Preventing inhibition of tyrosinase with modified electrodes

doi:10.1016/j.aca.2006.04.088

Analytica Chimica Acta

Volume 572, Issue 1, 14 July 2006, Pages 25-31

https://doi.org/10.1016/j.aca.2006.04.088 Get rights and content

Abstract

Wines, especially red wines, contain numerous biologically active compounds, the most important of which are polyphenols, whose nutritional importance is attributed to their antioxidant power. Because of this, the detection of the amount of phenolic compounds in red wines becomes extremely important. However, using free enzyme in the determination of phenolic compounds in wines cannot reflect the actual values since there are also naturally found inhibitors in red wines. In this study, benzoic acid, cinnamic acid, and sorbic acid were utilized to understand the behavior of immobilized polyphenol oxidase in the conducting polymer matrices toward inhibition. Cinnamic acid was found to be the most powerful inhibitor for both free and immobilized enzyme in copolymer matrix of poly(terephthalic acid bis-(2-thiophen-3-yl-ethyl) ester) (PTATE) with polypyrrole (PPy). In the case of immobilized enzyme in PPy matrix, it was observed that sorbic acid is a stronger inhibitor than cinnamic acid. The inhibitory effects of these inhibitors on PPO were compared with respect to both the structural differences of inhibitors and conducting polymer matrices.

Introduction

Tyrosinase (EC 1.14.18.1) is a copper containing oxidoreductase enzyme which catalyzes two different reactions via separate active sites: (a) the orthohydroxylation of monophenols, commonly referred to cresolase activity and (b) the oxidoreduction of orthodiphenols to orthoquinones, commonly referred to as catecholase activity. It is commonly found in yeast, mushroom, apples, and potatoes.

Tyrosinase has widespread applications in medical and industrial fields. Tyrosinase catalyzes the synthesis of melanin through the hydoxylation of tyrosine to dihydoxyphenylalanine (DOPA) and the subsequent oxidation of DOPA to dopaquinone. The unstable dopaquinone will polymerize and precipitate into melanin. The cresolase activity of tyrosinase is of particular importance because it synthesizes DOPA. DOPA is a precursor of dopamine, an important neural message transmitter. Patients who suffer from Parkinson's disease show a significant decrease in the concentration of dopamine found in the substantial nigra of the brain [1], [2]. Also, the production of epidermal hyperpigmentation of melanin causes some dermatological disorders such as melasma, freckles, ephelide, senile, lentignes, etc. [3]. It has been used as part of an enzyme–electrode system to detect catechols and assess catecholamines in the urine of patients with neural crest tumors [4].

As to the industrial aspects, tyrosinase is used in the determination of phenols and its derivatives, especially for cleaning surface water and the effluent of industrial discharges. Some of the industrial sources of phenol discharge include oil refineries, coke and coal conversion plants, plastics and petrochemical companies, dyes, textiles, timber, mining, and the pulp and paper industries. Virtually all phenols are toxic. Moreover, they have a high oxygen demand and can deplete oxygen [5]. As a consequence, this may affect the ecosystem of water sources where phenols are discharged. Tyrosinase causes the precipitation of phenols, which can then be filtered out from surface waters and industrial discharge sources. The enzyme has also been used as a sensor to detect the concentration of phenols in waste water [6], [7].

Tyrosinase is responsible for enzymatic browning of some fruits and vegetables during handling and storage by affecting the taste and nutrition value of them. Browning has been attributed to a rapid degradation of the red pigments by tyrosinase, producing brown-colored byproducts [8].

Enzyme immobilization has become an important aspect of biotechnology. The usage of tyrosinase is limited because of its instability and rapid inactivation. Construction of biosensors using tyrosinase as the bio-component and conducting polymers as the matrix has many advantages. Immobilization of an enzyme provides multiple and repetitive use by increasing stability of enzyme. It prevents contamination and protects enzyme against changes in pH, temperature and ionic strength in the bulk solvent.

Immobilization of enzyme in a conducting polymer matrix by using electropolymerization is an easy, one-step procedure. It provides accurate control of polymer thickness via electrical charge passing during the film formation. Localization of electrochemical reaction exclusively on the electrode surface allows precise modification of micro-electrodes, surfaces of complex geometry and the possibility to build up multi-layer structures [9], [10], [11], [12], [13], [14], [15], [16], [17], [18].

In this study, enzyme electrodes were constructed by immobilization of tyrosinase via entrapment during the electrolysis performed for the synthesis of conducting polymers. In the previous studies [19], [20], [21], amount of phenolic compounds found in red wines are determined by using different conducting polymer matrices. The results of these studies show that some compounds naturally found in red wines inhibit the enzyme by decreasing the rate of enzyme-catalyzed reaction.

Wine, especially red wine, is very rich in the amount of phenolic substances. The phenolics have a number of important functions. They affect the tastes of bitterness and astringency. Also, they are responsible for the color of red wine. They are also bactericidal agents and impart antioxidant properties, being especially found in the skin and seeds of the grapes. The chemical composition of a wine is influenced by the climatic and atmospheric conditions, soil type, vine cultivation, and the treatment to which it is subjected. Due to this reason, amount of phenolics vary from one brand and type of wine to another. Process difference causes red wines to contain almost ten times higher amount of phenolics.

A wide range of chemicals inhibit tyrosinase activity, especially aromatic aldehydes [22], aromatic acids [23], [24], and kojic acid [25], [26]. Among them, sorbic acid and some aromatic carboxylic acids of the benzoic, cinnamic, and phenylalkanoic series have been widely studied and proven to be inhibitors for the PPO activities of various origins [27], [28], [29], [30], [31], [32]. It is known that benzoates and cinnamates are naturally found in red wines with high concentrations. Thus, during the analysis performed for the determination of amount of phenolic compounds, we should consider the effect of these substances on the measurements.

The aim of this study is to construct a biosensor by using tyrosinase as the bio-component and a copolymer of poly(terepthalic acid bis-(2-thiophen-3-yl ethyl) ester) (PTATE) with pyrrole as the matrix. Then, the inhibitory effects of benzoic acid, cinnamic acid, and sorbic acid were determined by using this biosensor.

Section snippets

Materials

Tyrosinase (PPO) (EC 1.14.18.1) (50,000 units) was purchased from Sigma. Pyrrole was purchased from Aldrich and sodium dodecyl sulfate (SDS) from Sigma. Pyrrole was distilled before use. MBTH, acetone, and sulfuric acid used in spectrophotometric activity determination of PPO were also obtained from Sigma. For the preparation of citrate buffer, tri-sodium citrate-2 hydrate, and citric acid were used as received. Catechol was purchased from Sigma. All catechol solutions were prepared in citrate

Results and discussions

Fabricated enzyme electrodes were characterized according to kinetic parameters, temperature and pH stability, operational stability, and shelf life as reported previously [37]. Kinetic parameters which were obtained from Lineweaver–Burk plot [38] are the maximum reaction rate (V_max) of the enzymatic reaction and the Michaelis–Menten constant (K_m). K_m is the parameter that shows the affinity of enzyme toward substrate. Lower K_m value means high affinity of enzyme to substrate. While free PPO

Conclusions

This study shows that determination of phenolic amount in red wines by using free enzyme does not reflect the actual values because of the inhibitors. Enzyme immobilization in conducting polymer matrices provides better results by protection of enzymes toward inhibition. The matrix differences cause the conformational changes on enzyme and this situation give rise to the changes in inhibitory powers of inhibitors as compared to free enzyme inhibition.

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In these PPy based biosensors the enzyme was either electro entrapped inside the PPy matrix or attached to the surface of PPy film in pure or composite form. These include electro entrapment of glucose oxidase (GOX) (Fortier et al., 1990; Njagi and Andreescu, 2007; Umana and Waller, 1986) for glucose determination, electro entrapment of tyrosenase for determination of phenolic compounds (Narlı et al., 2006), surface attachment of acetylcholinesterase (AChE) on PPy and polyaniline (PANI) composite polymer film doped with multi-walled carbon nanotube (Du et al., 2010a,2010b) and on Au nanoparticles–polypyrrole nano wire composite film (Gong et al., 2009) for determination of OP and OC pesticides. Though the surface immobilized PPy-AChE-nanomaterial sensors can give high sensitivity, their durability is poor due to bio-fouling and washing out of the enzyme.
The work presented here describes a novel, easy and low-cost method of fabrication of a highly sensitive acetylcholinesterase biosensor and its application to detect organophosphate and organocarbamate pesticides. Acetylcholinesterase was electro-immobilized into a thick conducting layer of polypyrrole. Porcine skin gelatin and gluteraldehyde mixture was used for stabilizing the system. Acetylthiocholine chloride was used as the substrate. Polypyrrole catalyzed the electrochemical oxidation of thiocholine and promoted the electron transfer, thus lowering the oxidation potential and increasing the detection sensitivity. Electro oxidation of thiocholine in polypyrrole matrix occurred at 0.1 V under low potential scan rate. The thiocholine sensitivity of the electrode was found to be 143 mA/M. The sensor was applied to detect the sample organophosphate pesticide ethylparaoxon and organocarbamate pesticide carbofuran. The detection limit for paraoxon was found to be 1.1 ppb and that for carbofuran is 0.12 ppb. The sensor showed good intra and inter state precision with relative standard deviation (RSD) 0.742% and 6.56% respectively. Both dry and wet storage stability were studied. The sensor stored at 0 °C in dry condition had a good storage stability retaining 70% of its original activity for 4 months. During wet storage, the activity decrease followed the same trend, however, the operational stability at the end of the storage period was found to be less compared to the dry storage case. The developed biosensor is as a promising new tool for analysis of cholinesterase inhibitors.
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A change in the type of inhibition was reported and investigated in [24] for acetylcholinesterase inhibition by heavy metals. Similarly, in [30] which employs inhibition of polyphenol oxidase, it was demonstrated that immobilising the enzyme in polymeric films of different characteristics changes the inhibition type and the inhibition constant (affinity of the inhibitor for the enzyme). Therefore, the immobilisation procedure could be an explanation for obtaining different types of inhibition for the same metal ion, even when using the same enzyme.
Electrochemical enzyme sensors prepared from cobalt or copper hexacyanoferrate modified carbon film electrodes plus glucose oxidase (GOx) immobilised by crosslinking with glutaraldehyde were successfully applied to the determination of heavy metal cations using fixed potential amperometry. Sensor performance was optimised with respect to the applied potential and influence of pH of the electrolyte solution. Cadmium, cobalt, copper and nickel ions were detected in the presence of fixed amounts of glucose, and the response to glucose was tested in the absence and presence of a fixed concentration of inhibitor. Electrochemical impedance spectroscopy was used for the first time to characterise the response of glucose biosensors in the presence of the inhibitors. The enzyme inhibition mechanism is reversible and competitive and 10% enzyme inhibition was achieved with submicromolar or micromolar concentrations of the metal cations.
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A polyaniline–polyphenol oxidase (PANI–PPO) biosensor for detecting benzoic acid is reported. The biosensor is based on the inhibition of benzoic acid on the biocatalytic activity of the polyphenol oxidase (PPO). The Michaelis–Menten constant ( ${k^{'}}_{m}$ ) and maximum response current (I_max) in both the absence and presence of benzoic acid are also evaluated. The kinetic analyses show that the inhibition of benzoic acid on the PPO activity is reversible and competitive with an inhibition constant of 28.7 μmol dm⁻³. A limit of detection of 0.3 μmol dm⁻³ benzoic acid is obtained. The I_0.5 value is estimated to be 35 μmol dm⁻³. The biosensor has good sensitivity and high stability. So it can be used as a new approach for the detecting benzoic acid.
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Preventing inhibition of tyrosinase with modified electrodes

Abstract

Introduction

Section snippets

Materials

Results and discussions

Conclusions

Appl. Radiat. Isotopes

Anal. Biochem.

Biomaterials

React. Funct. Polym.

Int. J. Biol. Macromol.

Synth. Met.

React. Funct. Polym.

Synth. Met.

Eur. Polym. J.

Bioelectrochemistry

Int. J. Biol. Macromol.

Talanta

Anal. Chim. Acta

Polymer

Anal. Biochem.

Anal. Chim. Acta

Anal. Biochem.

J. Chromatogr. A

Lebensm-Wiss Technol.

J. Mol. Catal.

Clin. Neuropharmacol.

Molecular Aspects of Dermatology