Chern. Anal. (Warsaw), 43,135 (1998)

Analytical Applications of Inhibitionof Enzymatic Reactions

by Tadeusz Krawczynski vel Krawczyk

Departmenf(~fChemistry,University (~fWarsaw, Pasteura 1,02-093 Warsaw, Poland

REVIEW

Key words: enzymatic determination, pesticides, heavy metals, cyanides, fluorides,

biosensors, spectrophotometry, fluorimetry, amperometry, potentiometry

The analytical application of inhibition of enzymatic reactions is reviewed. The deter­mination of environmental pollutants, namely: organophosphorus, carbamate and chlo­roorganic pesticides, heavy metal ions, fluorides and cyanides is described. Thedetermination of pesticides is based mainly on their inhibition action on cholinesterases,while for the determination of heavy metals various enzymes (urease, invertase, xan­thine oxidase, peroxidase, glucose oxidase, butyrylcholinesterase and alkaline phospha­tase) are used. For the determination of cyanide its inhibition of mainly cytochromeoxidase and tyrosinase is applied, while fluoride inhibits mainly liver esterase (lipase).Numerous detection techniques were used, e.g. amperometry, potentiometry, spectrop­hotometry, fluorimetry or thermometry for detection of different substrates as well asproducts of enzymatic reactions in static and flow conditions. Applied enzymes \vereused first of all in immobilised form like biosensors or enzyme reactors. The examplesof determination of toxic pollutants in environmental (waters, soil extracts, riversediments) and biological (fruits, vegetables, plant extracts, body fluids) samples aregiven.

Dokonano przeglqdu zastosowan analitycznych inhibicji reakcji enzymatycznych. Opi­sano oznaczanie zanieczyszczen srodowiskowych: pestycyd6w fosforoorganicznych,karbaminianowych i chloroorganicznych, jon6w metali, cyjank6w i fluork6w. Oznacza­nie pestycyd6w opiera si~ na inhibicji gl6wnie cholinoesteraz, natomiast do oznaczaniametali stosuje si~ rMne enzymy (ureaz~, inwertaz~, oksydazy ksantyny i glukozy,peroksydaz~ chrzanowq, butyrylocholinosteraz~ i fosfataz~ alkalicznq). Do oznaczunia.cyjank6w wykorzystuje si~ gl6wnie inhibicj~ oksydazy cytochromowej i tyrozynazy,natomiast do oznaczania fluork6w -lipazy. Stosuje si~ r6zne metody detekcji substra­t6w i produkt6w reakcji, np. amperometri~, potencjometri~, spektrofotometri~, t1uory­metri~, termometri~ w warunkach statycznych i przeplywowycb. Podano przykl:adyoznaczania toksyn srodowiskowych w wodach, ekstraktach gleby, osadach dennychoraz materiaIe biologicznym (awace, warzywa, ekstrakty roslinne i plyny ustrojovvc)

136 T. Krawczynski vel Krawczyk

Inhibitors are substances decreasing the rate of enzyme-catalysed reactions.These compounds can be divided into two groups: reversible and irreversible ones.Reversibleinhibitors bind to an enzyme in a reversible way and can be removed bydialysis or simply dilution to restore full enzymatic activity. Irreversible inhibitorscannot be removed from an enzyme by dialysis, however, it may be possible toremove an irreversible inhibitor from an enzyme by introducing another componentto the reaction mixture.

Reversible inhibitors usually rapidly form an equilibrium system with an enzyme.An inhibition, depending on the concentration of enzyme, inhibitor and substrate,remains constant during the initial period, whereas the degree of inhibition byreversible inhibitors may increase over this period of time.

Among reversible inhibitors three mechanisms ofinhibition can be distinguished:competitive, uncompetitive and non-competitive one (Fig. 1). Competitive inhibitiontakes place, when an inhibitor exhibits structural similarity to the given substrates ofenzymatic reaction and may compete for the same binding site on the enzymeblocking reactive group of an enzyme or is held in an unsuitable position with respectto the catalytic site. In either case a dead-end complex is form, and the inhibitor mustdissociate from the enzyme and be replaced by a molecule of substrate. The degreeof competitive inhibition depends on the concentration of an inhibitor and a substrateas well as their relative affinity for the enzyme. A low concentration of substratefavours the inhibition process and vice versa - at high substrate concentrations theinhibitor is much less successful in competing with the substrate and the degree ofinhibition is less marked. At very high substrate concentrations the effect of theinhibition is negligible. The maximum rate (Vmax) of the reaction is unchanged,however, Michaelis constant Km is increased in the presence of inhibitor and calledthen an apparent Michaelis constant K'm.

Uncompetitive inhibitors bind only to the enzyme-substrate complex and not tothe free enzyme (see Fig. 1), when a favourable change of enzyme conformation aftersubstrate binding is formed, or the inhibitor can bind directly to the enzyme-boundsubstrate. The inhibitor does not compete with the substrate for the same activecentre, so the inhibition cannot be overcome by increasing the substrate concentra­tion. This kind of inhibition occurs rather rarely with single-substrate reactions and

presence of such a kind of inhibitor alters both Michaelis constant and maximumrate of enzymatic reaction.

Non-competitive inhibition is observed, when an inhibitor can combine with anenzyme molecule to produce dead-end complex, regardless of whether a substratemolecule 'is bound or not. An inhibitor destroys the catalytic activity of the enzymeei ther by binding to the catalytic site or as a result of a conformational changeaffecting the catalytic centre - not affecting substrate binding. The total enzymeconcentration is effectively reduced by the inhibitor decreasing the value of Vmax butnot altering Km, since neither inhibitor nor substrate affects the binding of the other.In many real cases one can observe mixed inhibition when more than one amongabove mentioned mechanisms occur depending on the relation between inhibitionconstants Ki and K1, describing the equilibrium of formation of enzyme-inhibitorcomplex due to reaction between enzyme and inhibitor and the enzyme-substrate-in-

l!-nzimatic inhibition in analytical applications

(A)

Q + ~ QJ=Q + LJpES E

Q + LJ +-~ QIIE

(B)

137

(C)

Q+LJ= 8J=Q +LJE S ES E P

-I T1 +1

@ESI

(D)

fiE+8

W=[t ~+

-8 ES E P

-I r 1+1 -I r 1+1

@ +8 @EI ~S ESI

Figure 1. Possible examples of mechanisms of reversible inhibition. (A), (B) - competitive inhibitionwith inhibitor (I) binding to the same (A) or different site (B) of enzyme (E) as substrate. (5);(C) - uncompetitive inhibition; (D) - non-competitive inhibition; P - product

138 T. Krawczynski vel Krawczyk

hibitor complex, when the inhibitor is bound to the enzyme-substrate complex. Alsosome other types of inhibition such as substrate inhibition or so called allostericinhibition are mentioned but the description of them exceeds the frames of thisreVIew.

In the contrary to reversible inhibition, the irreversible inhibition takes oftenplace in real systems. An irreversible inhibitor binds to the active site of the enzymeby an irreversible reaction and cannot subsequently dissociate from it. A covalentbond is usually formed and the inhibitor may act by preventing substrate-binding orit may destroy some component of catalytic site. The total recovery of the initialenzyme activity is then practically impossible.

Enzymes are frequently inhibited specifically by low concentration of certainchemical substances, and so enzymatic methods are commonly used for inhibitordeterminations [1-3]. The earliest analytical method based on enzyme inhibitiondates from 1908 [4] and concerns the possibility of determining fluoride by itsinhibition of liver esterase.

DETERMINATION OF PESTICIDES

The determination of traces of pollutants in biological materials and environmen­tal samples (natural water and air) has become increasingly important. One class ofthese pollutants are organophosphorus and carbamate pesticides, widely used inagriculture. They show an environmental persistence lower than the organochlorinecompounds but have a higher acute toxicity which can be a serious problem for theequilibrium of aquatic ecosystems. Another problem is food contamination whichcould have a serious impact on human health. A high acute toxicity of thesecompounds creates need for fast-responding detection systems in order to protecthuman health during manufacturing and application. Pesticides inhibit the action ofacetylcholinesterase, which hydrolyses the neurotransmitter acetylcholine, produc­ing acetic acid and choline, in order to re-establish the initial state of the postsynapticmembrane. When this enzyme is inhibited, nerve impulses are disrupted sinceacetylcholine remains present in the synaptic region. As a consequence some neuro­logical diseases (e.g. tetanic shock with eventual muscle paralysis) may occur.Therefore there is an urgent need for the development of rapid analytical techniquesfor the determination of organophosphorus and carbamate pesticides and othercompounds which might have similar toxicological behaviour (e.g. paralysing gases).

At the moment, the most commonly used techniques for pesticide determination,are gas chromatography and HPLC [5]. Although these techniques are available, theyare expensive and require skilled personnel to operate them. Analysis time is gener­ally considerable since these methods require laborious sample pre-treatment andpreconcentration steps limiting the frequency of analysis. The increasing use ofimmunoassays for the determination of pesticides is observed during the last decade[6]. It is a promising method for screening environmental contaminants (especiallyfor water quality control) with immunoaffinity chromatography or immunosensors.

Enzimatic inhibition in analytical applications 139

The chromatographic methods cannot be used for continuous on site analysis, asthe instruments used are in general large. From the other side pesticides can beanalysed by selective enzymatic procedures based on their ability to inhibit thecatalytic activity of certain enzymes such as lipase, acid and alkaline phosphatase,acylase and, most frequently, cholinesterases (acetylcholinesterase AChE and buty­rylcholinesterase BChE). The last one as less selective undergoes stronger inhibition.In general the selectivity of inhibition of cholinesterases by pesticides is not veryhigh, so chromatographic techniques (HPLC, TLC) have been recommended for theseparation of pesticides before their enzymatic determination. With regard to non­sufficient selectivity toward individual pesticides the most reasonable approachseems to be the determination of so called paraoxon equivalents (e.g. in water samples[7]) or the extent of enzyme inhibition can be used as index of the amount ofanticholinesterase pesticides present in real samples (fruits and vegetables [8]) toobtain rapid screening method of their determination.

Enzymatic methods based on the inhibition of cholinesterase activity haverecently been the object of intensive investigation due to their sensitivity andspecificity. The ease of performance of the assay and the low cost of the equipmentmake such methods highly attractive for laboratories. Enzyme inhibition tests are ofgreat interest in environmental pollution analysis because the toxicity of pollutantssuch as pesticides is the result of in vivo enzyme inhibition and biological testsinvolving enzyme inhibition used for the determination of environmental toxins seemto be more reliable than physical (e.g. chromatographic) methods.

Enzymatic methods based on the inhibition can be coupled to various analyticaltechniques by the use of different substrates, like acetyl- or butyrylcholine, acetyl­or butyrylthiocholine or indoxyl acetate. An inhibition reaction can be carried out insolution. Although this method as limited to single use of enzyme is expensive, itdoes not require a regeneration of inactivated enzyme. Enzymes are often immobi­lised in reactors or in the form of biosensors especially useful for this purpose. Beinga combination of a biological component with a sensing device (transducer), theyhave great versatility of design and a wide range of applications. The most advantageof biosensors is their high selectivity and simple use.

Among transducers, ion-selective electrodes, amperometric electrodes, ion-se­lective field effect transistors (ISFETs), optical fibres, fluorimetric devices, bulkwave and surface acoustic wave (SAW) piezoelectric transducers have been investi­gated. Potentiometric as well as amperometric devices are the most frequently used.Although they are not in thermodynamic equilibrium and therefore are unstable andhighly sensitive to interferences, they exhibit slow drift of the signal and a significantsensitivity to ionic strength and buffer capacity. The interface between the enzymelayer and the solid phase of an electrode may create also difficulties in electrontransfer. They offer, however, considerable advantages like high sensitivity, linearityin a broad concentration range, relatively short response time, and sufficient repro­ducibility and stability. Moreover, they can be applied in field conditions in flowmeasurements and in fast screening tests for hazardous pollutants in environmentaland food samples in order to protect human health.

140 T. Krawczynski vel Krawczyk

Cholinesterases can be inhibited more or less specifically depending on thesource of enzyme [9]. The measurement of the signal, proportional to the concentra­tion of substrate or product of enzymatic reaction, obtained after some incubationtime when the enzyme is in contact with pesticide depending on the kind of pesticideand its concentration, is carried out after the definite time or the rate of substrate orproduct changes during the initial period of enzymatic reaction is monitored. Thisvalues compared with obtained in the same conditions but without the interaction ofenzyme with inhibitor gives so called degree of inhibition and is proportional to theinhibitor concentration. The measurement is most often performed in non-flowconditions, however, recently also flow-injection method is more and more oftenutilised [10-19].

The inhibition of cholinesterases can be reversible (carbamate pesticides) orirreversible (organophosphorus pesticides). The proposed mechanism of irreversibleinhibition includes the interaction of organophosphorus pesticide with serine hydro­xyl groups of the enzyme protein. In the case of reversible inhibition the regenerationof the enzyme immobilised in the reactor or in the biosensor form can be obtainedby addition of buffer or substrate solution which indicates the competitive mechan­ism. However, when irreversible inhibition takes place, strong agents like someoximes, most frequently 2-pyridine aldoxime (2-PAM) [11,13,18-27], 1,1'-tri­methylene-bis(4-formylpyridinium bromide)-dioxime (TMB-4) [12,15,28] or obi­doxime [7,29] are used. One must, however, point out, that such regeneration ispractically never complete even after very long time. The efficiency of differentreactivating reagents was also compared [12]. The schemes of inhibition and reacti­vation mechanisms are shown in Figure 2.

Amperometric detection

As it was pointed out earlier, an amperometric detection is the most frequentlyused for the determination of pesticides based on their inhibition of cholinesterasesor other enzymes. This technique depends on the substrate of the enzymatic reactionused. Cholinesterases can catalyse hydrolytic decomposition of acetyl- (ATCh+)orbutyrylthiocholine:

Tiocholine (TChH+) can be monitored due to oxidation:

2TChH+--TCh-TCh2+ + 2H+ + 2e

on Pt electrode at +410 mV vs. Ag/AgCI [20,30], Ti-Au-Pt electrode at +700 mV vs.saturated calomel electrode (SCE) [15], on graphite at +800 mV vs. SCE [31] orCo-phthalocyanine-modified graphite electrode at +250 mV vs. Ag/AgCI [32-34].Also graphite paste (+700 mV vs. Ag/AgCI) [35] or graphite paste modified withCo-phthalocyanine (+300 mV vs. Ag/AgCI) [36] electrodes were used for this

(A)

Enzimatic inhibition in analytical applications 141

OR'./'

Enz.-Ser-OH + HO-P," OR"o

(B)

inhibition)

OR',/"

Enz.-Ser-O-P, + H 20II OR"o

o/I

2(RO}-P--{}-Enzyme +

Phosphorylated enzyme

1(; N WlL N~ "O-P-(ORJz+ I

CH3

Phosphorylated oxime

2-PAM

+ Enzyme-OH

Reactivated enzyme

Figure 2. Inhibition (A) and reactivation (B) mechanism of enzymes with organophosphotus pesticides

purpose as well as graphite powder biocomposite containing a non-conducting epoxyresin, the electronic mediator 7,7,8,8-tetracyanoquinodimethane and the enzyme(+300 mV vs. Ag/AgCI) [37]. Thiocholine was also used as depolariser in galvano­static amperometric system with two platinum electrodes polarised with 25 J.lAcurrent [38] or the reduction current ofthiocholine-mercury compound was measuredat -550 mV vs. SCE at mercury film-covered silver electrode [39].

Another possibility is to use acetyI- or butyrylcholine:

A t 1 h 1· H 0 Acetylcholinesterase Ch l' CH' C'100Hce y come + 2 ~ 0 me +" 3

Choline formed as a product of the above reaction can be then oxidised with theoxygen from the solution in the presence of another enzyme, choline oxidase:

. Choline oxidase .Cholme + 202 • Betame + H20 2

142 T. Krawczy,iski vel Krawczyk

The last reaction can be monitored due to detection of hydrogen peroxide on Ptelectrode at +600-+700 mV vs. Ag/AgCI [14,16,29,40,41] or with commercial H20 2

detector [42,43]. Also the measurement of the oxygen consumption can be carriedout with Clark oxygen electrode [44].

Besides choline or thiocholine esters, acetylcholinesterase can catalyse the de­composition of other esters like 4-aminophenol acetate giving 4-aminophenol oxi­dised on glassy-carbon electrode at +250 mV vs. SCE [21], or indoxyl acetate withformation of indoxyl which was oxidised on Pt electrode at +300 mV vs. Ag/AgCI[45].

Besides cholinesterases some other enzymes were rarely used for the determina­tion of pesticides due to their inhibition effect. In the case of aldehyde dehydrogenasecatalysing the oxidation of propionaldehyde by nicotine adenine dinucleotide (NAD):

P . ld h d NAD+ Aldehyde dehydrogenase P . . .d NADHroplOna eye + ~ roplOlllC aCl +

NADH formed was re-oxidised by feerricyanide in the presence of another enzymediaphorase:

NADH + Fe(CN)~- Diaphoras~ NAD+ + Fe(CN)t

and oxidation current of ferrocyanide to ferricyanide was then monitored at +81 mVvs. Ag/AgCI [30]. Also inhibition of tyrosinase catalysing oxidation of varioussubstrates (catechol, -dopamine, L-DOPA, epinephrine [46] or Catechol Violet [47])to quinone with consecutive quinone oxidation current monitoring on glassy-carbonpolypyrrole-coated [46] or gold-graphite-polypyrrole electrodes [47] at -200 mV vs.SCE was applied.

Potentiometric detection

As it was shown earlier, during hydrolysis of choline esters catalysed by choli­nesterases except choline the proper organic acid (acetic or butyric) is form changingthe pH of tested solution (and hence - the potential) during the course of enzymaticreaction (also inhibited in the presence of pesticide). This change can be monitoredwith various pH electrodes like glass electrode [13,18,22,26,48-51], metal oxide pHelectrodes (e.g. Pd/PdO or Ir/h02 [21,39]) and pH ISFETs [23-25]. Also a commer­cial enzyme field effect transistor (ENFET) with immobilised urease was used forthis purpose [28] as CO2 and NH3, both pH changing species, are formed during thehydrolysis of urea. Despite pH-sensitive sensors also change of concentration ofacetylcholine being the substrate of enzymatic reaction catalysed by acetylcholines­terase was measured with commercial (Corning Model 476200) liquid-membraneelectrode sensitive to acetylcholine [52]. Interesting measurement of change of redox

couple Fe(CN)~-/Fe(CN)~- potential was applied in the method elaborated by Ivanit­

skii and Rishpon [11]. They used acetylthiocholine as a substrate and thiocholine

formed in the enzymatic reaction for the reduction of Fe(CN)~-:

Enzimatic inhibition in analytical applications 143

2TChH+ + 2Fe(CN)~-_ TCh-TCh2+ + 2H+ + 2Fe(CN)t

so the decrease of acetylcholinesterase activity due to pesticide caused less decreaseof the potential after addition of substrate compared to the same situation when therewas no inhibitor in the solution.

Spectroscopic detection

Spectroscopic methods like spectrophotometry VIS or fluorimetry are moreseldom used than electrochemical ones described in previous sub-paragraphs. Themost popular is so called Ellman method of spectrophotometric detection of thio­choline formed during enzymatic hydrolysis of acetylthiocholine. As a colour de­veloping reagent 5,5'-dithiobis(2-nitrobenzoic acid) is used and the absorbance ismeasured at A=405 nm [7,16,17,53-55]. Another possibility of spectrophotometricdetermination of pesticides based on the inhibition of cholinesterase activity is themeasurement of coloured species formed by a-naphthol with p-nitrobenzenediazo­nium fluoroborate [10,12] in the broad range of wave-lengths from 440 to 560 nmdepending on pH. In this case a-naphthol is a product of hydrolysis of a-naphthylacetate catalysed by acetylcholinesterase. Inhibition of cholinesterases can be alsoutilised with sepctrofluorimetric detection, e.g. of N-methylindoxyl (f"ex = 430 nm,Aem =501 nm) formed during the decomposition of N-methylindoxyl acetate [9].Also fluorimetric detection of umbelliferone formed by decomposition of umbelli­ferone phosphate catalysed by alkaline phosphatase (determination of organophosho­rus pesticides) or acid phosphatase (determination of carbamate and organochlorinepesticides) [56] or its derivative 4-methyl-umbelliferone being the product of enzy­matic decomposition of 4-methylumbelliferone heptanoate in the presence of lipase[8] (Aex =330 nm, Aem =450 nm) was used.

Analytical characteristics of pesticide determination methodsbased on enzyme inhibition

The analytical parameters of enzyme determination methods based on enzymeinhibition like linearity range, accuracy or precision depend mainly on the pesticidedetermined and the method of detection and it is difficult to quote all the data forevery pesticide and method (not taking into account the enzyme used as more than90% papers cited in this review are based on inhibition of cholinesterases). Someimagination about this variety one can obtain from the data for the most oftendetermined organophosphorus pesticide paraoxon. For this model pesticide range ofconcentration determined was e.g. from 2 x 10-10 to 1 X 10-7 mol 1-1 [7], from 1 x10-9 to 1 X 10-6 mol I-I [22] and from 9 x 10-9 to 5 X 10-7 mol 1-1 [12] withpotentiometric, amperometric or spectrophotometric detection, respectively. Fromthe other hand higher and narrower concentration ranges, like from 4 x 10-8 to 4 X

10-7 mol 1-1 [52], from 1 x 10-7 to 4 X 10-7 mol 1-1 [41] or from 2 x 10-4 to 8 X

10-4 mol 1-1 [54] are also reported for the same detection methods, respectively. Theprecision of the determination of paraoxon was better than 2% (RSD) both foramperometric [43] or spectrophotometric [10,12] detection.

144 T. Krawczynski vel Krawczyk

The most frequently reported analytical parameter of the method is the detectionlimit, hence it seems to be the most representative parameter for the comparison ofdifferent detection techniques used for the determination of pesticides by enzymeinhibition. The values of detection limits for 5 most often determined organophos­phorus pesticides are given in Table 1, and in Table 2 these values are reported forcarbamate and organochlorine pesticides. In the second Table one from the chemicalwarfare agents group (sarin) is also reported.

Table 1. Application of esterases inhibition for the determination of organophosphorus pesticides*)

)Other organophosphorus pesticides determined or tested: azinphos methyl and ethyl, benzophos- phate,bromophos methyl, carbophos, chlorfenvinphos, chlorophos, 2,2'-dichlorovinyldimethyl phosphate, diiso­propyl fluorophosphate, ethylbromophos, ethyl dithiopyrophosphate, ethylparathion, fenitrothion, fenthion,fonofos, heptenophos, malaoxon, methylnitrophos, methylparathion, mevinphos, monocrotofos, parathionmethyl and ethyl, phosphamide, systox, tetram.

Pesticide Detection method EnzymeLimit of detection,

Referencemolr l

Paraoxon Amperometry AChE 1 x 10-10-1 X 10-7 19,21,29,30,35,37,41

AChE or BChE 5 x 10-9-3 X 10-7 32,40,44

BChE 3 x lO-1O~l X 10-8 33,37,42,43

Potentiometry AChE 1 x 10-9-4 x 10-8 18,22,52

BChE 7 x 10-7-1.5 X 10-6 27,49

Spectrophotometry AChE 2 x 10-10-5 X 10-5 7,10,12,54

BChE 2 x 10-7 55

Dichlorvos Amperometry AChE 9 x 10-9-1 X 10-7 15,35BChE 1.2 x 10-9 33

Potentiometry AChE 2 x 10-7-1 X 10-6 11,18,25,26

Parathion Amperometry AChE 3.5 x 10-9-2.5 x 10-7 40,44Potentiometry AChE 6 x 10-7 18,26

Fluorimetry BChE 9 x 10-9-5 X lO-R 9

Malathion Amperometry AChE or BChE 2 x 10-7-2.5 X 10-6 32,44BChE 2 x lO-R 43

Potentiometry AChE 1 x 10-10-1.5 X 10-9 18,22

Trichlorphon Amperometry AChE 1 x 10-9-1 X 10-6 14,31Potentiometry AChE 1 x 10-6 /23

AChE or BChE 1 x 10-7 24

*

Table 2. Determination of other pesticides based on enzyme inhibition

Pesticide Detection method EnzymeLimit of detection,

Reference(moll- l)

Aldicarb Amperometry AChE 1 x lO-R-2 X 10-7 30,41Potentiometry AChE 4 x 10-7 26

Carbaryl Amperometry AChE 1.3 x 10-10-1 x 10-7 19,21,35,37AChE or BChE 1 x 10-9-1 X 10-6 32,34

BChE 1.1 x lO-R 33

Enzimatic inhibition in analytical applications 145

Table 2 (continued)

Potentiometry AChE 2.5 x lO--7 18

BChE 2 x lO-5 49

Spectrophotometry AChE 2 x lO-R 17

Carbofuran Amperometry AChE 9 x lO-9-2.5 x lO-R 8,16-18,35,37

BChE 1 x lO-7 37

Potentiometry BChE 4 x lO-6 49

Urease 4 x lO-1O 28

Aldrin Fluorimetry Lipase 0.8IJ.g ml- 1 57

Alkaline 51J.g ml- 1 56phosphatase

Heptachlor Fluorimetry Lipase 1 x lO-7 57

Lindane Fluorimetry Lipase 3 x lO-6 6

Sarin Spectrophotometry BChE 0.01 ngml-1 53Amperometry BChE 2 x 10-4 IJ.g ml-1 38

Other compounds determined or tested: carbamate pesticides: butoxycarboxime, carbamoyl choline, maneb,methomyl, propham, propoxur, sevin, sulfometuronmethyl, tifensulfuron methyl; chloroorganic pesticides:DDT, dieldrin; nerve agents: soman, tabun.

The time of the analysis depends strongly on incubation time and ranges from3 min [35] to 1 h [42] depending on the concentration of paraoxon. However, in thecase of organophosphorus pesticides this time can be considerably longer if thereactivation procedure is carried out (e.g. even 6 h [19,21]).

There are also few papers describing the determination of pesticides or paralysinggases in real samples, e.g. in tap [7], drinking [16] or synthetic sea [18] water,_ innatural waters [43], in lake water and soil extracts [44], in lagoon water and kiwifruits [19], in river sediments [34], in plant extracts [9], and in fruits and vegetables[8]. Sarin was determined in air samples [53].

DETERMINATION OF METAL IONS

The study of the effect of metal ions on enzyme activity has attracted considerableinterest in analytical chemistry. Enzymes are frequently inhibited by low concentra­tion of metals, so enzymatic methods are commonly used for the determination ofsuch inhibitors. From the point of view of economy and ease of handling, conven­tional enzymatic procedures can be dramatically improved by using immobilisedenzymes and flow methods. The sensitivity and selectivity of enzymatic methodstogether with simplicity, speed and ease of automation make these methods highlypromising for the use in environmental protection. Detection of enzyme inhibitorscan be performed in a very sensitive way, since the interaction of a single inhibitormolecule with an enzyme can result in a large reduction of the enzyme activity, theenzyme thus acting as an amplifier. Enzyme electrodes are relatively easily con­structed and can be used for in situ experiments. The primary aim for using them ismonitoring and screening. The literature data describing effects of various metal ions

146 T. KrawcZyfiski vel Krawczyk

on enzymes activity indicate that inhibition results from metal interaction withsulphydryl groups of the enzyme which contributes to non-stabilisation of theenzyme-substrate complex.

The inhibition of urease was the most frequently studied from the point of viewof metal determination with various detection methods. It is inhibited by mercury[58-65], copper [15,28,63-67], silver [62,63], cadmium [63,65], lead, nickel, cobaltand manganese [63], zinc and chromium(VI) [65]. The electrochemical detection wasapplied in examination of other enzymes inhibition by heavy metals, e.g. glucoseoxidase (Hg, Cu, Ag [68] or Cu and Mn [69]), peroxidase (Ni, Co, Mn [70]), ,acetyl­(Cu, Hg [51]) or butyrylcholinesterase (Hg, Pb, Cd [43] or Cu, Bi, Pb, and TI [39])and oxalate oxidase (Cu [71]). However, it should be noticed that electrochemicalmethods were used less frequently than e.g. spectrophotometric one. In this methodof detection except of inhibition of urease for the determination of Hg, Ag, Cu, Zn,Pb, Cr(In) and Co [58], Cu [66], Cu and Hg [64] or Hg, Cu, Cr(VI), Zn, Cd andFe(HI) [65] also other enzymes were used. Alkaline phosphatase inhibition was testedfor the determination of Be and several other metal ions [66], Mg, Cd, Ca, Ba and Pb[72], and Be and Zn [73]. Xanthine oxidase was found to be inhibited by Ag, Hg, Cu,Cr(VI), V(V), Au(III) and TI(I) [74] and glucose oxidase by Ag, Hg, Pb or As(II!)[75] while some dehydrogenases, like isocitric or lactate were inhibited by micro­molar amounts of Zn, Mn, Cu, Cd, Ni and Co [76] or by Hg, Cu, Zn and Cd [77],respectively. Spectrophotometric detection was also used for the determination ofHgusing ~-fructofuranosidase (invertase) [78]. In the case of this enzyme also thechange of optical rotation was the measure of enzyme activity and its inhibition byAg [79,80] as well as Hg [79] was used for the determination of these metal ions.Fluorimetric detection was applied for the determination of Hg [56] or Hg and Ag[62] based on urease activity inhibition, and for the determination of Bi and Be(inhibition of alkaline phosphatase [56]). Enzyme thermistor as well as SAW/imped­ance enzyme transducer, both with immobilised urease, were used for the determi­nation ofHg [81] or Cu [67,81], and Hg [82], respectively.

It should be also mentioned that some enzymes contain metal ions being thecomponents of their active centres. After removing of metal ion, e.g. by treating withstrongly complexing agent, the obtained apo-enzyme form is inactive. However, itsactivity increases rapidly after introducing traces of metal ions and this increase wasalso applied for the determination of some metal ions due to activation of enzyme.In this way Zn or Ca were determined with apo-enzyme of alkaline phosphatase [83]or Zn with aminopeptidase from pig kidney [84].

Amperornetric detection with immobilised enzymes

In amperometric biosensors with immobilised enzymes oxidoreductases aremostly employed as they catalyse redox reactions where redox species (e.g. H20 2)

are formed. These products of enzymatic reaction can be monitored amperometri­cally. Potential inhibitors like heavy metals decrease the formation of redox productand hence amperometric signal decreases proportionally to the concentration ofinhibitor. Butyrylcholinesterase, which catalyses the hydrolysis of choline esters

Enzimatic inhibition in analytical applications 147

belongs to the group of enzymes which are inhibited by heavy metals. In the presenceof choline oxidase the final product of enzymatic reaction is hydrogen peroxideformed due to the oxidation of liberated choline. The mixture of these enzymes wasimmobilised on nylon net at the surface of commercial amperometric H20 2 detectorforming biosensor [43]. The inhibition of BChE by Hg, Pb and Cd was examinedand the minimum concentration of these metals at which the decrease of the ampe­rometric signal started was evaluated as 30, 10 and 60 J-lmol 1-1, respectively. Inanother biosensor, where BChE was immobilised in nitro-cellulose membrane, thedecrease of amperometric signal of the oxidation of hydrolysis product thiocholinedue to heavy metals was utilised for the determination of Cu and Pb in the range from1 x 10-9 to 1 X 10-5 and from 5 x 10-6 to 1 X 10-3 mol I-I, respectively [39].Reactivation of the enzyme activity was obtained by the use of 0.1 moll-1 EDTA or0.02 moll-1 hydroxylamine solutions. The enzyme was also inhibited by Tl(I), Cdand Bi. Also inhibition effect of glucose oxidase immobilised on nylon membrane atthe Pt electrode surface was investigated for 16,metal ions [68]. Only Cu, Hg and Agexhibited significant inhibition. The enzyme electrode could be reactivated byEDTA, the reactivation being most effective for Cu. The ability to restore the enzymeactivity following Cu inhibition, and the linear response of the detector between 2.5x 10-4 and 5 x 10-3 moll-1 indicated a prospect for the use of a flow system fordetermining this enzyme inhibitor. In another work glucose oxidase was immobilisedin polyaniline conducting polymer [69], so this detector could be considered aschemically modified electrode. The inhibition by 15 metal ions including also heavymetals like Zn, Cd, Cr(HI), Cu, Mn, Fe(H), Co, Ni and AI, was investigated. Only Cuinhibited the enzyme and Mn in low concentration exhibited an activation effect. Itwas also demonstrated that another enzyme from oxidase group, horseradish perox­idase was inhibited by some metal ions. The enzyme was immobilised in carbon pasteand the inhibition effect of Ni, Co and Mn was eliminated by EDTA being also thecomponent of electrode matrix [70]. Cu was found also to inhibit oxalate oxidaseinterfering the determination of oxalate [71]. The inhibition effect was removed byEDTA. Compagnone et al. [85] have tested amperometric biosensors with 15enzymes from oxidases group for the determination of 12 metal ions. The best resultswere obtained for Cd with D-amino acid oxidase (range 5-20 ppm, detection limit 2ppm), for Cu with alcohol oxidase from Pichia Pastoris (range 0.05-0.5 ppm,detection limit 0.03 ppm), Hg with glycerol-3-P oxidase (range 0.05-0.5 ppm,detection limit 0.02 ppm), Ni with sarcosine oxidase (range 1-10 ppm, detection limit1 ppm), Se(IV) with glutathione oxidase in the same range and with detection limitof 0.5 ppm and for V(V) with glutathione oxidase (determination in the range 0.3-2ppm). Hg compounds were also determined due to inhibition of invertase [86] withindirect amperometric detection of hydrogen peroxide formed after oxidation ofglucose in the presence of glucose oxidase on Pt electrode at +650 mV vs. Ag/AgCl.As glucose was formed during the reaction:

InvertaseSucrose + H20 I D-Glucose + D-Fructose

148 T. KrawcZy'lski vel Krawczyk

the decrease of the amperometric signal was observed in the presence of invertaseinhibitor. The method with enzyme immobilised on nylon net at the electrode surfacewas applied for the determination of Hg compounds, MetHg and EtHg in the range1-50 ppb. The biosensor used was reactivated by dipping in 10 mmoll-I cysteinesolution for 10 min. In contrast to free enzyme in solution, its immobilisationremoved interferences of Ag.

Potentiometric detection with immobilised enzymes

Also potentiometric biosensors with immobilised enzymes (mainly urease) weretested for the inhibition effect of metal ions. Urease catalyses hydrolysis of ureaforming ammonia and carbon dioxide. Both change the pH, so every pH detector,like glass or metal oxide electrodes as well as pH-sensitive ISFETs may be used formonitoring the analytical signal. However, in the earliest analytical works, in whichthe inhibition of urease was the base for heavy metals determination [63], the enzymein solution was utilised. The determination with pH-stat detection was appliedfor Ag(2 x 10-8-1 x 10-7 and 2 x 10-7-1 X 10-6 moll-I), Hg (2 x 10-7-1 x 10-6 moll-I), Cu,Cd, Co, Ni, Mn and Pb in the range between 2 x 10-6 and 1 x 10-5 moll-I. Picogramto nanogram amounts of these metals were determined in water solution with relativeerror < ± 20%. Also Winquist eta!' [61] used urease in solution or immobilised ontest plates containing dry reagent strips with all necessary chemicals for the verysensitive evaluation of Hg content down to 5 nmol 1-1. In their work an ammoniagas-sensitive iridium thin metal oxide semiconductor detector was used. Zurn andMuller [15] used photolithographically patterned enzyme membranes with ureaseimmobilised onto the pH-sensitive gate area of a miniaturised transducer (ISFET) forthe detection of Cu based on enzyme inhibition. Such a biosensor was able to detectCu(ll) in water in the ppm-range without preconcentration. The inhibition of ureaseimmobilised in enzyme reactor was also applied to the determination of Hg in therange up to 7 nmol [59]. Ammonia formed in enzymatic reaction was detected by anammonia gas electrode. The sample volumes were 5 or 25 ml and the total Hgconcentrations were up to 1.5 X 10-7 mol 1-1 and up to 3 X 10-8 moll-I, respectively.The reactor was regenerated by thioacetamide and EDTA between the measurements.Mercury was strongly bonded to the urease, so the method should be useful fordetermination of free as well as complex Hg(ll) ions. Only silver and copperinterfered. Also potentiometric detection (pH electrode) of acetic acid formed duringhydrolysis of acetylcholine catalysed by acetylcholinesterase was applied for thedetermination of Cu and Hg down to 1 mg 1-1 due to inhibition effect [51]. Theenzyme was immobilised on acetate cellulose membrane. Similar results werepresented for acid phosphatase [51].

Other detection methods

In the mentioned above methods, based on other than electrochemical detectiontechniques, mainly immobilised enzymes were used for the determination of metalions based on inhibition. Metal inhibition studies of urease immobilised on controlledpore glass (CPG) through different bifunctional coupling reagents were performed

Enzimatic inhibition in analytical applications 149

using a fibre-optic biosensor configuration, wherein the pH change resulting fromthe biocatalytic hydrolysis of urea was compared before and after exposure to themetal ion solutions [58]. The strongest inhibition by Hg, Ag, Cu, Zn, Pb, Cr(III) andCo was 'observed for the urease immobilised with cyanuric chloride- and glutaralde­hyde-activated support, however, only for Hg the calibration is given in the rangefrom 1 x 10-7 to Ix 10-3 moll-I. The biosensor was reactivated with 0.75 mol I-Idiethylenetriaminepentaacetic acid (DTPA) (pH 6). Urease immobilised on apolymer support (VA Epoxy) in the reactor was also inhibited by Cli [66]. Ammoniaconcentration formed in enzymatic reaction was measured photometrically withindophenol method. The elaborated method was applied for the Cu speciation indrinking and surface water samples. A solution of EDTA was able to reactivate theinhibited enzyme. The use of urease in solution with the same indophenol spectro­photometric detection enabled the determination of Hg and other heavy metals inaqueous soil extracts [64] (Hg down to 10 ppb and Cu down to 5 ppb) and in surfacewater, ground water and waste water samples [65] (Hg down to 0.05 ppb). Alsoinhibition of urease immobilised in column enzyme thermistor [81] as well as onSAW/impedance enzyme resonator [82] was applied for the determination of Hgdown to 1 x 10-9 mol I-I and 1 x 10-7 m'oll- l , respectively. The last l11ethod wassuccessfully applied to the determination of Hg in waste water and the results agreedwell with those obtained by AASmethod.

Fluorimetric determination of Hg based on the inhibition of enzymatic activityof urease immobilised on CPG in a flow-injection configuration was described byBryce et ai. [60]. The linear determination range was between 0.5 and 100 ppb andthe sampling frequency was 6 h- l. The immobilised enzyme reactor was regeneratedby L-cysteine injected between samples. Cu was found to interfere strongly~. The sameconditions, i.e. acid urease immobilised on CPG in column reactor and flow-injectiontechnique but with thermometric detection, were applied for the determination of Cuin the range from 5 x 10-6 to 1 X 10-4 moll~l with average standard error 2% [67].The mean advantages of the method were: 20-fold higher sensitivity of acid ureaseto Cu than urease (from jack beans) commonly used for metal sensing, regenerationof enzyme did not require any metal chelating agent, no decrease in enzyme activityis observed because of irreversible inhibition and of performing intermittent moni­toring of Cu using a thermistor device is possible. Screening method for trace Hganalysis using flow-injection with urease inhibition and a fluorescence detection wasdescribed by Narinesingh et ai. [62]. In their method a urease in flowing solution wasused. Calibration curves were found to be linear up to 22 ppb and the detection limitof 0.2 ppb could be achieved. Hg was determined in soil samples with the results wellcomparable with AAS cold vapour technique.

The spectrophotometric studies of alkaline phosphatase inhibition were based onthe determination of p-nitrophenol at A = 400-405 nm formed in the catalyticdecomposition ofp-nitrophenyl phosphate substrate [73,87]. The method was appliedfor the determination of Be in a flow-injection system with enzyme immobilised onCPG in the range from 1.5 x 10-5 to 1.5 x 10-4 mol I-I as well as for the determinationof other metal ions (Cu, Zn, Cd, AI, Fe(III), Ni and Co) [87], or Be and Zn in the18-90 ng and 0.6-6 g range, respectively [73]. In the case of fluorimetric study of

150 T. Krawczynski vel Krawczyk

inhibition of alkaline phosphatase by metal ions, umbelliferone phosphate was usedas a substrate of enzymatic reaction [56]. The inhibition by Be and Bi (without anyincubation) allowed the determination of these metals in the range 12-130 ppb withmean error ±1.59'0 and 3-63 ppm with mean error ±1.3%, respectively.

Oxidation of o-dianisidine with hydrogen peroxide catalysed by horseradishperoxidase, inhibited by Hg, was used for the determination of this metal down to 3x 10-7 J-Lg ml-1 [73]. The product of the oxidation was monitored spectrophotome­trically at A=460 nm. In the same paper [72] the inhibition of alkaline phosphatasewas used for the determination of Mg 00-3-10-1 J-Lg ml- I), Cd (10-3-10-z J-Lg ml-I)or Ca and Ba in the presence of Sr. Lead could be determined down to 6 x 10-4 J-Lgml-I. The same reaction, i.e. oxidation of o-dianisidine by HzOz was used for thespectrophotometric measurement of glucose oxidase inhibition by metal ions.In thiscase hydrogen peroxide oxidising o-dianisidine was formed as a product of catalyticoxidation of glucose in the presence of glucose oxidase. The method was used forthe determination of Ag in the range from 2 x 10-6 to 10-5 mol 1-1 [75]. Also Hg inthe range 0.1-0.4 f-lg was determined with standard deviation ±8 ng and error < 34%.

The effect of metal ions on the catalytic conversion of xanthine into uric acid inthe presence of xanthine oxidase was studied spectrophotometrically [74]. Utilisationof the linear relationship between relative enzyme activity and inhibitor concentrationallowed sensitive and selective determination of Ag and Hg (10-9-10-8 mol 1-1 ), andof Cu and Cr(VI) 00-7-10-6 moll-i) with mean RSD = 2.5% and relative error 4%.The effect of various metals on the enzyme-catalysed conversion of isocitric acid toa-ketoglutarate was studied in the presence of triphosphopyridine nucleotide [76].Mn, Mg and Co were activators for the reaction while many other metals (Be, Ca,In(Ill), Sr, Ba, AI, Ce(III), Fe(III), Ni, Cu, Ag, Cd, Hg, Pb) act as inhibitors. Znactivated the enzyme at low concentrations but inhibited at above 10-4 moll-I.

The limits of detection of heavy metals using enzyme inhibition are given inTable 3.

Table 3. Determination of metals based on enzyme inhibition

MetalEnzyme inhibited Detection method

Limit of detection,Reference

determined mol 1-1

Mercury Urease Potentiometry 5 x 10-9-2 X 10-7 59,61,63

Spectrophotometry 2.5 x W-IO-1 x lO-R 64,65,69

Fluorimetry 2.5 x 10-9-1 x lO-R 60,62

SAW resonator 1x 10-7 82Invertase Spectrophotometry lxlO-1l 71

Polarimetry 2 x lO-R 79

BChE Amperometry 3 x 10-5 43Xanthine oxidase Spectrophotometry 1 x lO-R 74Peroxidase Spectrophotometry 5 x 10-6 72Glucose oxidase Amperometry 1 x 10-5 68

Copper Urease Spectrphotometry 1.5 x 10-7-1 x lO-R 64-66

Potentiometry 2 x 10-6-3 X 10-6 15,63Thermometrv 1 x 10-6 67

Enzimatic inhibition in analytical applications 151

Table 3 (continued)

BChE Amperometry 1 x 10-9 39

Alkaline phosphatase Spectrophotometry 1.5 x 10-5 87

Xanthine oxidase Spectrophotometry 1 x 10-7 74

Glucose oxidase Amperometry 5 x 10-5 68

Silver Invertase Spectrophotometry 8 x 1O-R-2 X 10-7 72,79

Polarimetry 1 x 10-7 80

Urease Fluorimetry 9 x 10-10 ~ 62

Potentiometry 2 x 10-8 63

Glucose oxidase Amperometry 1 x 10-4 68

Spectrophotometry 2 x 10-6 75

Lead BChE Amperometry 5 x 10-7-1 X 10-5 39,43Peroxidase Spectrophotometry 5 x lO-R 72Alkaline phosphatase Spectrophotometry 3 x 10-9 72Urease Potentiometry 2 x 10-6 63

Zinc Alkaline phosphatase Spectrophotometry 1 x 10-6-1 X 10-5 73,87

Peroxidase Spectrophotometry 2 x 10-6 72Urease Spectrophotometry 8 x 10-4 65.

Cadmium BChE Amperometry 5 x 10-5-6 X 10-5 39,43Alkaline phosphatase Spectrophotometry 9 x 10-6-1 X 10-4 72,87

Peroxidase Spectrophotometry 5 x 10-6 72

Beryllium Alkaline phosphatase Fluorimetry 1.3 x 10-6 56Spectrophotometry 3 x 10-7-1.5 X 10-5 73,87

Bismuth Alkaline phosphatase Spectrophotometry 1 x 10-6 73BChE Fluorimetry 1.4 x 10-5 56

Amperometry 1 x 10-5 39

Nickel Urease Potentiometry 2 x 10-6 63Alkaline phosphatase Spectrophotometry 2 x 10-6 87

Cobalt Urease Potentiometry 2 x 10-6 63Alkaline phosphatase Spectrophotometry 1.5 x 10-5 87

Chromium Urease Spectrophotometry 1.8 x 10-4 65Xanthine oxidase Spectrophotometry 3 x 10-7 74

V(V), Au(III) and Tl(I) as well as Fe(III), AI, Mg, Ca and Ba were also determined by inhibition of xanthineoxidase and alkaline phosphatase, respectively. Hg, Cu, Zn and Cd exhibited the effect oflactate dehydroge­nase inhibition.

DETERMINATION OF OTHER INHIBITORS

Cyanide

Due to its toxicity as a respiratory inhibitor, much research has been devoted tothe analysis of cyanide. Cyanide expresses its toxicity by binding to the terminalcomponent in the electron transport chain in the mitochondria, cytochrome oxidase.It blocks an intermolecular electron transfer thus stopping terminal electron transport

152 T. Krawczynski vel Krawczyk

to oxygen. Cytochrome oxidase inhibition by cyanide is known to be uncompetitivetoward 02'

As toxicity of cyanide manifests itself in inhibition of cytochrome oxidase inliving organisms, it was obvious to use the inhibition of this enzyme for thedetermination of cyanide. In this determination amperometric detection (see Fig. 3for the principle of the method) of reduction current of cytochrome c on carbon pasteelectrode at -150 mV vs. Ag/AgCI [88] or decrease of oxygen consumption withClark electrode [89] were utilised. In the first method cyanide could be determinedin the range 1 x 10-6-1.4 X 10-5 mol I-I with limit of detection 5 x 10-7 mol I-I [88]while the second one allowed the determination in very similar range (0-1.2 x 10-6

moll-I) and detection limit (4 x 10-7 moll-I) [89]. Also amperometric detection wasapplied for the determination of cyanide due to inhibition of another enzyme,tyrosinase [46,90]. For this purpose biosensor with tyrosine immobilised on glassy­carbon electrode was used [90]. The analogy between the inhibition of tyrosinase andthat of cytochrome oxidase restraining metabolic respiration is shown in Figure 4.The amperometric detection was based on the measurement of reduction current offerricyanide used as mediator instead of cytochrome c in the case of cytochromeoxidase:

Electrode reaction: Fe(CN)~- + e -+Fe(CN)~-

at 0 mY. Cyanide could be determined in the range up to 1 X 10-3 mol 1-1 withdetection limit below 5 x 10-5 moll-I. The binding of cyanide to tyrosinase was alsoreversible. In another method of determination of cyanide due to tyrosinase inhibitionwith amperometric detection the reduction current of o-dichinone at -200 mV vs.SCE was measured (Fig. 5). The biosensor used consisted of glassy-carbon electrodewith tyrosinase immobilised in polypyrrole layer [46]. The determination of cyanideup to 1 X 10-6 mol I-I with very low detection limit of 2 x 10-8 mol I-I was possiblemaking this device particularly interesting for monitoring cyanide, e.g. in drinkingwater. Also amperometric biosensor with horseradish peroxidase immobilised onglassy-carbon disc of rotating platinum and glassy-carbon ring-disc electrode wasused for the determination of cyanide in ppb range [91]. The principle of the methodis shown in Fig. 6. In this method the reduction current of ferrocenium ions on Ptring at 0 V vs. SCE was measured. Indirect determination of traces of cyanide was

Graphite r - •. - - - .• _. - - - . - - .. - - .. -,

particle=t-(cyt. c).)~(cyt. a-CU.l". ~(cyt. a.-cu"I.,!,,(H:Pe-/ Ccyt. C)red \icyt. a-CUa)ox './\.ccyt. a3-~)red:) O2

'-. __ ••. __ •• , •.••.••. _. __ •••••. J

Cytochrome oxidase

Figure 3. Principle of amperometric detection of cyanide based on inhibition of cytochrome oxidase[88]

Enzimatic inhibition in analytical applications 153

D(ox)~ Cyt c (redlXCyt OX (OX)X lip (sl

0''''1~,~ Cyte 10XI Cyl Ox (..., Oz

reN

!M(red)X

pPO(OXIX lip. lb'

M (ox) PPO (red) ~

T

Figure 4. Comparison of the terminal sequence of electron transport in aerobic respiration chain (a) withamperometric sensor used for the determination of cyanide based on tyrosinase inhibition (b)[90]. D - electron donor (cytochrome reductase), Cyt c - cytochrome c (electron mediator),Cyt Ox - cytochrome oxidase, M - electron mediator (e.g. ferricyanide), PPO - tyrosinase

catecholquinone

- ..._ ...._ ...-..~..._"-r-'~...;l........Jl,__ -..... - 200 mVvs SeE

phenol or catechol + Oz

Figure 5. Schematic description of the electroenzymatic cycle for the detection of phenol or catechol assubstrate during amperometric determination of cyanide based on inhibition of tyrosinase [46]

also carried out utilising the decrease of inhibition effect of Hg or Ag (due tocomplexation) of invertase [92]. In the presence of 2 X 10-8 mol I-I Hg cyanide fromthe range 2 x 10-8-1 X 10-7 mol I-I could be determined, whereas higher concentra­tions (1 x 10-6-1 x 10-5 mol 1-1) were determined in the presence of 2 x 10-7 mol 1-1Ag with accuracy ±3%.

154 T. Krawczynski vel Krawczyk

2 Fe(cp)2

2Fe(cp);

Figure 6. Schematic representation ofthe principle of amperometric detection of horseradish peroxidase(HRP) activity with two working electrodes during cyanide determination based on HRPinhibition [91]. Fe(cph - ferrocene (electron mediator)

Fluoride

Fluoride content is a parameter that should be controlled in waters occurring therefrom phosphate fertilisers manufacture, the aluminium and steel industries, oil wellsand effluents from atomic energy plants. Fluoride may also be added to natural watersupplies and pharmaceutical products, and it plays an important role in dental health,so that fluoride determination in teeth and body fluids can also be required.

Similarly like in the case of the first analytical paper describing the utilisation ofenzyme inhibition for the determination of fluoride with liver esterase (lipase) [4],also further methods of enzymatic fluoride determination were based on the inhibi­tion of this enzyme. In those methods the following reactions are utilised:

Ethyl butyrate~Ethanol+ Butyric acid

Ethanol + NAD+ Alcoholdehydrogenas~ Acetaldehyde + NADH

The methods of detection include the potentiometric detection of butyric acid [93,94]or spectrophotometric detection of NADH at A = 340 nm [95]. The last methodallowed the determination of fluoride in the linear calibration range from 8 x 10-7 to8 X 10-6 mol 1-1 with limit of detection of 1.6 x 10~6 mol 1-1 in water [95], whereaswith previous method fluoride in blood and urea as well as in water were determined[93]. The inhibition of acid phosphatase by fluoride was applied for their determina­tion, too [96]. The principle of the determination was based on the followingreactions:

Acid phosphatase 2-Glucose 6-phosphate + H20 ~ Glucose + HP04

Glucose oxidaseGlucose + O2 • Gluconolactone + H20 2

Enzimatic inhibition in analytical applications 155

An amperometric measurement of oxygen consumption with Clark electrode wasapplied as a detection method. Fluoride were determined in the range up to 6x 10-3

mol 1-1 with detection limit 1 x 10-4 mol 1-1 and precision of 6.5% (variationcoefficient for n = 12 at 2 x 10-3 mol 1-1). Non-competitive inhibition mechanismwas postulated.

Another enzyme used for the determination of fluoride was urease [97]. In thismethod CO2 formed during enzymatic reaction was detected potentiometrically withgas sensitive electrode. Fluoride in the range 3 x 10-4-1 X 10-2 mol 1-1 could bedetermined.

Other species

The methods mentioned above were also applied for the determination of otherthan cyanide or fluoride species. Gaseous hydrogen sulphide was determined up to40 ppm with limit of detection 1 ppm due to inhibition of cytochrome oxidase withamperometric detection [89]. Sulphide was also determined due to decrease ofinhibition effect of Hg on invertase by the method analogous to described for cyanide[92] in the narrow range 1 xl 0-7-2.5 x 10-7 moll-I. The same method allowed thedetermination of iodide in the range from 1 X 10-7 to 7 X 10-7 mol 1-1 [92] and theinhibition of tyrosinase was used for the determination of chlorophenols in the rangefrom 4 x 10-7 to 2 X 10-6 mol 1-1 [46]. Inhibition of acid phosphatase by phosphatewas applied for their determination in the range up to 2 X 10-4 mol 1-1 with limit ofdetection 2.5 X 10-5 mol 1-1 by method described for fluoride [96].

Organic-phase biosensors suitable for monitoring low levels ofenzyme inhibitorsin non-aqueous media, were described by Wang et ai. [98]. The inhibition oftyrosinase or horseradish peroxidase by thiourea, benzoic acid, diethyldithiocarba­mate, hydroxylammonium sulphate and mercaptoethanol was exploited for highlysensitive amperometric measurements in organic media. Fast on-line monitoring ofvarious inhibitors was illustrated in a flow-injection operation, with a detector basedon enzyme inhibition and an acetonitrile carrier solution.

Limits of detection of methods elaborated for the determination of some from theabove mentioned species are reported in Table 4.

Table 4. Determination of other enzyme inhibitors

InhibitorEnzyme inhibited Detection method

Limit of detection,Reference

determined molr1

Cyanide Tyrosinase Amperometry 2 x 1O-x-5 X 10-5 46,90Cytochrome oxidase Amperometry 4 x 10-7-5 X 10-7 88,89Peroxidase Amperometry ppb 91Invertase Polarimetry 2 x lO-x 92

Fluoride Liver esterase Spectrophotometry 95(lipase)

Potentiometry 2 x 1O-x-5 X 10-5 93,94AChE Potentiometry 1.6 x 10-5 22Urease Potentiometry 1 x 10-4 97Acid phosphatase Amperometry 1 xlO-4 96

156 T. Krawczynski vel Krawczyk

Table 4 (continued)

Hydrogen Cytochrome oxidase Amperometry 3 x 10-5 89sulphide

Invertase Polarimetry 1 x 10-7 92

Iodine Invertase Polarimetry 3 x 10-7 92

Phosphate Acid phoshatase Amperometry 2.5 X 10-5 96

Acknowledgement

The author wish to greatly appreciate the help (~f PT(~f M. Trojanowicz in critical review (~f themanuscript.

This work was performed underfinQncial support (~fBST-562/9197 Grant.

REFERENCES

1. Tran-Minh c., Ion Selective Electrode Rev., 7,41 (1985).2. Jain M.K., Handbook (~fEnzyme Inhibitors (1965-1977), John Wiley & Sons, Inc., New York, 1982.3. Zollner H., Handbook ofEnzyme Inhibitors, VCH Verlagsgesellschaft, Weinheim, 1989.4. Ambergand S. and Loevehart A.S., J. Biol. Chem., 4,149 (1908).5. Sherma J. and Zweig G., Anal. Chem., 55, 57R (1983).6. Meulenberg E.P., Mulder W.H. and Stoks P.G., Environ. Sci. Technol., 29,553 (1995).7. Bhattacharya S., Alsen c., Kruse H. and Valentin P., Environ. Sci. Technol., 15, 1352 (19818. Pa1chetti 1., Cagnini A., Del Carlo M., Coppi C., Mascini M. and Turner A.P.F., Anal. Chim. Acta, 337,

315 (1997).9. Guilbault G.G, Sadar M.H., Kuan S.S. and Casey D., Anal. Chim. Acta, 52, 75 (1970).

10. Leon-Gonzales M.E. and Townshend A., Anal. Chim. Acta, 235, 267 (1990).11. Ivanitskii D.M. and Rishpon J., Biosensors & Bioelectronics, 9,569 (1994).12. Garcia de Marfa C., Manzano Munoz T. and Townshend A., Anal. Chim. Acta, 295, 287 (1994).13. Tran-Minh C., Anal. Proc., 30, 73 (1993).14. Wollenberger V., LOffler V., GruB R., Gapel W. and Scheller F.W., in Biosensors, Fundamentals,

Technologies and Applications, Scheller F. and Schmidt RD., eds., GBF Monographs, Vol. 17, p. 35(1991).

15.Zurn A and Muller H., Fresenius J. Anal. Chem., 346, 589 (1993).16. Kindervater R, Kunnecke W. and Schmid RD., Anal. Chim. Acta, 234, 113 (1990).17. Quintero M.C., Silva M. and Perez-Benditto D., Talanta, 38, 1273 (1991).18. Kumaran S. and Tran-Minh C., Anal. Biochem., 200,187 (1992).19. La Rosa C., Pariente F., Hernandez L. and Lorenzo E., Anal. Chim. Acta, 308, 129 (1995).20. Mionetto N., Marty J.-L. and Karube 1., Biosensors & Bioelectronics, 9,463 (1994).21. La Rosa C., Pariente F., Hernandez L. and Lorenzo E., Anal. Chim. Acta, 273, 273 (1994).22. Tran-Minh c., Pandey P.c. and Kumaran S., Biosensors & Bioelectronics, 5,467 (1990).23. Sakai H., Kaneki N., Tanaka H. and Hara H., Sensors & Materials, 3,145 (1992).24. Nyamsi Hendji A.M., Jaffrezic-Renault N., Martelet C., Clechet P., ShulgaAA,Strikha V.I, Netchipo-

ruk L.L, Soldatkin A.P. and Wlodarski W.B., Anal. Chim. Acta, 281, 3 (1993).25. Dumschat c., Muller H., Stein K. and Schwedt G., Anal. Chim. Acta, 252, 7 (1991).26. Schwedt G. and Hauck M., Fresenius Z. Anal. Chem., 331, 316 (1988).27. Trojanowicz M. and Hitchman M.L., Chem. Anal. (Warsaw), 40,609 (1995).28. Zurn A, Rabolt B., Grafe M. and Milller H., Fresenius J. Anal. Chem., 349, 666 (1994).29. Marty J.-L., Sode K. and Karube 1., Electroanalysis, 4, 249 (1992).30. Marty J.-L., Mionetto N., Noguer T., Ortega F. and Roux C., Biosensors & Bioelectronics, 8, 273 (1993)31. Stoyecheva M., Anal. Lett., 27, 3065 (1994).32. Skladal P. and Mascini M., Biosensors & Bioelectronics, 7, 335 (1992).33. Skladal P., Anal. Chim. Acta, 269, 281 (1992).34. Skladal P., Pavlfk M. and Fiala M., Anal. Lett., 27, 29 (1994).

Enzimatic inhibition in analytical applications 157

35. Martorell D., Cespedes F., Martinez-F<ibregas E. and Alegret S., Anal.Chim. Acta, 290, 343 (1994).36. Sk1<idal P., Anal. Chim. Acta, 252, 11(1991).37. Martorell D., Cespedes F., Martfnez-Fabregas E. and Alegret S.,Anal. Chim. Acta, 337, 305 (1997).38. Guilbault G.G., Kramer D.N. and Cannon, Jr. PL., Anal. Chem., 34, 1437 (1962).39. Budnikov H.C., Medyantseva E.P. and Babkina S.S., J. Electroanal. Chem., 310, 49 (1991).40. Palleschi G., Bernabei M., Cremisini C. and MasciniM., Sensors & Actuators B, 7, 513 (1992).41. Bernabei M., Cremisini c., Mascini M. and Palleschi G., Anal. Lett., 24,1317 (1991).42. Bernabei M., Chiavarini S., Cremisini C. and Palleschi G., Biosensors & Bioelectronics, 8, 265 (1993).43. Campanella L., Cocco R, Sammartino M.P. and Tomassetti M., Sci. Total Environ., 123/124, 1 (1992).44. Campanella L., Achilli M., Sammartino M.P. and Tomassetti M., Biochem. Bioenerg., 26, 237 (1991).45. Razumas VJ., Kulys J.J. and Malinauskas A, Environ. Sci. Technol., 15,360 (1981).46. Besombes J.-L., Cosnier S., Labbe P. and Reverdy G., Anal. Chim. Acta, 311, 255 (1995).47. McArdle F.A. and Persaud K.C., Analyst, 118,419 (1993).48. Kumaran S. and Morita M., Talanta, 42, 649 (1995).49. Kumaran S. and n'an-Minh C., Electroanalysis, 4, 949 (1992).50. Stein I<. and Schwedt G., Anal. Chim. Acta, 272, 73 (1993).51. Danzer T. and Schwedt G., Anal. Chim. Acta, 318, 275 (1996).52. Baum G. and Ward F.B., Anal. Chem., 43, 947 (1971).53. Alfthan 1<., Kenttamaa H. and Zukale T., Anal. Chim. Acta, 217,43 (1989).54. Itakruni LA, Almuaibed AM. and Townshend A,Anal. Chim. Acta, 282, 307 (1993).55. Breuer H.,J. Chromatogr., 243,183 (1982).56. Guilbault G.G., Sadar M.H. and Zimmer M., Anal. Chim. Acta, 44, 361 (1969).57. Guilbault G.G. and Sadar M.H., Anal. Chem., 41, 366 (1969).58. Andres RT. and Narayanaswamy R, Analyst, 120, 1549 (1995).59. Ogren L. and Johansson G., Anal. Chim. Acta, 96, 1 (1978).60. Bryce D.W., Fernandez-Romero J.M. and Luque de Castro M.D.; Anal. Lett., 27, 867 (1994).61. Winquist F., Lundstrom I. and Danielsson B., Anal. Lett., 21, 1801 (1988).62. Narinesingh D., Mungal Rand Ngo T.T., Anal. Chim. Acta, 292,185 (1994).63. Toren, Jr. E.C. and Burger FJ., Mikrochim. Acta [Wien], 1049 (1968).64. Prell-Swaid A. and Schwedt G., Acta Hydrochim. Hydrobiol., 22, 70 (1994).65. Wittekind E., Werner M., ReinickeA, Herbert A and Hansen P., Environ. Technol., 17, 597 (1996).66. Schwedt G., Waldheim D.-G., Neumann K.-D. and Stein 1<., Fresenius J. Anal.Chem., 346, 659 (1993).67. Preininger C. and Danielsson B., Analyst, 121, 1717 (1996).68. Donlan AM., Moody GJ. and Thomas J.D.R, Anal. Lett., 22, 1873 (1989).69. Shaolin M. and Jinqing 1<., Electrochil11. Acta, 40, 241 (1995).70. Wang J. and Chen Q., Anal. Chem., 65, 2698 (1993).71. Almuaibed AM. and Townshend A., Anal. Chim. Acta, 218, 1 (1989).72. Dolmanova I.F., Shekhovtsova T.N. and Kutcheryaeva V.V., Talanta, 34, 201 (1987).73. Townshend A and Vaughan A, Talanta, 16, 929 (1969).74. Ghe AM., Stefanelli C. and Carati D., Talanta, 31, 241 (1984).75. Toren, Jr. E.C. and Burger F.J., Mikrochim. Acta [Wien], 538 (1968).76. Kratochvil B., Boyer S.L. and Hicks G.P., Anal. Chem., 39, 45 (1967).77. Hang YD. and Woodams E.E., Food Chem., 45,15 (1992).78. Maslowska J. and Leszczynska J., Talanta, 32, 883 (1985).79. Mealor D.and Townshend A, Talanta, 15,747 (1968).80. Mealor D. and Townshend A., Talanta, 15,1371 (1968).81. Mattiasson B., Danielsson B., Hermansson C. and Mosbach K., FEBS Lett., 85, 203 (1978).82. Liu D., Yin A, Chen K., Ge 1<., Nie L. and Yao S., Anal. Lett., 28, 1323 (1995).83. Townshend A. and Vaughan A., Talanta, 17, 289 (1970).84. Lehky P. and Stein B.A, Anal. Chim. Acta, 70, 85 (1974).85. Compagnone D., Palleschi G., Varallo G. and Imperiali PL., in Environmental Monitoring and

Hazar-dous Waste Site Remediation, Vo-Dind T. and NieBner Reds., SPIE Series, Vol. 2504, p.141(1995).

86. Amine A, Cremisini C. and Palleschi G., in Environmental Monitoring and Hazardous Waste SiteRemediation, Vo-Dind T. and NieBner Reds., SPIE Series, Vol. 2504, p. 209 (1995).

87. Marcos J. and Townshend A, Anal. Chim. Acta, 299,129 (1994).88. Amine A, Alafandy M., Kauffmann J.-M. and Novak Pekli M., Anal. Chem., 67, 2822 (1995).89. Albery WJ., Cass AE.G., Mangold B.P. and Shu Z.X., Biosensors & Bioelectronics,S, 397 (1990).90. Smit M.H. and Rechnitz G.A., Anal. Chem., 65, 380 (1993).

158 T. KrawcZyliski vel Krawczyk

91. Smit M.H. and Cass AE.G., Anal. Chern., 62, 2429 (1990).92. Mealor D. and Townshend A, Talanta, 15, 1477 (1968).93. Linde H.W., Anal. Chern., 31, 2092 (1959).94. McGaughey C. and Stowell E.C., Anal. Chern., 36, 2344 (1964).95. Marcos 1. and Townshend A, Anal. Chirn. Acta, 310, 173 (1995).96. Schubert F, Renneberg R., Scheller F.W. and Kirstein L., Anal. Chern., 56, 1677 (1984).97. Tran-Minh C. and Beaux J., Anal. Chern., 51,91 (1979).98. Wang J.,Dempsey E., Eremenko A and Smyth M.R., Anal. Chirn. Acta, 279, 203 (1993).

Received August 1997Accepted January 1998