Hydrazines probe of interaction of horseradish peroxidase with cryo-hydrogel

ANALYTICA

CHIMICA ACTA

ELSEVIER

Analytica

Chimica Acta 353 (1997) 45-52

Hydrazines probe of interaction of horseradish peroxidase with cryo-hydrogel Yimin Zhu’, Jingzhong Laboratory

of Electroanalytical

Received

Zhang, Shaojun Dong*

Chemistry, Chmgchun Institute of Applied Chemistry, Chinese Academy of Sciences, Chmgchun 130022, China

18 December

1996; received in revised form 7 May 1997; accepted 2 June 1997

Abstract The interaction between horseradish peroxidase (HRP) and the cryo-hydrogel was probed by using hydrazines which show high specificity of the reaction of the edge in the prosthetic heme of horseradish peroxidase. For comparison, the interaction of hydrazine with the horseradish peroxidase adsorbed on graphite electrode was also carried out by using steady-state response of the enzyme electrode and cyclic voltammetry. In order to obtain a proper explanation of the kinetic parameters for the enzymatic reaction, the theoretical expressions of I,, and KM’ in the Michaelis-Menten equation for the experimental system were provided. 0 1997 Elsevier Science B.V. Keywords:

Hydrazines

probe; Horseradish

peroxidase;

Kinetic expression;

1. Introduction By nature, many enzymes are highly efficient at recognizing specific analytes or catalyzing reactions in aqueous biological media. These characteristics make enzymes to be desirable reagents, but the aqueous medium being necessary for enzymatic reactions limits their commercial viability. Drastic changes of enzymes in the preferred buffered aqueous medium often lead them to partial or total denaturation and loss of reactivity. Also, most redox enzymes lack pathways that can transport electrons from their embedded redox sites to an electrode [ 1,2]. Efforts are being

*Corresponding author. Fax: +86 (431) 5685653; e-mail: [email protected]. ‘Present address: Jilin Institute of Technology, Changchun 130012, China. 0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PI1 SOOO3-2670(97)00385-l

Cryo-hydrogel

made to utilize these reagents in biosensors by immobilizing them in alternative environments that stabilize them and preserve their activities [3]. Currently, biosensors are being used primarily in clinical testing, but the potential application of biosensors as probes for monitoring and controlling the industrial processes seems promising. One of the most important researches in biosensor is the immobilization of enzymes. Conventional methods of enzyme immobilization including covalent binding, physical adsorption, and cross-linking to a suitable carrier matrix have been used. Alternatively, enzymes can be physically entrapped and microencapsulated in polymeric matrices [4-71, and even can be reconstituted with a modified prosthetic group [8]. Though entrapment of purified enzymes in sol-gel matrices has been reviewed [9], enzymes often fail to retain their native stabilities and activities upon immo-

46

Y. Zhu et al./Analytrca

Chimico Actu 353 (1997) 45-52

bilization, a flaw that results in low stabilities or altered functional responses of biosensors must be overcome. Recent researches [lo, 111 in our laboratory have demonstrated that a cryo-hydrogel obtained by the lyophilized method can provide a semi-interpenetrating network and that enzymes immobilized by this method can retain their functional characteristics to a large extent even in organic solvents. In general, the gel layer itself contains the essential water required for enzyme catalytic activity, which allows the enzyme to be operated in pure or neat organic phase. Although the exact role of water is not yet fully understood, its effect has been attributed to the maintenance of the hydration layer [12]. However, the nature of the interaction of enzyme with the cryo-hydrogel still remains unclear. Several hydrazine analogues have been succesfully used as pharmaceutical agents [13]. However, hydrazines are a carbonyl trapping agents which can deactivate many oxidative enzymes, including metalloproteins [14-171, and are therefore, generally cytotoxic. The deactivation of horseradish peroxidase (HRP) by hydrazines recently has shown that hydrazines were oxidized by electron transfer to the heme edge rather than to the ferry1 oxygen, because of the presence of high specificity of the reaction of the 6meso position and the &methyl group in the solventexposed heme edge of HRP, with hydrazines metabolite, and the absence of the hydrazines adducts expected from reaction with the iron or nitrogen atoms of the heme [15-171. Here, we discuss the interaction between HRP and the cryo-hydrogel by using hydrazine probe. Besides, there are well developed models for the amperometric enzyme electrode function [ 182 11too. However, we are interested in providing the kinetic explanation for electrocatalysis of the enzyme immobilized by cryo-hydrogel method.

2. Experimental

rate(B) (Analytical grade) were from Beijing Chemical Reagent Co. All enzyme incubations and assays were carried out in 50 mM sodium phosphate buffer (pH 7.0). All solutions were prepared with deionized water and purged with high purity nitrogen. 2.2. Immobilization

method

The pyrolytic graphite electrode (5 mm diameter) was polished on wet, fine emery paper, ultrasonicated in deionized water and acetone, successively. Preparation of the cryo-hydrogel-HRP electrode was based upon our previous reports [ 10,111. A kind of polyhydroxy cellulose (PHC) was prepared in our laboratory, which is a mixture of polyvinyl alcohol (PVA) and carboxymethyl hydroxyethyl cellulose (CMHEC) (with CMHEC/PVA from l/5 to l/3 w/w). 5 mg HRP was dissolved in 0.2 ml of 10% PHC aqueous solution, an aliquot (0.02 ml) of the mixture was spread over the pyrolytic graphite electrode surface. The electrode then underwent the repeated process of freezing and thawing for 12 to 24 h to form a threedimensional cross-linked cryo-hydrogel on the surface, and kept in refrigerator at 4°C before use. The enzyme electrode was rehydrated with 0.01 ml of water, dried in ambient atmosphere for 30 min, and then used. The adsorbed HRP electrode was prepared by immersing the pyrolytic graphite electrode in 1 mg/ ml HRP solution for 20 min and then washing out the unadsorbed species from electrode surface with deionized water and subsequently with buffer solution. 2.3. Electrochemistry A three-electrode cell configuration with a Pt wire counter electrode and Ag/AgCl (saturated KCI) reference electrode was employed for all experiments. An electrochemical system (PAR model 370) and a Bioanalytical system (model NOB, West Lafayette, IN) were used to perform electrochemical experiments.

2. I. Reagents 3. Results and discussion Hydrazine solutions were prepared from hydrazine hydrochloride (Sigma Chemical Co.) and methylhydrazine (Fluka AG Co.). While HRP (E.C. 1.11. I..7, Type VI, 318 U/mg) was obtained from Sigma, 30% hydrogen peroxide and potassium hexacyanofer-

3.1. Response

of the enzyme

electrode

In order to exhibit the interaction of HRP with the cryo-hydrogel, we chose the classic oxidation of the

E Zhu et al. /Analytica

hexacyanoferrate(I1) presence of HRP:

ion by hydrogen

Chimica Acta 353 (1997) 45-52

peroxide in the

In aqueous solution the hexacynoferrate(II1) ion produced in the HRP-catalyzed reaction is reduced at potentials of 30 mV [22,23]. When the adsorbed-HRP electrode was immersed in pH 7.0 buffer solution containing 5 mM hexacynoferrate(I1) ion, a stable base current of about 10 nA was recorded after an equilibration time of about 5-15 min (curve A in Fig. 1). As a sample containing hydrogen peroxide was injected, the current increases thereby reflecting the electrochemical reduction of the hexacynoferrate(II1) ion. The magnitude of the increasing current depends on the concentration of hydrogen peroxide, as shown in curve B in Fig. 1. Its kinetic results can be analyzed in terms of Lineweaver-Burk form of the Michaelis-Menten equation. The result gives Z,, 5.260f0.003 n&cm’ and KM’ =18.80f0.02 n&I, where Z,, is the saturation current and KM’ is the apparent Michaelis-Menten constant. The adsorbedHRP electrode was immersed in pH 7.0 buffer solution containing 5 mM hexacynoferrate(I1) ion and 0.383 mM hydrazine for 1 min and later, hydrogen peroxide was injected. The current did not increase observably (curve C in Fig. 1) thereby reflecting the deactivation of the adsorbed-HRP by hydrazine.

___-_--

10pA

--

41

Meanwhile, the deactivation of the adsorbed-HRP by metbylhydrazine is similar to the result by hydrazine. The inhibitory action of the hydrazines is irreversible. Recent studies for the deactivation of HRP by methylhydrazines [15-171 have indicated that 6meso-methylated heme can be formed and the substrates interacted primarily or exclusively with the heme edge rather than the ferry oxygen of HRP. The absorbance spectrum study of deactivation of HRP by methylhydrazine exhibits that the soret absorbance of HRP shifts to red from its initial position at 402 nm and decreases in intensity. The soret band shift is accompanied by the appearance of an absorbance peak at 830 nm. The formation of a methylhydrazineHRP interaction intermediate with an absorption band at 830 nm is reminiscent of the formation of a similar chromophoric species in the reaction of HRP with cyclopropanone hydrate [24]. The intermediate may thus, be a key species in the deactivation. The cryo-hydrogel-HRP electrode, under comparable conditions in the absence of hydrazine, gives KM’ = 19.50f0.02 mM and Z,, = 0.181f0.003 mAZcm’ from curve A in Fig. 2. Curves B and C in Fig. 2 yield KM’ = 19.70f0.02 mM and I,,, = 0.164f0.003 mA/ cm2, and KM’ = 19.40f0.02 mM and Z,,, = 0.130f0.003 mAZcm2 (Table 1) in the presence of 0.383 mM and 2.550 mM hydrazine, respectively. There is a little deactivation of the cryo-hydrogelHRP even in a large amount of hydrazine, showing the non-competitive inhibition [25] of the HRP by hydrazine. After the cryo-hydrogel-HRP electrode was exposed to hydrazine 2 min, the extent of the inhibition was not found to depend on the time of exposure. During electrode preparation the enzyme underwent a lyophilization and the semi-interpenetrating network of the polymer formed gradually, which in turn, results in the active conformation of the enzyme retained in hydrogel. Further, the active conformation might be

C A

100s Fig. 1. Steady-state response of the adsorbed-horseradish peroxidease (HRP) electrode in pH 7.0 phosphate buffer solution containing 5 mM hexacynoferrate(I1) ion: (A) in absence of hydrogen peroxide, (B) injecting hydrogen peroxide on increasing the concentration 0.267 mM steps, (C) repeating (B) in presence of 0.383 mM hydrazine. Operating potential: 30 mV; stirring rate: 300 ‘pm.

Table 1 Kinetic parameters

for the enzymatic

Cryo-hydrogel-HRP No hydrazine With 0.383 mM With 2.550 mM With 0.383 mM With 2.550 mM

hydrazine hydrazine methylhydrazine methylhydrazine

reaction J&t’ (mM)

Lu

19.50 19.70 19.40 19.60 19.20

0.181 0.164 0.130 0.153 0.128

(&cm?

48

K Zhu er al. /Analyrica Chimica Acta 353 (1997) 45-52

soluble enzyme (typically 0.1 mM), an observation suggesting mass transport control of the current. Moreover, the adsorbed enzyme has a greater maximal current than the entrapped preparation, an observation suggesting the greater resistance in the latter case. On the other hand, the greater resistance of the entrapped enzyme may be due to the production of smaller amounts of the inactivating products from hydrazines due to slower turnover. i.e. smaller currents are

generated. 3.2. CycEic voltununetric

Fig. 2. Electrochemical Lineweaver-Burk plot for the cryohydmgel-HRP electrode to hydrogen peroxide in pH 7.0 phosphate buffer solution containing 5 mM hexacynofermte(l1) ion. Experimental conditions: injecting hydrogen peroxide on increasing the concentration 0.133 mM steps in absence of hydrazine (A), in presence of 0.383 mM (B), and 2.550mM (C) hydrazine. Operating potenrial: 30 mV; stirring rate: 300 rpm.

“locked in” by the hydrogen bonds between the hydroxyl groups on the polymer chains and the carboxy1 groups and amino-groups of the enzyme. Similarly, KM’ = 19.60&0.02 mM and I,,, = 0.1531tO.003 mA/cm2. and KM’ = 19.2010.02 mM mA/cm’ were obtained, and I,, = 0.128f0.003 respectively (Table I), in the presence of 0.3X3 mM and 2.550 mTvl methylhydrazine. Comparing the results with that of the adsorbed-HRP, only a little deactivation of the cryo-hydrogel-HRP can be observed even in the presence of a large amount of methylhydrazine. This indicates that the solventexposed heme edge (such as &mesn-position) of HRP may also be “locked in” by the cryo-hydrogel. The PVA gel entrapped HRP (no freezing process) showed the behavior similar to the adsorbed HRP, because the PVA gel was gradually solved. Alternatively, the immobilized enzyme whether entrapped or simply adsorbed shows a very high KM’ (1020 mM) for peroxide when compared to the

measurements

It is important to obtain information about the permeation of hydrazine in the cryo-hydrogel. The cyclic voltammogram of the cryo-hydrogel-HRP electrode in the blank buffer solution is shown in curve A in Fig. 3. When the cryo-hydrogel-HRP electrode was immersed in a solution containing an appropriate concentration of hydrazine, an irreversible oxidation wave with peak potential +0.74 V occurred as shown in curves B and C in Fig. 3. The peak current increases with increasing the concentration of hydrazine. This indicates that hydrazine can permeate through cryohydrogel layer to the graphite electrode surface and be oxidized over the potential 0.40 V. Similarly, an irreversible oxidation wave witi peak potential of 0.85 V of methylhydrazine was produced at the cryo-hydrogel-HRP electrode (Table 2). For comparison, the cyclic voltammogram of the polished graphite electrode in the blank buffer solution is shown in curve A in Fig. 4. When the graphite electrode was immersed in a solution containing 0.128 mM hydrazine, an irreversible oxidation wave with a peak potential +0.45 V was achieved as shown in curve B of Fig. 4. Obvious difference between +0.74 V in curve B in Fig. 3 and 0.45 V in curve B in Fig. 4 should be noticed. However, when HRP with an appropriate concentration of hydrazine was added into the solution, the oxidation peak potential of hydrazine shifted towards more positive side with Table 2 Anodic peak potential (V) of hydratines

Methylhydrazine Hydrazine

On graphite

On the cryo-hydmgel-HRP

0.60 0.45

0.85 0.74

E Zhti et al./Analytica

49

Chimica Acta 353 (1997) 45-52

IW I

I

0

I

1

0.4

I

1

1

0.8

EN Fig. 3. Cyclic voltammograms for the eryo-hydrogel-HRP electrode in pH 7.0 phosphate buffer solution (A), and in a solution containing an appropriate concentration of hydmzine, 0.128 (B), 0.192 (C), 0.255 (D), and 0.320 mM (E). Sean rate, 10 mV/s.

decrease in the peak current. This can be attributed to the effect of adsorption of HRP on the electrode surface. Finally, an oxidation wave with peak potential +0.70 V was obtained, which is similar to the result in Fig. 3. These results indicate that the slight deactivation of the cryo-hydrogel-HRP by hydrazine is not due to non-permeation of hydrazine in the cryo-hydrogel layer. Meanwhile, an irreversible oxidation of methylhydrazine gives the peak potential 0.60 V (Table 2) at the bare graphite electrode. The peak current increases by increasing the concentration of methylhydrazine, thereby showing that merbylhydrazine can permeate the cryo-hydrogel layer too

EN Fig. 4. Cyclic voltammograms for the graphite electrode in pH 7.0 phosphate buffer solution (A), in a solution containing 0.12s mM hydrazine (B), and on adding: 0.033 mg/ml HRP (C). 0.066 mgknl HRP (D), 0.099 mg/ml HRP (E). 0.132 mg/ml HRP (F) in the (B) solution. Scan rate: 10 mV/s.

3.3. Kinetic analysis of enzyme reaction the cryo-hydrogel layer

in

The results show that at the adsorbed-HRP electrode, Z,, =5.260 mA/cm’ and KM’ = 18.80 mM, while at the cryo-hydrogel-HRF’ electrode, Z,,, = 0.181 mA/cm* and KM’ = 19.50 KIM. There is very large difference between 5.260 and 0.181 n-A/cm2 in saturation current, while there is a small difference between 18.80 and 19.50 mM in apparent MichaelisMenten constant. For a proper explanation of these results, the theoretical expression of I-, and KM’ in the cryo-hydrogel-HRP layer seems to be essential.

Y Zhu et al. /Analytica

50

Chimica Acta 353 (1997) 45-52

steady-state

3.4. Nature of the enzyme sensor reaction From the results in Fig. 2, the reaction simple Michaelis-Menten kinetics, l/Z =

~M’/&ax~S)

+

follows

where Z and Zmax are the current density and the saturation current density, respectively, Z&r’ is the apparent Michaelis-Menten constant, and Cs is the concentration of the substrate. When the reaction rate is limited by the first order of decomposition of the enzyme-substrate complex (ES) to the product (P), and when this complex is formed rapidly and reversibly from the enzyme (Ened) and substrate (S), the rate expression has the same form as Eq. (1) [25]: kl ERed + S z ES%Eox+P

(2)

The oxidized enzyme is reduced mediator (Mad) as follows: Eox + bed

5

@%,hl(dCMc&+,,

for MoX at x = &:

+D&,dl(dCh&/d&

+ k4%~,h&&,hl (1)

l/&ix

conditions

by the electron

(9)

= 0

where Dao,,~ is the diffusion coefficient of Max in Nernst diffusion layer (dl). At the hydrogel layerNemst diffusion layer interface a jump in the concentration can take place [6]. The partition coefficient (a) is defined by a = Cd/CM Considering x = dhl,

(10) steady-state

k&&,,hlCEo,,hl

conditions

for enzyme

(11)

= WER~,hJs,hllKM

As the total concentration layer CE = CErccd,hl + c,,,hl

From Eq. (7) obtain

of enzyme in the hydrogel

(12)

+ CES,hl

and

at

considering

Eqs. (8)-(12),

we

(3)

Eyed + Max

Z = nFk3CECs,hlDMo,,hlddlk51(KM The oxidized electron mediator (MoX) is electrochemically reduced at the electrode surface,

+ C.S,hl(l + k3/k4CM~,hl)}((YDM~,,dlDhlo,,hl + ~dh@m,&s

k4 MOx

+ ne

(4)

---) MRed

3.5. Kinetic expression of the enzyme sensor reaction From the reaction 4, the cathodic current density (I) is proportional to the reaction rate [26], Z = nFks&,,,,

(5)

where Cr,,rorOis the concentration at electrode surface. According to Fick’s first law, the flux Q is proportional to the concentration gradient (dC/dr), -ZMox = D&X cd%.

where D is the diffusion layer (hl), Z/nF = -.&

(6)

/&) coefficient.

For the hydrogel (7)

Considering a diffusion through a linear concentration gradient, we have C&,o = &$,x,hl’k,hl/(Dr&,~ where

di.,i is the hydrogel

+&&I

layer

thickness.

(8)

Under

(13)

kdd&iti,hl)

Considering lower part of the substrate concentrations in the Z-Cs relationship, we may obtain KM Cs,hl( 1+k3/k&M,,hl). Under steady-state conditions for S at x = dhl, Ds,hl(dCs/dX)hl+Ds,dl(dCs/dX)dl-k3CECs,hl/Khl=O (14) and at x =O, Ds,hl(dCs/d-&O

(15)

= 0

Thus, Z = nFksCnC+J&,&t&s/ X KM(~

+ dd&E/&,dM)

X (~&c,+IIDM~,~

= D&,t,t(dCM&l&a

-

+Qd&Mox,&

-~&@M,,JII) (16)

Eq. (16) can express the experimental current density because the concentration of hydrogen peroxide (Cs) is smaller under the experimental conditions. In addition, we need to obtain I,,, in order to explain the experimental results. Considering higher part of the

E Zhu et al. /AnaZyfica

Chimica Ada 353 (1997) 45-52

substrate concentrations in Z-C, relationship, we may obtain KM < Cs,h1(1 + /Q/~&M,_&. Under steadystate conditions for MRed at n = dhl ~M,,hl(dCM~/‘i& -

+ &~,dl(dCM,w/~)d,

k3CERd,dlG,hllKM

=

Under a linear concentration KM

<

CS,hl(

(17)

0

1 + k3/k4CMRd,hl)

profile, and considering and

CM,,O

to zero under the applied potential, I max -

nFWE~M,,,~~dlks/{1 x

(~~M~~,dl&vl&l

-

k&&,.,hl)

As Z = Z,,Cs,,JK~‘, KM’

=

KM(a X (1

+ +

approaches

we have

Q’~~/(~~CM~+T~))

51

I-RP may be “locked in” by the cryo-hydrogel. Because a general carbonyl trapping agent (hydrazine) and methylhydrazine which shows the high specificity of the reaction of the solvent-exposed heme edge in HRP, do not markedly depress the catalytic activity of HRP immobilized in the cryo-hydrogel. Meanwhile, the primary theoretical explanation of the kinetic parameters for the enzymatic reaction occurred in the immobilized enzyme layer, is provided.

Acknowledgements

QdhlDMti,dlkS (W

and from Eqs. (16) and (18),

The support of this project by the National Natural Science Foundation of China is greatly appreciated.

+ ddlk3CE/&,dl&)/ +

~k3/(k4CMnti,ca))

(19)

For the adsorbed-HRP electrode, & = d&s (the adsorbed enzyme layer thickness), a = 1, and &&,h, = &,,,& Eqs. (18) and (19) Can be alSO used to analyze its I,,, and KM’. From Eq. (lg), parameters & and k,hi (referred to the cryo-hydrogel layer) do not affect on KM’. Thus only a small difference between 18.80 mM for the adsorbed-HRP and 19.50 n&l for the cryo-hydrogelHRP can be observed. But the parameters are involved in Eq. (18), so their effect on Z,, (5.240 mA/cm’ for the adsorbed-HFW, 0.18 1 mA/cm’ for the cryo-hydrogel-HRP) is very large. For the enzyme electrodes, the enzyme layer (the adsorbed layer or the cryo-hydrogel layer) is a key layer that we need to notice. of course, other factors in Eqs. (18) and (19) can also determine the values of I,., and KM’, such as the kinetic factors k3, k4 and k5, the partition coefficient a, the diffusion properties &, and L&.,dl, and the concentrations of enzyme and mediator (C, and CM~). and so on. The most troublesome thing in all factors is the wet membrane thickness (&), which lies below 0.1 cm for most membranes, and there are no precise methods for their measurement as yet. At present, Eqs. (18) and (19) can not be experimentally confirmed. Their model calculations have not been published. Many further work would be the subject of detailed investigations. In summary, the present results suggest that the carbonyl group and the solvent-exposed heme edge of

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[25] A. Fersht, Enzyme Structure and Mechanism, 2nd ed., W.H. Freeman and Company, 1985. [26] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, Wiley, New York, 1980.