products Industries, Eastman Organic Chemicals Department, Rochester 3, N. Y., private communication, 1964. (8) Harlow, G. A., i l N A L . CHEY. 34, 148 (1962). (9) Harlow, G. A., Noble, C. XI., Wyld, G. E. A., Ibid., 28, 787 (1956). (10) Harlow, G. A., Wyld, G. E. A., Ibid., 34, 172 (1962). (11) Helfferich, F., “Ion Exchange,” McGraw-Hill, Yew York, 1962. (12) Kolthoff, I. RI., Lingane, J. J., “Polarography,” 2nd ed., 1-01, 11, p. 423, Interscience, Sew York, 1952 (13) Marple, L. W., Fritz, J. S.,. ~ N A L . CHEM.34, 796 (1962). (14) Olsen, E. D., Sobel, H. R., Talanta 12, 81 (1965). (15) Olsen, E. D., Sobel, H. R., Franklin and Marshall College, Lancaster, Pa., unpublished data, 1962. (16) Peracchio, E. S., Neloche, V. W., J . Am. Chem. SOC.60, 1770 (1938). (17) Rossotti, F. J . C., Rossotti, H., (‘The Determination of Stability Constants,” p. 61, llcGraw-Hillj New York, 1961. (18) Supin, G. S.,Zh. Fiz. Khim. 34, 924 (1960); C.A. 57, 44669 (1952). (19) Walker. J., Johnson, J., J . Chem. SOC.87, 957 (1905). (20) Zlotowski, I., Kolthoff, I. M.,IYD. ENG.CHEM.,ANAL.ED. 14, 473 (1942). RECEIVEDfor review May 14, 1965. Accepted July 15, 1965. Work supported in part by the National Science Foundation (NSF-GP-3482). Division of Analytical Chemistry, 150th Meeting, ACS, Atlantic City, N. J., September 1965.
Preparation of Immobilized Cholinesterase for Use in Analytical Chemistry E. K. BAUMAN AND L. H. GOODSON Midwest Research Institute, Kansas City, Mo.
GEORGE G. GUILBAULT AND D. N. KRAMER Defensive Research Division, Edgewood Arsenal, Md.
b This report describes the preparation of an immobilized (insolubilized) cholinesterase. The enzyme, immobilized by the use of a starch matrix and placed on a urethane foam pad, is stable and active for 12 hours. The substrate solution used, butyrylthiocholine iodide, is stable a t room temperature for two weeks. The activity of the enzyme is monitored electrochemically, using two platinum electrodes and an applied current of 2 pa. As long as the enzyme is active, the electrochemical system will indicate a low potential, ca. 150 mv., because of oxidation of the thiocholine (produced by enzymatic action) to the disulfide at the platinum anode. 1378
ANALYTICAL CHEMISTRY
As the activity of cholinesterase is decreased, the potential will rise to a higher value, 350 to 400 mv. (the potential of oxidation of iodide in the original substrate to iodine).
0
disadvantages of (objections to) the use of enzymes in analysis is the high cost of materials. A continuous or semicontinuous routine analysis using enzymes would require large amounts of these materials, quantities considerably greater than can be reasonably supplied, and quantities that would represent a prohibitive expenditure in many cases. If, however, the enzyme could NE OF THE PRIMARY
be prepared in a n insolubilized (immobilized) form without loss of activity so that one sample could be used continuously for many hours a considerable advantage would be realized. A search of the literature revealed that two primary techniques could be used to insolubilize an enzyme, such as horse serum cholinesterase: Nitz (8) and Bar-Eli and Katchalski (I) have chemically modified the enzymes chymotrypsin, trypsin, and urease by the introduction of insolubilizing groups. McLaren ( 7 ) , Xikolaev (9) and Barnett ( 2 ) , among others, have attempted physical entrapment of the enzymes asparaginase, ribonuclease, and chymotrypsin by adsorption, absorption, or
,,/
THICKNESS ,003
n n
.PLATINUM
CATHODE
uu
Figure 1. Details of enzyme pad, ring, and grid elec:trode assembly
0-
ion exchange. Bernfield and Wan (3) succeeded in the immobilization of chymotrypsin and trypsin in acrylamide gels. Recently, Vasta and Usdin (10) have shown that cholinesterase could be insolubilized by entrapment in a starch gel. This paper describes the preparation of immobilized cholinesterase on a urethane foam pad. The enzyme has been shown to maintain its activity for up to 12 hours. Crood reproducibility and uniformity are achieved in pad preparation. EXPERIMENTAL
Immobilized Cholinesterase. Insolubilized cholinesterase was prepared by a modification of the procedure of Vasta and Usdin (10). Four grams of Connaught starch (Connaught Medical Research Labs, University of Toronto, Canada) is placed into 10 ml. of 0.1M tris(hydroxymethy1)-aminomethane buffer, pH 7.4, and the cool slurry is poured into a boiling mixture of 28 ml. of tris buffer and 2 ml. of U.S.P. glycerine. The resulting mixture is boiled until a clear solution is obtained after which it is covered and
allowed to cool t o 47" C. I n another beaker, 400 mg. of horse serum cholinesterase (Armour and Co., activity 3.0 units per mg.; one unit = 1 pmole of acetylcholine hydrolyzed per mg. of enzyme per minute) is dissolved in 5 ml. of tris buffer, and this solution is poured into the starch solution a t 47' C. The beaker is washed with 5 ml. of tris buffer making a total volume of 50 ml. This enzyme starch solution is gently stirred for 10 seconds, and immediately poured onto 1 sq. ft. of 1/4 inch thick open cell urethane foam (Scottfoam, Scott Paper Co., Philadelphia, Pa.) which had been previously washed with Alconox detergent, rinsed with distilled water, then dried. The enzyme-starch solution is gently worked into the urethane foam with special care to minimize foaming; then the urethane pad is gently squeezed to remove the excess liquid. The pad is set in the refrigerator a t 40' F. for an hour to gel, after which it is placed in a vacuum desiccator (containing no desiccant), and pumped dry overnight with a mechanical pump. The large pad is cut into individual circular pads of 3/4 inch diameter. Each pad contains approximately 12 mg. of starch and three units of cholinesterase. Apparatus. The apparatus used to monitor the activity of enzyme in the urethane pads is indicated in Figures 1 and 2. Figure 1 shows the details of the enzyme pad, O-ring, and disk electrode assembly. The disk electrodes were prepared by punching 1/16-inch diameter holes into a 1-inch circular piece of 0.003 inch thick platinum sheet that has a 3/8 X 1/4 inch handle (available by special order from J. Bishop and Co., Malvern, Pa.). A pad, ,3/4 inch in diameter, prepared as described above, is then placed into a 1 X 3/4 X 1/8 inch O-ring, the electrodes are placed above and below the pad, and the pad and electrodes are placed into a Millipore micro analysis filter holder. The filter holder was held together
500
with the clamp provided with the filter, and the waste was collected in a 250-ml. filter flask. The substrate, 5 X l O - 4 X butyrylthiocholine iodide in 0.1X tris buffer, pH 7.4, is pumped over the pad using a Holter (Holter Co., Bridgeport, Pa., Model R D 134) positive displacement liquid pump, with a delivery rate of 1.0 ml./minute. Air and water were sucked through the enzyme pad by means of a Brailsford blower (Type TD-1, Brailsford and Co., Inc., Rye, N. Y.). The circuitry that was used in conjunction with the electrode assembly is shown in Figure 2. The 24-volt input was provided by a Kepco constant current power supply and the cell voltage was monitored with a Keithley high impedance electrometer and was automatically recorded on a Brown recorder. Alternatively, the current applied could be read using the Keithley electrometer. Using the circuit described, with R1 = 4.7 megohm and Rz = 10 megohm, a current of 2 pa. was observed. Assay of Enzyme Activity. I n all assay operations, the following conditions were used: substrate, 5 X lO-4-V butyrylthiocholine iodide in tris buffer, pH 7.4; substrate flow rate, 1.0 ml./ minute; enzyme, ea. three units of horse serum cholinesterase per pad; air flow, 1 liter/minute; cell current, 2 pa. The following steps were used in assay of the enzyme. INSERTION OF ENZYME PAD. d pad containing enzyme is placed in the 0ring between the two electrodes in the filter holder, and the electrode assembly (Figure 1) is assembled. INITIATION OF OPERATION.With an enzyme-containing pad in place, the liquid and air pumps are turned on. The current is then applied, and the cell voltage is automatically recorded. A steady base potential, usually about 150 mv., will be established in about 10 minutes ( E o ,Figure 3). ASSAY PROCEDURE. To initially check the operation of the electrodes,
-
I i 4% I R2
400
4
6in
d300 2 d I
200
ELECTIlODES
loo C E L L 'VOLTAGE
24 V INPUT
Figure 2. panel
Experilmental apparatus circuitry and control
1 13
00
BLOWER
-
1
14
1
15 TIYE,
16 MINUTES
Figure 3. Typical operation and response curves of experimental apparatus A. B.
Flow r a t e , 1 ml./min. Flow rate, 1 ml./min.
Substrate solution, Off; KI solution, O n S u b s t r a t e solution, O n ; KI solution, Off
VOL. 37, NO. 1 1 , OCTOBER 1965
1379
a second liquid stream of 10-JM potassium iodide is pumped over the enzyme pad at the same time as the substrate. The reference or hydrolysate potential will not be affected. Then the suhstrate stream is switched off. The potential should immediately rise t o a substrate level (the potential of the iodide/iodine couple), 400 mv, if the assay apparatus is operating properly (Figure 3, A ) . If the potential fails t o rise, the current is checked, and, if necessary, the platinum electrodes are cleaned by soaking in 3N nitric acid for an hour, followed by a rinse in distilled water, and conditioning in a constant flow of substrate in the system for 5 to 10 minutes. To check the activity of enayme, the substrate is again passed over the pad. If the enzyme pad is completely active, a reading of 150 mv. will he observed. Any loss of enzyme from the pad will result in a proportional rise in the observed voltage until a reading of 400 mv. is obtained with no enzyme present. REPLACEMENT OF PAD%After 12 hours of operation, the enzyme pads must he replaced. The applied current is turned off, then the liquid and air pumps; and the pad is replaced with a fresh one. The liquid and air streams, then the current, are turned on, and the procedure may be continued as above. RESULTS AND DISCUSSION
Theory of Operation. As long as the enzyme cholinesterase is active in the pad, t h e butyrylthiocholine iodide will be hydrolyzed t o t h e easily oxidizable thiocholine iodide. At a constant current of 2 pa., a potential of ca. 150 mv. (Table I) will be established across the cell assembly pictured in Figure 1. Since the electrooxidation of the thiol takes place at the anode, it is important that the anode be located a t the downstream surface of the pad where the concentration of hydrolysis product is greatest:
Table I. Effect of Cell Current and Substrate Concentration on Cell Voltage (Liquid flow = 0.5 ml./min.) Cell Voltage, mv. current, Substrate Hydmly- Sub(*.) concn., M sate strate 2.0 2.0
x x x
1010-4
in-"
200
150
150 in-* 350 in-' 525 1 x 10-8 605 Flow rate = 1.0 ml./min.
2.0
4.3 8.3 14.0 a
1
5 5
x 1x 1
440 400 400 525 610 6411
PAD B
Reparation of Immobilized Enzyme. The procedure of Vasta and Usdin (20)for immobilization of cholinesterase was unsatisfactory because of B lack of satisfactory air and liquid flow at differentialpressures of less than 6 inches of Hg, and of satisfactory air and liquid to enzyme contact. Preliminary experiments indicated that open cell urethane foam could be used as a support for the starch gel containing enzyme. Using foam pads a/, inch in diameter containinch ing 8% starch gel in a 1 X 3/4 X O-ring (to prevent complete crushing of the pads and to ensure good contact between the platinum grids and the pad surfaces), up t o 2 liters of air and 5 ml. of liquid could be passed over the pads simultaneously, at differential pressures of less than 2 inches of Hg. I n order to gain some idea of the uniformity of the starch (and hence of the enzyme) on the pads, and the resistance of the starch t o desorption by the liquid stream, several pads were stained with a O.lMIr0.1111 K I solution. Photographs of three of these pads are shown in Figure 4. The pads A and B in Figure 4 were prepared as previously described. Pad C w&s not treated with
CHJ I-
\ /
CHsNC-CHrCHrS--CCsH7 CHI
II
+ Hs0
ChE --t
0
CH, I-
\
CHsN+-CHrCH2--SH /
If no enzyme is present in the pad, no thiol will be formed and the potential will rise to that of the iodide/iodine couple, 350 to 400 mv. (Figure 3, E,). Polarograms of 5 X 10-'M substrate and hydrolysis products using grid electrodes revealed that at 2 pa., a voltage change of ea. 200 mv. could be expected; the potentials found agree well with those observed in actual operation. The electrochemical hasis for the mode of operation of this system has been extensively discussed (6, 6). 1380
.
ANALYTICAL CHEMISTRY
PAD A
+ CaH7COO-
starch gel, and is included for visual comparison. Pad A is unused, and illustrates the uniform distribution of the starch gel on the urethane foam. The color after application of the IyKI solution was the typical purple-black of the starch-iodine complex. Pad B was tested in the electrochemical apparatus for approximately 1 hour during which time it was exposed to a flow of 1.5 liters of air and 1.0 ml. of substrate solution per minute. Thereupon, the pad was stained with iodine-KI solution, and
re" c
Figure 4. Photographs of lz.KI stained urethane foam pads before and after use. See text
again the typical purple-black color is observed. This test indicates that much of the starch is held in the pad during the test. Part of the enzyme (about 15%) is washed off the pads in the first 15 to 20 ml. of effluent, and essentially none is lost thereafter. After 12 hours of operation, there is significant activity in the enzyme pads, as indicated by a low potential reading. Addition of 5% glycerine to the starch gel produced pads that were less subject to mechanical damage, and which were able to rehydrate more quickly than pads without glycerine. Also, air drying of the impregnated urethene pads produced pads that were more uniform and higher in enzyme content than those obtained from a freeee drying technique. Electrode Reproducibility. When operation of the test apparatus is initiated, t h e platinum electrodes assume a steady, equilibrium potential in about 10 minutes, and thereafter will reach essentially t h e same potential, ea. 150 my., rapidly after t h e pads are changed. I n 40 hours of operation, no difficulties were encountered with the electrodes, and reproducible potentials and potential changes were recorded. If the electrodes should become contaminated (as indicated hy a poor response), they may
be easily cleaned by soaking in 3N nitric acid. Also, the electrodes were found to function better, if the current is switched off when changing the pads and during nonoperation of the apparatus. Stability of Substrate. Figure 5 shows t h e per cent hydrolysis of butyrylthiocholine iodide as a function of time; the method of Ellman (4) was used for the assay. When the solution is stored under Nz the true hydrolysis rate of butyrylthiocholine iodide to butyric acid and thiocholine is about 870 per day. When the solution is exposed to the oxygen of the air, the thiocholine is oxidized to the disulfide and a n apparent hydrolysis of 2 to 370 is observed which is almost independent of time after the first 24 hours. Because the electrode reaction which is monitored is the oxidation of thiol to disulfide, the disulfide in the substrate supply is of little consequence. These experiments indicate that the stability of the substrate will not be a problem. Although some spontaneous hydrolysis occurs (5% per day a t 25' C. for 1.5 x 10-3Jf solutions), the thiol formed will be rapidly oxidized to disulfide by the air. Substrate solutions have been used in these laboratories after being left a t 25' C. for up to 10 days, with no appreciable differences in electrochemical and enzyme responses. Likewise, elevated temperatures will not cause significant decomposition of the substrate. A 1 X 10-3AW substrate solution was heated a t 60" C. for 3 hours, then cooled to 25' C. No flee thiol was observed, and the substrate was hydrolyzed by the enzyme. Applications of' Immobilized Enzyme. T h e immobilized cholinesterase can be used a,nalytically in much the same may t h a t the soluble enzyme is used, t h a t is, t o determine the concentration of a substrate t h a t is acted upon b y t h e enzyme, a n inhibitor t h a t inactivates the enzyme, or a n activator that provides a n acceleration in enzyme activity. T h e advantage of the insolubilized cholinesterase lies in the fact that it, unlike the soluble enzyme, is not used up in a n analysis, but the same material can, in fact, be used for up to 12 hours of continuous operation For example, immobilized cholinesterase has been used to determine the concentration of the substrates acetyl- and butyrylthio-
I? >
-I
K
n
> I
c W z K
2 4
c W
K
a
DAYS
Figure 5. Autohydrolysis of 1.5 X 10-3M butyrylthiocholine iodide in tris buffer, pH 7.4, stored at room temperature (25'C.) under air and NP
choline iodide. A sample of the substrate is passed over the enzyme, and the voltage is recorded. From calibration plots, of the log of the substrate concentration us. voltage, the concentration of the thiol ester can be calculated. This theory was discussed in a previous publication ( 5 ) . I n a similar manner, a continuous monitoring of the substrate concentration can be effected. I n addition, the enzyme can be used to detect anticholinesterase inhibitors, such as insecticides, as will be shown in a future publication. By immobilization of other enzymes, such as glucose oxidase, the concentration of a substrate (glucose) may be continually determined by observing the potentials produced. Preliminary experiments revealed that the glucoseglucose oxidase system can be followed using the described device. Sharp changes (200 mv./minute) were observed in switching from peroxide (produced in normal enzymic action) to glucose (no enzyme). The use of immobilized enzyme in analytical applications should have a great potential. By this technique, the
quantity of enzyme is conserved, and the same material may be used over and over. The application of this technique to other enzyme systems should be possible. LITERATURE CITED
(I) Bar-Eli, A,, Katchalski, E., J . Biol. Chem. 238, 1690 (1963). (2) Barnett, L. B., Bull, H. B., Biochim. Biophys. Acta 36, 244 (1959). (3) Bernfield, P., Wan, J., Science 144, 678 (1963). (4) Ellman,' G. L., Biochem. Pharnzacol. 7,88 (1961). (5) Guilbault, G. G., Kramer, D. N., Goldberg, P., J . Phys. Chem. 67, 1747 (1963). (6) Kramer, D. N., Guilbault, G. G., Cannon. P. L.. ANAL.C n m . 34. 842 (1962). (7) McLaren, A. D., Peterson, G. H., Soil Sci. SOC.Am. Proc. 22, 239 (1958). (8) Mitz, M. A., Summaria, L. J., Nature 189, 576 (1961). (9) Nikolaev, A., LIardashev, S. R., Biokhimya 26, 565 (1962). (10) Vasta, B., Usdin, V., hlelpar, Inc., Falls Church, Va., Final Report, Contract DA 18-108-405-CML-828, Section 3.3.4, p. 3.102 (Oct. 1963). RECEIVED for review July 2, 1965. Accepted August 5, 1965.
VOL. 37, NO. 1 1 , OCTOBER 1965
1381
