Improved Enzyme Electrode for Amygdalin R. A.
Llenado and G. A. Rechnitz Department of Chemistry, State University of New York, Buffalo, N. Y . 14214
An enzyme electrode for amygdalin is constructed by immobilizing 0-glucosidase in a polymer gel layer coupled to a cyanide-sensing membrane electrode. The characteristics of a novel inverted electrode configuration are shown to be superior to earlier designs and to permit analyses of samples as small as 0.2 ml. Optimum operating conditions for the enzyme electrode system are described.
THEARRAY of useful ion-selective membrane electrodes and their applications continues to multiply (1-3). Recently the use of biological materials in membrane electrodes has resulted in the development of the valinomycin-based potassium electrode (4-6) and enzyme electrodes for urea (7) and for amino acids (8). In a recent preliminary communication (9), we reported a new kind of enzyme electrode made by coupling the enzyme P-glucosidase to a cyanide-responsive membrane electrode. This novel electrode, responsive to amygdalin, is the first successful example of a potentiometric enzyme electrode utilizing a nonglass membrane and will find applications in agricultural chemistry and plant biochemistry. The electrode is prepared by mechanically coupling a thin membrane of polyacrylamide gel containing immobilized @-glucosidase with the polycrystalline sensing element of the solid state cyanide electrode. When this electrode system is exposed to aqueous solutions of amygdalin, the immobilized P-glucosidase catalyzes the hydrolysis of amygdalin at the membrane according to
EXPERIMENTAL
Chemicals and Reagents. The enzyme &glucosidase, emulsin type, prepared from almonds, was obtained from Sigma Chemical Co., St. Louis, Mo. 63118. Its activity was reported as 3.7units per mg. This enzyme can be stored under refrigeration with little loss of activity. Solutions of amygdalin (Sigma) were prepared by sequential dilution of a stock solution with borax/NaOH buffer, O.lM, t o keep pH and ionic strength constant. Solutions were freshly prepared for each measurement except when used for studying solution aging effects. Solution deterioration can also be minimized by refrigeration. Polymer solutions with varying percentages of total monomer (5-15x) and cross-linking agent (4-16x) of the total monomer) were made by dissolving appropriate amounts of acrylamide (Eastman) and N,N’-methylenebisacrylamide(East man) in deaerated distilled and deionized water. Dissolved oxygen, which inhibits polymerization, was removed by nitrogen bubbling. Potassium persulfate and riboflavin (Eastman) were added as redox catalysts. Polymer solutions are stable when kept in the dark and refrigerated except when the redox catalysts have already been added. Preparation of the Enzyme Membrane. Normally, 100 mg of P-glucosidase was dissolved per milliliter of polymer solution. The solution was kept in the dark and refrigerated until dissolution of the enzyme was complete. The enzymepolymer solution was then transferred into a PlexiglasTeflon (Du Pont) syringe-type chamber (Figure l a ) where photopolymerization was carried out (IO-12) to form the membrane gel. After photopolymerization, slices of polyacrylamide membranes containing immobilized enzyme are obtained by cutting the material as it is extruded by the Teflon plunger (Figure lb). With practice one can conveniently and uniformly slice 100 p. membranes having thicknesses as thin as 300 No attempts were made to cut with better precision because swelling of the gel contributes significantly to the uncertainty. With 1 ml of polymerized material, one can cut 10-20 slices depending on the thickness desired. The sliced membrane is transferred onto a porcelain spot plate and stored under refrigeration until used (Figure lb). Preparation of the Enzyme Electrode. A thin enzyme membrane is mechanically coupled to the sensing element of an Orion 94-06 cyanide electrode (Figure 2a). The Plexiglas cap serves to hold the enzyme membrane rigidly in place and is also the sample cell for the potentiometric measurement. When properly prepared and preconditioned, the enzyme membrane swells to fit its space snugly and prevents solutions from creeping into the sides of the holder. Silicone oil applied to the body of the electrode facilitates the positioning and removal of the Plexiglas cap. This method of assembly was superior to direct polymerization of the gel on the electrode sensing element as described in our preliminary communication (9). Potentiometric Measurements. Figure 2b shows how potentiometric measurements were carried out with the enzyme electrode connected to the imput terminal of a Corning Model 10 pH meter. The reference electrode, Orion 90-01 sleeve type, fits precisely into the assembly to form a solution
*
0
The cyanide ion, produced in stoichiometric proportion to the concentration of amygdalin in the sample solution, gives rise to the potentiometric response of the electrode system. We now report a detailed study of the properties of this enzyme electrode and propose an improved electrode system configuration designed for optimum convenience and utility. It will be seen that the characteristics of the present electrode system are even more attractive than those described in our preliminary report (3). ( I ) G. A. Rechnitz, Accourifs Cltem. Res., 3, 69 (1970). (2) R. A. Durst, Ion Selective Electrodes, NBS Special Publication No. 314, U. S. Government Printing Oflice, Washington, D. C., 1969. (3) E. Pungor and K. Toth, Auulyst, 95, 625 (1970). (4) M. S. Frant and J. W. Ross, Jr., Sciertce, 167,987 (1970). (5) G. A. Rechnitz, ANAL. CHEM., 41 (12), 109A (1969). (6) G. A . Rechnitz and M. S. Mohan, Scierice, 168, 1460 (1970). (7) G. G. Guilbault and J. G. Montalvo, Jr., J . Amer. Cliem. Soc., 92, 2533 (1970). (8) G. G. Guilbault and E. Hravankova, ANAL. CHEM., 42, 1779 (1970). (9) G. A. Rechnitz and R. A. Llenado, ibid., 43, 283 (1971).
(10) P. Bernfeld and J. Wan, Scierrce, 142, 678 (1963). ( I 1) G. Hicks and S . Updike, Nature, 214,986 (1967). (12) G. G. Guilbault and J. Das, A w l . Biochem., 33, 341 (1970).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
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A
--
9
Enzyme-polyocrylamido mom rone B Sensing dement C Rubber washer D Plexiglas Cap E-Orim9196ektrode F Sample solution 0 Orion 90-01 reference electrode H lo pH meter
--
-
n
Figure 1. (a) Photopolymerization of enzyme-acrylamide solution (b) Slicing of enzyme-polycrylamide membrane space requiring as little as 0.20 ml of sample. Millivolt readings are taken at 25 "C with the amygdalin sample solutions preequilibrated at 25.0 -f 0.1 "C in a constant temperature bath. Readings were monitored on a Varicord Model 43 recorder.
RESULTS AND DISCUSSION Effect of Substrate Concentration. The electrode system senses the cyanide produced at the membrane via the pglucosidase catalyzed hydrolysis of amygdalin. Because of the stoichiometry of Reaction 1, the potential of the electrode is dependent on the amygdalin concentration.
E
E o - 2.3RT/F Iog'TN
(2)
E" - 2.3RT/F log[Amygdalin]
(3)
=
and
E
>
180
-
140
-
=
Figure 2. ( a ) Cross-section of enzyme electrode (b) Potentiometric measurement setup However, the response is not completely Nernstian, having a slope of 48 mV per decade change of amygdalin concentration at the linear portion of the calibration plot shown in Figure 3. This is so because not all of the cyanide produced reaches the electrode surface. Reasonable reproducibility of potentials is observed as shown in Table I. Below lO-5M amygdalin concentration, enzymatic hydrolysis is extremely slow and the electrode response is too sluggish to be of analytical value. Above 10-*M,the calibration curve levels off because a steady state is reached and the reaction is zero order with respect to the substrate concen-
Figure 3. Amygdalin concentration curve
E
Y
'
pH 10.4, 25 "C, and 10 mg of enzyme per membrane. Curve ( A ) fresh, ( E ) 3 days old, (C)18 days old
100-
60
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, S E P T E M B E R 1971
240-
200
Figure 4. Comparison of Orion 94-06 cyanide electrode response with and without a 600-g polyacrylamide membrane All measurements were made at pH 10.4, 25 "C, and ionic strength held constant at 0.1M
-
>1-
E
w
-
I
1iQ-
86-
-
2Wr
180-
80 -
100
60
40
9' I
6, - d/
40-
I
I
I
2
0
3 -100 [CN-]
I
I
4
5
M
10-2hl
Figure 5. Elecrode response times with fresh amygdalin solutions and 10 mg of enzyme per membrane
/--W-lM
Q o -
10-5111
/-IO
20 TIME,
40
minutn
~~
tration. The absolute validity of our amygdalin calibration curve is shown by comparison with the calibration of the electrode using aqueous solutions of cyanide (Figure 4). Typical electrode response times are shown in Figure 5. Effect of Solution and Enzyme Age. Amygdalin solutions deteriorate with time to yield progressively lower potentials and lower calibration slopes. These effects are shown in Figures 6 and 3. Solution deterioration is retarded by storing under refrigeration. When older solutions are measured for cyanide ion, no free cyanide ion is observed showing that other hydrolytic products are formed, extensive solvation occurs, or conformational changes retard the rate of the enzymatic reaction. The results of continuous electrode use are shown in Figure 7. The enzyme electrode was satisfactory only for four days under such extreme use because of leaching of the enzyme from the gel layer. Longer lifetimes can be achieved by increasing the concentration of the enzyme immobilized in the gel layer or by storing the electrode between measurements. It is also possible to increase electrode lifetimes by interposing a cellophane film between the sample and the electrode, but this leads to retardation of electrode response
~~
~~
-
~~
Table I. Reproducibility of Potentials Amygdalin concentration Trials 1 2 3
4
IO-*M
-195mV -196 -191 -194 -191 -193
10-3~
-148mV - 148 -150 - 147
10-4~
-99mV - 100
-96 -101
1 0 - 5 ~
-58mV - 58 - 56 - 54
5 - 148 -104 - 52 Mean - 148 - 100 - 56 Std dev 2 35 1 12 2 92 2 6 Relstddev 1 22% 0 756% 2 92% 4 6% a Readings were made at pH 10.4, T = 25 "C, 10 mg of enzyme per membrane with freshly prepared amygdalin solutions.
(7, 13). Because the immobilized enzyme can be stored indefinitely under refrigeration (I2), it is easily possible to prepare enough gel material for 100 determinations or more by immobilizing 100 mg of enzyme in 1ml of polymer. (13) K. K. Stewart and L. C. Craig, ANAL. CHEM., 42, 1257 (1970).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11. SEPTEMBER 1971
e
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140
Figure 6. Effect of solution age and storage of immobilized enzyme membrane pH 10.4, 25 "C, and 6 mg of enzyme per membrane. Curve ( A ) fresh, ( B ) 4 days old, (C)14 days old
'
100
60
-
20
2
180
140
>
[Amygdalin],
5
M
-
E
'
4
3
- 1-
Figure 7. Continuous measurements
Y
( A ) first day, ( B ) second day, (C) third day, ( D )
100-
60
fourth day
-
-Log
[Amygdalin],
M
Effect of Amount of Enzyme in the Membrane. Little difference was observed when the amount of enzyme in the membrane was changed from 3 mg to 6 mg to 10 mg. This is so because the amount of enzyme is always much greater than the amount of substrate per 0.20 ml of sample, except at 10-*M amygdalin where the amount of substrate and enzyme are such that the response becomes zero order with respect to the substrate and the curve levels off. Effect of Buffer, Temperature, and pH. The behavior of the enzyme electrode is dependent on the pH of the solution to which the system is exposed because the sensing element of the cyanide electrode responds only to the activity of the cyanide ion (14) and not to any HCN formed ( K . = 4 X 10-lo). At pH 9.2, 50% of the cyanide is free; above pH 10.3, 90% of the cyanide is free; and at pH 12, virtually all the cyanide is in the free form. For this enzyme electrode, the pH range of 10-11 seems to be optimum. Working above pH 11 exposes the immobilized enzyme to irreversible deactivation while resulting in only negligible improvements on the overall electrode
Cyanide Electrode Instruction Manual, Orion Research Inc., Cambridge, Mass. 02139.
(14) Orion
1460
response and at pH < 10 sensitivity is lost because of HCN formation. Borax/NaOH, NaHC03/NaOH, and Na2HP04/NaOH buffer systems were investigated. The borax system was best for use with the electrode. The others gave high blanks and poor reproducibility due to reaction with silver ion from the electrode crystal or to inhibition of the enzyme. Best results were obtained by working at room temperature. While higher temperatures speed up electrode response somewhat, they also result in more rapid deterioration of amygdalin solutions and enzyme gels. Effect of Diverse Ions. Ions capable of forming insoluble silver salts will interfere because of the formation of a precipitate on the membrane surface; substances capable of reducing silver ion will also interfere. Thus, direct enzyme immobilization on the sensing elements is less attractive than mechanical coupling of the gel membrane because the latter permits rapid cleaning of the crystal surface and replacement of the enzyme layer when necessary. Certain transition and heavy metal ions form very stable cyanide complexes and, hence, will interfere. Some metals like Cu, Cd, and Hg may serve to inactivate the enzyme. However, common impurities like CI- and Br-, and the ordinary constituents of many biological samples can be tolerated.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
CONCLUSION
The electrode described represents a new variation of potentiometric enzyme systems, We have succeeded in coupling an enzyme membrane to a polycrystalline non-glass membrane. The principle involved is generally applicable since there are literally thousands of enzymes with high activity and selectivity which can be coupled to existing potentiometric sensors (5). It may also be possible to adapt these systems to the determination of the @-glucosidase
enzyme rather than the substrate. Such efforts are currently under way and, if successful, would lead to new sensors useful for enzyme analysis and for the diagnosis of certain disease states involving enzyme abnormalities.
RECEIVED for review March 25, 1971. Accepted June 8, 1971. We gratefully acknowledge the support of the National Institutes of Health.
Correction Identification of Barbiturates by Chemical Ionization Mass Spectrometry In this article by H. M. Fales et al. [ANAL.CHEM., 42, 1432 (1970)l there is an error on page 1434. Figure 2 shows a line
in the E.I. spectrum at mje 140. This should be mje 141, and therefore a revised Figure 2 is shown below. 24 I
Figure 2. Electron impact and chemical ionization mass spectra of ortal
ORTAL, M
= 240, C.1. 1
141
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