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Amperometric determination of zinc with an apoenzyme-treated graphite electrode
Analytica Chimica Acta, 152 (1983) 271-274 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Short Communication
AMPEROMETRIC DETERMINATION APOENZYME-TREATED GRAPHITE
J. J. JASAITIS, Institute
OF ZINC WITH AN ELECTRODE
V. J. RAZUMAS and J. J. KULYS*
of Biochemistry,
Lithuanian
Academy
of Sciences,
Vilnius (U.S.S.R.)
(Received 21st February 1983)
Summary. Zinc can be determined in the range 0.8-12 PM by activation of apo-alkaline phosphatase immobilized through covalent binding on a graphite electrode. o-Hydroxyphenylphosphate is used as substrate. Current is measured at an applied potential of 0.3 V vs. SCE. Very few studies have been made of the use of enzyme electrodes for the determination of metal ions. Liu et al. [l] constructed a potentiometric enzyme electrode sensitive to mercury(I1) and silver(I) ions, based on glucose oxidase and catalase immobilized in a polyacrylamide gel [ 11. The lower limits of determination were 5 X lo4 M Hg2+ and 4 X 1c6 M Ag’, respectively. The present communication describes the development of an amperometric method for the determination of zinc ions with use of alkaline phosphatase from E. coli immobilized by covalent binding on a graphite electrode. The zinc ions present in alkaline phosphatase may be removed by complexing with EDTA. Selective binding of zinc ions by the resulting apoenzyme reactivates enzyme activity, and allows very sensitive detection [2] and determination [3] of zinc. Experimental Reagents. Alkaline phosphatase (E.C. 3.1.3.1) from E. coli (All-Soviet Research Institute of Applied Biochemistry, Olaine) had an activity of 7.9 U mg-' . Enzymatic activity was determined spectrophotometrically [ 41 with disodium p-nitrophenylphosphate (Fluka) as a substrate in 0.1 M Tris--HCl10 mM magnesium sulphate buffer, pH 8.0. Water-soluble carbodiimide-lcyclohexyl-3(2-morpholinoethyl)carbodiimid~ethoptoluenesulfate (Serva, G.F.R.) was used. o-Hydroxyphenylphosphate was synthesized from phosphorus(V) oxide and catechol [ 51. All other reagents were chemically pure or of the highest purity available. Regeneration of the activity of immobilized alkaline phosphatase was studied in 0.1 M Tris-HCl buffer (pH 8.0) containing no magnesium ions. Enzyme immobilization was done in 0.05 M acetate buffer (pH 5.6). All solutions were prepared in twice-distilled water, which was also used for all washings. 0003-2670/83/$03.00
o 1983 Elsevier Science Publishers B.V.
272
Apparatus. Electrochemical measurements were done with a OH-105 polarograph (Radelkis, Hungary) in a 25cm3 glass cell thermostatted at 25 f O.l”C, using a three-electrode circuit. A graphite disc (0.6 cm in diameter) pressed into teflon was used as the working electrode. The electrode surface area after activation and immobilization of the enzyme (0.89 cm?) was determined by the dependence of the limiting current of potassium hexacyanoferrate(II1) reduction in 1 M potassium chloride on the electrode rotation speed [6] . A saturated calomel electrode (Radiometer, Denmark) was used as the reference. A platinum plate having a geometrical surface area of 12.4 cm2 served as the auxiliary electrode. During measurements the solutions were stirred by a disc magnetic stirrer (Radiometer). A Specord M-40 spectrophotometer and quartz cells (1 cm) were used for spectrophotometric measurements. Immobilization of alkaline phosphatase on graphite. The enzyme was covalently attached to graphite using the water-soluble carbodiimide [ 71. For this purpose, the graphite electrode was polished with fine emery paper, and treated ultrasonically for 5 min (22 kHz, 0.2 A) in absolute methanol and water. It was anodized in 10% nitric acid-2.5% potassium dichromate at 2.2 V vs. SCE for 30 s and washed with water. The activated graphite electrode was kept in 0.05 M carbodiimide solution in 0.05 M acetate buffer (pH 5.6) for 30 min and then immersed in the enzyme solution (alkaline phosphatase, 1 mg ml-‘, in 0.05 M acetate buffer, pH 5.6) for 12 h at 4°C The electrode was washed with 1 M sodium chloride solution (30 min) and stored in 0.1 M Tris-HCl buffer (pH 8.0) for 48 h at 4°C to eliminate any adsorbed protein. Removal of zinc from the immobilized enzyme. Zinc was removed as described previously [8] by immersing the enzyme electrode for 48 h in 0.05 M EDTA solution at pH 5.5. The process was monitored spectrophotometrically by following the hydrolysis rate of p-nitrophenylphosphate [ 41. Determination of zinc. The enzyme electrode with apo-alkaline phosphatase was placed in a cell containing 0.1 M Tris-HCl buffer (pH 8.0) and kept at +0.3 V vs. SCE until a constant background current was established (about 5 min) [9]. The solution of o-hydroxyphenyl phosphoric acid in the same buffer was introduced into the cell to give an overall concentration of lo4 M. Then the buffer solution containing zinc acetate (l-12 PM) was introduced and the value of the constant catalytic current resulting from oxidation of the hydrolysed substrate was recorded. Results and discussion When stored in the solution of EDTA (pH 5.5) for 2 days, the enzyme electrode based on covalently attached alkaline phosphatase completely loses its catalytic properties with reference to p-nitrophenylphosphate. It is also inactive in the hydrolysis reaction of the synthesized substrate o-hydroxyphenylphosphate as indicated by the absence of an electrode current at 0.3 V vs. SCE. The current is generated on platinum or carbon electrodes after catechol ester hydrolysis to catechol, which may be detected at potentials (<0.35 V vs. SCE) lower than that for direct ester oxidation [lo].
273
Introduction of zinc ions at low concentrations into the electrochemical cell containing lOa M catechol ester and an apo-phosphatase electrode produces a stationary current after about 30 s; the magnitude of the current depends on the concentration of zinc ions in the solution (Fig. 1). The lower limit of determination is 0.8 PM. A plot of inverse current against inverse zinc concentration is linear. Its equation may be expressed as
i-’ (MA-‘) = (0.051 rt 0.012) + (2.264 + 0.032) [Zn’+]-’ (PM-‘) It has a correlation coefficient of 0.9998 (12 results). The electrode based on immobilized alkaline phosphatase may be reused if, after a determination, it is immersed in EDTA solution (pH 5.5) for 1 h and stored in 0.1 M Tris-HCl buffer (pH 8.0) at 4°C. Under these conditions, the data in Fig. 1 can be reproduced over three weeks. The activation of alkaline phosphatase by zinc ions and the catalytic process can be presented in a simplified form: a-E + Zn2+
“,zn
E;E+S
2
E*S
5
E+P
where a-E and E are the apo-enzyme and active enzyme, respectively, S is o-hydroxyphenylphosphate, P is catechol, KZn is the dissociation constant for zinc in the enzyme active site, KS is the dissociation constant of the enzyme-substrate complex and k is the first-order rate constant for product formation. From this scheme, if the overall process rate is limited by the catalytic reaction, the stationary rate of catechol formation (R) may be expressed as R =
WI dsl d Wdl + Kznl[Zn2+l I+ PI 01
(1)
where [E] ,,, [S] 0, and [Zn”‘] are the overall concentrations of enzyme, substrate and zinc ions. Under stationary-state conditions, the substrate flow to the electrode surface equals the enzymatic reaction rate because the thickness of the catalytic layer is considerably smaller than that of the diffusion layer 6 [ 111 :
03 0
2
L
6
8
10 iZn”ll_u
12 M)
Fig. 1. Dependence of the current in the presence of o-hydroxyphenylphosphate on zinc concentration (0.1 mM substrate in 0.1 M Tris-HCI buffer, pH 8.0, E = 0.3 V vs. SCE, 25°C).
274
DUSI o DFW
-
[Sl,W = VmaASl,I&(1 + KznWn2+lI+ PI 3 = VmaxKWUMl + KdtZn*+l) + [%I -WI,
(2) (3)
where D = Ds = Dp is the diffusion coefficient for substrate or product, [S] o and [S] s are the substrate bulk and surface concentrations, respectively, [P] 8 is the concentration of the enzymatic reaction product at the surface, V,,, is the maximal rate of the enzymatic reaction, and k, is the heterogeneous rate constant for catechol oxidation. In the derivation of Eqns. (2) and (3), it is assumed that the product concentration outside the diffusion layer is zero. If KM = &(l + K,,/[ Zn*+] ) where KM is the apparent Michaelis constant, k = V,,,/K,, /3 = D/S, and if during the experiment KM/[ S] o %-1 the solutions to Eqns. (2) and (3) are [S] 8 = p [ S] o/(/3 + iz) and [P] s = kB [S] o/ (12 + @(k, + 0). The constant electrode current is i = nFAFz,[P], = nFAk.&3[S] ,/(k + @(k, + p), where n is the number of electrons involved in the electrode process, F is the Faraday constant and A is the area of the electrode. Thus the inverse current is directly proportional to the inverse zinc concentration as observed experimentally: i-’ = (k, +
P)W’Ak,[Sl
OW
+
&IV,,,
+
KsKz,/Vm,,[Zn2+l1
(4)
This equation indicates that at low zinc concentrations the current is directly proportional to the immobilized enzyme activity and inversely proportional to the product of the dissociation constants. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
C. C. Liu, F. M. Fryburg and A. K. Chen, Bioelectrochem. Bioenerg., 8 (1981) 703. A. Townshend and A. Vaughan, Anal. Chim. Acta, 49 (1970) 366. A. Townshend and A. Vaughan, Talanta, 17 (1970) 289. H.-U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1965, p. 783. K. J. Humphries and G. Scott, J. Chem. Sot., Perkin Trans. 2,6 (1973) 831. R. N. Adams, Electrochemistry at Solid Electrodes, Dekker, New York, 1969, p. 89. C. Bourdillon, J.-P. Bourgeous and D. Thomas, Biotechnol. Bioeng., 21 (1979) 1877. M. L. Applebury and J. E. Coleman, J. Biol. Chem., 244 (1969) 709. V. J. Razumas, J. J. Kulys and A. A. Malinauskas, Anal. Chim. Acta, 117 (1980) 387. J. Kulys, V. Razumas and A. Malinauskas, Bioelectrochem. Bioenerg., 7 (1980) 11. V. G. Levich, Physicochemical Hydrodynamics, Prentice-Hall, Englewood Cliffs, N. J., 1962, p. 60.
Short Communication
AMPEROMETRIC DETERMINATION APOENZYME-TREATED GRAPHITE
J. J. JASAITIS, Institute
OF ZINC WITH AN ELECTRODE
V. J. RAZUMAS and J. J. KULYS*
of Biochemistry,
Lithuanian
Academy
of Sciences,
Vilnius (U.S.S.R.)
(Received 21st February 1983)
Summary. Zinc can be determined in the range 0.8-12 PM by activation of apo-alkaline phosphatase immobilized through covalent binding on a graphite electrode. o-Hydroxyphenylphosphate is used as substrate. Current is measured at an applied potential of 0.3 V vs. SCE. Very few studies have been made of the use of enzyme electrodes for the determination of metal ions. Liu et al. [l] constructed a potentiometric enzyme electrode sensitive to mercury(I1) and silver(I) ions, based on glucose oxidase and catalase immobilized in a polyacrylamide gel [ 11. The lower limits of determination were 5 X lo4 M Hg2+ and 4 X 1c6 M Ag’, respectively. The present communication describes the development of an amperometric method for the determination of zinc ions with use of alkaline phosphatase from E. coli immobilized by covalent binding on a graphite electrode. The zinc ions present in alkaline phosphatase may be removed by complexing with EDTA. Selective binding of zinc ions by the resulting apoenzyme reactivates enzyme activity, and allows very sensitive detection [2] and determination [3] of zinc. Experimental Reagents. Alkaline phosphatase (E.C. 3.1.3.1) from E. coli (All-Soviet Research Institute of Applied Biochemistry, Olaine) had an activity of 7.9 U mg-' . Enzymatic activity was determined spectrophotometrically [ 41 with disodium p-nitrophenylphosphate (Fluka) as a substrate in 0.1 M Tris--HCl10 mM magnesium sulphate buffer, pH 8.0. Water-soluble carbodiimide-lcyclohexyl-3(2-morpholinoethyl)carbodiimid~ethoptoluenesulfate (Serva, G.F.R.) was used. o-Hydroxyphenylphosphate was synthesized from phosphorus(V) oxide and catechol [ 51. All other reagents were chemically pure or of the highest purity available. Regeneration of the activity of immobilized alkaline phosphatase was studied in 0.1 M Tris-HCl buffer (pH 8.0) containing no magnesium ions. Enzyme immobilization was done in 0.05 M acetate buffer (pH 5.6). All solutions were prepared in twice-distilled water, which was also used for all washings. 0003-2670/83/$03.00
o 1983 Elsevier Science Publishers B.V.
272
Apparatus. Electrochemical measurements were done with a OH-105 polarograph (Radelkis, Hungary) in a 25cm3 glass cell thermostatted at 25 f O.l”C, using a three-electrode circuit. A graphite disc (0.6 cm in diameter) pressed into teflon was used as the working electrode. The electrode surface area after activation and immobilization of the enzyme (0.89 cm?) was determined by the dependence of the limiting current of potassium hexacyanoferrate(II1) reduction in 1 M potassium chloride on the electrode rotation speed [6] . A saturated calomel electrode (Radiometer, Denmark) was used as the reference. A platinum plate having a geometrical surface area of 12.4 cm2 served as the auxiliary electrode. During measurements the solutions were stirred by a disc magnetic stirrer (Radiometer). A Specord M-40 spectrophotometer and quartz cells (1 cm) were used for spectrophotometric measurements. Immobilization of alkaline phosphatase on graphite. The enzyme was covalently attached to graphite using the water-soluble carbodiimide [ 71. For this purpose, the graphite electrode was polished with fine emery paper, and treated ultrasonically for 5 min (22 kHz, 0.2 A) in absolute methanol and water. It was anodized in 10% nitric acid-2.5% potassium dichromate at 2.2 V vs. SCE for 30 s and washed with water. The activated graphite electrode was kept in 0.05 M carbodiimide solution in 0.05 M acetate buffer (pH 5.6) for 30 min and then immersed in the enzyme solution (alkaline phosphatase, 1 mg ml-‘, in 0.05 M acetate buffer, pH 5.6) for 12 h at 4°C The electrode was washed with 1 M sodium chloride solution (30 min) and stored in 0.1 M Tris-HCl buffer (pH 8.0) for 48 h at 4°C to eliminate any adsorbed protein. Removal of zinc from the immobilized enzyme. Zinc was removed as described previously [8] by immersing the enzyme electrode for 48 h in 0.05 M EDTA solution at pH 5.5. The process was monitored spectrophotometrically by following the hydrolysis rate of p-nitrophenylphosphate [ 41. Determination of zinc. The enzyme electrode with apo-alkaline phosphatase was placed in a cell containing 0.1 M Tris-HCl buffer (pH 8.0) and kept at +0.3 V vs. SCE until a constant background current was established (about 5 min) [9]. The solution of o-hydroxyphenyl phosphoric acid in the same buffer was introduced into the cell to give an overall concentration of lo4 M. Then the buffer solution containing zinc acetate (l-12 PM) was introduced and the value of the constant catalytic current resulting from oxidation of the hydrolysed substrate was recorded. Results and discussion When stored in the solution of EDTA (pH 5.5) for 2 days, the enzyme electrode based on covalently attached alkaline phosphatase completely loses its catalytic properties with reference to p-nitrophenylphosphate. It is also inactive in the hydrolysis reaction of the synthesized substrate o-hydroxyphenylphosphate as indicated by the absence of an electrode current at 0.3 V vs. SCE. The current is generated on platinum or carbon electrodes after catechol ester hydrolysis to catechol, which may be detected at potentials (<0.35 V vs. SCE) lower than that for direct ester oxidation [lo].
273
Introduction of zinc ions at low concentrations into the electrochemical cell containing lOa M catechol ester and an apo-phosphatase electrode produces a stationary current after about 30 s; the magnitude of the current depends on the concentration of zinc ions in the solution (Fig. 1). The lower limit of determination is 0.8 PM. A plot of inverse current against inverse zinc concentration is linear. Its equation may be expressed as
i-’ (MA-‘) = (0.051 rt 0.012) + (2.264 + 0.032) [Zn’+]-’ (PM-‘) It has a correlation coefficient of 0.9998 (12 results). The electrode based on immobilized alkaline phosphatase may be reused if, after a determination, it is immersed in EDTA solution (pH 5.5) for 1 h and stored in 0.1 M Tris-HCl buffer (pH 8.0) at 4°C. Under these conditions, the data in Fig. 1 can be reproduced over three weeks. The activation of alkaline phosphatase by zinc ions and the catalytic process can be presented in a simplified form: a-E + Zn2+
“,zn
E;E+S
2
E*S
5
E+P
where a-E and E are the apo-enzyme and active enzyme, respectively, S is o-hydroxyphenylphosphate, P is catechol, KZn is the dissociation constant for zinc in the enzyme active site, KS is the dissociation constant of the enzyme-substrate complex and k is the first-order rate constant for product formation. From this scheme, if the overall process rate is limited by the catalytic reaction, the stationary rate of catechol formation (R) may be expressed as R =
WI dsl d Wdl + Kznl[Zn2+l I+ PI 01
(1)
where [E] ,,, [S] 0, and [Zn”‘] are the overall concentrations of enzyme, substrate and zinc ions. Under stationary-state conditions, the substrate flow to the electrode surface equals the enzymatic reaction rate because the thickness of the catalytic layer is considerably smaller than that of the diffusion layer 6 [ 111 :
03 0
2
L
6
8
10 iZn”ll_u
12 M)
Fig. 1. Dependence of the current in the presence of o-hydroxyphenylphosphate on zinc concentration (0.1 mM substrate in 0.1 M Tris-HCI buffer, pH 8.0, E = 0.3 V vs. SCE, 25°C).
274
DUSI o DFW
-
[Sl,W = VmaASl,I&(1 + KznWn2+lI+ PI 3 = VmaxKWUMl + KdtZn*+l) + [%I -WI,
(2) (3)
where D = Ds = Dp is the diffusion coefficient for substrate or product, [S] o and [S] s are the substrate bulk and surface concentrations, respectively, [P] 8 is the concentration of the enzymatic reaction product at the surface, V,,, is the maximal rate of the enzymatic reaction, and k, is the heterogeneous rate constant for catechol oxidation. In the derivation of Eqns. (2) and (3), it is assumed that the product concentration outside the diffusion layer is zero. If KM = &(l + K,,/[ Zn*+] ) where KM is the apparent Michaelis constant, k = V,,,/K,, /3 = D/S, and if during the experiment KM/[ S] o %-1 the solutions to Eqns. (2) and (3) are [S] 8 = p [ S] o/(/3 + iz) and [P] s = kB [S] o/ (12 + @(k, + 0). The constant electrode current is i = nFAFz,[P], = nFAk.&3[S] ,/(k + @(k, + p), where n is the number of electrons involved in the electrode process, F is the Faraday constant and A is the area of the electrode. Thus the inverse current is directly proportional to the inverse zinc concentration as observed experimentally: i-’ = (k, +
P)W’Ak,[Sl
OW
+
&IV,,,
+
KsKz,/Vm,,[Zn2+l1
(4)
This equation indicates that at low zinc concentrations the current is directly proportional to the immobilized enzyme activity and inversely proportional to the product of the dissociation constants. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
C. C. Liu, F. M. Fryburg and A. K. Chen, Bioelectrochem. Bioenerg., 8 (1981) 703. A. Townshend and A. Vaughan, Anal. Chim. Acta, 49 (1970) 366. A. Townshend and A. Vaughan, Talanta, 17 (1970) 289. H.-U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1965, p. 783. K. J. Humphries and G. Scott, J. Chem. Sot., Perkin Trans. 2,6 (1973) 831. R. N. Adams, Electrochemistry at Solid Electrodes, Dekker, New York, 1969, p. 89. C. Bourdillon, J.-P. Bourgeous and D. Thomas, Biotechnol. Bioeng., 21 (1979) 1877. M. L. Applebury and J. E. Coleman, J. Biol. Chem., 244 (1969) 709. V. J. Razumas, J. J. Kulys and A. A. Malinauskas, Anal. Chim. Acta, 117 (1980) 387. J. Kulys, V. Razumas and A. Malinauskas, Bioelectrochem. Bioenerg., 7 (1980) 11. V. G. Levich, Physicochemical Hydrodynamics, Prentice-Hall, Englewood Cliffs, N. J., 1962, p. 60.