Electrocatalytic oxidation of catechol phosphate by immobilized alkaline phosphatase

Bioelectrochemistry and Bioenergetics, 10 (1983) 427-439 A section of J. Electroanal. Chem., and constituting Vol. 155 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

566-ELECTROCATALYTIC IMMOBILIZED ALKALINE

V. RAZUMAS, Institute

J. JASAITIS

of Biochemistry,

(Manuscript

received

OXIDATION OF CATECHOL PHOSPHATASE

427

PHOSPHATE

BY

and J. KULYS

Lithuanian

December

Academy

of Sciences, Vilnius (U.S.S.R.)

22nd 1982)

SUMMARY Electrocatalytic oxidation of catechol phosphate (dihydrogen o-hydroxyphenyl phosphate) on cylindrical glassy carbon electrodes containing 0.3, 1.5 or 2 layers of covalently attached alkaline phosphatase proceeds on diffusive conditions at substrate concentrations lower than 1 mM. The half-wave potential of the catalytic current is equal to U,,a of catechol. At substrate concentrations of 2.5-10 mM and a rotation speed of 34.7-259.8 rad/s, the enzyme electrode with the lowest biocatalyst concentration operates in the kinetic mode and K,(,,,j of the process is equal to 20.2 mM. Macrokinetic behaviour of phosphatase electrodes and the effect of the enzyme inhibitor-l,lOphenanthroline-on the catalytic current are studied.

INTRODUCTION

Enzymes catalyse the electrochemical conversion of molecular gases and a number of organic compounds [l]. Acceleration of the electrochemical conversion of substrates by enzymes which do not catalyse the electron transfer directly is studied [2]. This phenomenon found application in developing new methods for the determination of inorganic ions [3], insecticides [4] and enzymatic activity [5]. However, macrokinetic behaviour of the systems mentioned has not yet been studied. A rotating electrode containing adsorbed or covalently immobilized enzymes serves as a simple and convenient means of analysing the mass transfer effects. The behaviour of rotating enzyme electrodes based on glucose oxidase has already been investigated [6-81. The results on the electrocatalytic oxidation of dihydrogen o-hydroxyphenyl phosphate on glassy carbon cylindrical rotating electrodes containing alkaline phosphatase (E.C. 3.1.3.1) covalently attached by means of cyanuric chloride are presented in this paper.

0302-4598/83/$03.00

0 1983 Elsevier Sequoia

S.A.

428 EXPERIMENTAL

Reagents

Alkaline phosphatase (E.C. 3.1.3.1) from E. coli (All-Soviet Research Institute of Applied Biochemistry, Olaine, U.S.S.R.) was used for immobilization. The enzyme activity (7.9 U/m@ was determined spectrophotometrically [9] using disodium salt of p-nitrophenyl phosphate (Fluka, Switzerland). Dihydrogen o-hydroxyphenyl phosphate was synthesized from catechol and P,O, [lo]. The other reagents used were analytically pure or of highest purity. Spectrophotometrical and electrochemical measurements were carried out in 0.1 M Tris-HCl buffer, pH 8.0, containing 10 mM MgSO,. Inhibition of alkaline phosphatase by I,lO-phenanthroline was studied without the addition of MgSO, to the buffer solution. Phosphate buffer (0.01 M, pH 7.5) was used for enzyme immobilization on glassy carbon activated by cyanuric chloride. Electrodes

Glassy carbon rods (0.28 cm diam., SU-1200, U.S.S.R.) pressed in Teflon were used in constructing disc and cylindrical electrodes. The length of cylindrical electrodes was 0.8 cm. The electrode surface and its change on the activation of glassy carbon are determined by the dependence of the K,[Fe(CN),] reduction limiting current in 1 M KC1 on the r.d.e. according to the method given in Ref. 11. A saturated calomel electrode (s.c.e.) (+ 0.244 0ersu.s n.h.e.; Radiometer, Denmark) was used as a reference electrode. The platinum plate (surface area 12.4 cm2) was used as an auxiliary. Apparatus

and measurement

technique

A spectrophotometer (Specord UV-VIS, G.D.R.) and quartz cells (1 cm thick) thermostated at 25 + 0.1 “C were used for spectral measurements. Potentiostatic and amperometric I versus t curves at a constant potential were recorded by a OH-105 polarograph (Hungary) in a three-electrode circuit. Electrochemical measurements were carried out in a glass cell (25 cm3) thermostated at (20-50) f 0.1 “C. Construction of a rotating electrode enabled us to perform electrochemical measurements in the w interval of 34.7-259.8 rad/s. Potentiostatic polarization curves were recorded from U,,, at a potential sweep rate of 2 mV/s. The enzymatic activity of immobilized alkaline phosphatase was determined by rotating the enzyme electrode at 34.7-259.8 rad/s in 1 mM solution of disodium salt of p-nitrophenyl phosphate in 0.1 M Tris-HCl buffer (pH 8.0) containing 10 mM MgSO,. The accumulation of p-nitrophenol in the cell was determined spectrophotometrically at 400 nm (c = 16,700 M-’ cm-’ [ 121). The rate dependence of enzymatic reaction on time and the substrate concentration for homogeneous enzyme were determined electrochemically at +0.3 V uerstcT

429

s.c.e. on the rotating glassy carbon cylindrical electrode (145 rad/s) by introducing the enzyme into the cell containing the buffer solution of catechol ester. The kinetics of the catalytic anodic current change of phosphatase electrode was recorded by rotating the electrode in the buffer solution at a constant value of U (0.3 V versus s.c.e.) until a stationary background signal was established and subsequently introducing the substrate solution into the electrochemical cell. The inhibition of homogeneous enzyme by l,lO-phenanthroline was carried out in the following two ways: incubating alkaline phosphatase in the inhibitor solution and recording the residual activity of the biocatalyst electrochemically at U = 0.3 V versus s.c.e., or studying the initial rate change of the catalytic current rise in the presence of an inhibitor in the substrate solution. The inhibition of immobilized alkaline phosphatase was studied according to the decrease in the stationary catalytic current of catechol phosphate oxidation.

Enzyme immobilization

on glassy carbon

Alkaline phosphatase was covalently attached to glassy carbon using cyanuric chloride according to the methods given in Refs. 7, 13 and 14. For this purpose the rods from glassy carbon were polished by a thin emery paper, subjected to ultrasound for 5 min (22 kHz, 0.2 A) in twice-distilled water and absolute methanol, then extracted by absolute methanol for 24 h and stored (2 h) in vacuum at room temperature. Pretreated glassy carbon was electrochemically oxidized in 10% HNO, + 2.5% K,Cr,O, at 2.2 V versus s.c.e. for 1 min and washed with twice-distilled water. Some rods were additionally heated for 1 h at 500 ‘C. Further glassy carbon was refluxed with 2 g LiAlH, in 100 cm3 of anhydrous ether for 3 h, washed with ether, 1 M HNO,, twice-distilled water, stored for 24 h in 2 M NaOH and then heated for 4 h at 120 “C. The attachment of cyanuric chloride to the surface of glassy carbon was made by keeping the rods in the reagent alloy for 30 min at 190 ‘C. The rotating electrodes were then assembled, pressing the rods into Teflon couplings. Gallium was used as a contacting material between glassy carbon and the steel rod on which a Teflon coupling was mounted. The cylindrical electrodes made were washed successively with anhydrous acetone, twice-distilled water and again with anhydrous acetone, rotating them at 145 rad/s to eliminate any cyanuric chloride excess. Every step was performed at 4 “C in the course of 15 mm. The enzyme was attached to cyanuric chloride residues by keeping the electrodes in the enzyme solution (0.4 mg or 4 mg of alkaline phosphatase per 1 cm3 of 0.01 M phosphate buffer, pH 7.5) at room temperature for 1 h or 12 h, respectively. Between the activation steps the glassy carbon rods were stored in a desiccator under P,Os at 4 ‘C, whereas the enzyme electrodes were kept in 0.1 M Tris-HCl buffer (pH 8.0) containing 10 mM MgSO, at 4 “C. Adsorbed protein was removed by rotating the enzyme electrode at 145 rad/s for 20 min in 1 M NaCl solution.

430

RESULTS

AND

DISCUSSION

Catalytic parameters

of immobilized

alkaline phosphatase

The data on the enzymatic activity of immobilized alkaline phosphatase with respect to the activation means of glassy carbon and the attachment conditions of the biocatalyst are given in Table 1. Tabulated results are given referring to the fact that the glassy carbon activation leads to a two- and fourfold increase in the electrode surface following anodization and additional heating at 500 “C. This is indicated by the increase in the Slimvalue of the reduction of [Fe(CN),13- anions on the rotating glassy carbon disc electrode. Since on determining the catalytic activity the substrate concentration is almost two orders higher than the value of K, for native enzyme (1.1 X low5 A4 [ 151) and the slope of the experimental lines-solution optical density versus time-is not dependent on the enzyme electrode rotation rate over the interval 34.7-259.8 rad/s, it becomes evident that the activities obtained are not affected by diffusive phenomena and correspond to the surface maximal rate of enzymatic reaction. The amount of immobilized enzyme for electrode I is 1.4, for electrode II it is 6.9 and for electrode III it is 9.7 pmole/cm2 (k,,, for the enzyme is equal to 11.4 s-l, its molecular weight is 86,000 [12]). The concentration of alkaline phosphatase on electrode I is 3.3 times lower than the monolayer filling when the enzyme globule diameter equals 6 nm [16]. Correspondingly, electrodes II and III contain 1.5 and 2 layers of enzyme. It should be noted that the heating of the glassy carbon (500 “C) during activation results in a l.Cfold decrease of the immobilized enzyme surface concentration (Table 1). Clearly, this is due to the destruction of glassy carbon and the degradation of surface functional groups at increased temperatures. The fact that

TABLE

1

Catalytic activity of immobilized alkaline phosphatase in hydrolysis depending on the enzyme electrode preparation conditions 0 Electrode type

I

II

III u p-Nitrophenyl MgSO.,.

Glassy carbon activation conditions

Anodization heating at 500 ‘C Anodization heating at 500 “C Anodization phosphate

Enzyme solution concentration, mg/cm3 +

0.4

+

reaction

Immobilization time, h

of p-nitrophenyl

Enzymatic activity x lo”, mole cm-*

1

1.64

4

12

7.87

4

12

concentration:

1 mM in 0.1 M Tris-HCl

phosphate

s-’

11.1 buffer (pH 8.0) containing

10 mM

431

(/ J , , 0.1

(/(Vvs ;.c.e.)

0.2

0.3

Fig. 1. Dependence of the stationary catalytic current of dihydrogen o-hydroxyphenyl phosphate oxidation on rotating phosphatase electrode I upon the potential. (1 mM substrate solution in 0.1 M Tris-HCl buffer containing 10 mM MgSO,, pH 8.0, 25 “C, o = 259.8 rad/s).

we did not succeed in immobilizing alkaline phosphatase on unanodized electrodes supports the conclusion given above. Catechol and dihydrogen

o-hydroxyphenyl

phosphate

oxidation on modified electrodes

Catechol oxidation on the electrodes modified by alkaline phosphatase proceeds with I& = 0.15 f 0.01 V versus s.c.e., and the transfer coefficient OL,obtained from curves in IlkoviE coordinates U the linearization data of polarization oersw ln[Z/(Z,i, - Z)], equals 0.52 + 0.02. The limiting current is recorded at U values exceeding 0.25 V. The increase in the rotation speed of the cylindrical enzyme electrode from 34.7 to 259.8 rad/s results in an increase in the Z,imvalue proportionally to &*. Such a dependence is characteristic of cylindrical electrodes [ 171. With electrode I the line slope of 9.6 PA s’/* rad- ‘I* enables the empirical expression of the diffusive layer (cm) to be obtained: a,,,, = 0.67 w- ‘1’. However, for cylindrical electrodes a turbulent convection of the solution is observed even at low values of w [7]. While calculating, we assumed that the number of transferred electrons (n) is equal to 2, the electrode area (A) is 3.31 cm*, the diffusion coefficient (D) is 10m5 cm*/s and the catechol concentration is 10e6 mole/cm. Over the rotation rate interval used $,,, varies from 0.12 to 4.2 x lo-* cm. After the immersion of rotating phosphatase electrodes I, II and III into the dihydrogen o-hydroxyphenyl phosphate solution the stationary anodic current is established in approximately 30 s, and its value depends on the potential (Fig 1). The current obtained is catalytic since the catechol ester is not oxidized on glassy carbon over the U interval studied [2]. The dependence of Z,, on U for all electrodes

432

20

z .5

4

10

0

Fig. 2. Dependence of the stationary catalytic current upon the rotation speed of enzyme electrodes. Electrode types: I (I), II (2), III (3, 3’). [ 1 mM dihydrogen o-hydroxyphenyl phosphate solution in 0.1 A4 Tris-HCl buffer containing 10 mM MgSO., (1, 2, 3) or without MgSO, (31, pH 8.0, 25”C, U = 0.3 V 0ersu.s s.c.e.1.

is characterized by U,,z = 0.16 + 0.01 V versus s.c.e., and (Y= 0.50 f 0.02. These parameters of the catalytic process correspond to those observed for the reactions of direct catechol oxidation. Hence, the generation of catalytic current is determined by the pre-electrode reaction of the substrate enzymatic hydrolysis up to catechol and its subsequent oxidation to o-quinone. Macrokinetic behaviour of enzyme electrodes The dependence of the stationary catalytic current on the rotation speed of cylindrical phosphatase electrodes is given in Fig. 2. Here, I,, is directly proportional to @‘I2 only in the case of electrode II. The data for electrode III linearize well in reverse coordinates (Fig. 3). With modified electrodes the dependences of IS, on substrate concentrations up to 1 mM do not linearize in Lineweaver-Burk coordinates. However, the increase in substrate concentration [S], up to 2.5-10 mM shows the Michaelis dependence of the electrode I stationary current on the concentration of catechol ester (K,,,(appj = 20.2 f 0.5 mM). In this case I,, is not the function of the electrode rotation rate.

433 1

24

20

/

16

3' 3 1;

2

f

1

(

4

Fig. 3. Dependence reverse coordinates.

8

12

16

of the stationary catalytic current on the rotation Experimental conditions and notation as in Fig. 2.

speed of enzyme

electrodes

in

Fig. 4. Illustration of the stationary current calculation for enzyme electrodes. [x is the distance from the electrode surface and 6 the diffusion layer thickness. For the construction of graphs [S],/[S], (1) according to equation (7) and [P]s/[S], (2) according to equation ,(8) the following values were used: mole/en-?, [S], = 10e6 mole/cm3, D = 2~ 10e6 Vi,, = 6.9~ lo-” mole cm-’ s-‘, K,,, = 1.8~ lo-’ cm2/s, k, = 1.2~ lOA cm/s, 6 = 0.12 cm.]

434

To explain the results obtained the pre-electrode-layer model given in Fig. 4 may be used. Characteristic features of this model are as follows: the thickness of the biocatalytic layer is comparable to the enzyme globule diameter (6 nm), the diffusion is uniform along the surface of the cylindrical electrode and is four to five layer aOopp orders higher than that of immobilized alkaline phosphatase. It is also supposed that the product concentration of enzymatic reaction at the boundaries of the diffusion layer with the solution equals zero. Assuming this, on stationary conditions equations (1) and (2) are valid:

0) (2) where D = D, = Dp are the diffusion coefficients, [S], the substrate bulk concentration, [S], the substrate concentration on the modified electrode surface, [Pls the surface concentration of the enzymatic reaction product, i.e. catechol, Vz,, the surface maximal rate of enzymatic reaction, K,,, the Michaelis constant, k, the reaction rate,constant of catechol on the electrode surface and 6 the diffusion layer thickness. Supposing that the current of enzyme electrodes is expressed by equation (3): I,, = &4k,[Pls

(3) then the solution of equations (l)-(3) gives a complicated dependence of I,, on [S],, mass transfer and enzymatic reaction constants. When K,/[S], x 1 or K,J[S], X= 1 the solution becomes simpler. In the first case,

[S], = [S], - + and

[p]s,SL

k, + P

(5)

where /I = D/S. Then n6Ak,V& ” =

k, + j3

(6)

At K,A% * 1,

PM

[‘I’=(k+fl)

(7)

435

and k/3 1% [‘I’=

(8)

(k+/?)(k,+j?)

where k = VAJK,.

Taking into consideration equations (3) and (8) we obtain

n%4kk,~[S], I”=

(k+B)(k,+p)

(9)

Inspection of equations (6) and (9) indicates that the catalytic current rise of phosphatase electrodes with the increase in o (Fig. 2) is possible only in the case of equation (9) since 6 = 0.670~‘/‘. The simplified equation (9) is not applicable for electrode I since the maximal value of k (5.7 x 10e5 cm/s) can be compared with p (1.7 X lop5 - 4.8 X 10s5 cm/s), hence, as follows from equation (7) the relation K,/[S], Z+ 1 will not be observed. (The values were estimated assuming that D = 2 X 10e6 cm2/s, k,,, of the hydrolysis reaction of catechol ester is 1.6 times lower than that of the p-nitrophenol derivative [2] and K,,, for the homogeneous enzyme is equal to 1.8 X 10:’ mole/cm3 [2] and remains the same after immobilization). Experimental curve 1 (Fig. 2) is characterized by a complicated dependence of I,, on [S],, D, 6, VA,, and K,,, obtained on the solution of equations (l)-(3). In the case of electrodes II and III with respect to the data in Table 1 the maximal values of k equal 2.7 x 10m4 and 3.9 X 10e4 cm/s respectively, this enables equation (9) to be used for interpreting experimental results. Evidently, electrode II, as follows from the data given in Figs. 2 and 3, operates in the outer-diffusion mode. An analogous conclusion can be made from the I,, dependence on temperature according to which the activation energy determined (4.6 kcal/mole) is characteristic of diffusion processes in water [ 181. The activation energy of enzymatic hydrolysis equals 7-10 kcal/mole [ 16,191. The catalytic current described requires that k//3 z+ 1, k,/P B=-1 and equation (9) transformed into equation (10): = n%4D[S]o&2

I Sf

0.67

Following equation (10) and the data from Figs. 2 and 3, the value of D calculated (2 X 10e6 cm2/s). The amount of active enzyme is 1.4 times greater on electrode III compared electrode II (Table 1). Hence, it must also obey the condition that k/B However, curve 3 in Fig. 2 shows a decrease in the relation k,//?. Supposing k,/@ = 1, equation (9) can be expressed in the following way: 1

1

I,t = “%k,[S],

0.67 + n6AD[S]o&2

was with x=-1.

that

(11)

Using equation (11) and the line 3 intercept on the ordinate (Fig. 3), the k, value of 1.2 x low4 cm/s was determined. The decrease in k, may be explained by a lower

436

catechol permeability through the layer of immobilized alkaline phosphatase on increasing the enzyme surface concentration going from electrode I to electrode III. The removal of Mg2+ ions from the buffer solution results in a decreased I,, value of electrode III (curve 3’, Fig. 2). This may be accounted for by the decreased catalytic activity of the enzyme making up 16% for homogeneous alkaline phosphatase. In this case equation (9) should be considered, assuming that k and k, exceed p only insignificantly. The reciprocal value of the catalytic current is expressed as follows: 1 -=

I SI

+

0.67 D&P2

(12)

indicating a decreased effect of the electrode rotation rate on the Ist value which was observed experimentally. The relations kk,/(k + k,) = 7.6 X lo-’ cm/s and k = 2.1 X 10m4 cm/s were determined using curve 3’ (Fig. 3) and assuming that k, retains its value of 1.2 X 10m4 cm/s. Therefore, it follows that, in the absence of Mg’+, V” max is decreased by 46%. However, it is possible that Mg2+ ions influence the catechol permeability through the immobilized enzyme layer and also on the k, value, since magnesium determines the conformation of protein globule [20]. The results obtained explain the absence of Michaelis dependence between I,, of the three electrodes and [S], when the latter value does not exceed 1 mM. This is accounted for by the fact that in all cases the relation V;_./K,,, exceeds the mass

Fig. 5. Dependence of the stationary catalytic current of electrode I upon [S], in Lineweaver-Burk coordinates at various l,lO-phenanthroline (mM) concentrations: 0 (1); 0.5 (2); 1 (3); 1.5 (4). [O.l M Tris-HCl buffer, pH 8.0, 25 “C, U = 0.3 V oersu~ s.c.e., w = 259.8 rad/s, determination time of I,, decrease after the addition of l,lO-phenanthroline-30 s.]

431

transfer coefficient p and, consequently, under the conditions mentioned none of the systems operates in the kinetic mode. On increasing the catechol ester concentration up to 2.5-10 mM the diffusive mode was changed to kinetic only for electrode I. In this case the stationary current is no longer dependent on w, and the data linearize in Lineweaver-Burk coordinates (curve 1, Fig. 5). However, the Michaelis constant determined is 112 times higher than the K, value for homogeneous enzyme at [S], values lower than 2 mM [2]. Increase in the K,,, and V,,, values on the concentration rise of disodium salt of p-nitrophenyl phosphate ([S], > 1 mM) for the enzyme from E. coli was observed by Heppel et al. [21]. In this connection the dependence of the homogeneous reaction rates at [S], > 2.5 mM was studied. It was found that at given substrate concentrations KmCoPPj= 21 + 0.5 mA4, i.e. in agreement with KmCappj for immobilized alkaline phosphatase. The phenomenon observed is evidently related to the dimeric structure of alkaline phosphatase. Since electrode I operates in kinetic model at [S], > 2.5 mM the catalytic current may be expressed by equation (13):

~wAz,k%~ I”=

K,+

(13)

[S],

according to which the value of Vi,, = 2.7 x lo-*’ mole cme2 s-’ was calculated. This value is 26.3 times higher than VA,, at [S], -C 1 mM. On storing electrode I for a month the catalytic activity of the enzyme decreases by 78%. Inhibition of immobilized alkaline phosphatase by I,lO-phenanthroline The introduction of l,lO-phenanthroline (OP) into the electrochemical cell containing the catechol ester and the rotating phosphatase electrode I results in a decrease of the stationary catalytic current. The fall of I,, proceeds at first sharply (instantaneous inhibition) and later (after 20-30 s) the process rate slows down (time-dependent inhibition). The inhibition of alkaline phosphatase is fully reversible in the first step, and phenanthroline acts as a competitive effector (Fig. 5). The processing of the data from Fig. 5 in Dixon derivative coordinates l/Z,, versus inhibitor concentration [I], (Fig. 6) following from equation (14) 1 z -

1 nGAV&

I

04)

enables one to determine the value of complete competitive inhibition constant K, = 1.8 f 0.05 mM according to the abscissa values of the intersection of lines 1-4. The inhibition of immobilized alkaline phosphatase activity is irreversible in the second step and obeys the laws of the first-order reactions with the rate constant k ohs= (6.7 f 0.2) X 10e3 rnin-’ at [I], = 1 mM. The results obtained indicate that the inhibition of alkaline phosphatase by l,lO-phenanthroline can be described by the following scheme for a homogeneous

438

Fig. 6. Processing of the data from Fig. 5 in Dixon derivative coordinates. Catechol ester concentration (mM): 2.5 (1); 5 (2); 7.5 (3); 10 (4).

[22,23] and also for an immobilized enzyme:

K, E.Zn+OP~E*Zn.OP~Ei,

k

(15)

Therefore, it follows, that at first a quick complex formation of chelate agent with zinc takes place in the enzyme active centre and then the complex E * Zn * OP with the rate constant kin changes to the inactive form of alkaline phosphatase. Taking into consideration scheme (15), a kinetic equation of the inhibition process can be derived: I0

kin[IIot

lnff=K, + [Ilo

(16)

where IS: is the electrode catalytic current determined after a quick reversible interaction of the enzyme with the inhibitor, and I,, is the electrode current during the irreversible inhibition of alkaline phosphatase. It is evident that kobs for electrode I must be equal to the relation kJI],/(K, + [I],,) according to which kj, = (1.88 f 0.06) x 10e2 mm-‘. The K, and k,, values are 2.3-fold lower and 1.9-fold higher respectively, for homogeneous enzyme as compared to immobilized alkaline phosphatase. This indicates that the covalent attachment of the biocatalyst exerts a certain stabilizing effect on its inactivation process by chelate reagents, namely l,lO-phenanthroline.

439

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