REVIEW
Chern. Anal. (Warsaw), 43,135 (1998)
Analytical Applications of Inhibition
of Enzymatic Reactions
by Tadeusz Krawczynski vel Krawczyk
Departmenf(~fChemistry, University (~fWarsaw,
Pasteura 1,02-093 Warsaw, Poland
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. The
determination 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 cytochrome
oxidase 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 as
products of enzymatic reactions in static and flow conditions. Applied enzymes \vere
used first of all in immobilised form like biosensors or enzyme reactors. The examples
of determination of toxic pollutants in environmental (waters, soil extracts, river
sediments) and biological (fruits, vegetables, plant extracts, body fluids) samples are
given.
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 oznaczania
metali 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:ady
oznaczania toksyn srodowiskowych w wodach, ekstraktach gleby, osadach dennych
oraz 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 by
dialysis or simply dilution to restore full enzymatic activity. Irreversible inhibitors
cannot be removed from an enzyme by dialysis, however, it may be possible to
remove an irreversible inhibitor from an enzyme by introducing another component
to 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 by
reversible inhibitors may increase over this period of time.
Among reversible inhibitors three mechanisms of inhibition can be distinguished:
competitive, uncompetitive and non-competitive one (Fig. 1). Competitive inhibition
takes place, when an inhibitor exhibits structural similarity to the given substrates of
enzymatic reaction and may compete for the same binding site on the enzyme
blocking reactive group of an enzyme or is held in an unsuitable position with respect
to the catalytic site. In either case a dead-end complex is form, and the inhibitor must
dissociate from the enzyme and be replaced by a molecule of substrate. The degree
of competitive inhibition depends on the concentration of an inhibitor and a substrate
as well as their relative affinity for the enzyme. A low concentration of substrate
favours the inhibition process and vice versa - at high substrate concentrations the
inhibitor is much less successful in competing with the substrate and the degree of
inhibition is less marked. At very high substrate concentrations the effect of the
inhibition is negligible. The maximum rate (Vmax) of the reaction is unchanged,
however, Michaelis constant Km is increased in the presence of inhibitor and called
then an apparent Michaelis constant K'm.
Uncompetitive inhibitors bind only to the enzyme-substrate complex and not to
the free enzyme (see Fig. 1), when a favourable change of enzyme conformation after
substrate binding is formed, or the inhibitor can bind directly to the enzyme-bound
substrate. The inhibitor does not compete with the substrate for the same active
centre, 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 maximum
rate of enzymatic reaction.
Non-competitive inhibition is observed, when an inhibitor can combine with an
enzyme molecule to produce dead-end complex, regardless of whether a substrate
molecule 'is bound or not. An inhibitor destroys the catalytic activity of the enzyme
ei ther by binding to the catalytic site or as a result of a conformational change
affecting the catalytic centre - not affecting substrate binding. The total enzyme
concentration is effecti vely reduced by the inhibitor decreasing the value of Vmax but
not 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 among
above mentioned mechanisms occur depending on the relation between inhibition
constants Ki and K 1, describing the equilibrium of formation of enzyme-inhibitor
complex due to reaction between enzyme and inhibitor and the enzyme-substrate-in-
l!-nzimatic inhibition in analytical applications
137
(A)
Q
+
Q
+
E
QJ=Q LJ
~
LJ
+
ES
+-~
I
p
E
QI
(B)
(C)
Q+LJ= 8J=Q +LJ
S
E
ES
E
P
-I T1 +1
@
ESI
(D)
fiE
-I
+8
-8
r 1 +1
@
EI
W=[t
ES
-I
+8
~S
+
E
~
P
r 1 +1
@
ESI
Figure 1. Possible examples of mechanisms of reversible inhibition. (A), (B) - competitive inhibition
with 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. Also
some other types of inhibition such as substrate inhibition or so called allosteric
inhibition are mentioned but the description of them exceeds the frames of this
reVIew.
In the contrary to reversible inhibition, the irreversible inhibition takes often
place in real systems. An irreversible inhibitor binds to the active site of the enzyme
by an irreversible reaction and cannot subsequently dissociate from it. A covalent
bond is usually formed and the inhibitor may act by preventing substrate-binding or
it may destroy some component of catalytic site. The total recovery of the initial
enzyme activity is then practically impossible.
Enzymes are frequently inhibited specifically by low concentration of certain
chemical substances, and so enzymatic methods are commonly used for inhibitor
determinations [1-3]. The earliest analytical method based on enzyme inhibition
dates from 1908 [4] and concerns the possibility of determining fluoride by its
inhibition 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 of
these pollutants are organophosphorus and carbamate pesticides, widely used in
agriculture. They show an environmental persistence lower than the organochlorine
compounds but have a higher acute toxicity which can be a serious problem for the
equilibrium of aquatic ecosystems. Another problem is food contamination which
could have a serious impact on human health. A high acute toxicity of these
compounds creates need for fast-responding detection systems in order to protect
human health during manufacturing and application. Pesticides inhibit the action of
acetylcholinesterase, which hydrolyses the neurotransmitter acetylcholine, producing acetic acid and choline, in order to re-establish the initial state of the postsynaptic
membrane. When this enzyme is inhibited, nerve impulses are disrupted since
acetylcholine 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 techniques
for the determination of organophosphorus and carbamate pesticides and other
compounds 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, they
are expensive and require skilled personnel to operate them. Analysis time is generally considerable since these methods require laborious sample pre-treatment and
preconcentration steps limiting the frequency of analysis. The increasing use of
immunoassays for the determination of pesticides is observed during the last decade
[6]. It is a promising method for screening environmental contaminants (especially
for 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, as
the instruments used are in general large. From the other side pesticides can be
analysed by selective enzymatic procedures based on their ability to inhibit the
catalytic 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 very
high, so chromatographic techniques (HPLC, TLC) have been recommended for the
separation of pesticides before their enzymatic determination. With regard to nonsufficient selectivity toward individual pesticides the most reasonable approach
seems 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 of
anticholinesterase pesticides present in real samples (fruits and vegetables [8]) to
obtain rapid screening method of their determination.
Enzymatic methods based on the inhibition of cholinesterase activity have
recently been the object of intensive investigation due to their sensitivity and
specificity. The ease of performance of the assay and the low cost of the equipment
make such methods highly attractive for laboratories. Enzyme inhibition tests are of
great interest in environmental pollution analysis because the toxicity of pollutants
such as pesticides is the result of in vivo enzyme inhibition and biological tests
involving enzyme inhibition used for the determination of environmental toxins seem
to be more reliable than physical (e.g. chromatographic) methods.
Enzymatic methods based on the inhibition can be coupled to various analytical
techniques by the use of different substrates, like acetyl- or butyrylcholine, acetylor butyrylthiocholine or indoxyl acetate. An inhibition reaction can be carried out in
solution. Although this method as limited to single use of enzyme is expensive, it
does not require a regeneration of inactivated enzyme. Enzymes are often immobilised in reactors or in the form of biosensors especially useful for this purpose. Being
a combination of a biological component with a sensing device (transducer), they
have great versatility of design and a wide range of applications. The most advantage
of 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, bulk
wave 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 and
highly sensitive to interferences, they exhibit slow drift of the signal and a significant
sensitivity to ionic strength and buffer capacity. The interface between the enzyme
layer and the solid phase of an electrode may create also difficulties in electron
transfer. They offer, however, considerable advantages like high sensitivity, linearity
in a broad concentration range, relatively short response time, and sufficient reproducibility and stability. Moreover, they can be applied in field conditions in flow
measurements and in fast screening tests for hazardous pollutants in environmental
and food samples in order to protect human health.
140
T. Krawczynski vel Krawczyk
Cholinesterases can be inhibited more or less specifically depending on the
source of enzyme [9]. The measurement of the signal, proportional to the concentration of substrate or product of enzymatic reaction, obtained after some incubation
time when the enzyme is in contact with pesticide depending on the kind of pesticide
and its concentration, is carried out after the definite time or the rate of substrate or
product changes during the initial period of enzymatic reaction is monitored. This
values compared with obtained in the same conditions but without the interaction of
enzyme with inhibitor gives so called degree of inhibition and is proportional to the
inhibitor concentration. The measurement is most often performed in non-flow
conditions, however, recently also flow-injection method is more and more often
utilised [10-19].
The inhibition of cholinesterases can be reversible (carbamate pesticides) or
irreversible (organophosphorus pesticides). The proposed mechanism of irreversible
inhibition includes the interaction of organophosphorus pesticide with serine hydroxyl groups of the enzyme protein. In the case of reversible inhibition the regeneration
of the enzyme immobilised in the reactor or in the biosensor form can be obtained
by addition of buffer or substrate solution which indicates the competitive mechanism. However, when irreversible inhibition takes place, strong agents like some
oximes, 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 is
practically never complete even after very long time. The efficiency of different
reactivating 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 frequently
used for the determination of pesticides based on their inhibition of cholinesterases
or other enzymes. This technique depends on the substrate of the enzymatic reaction
used. Cholinesterases can catalyse hydrolytic decomposition of acetyl- (ATCh+)or
butyrylthiocholine:
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] or
Co-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 with
Co-phthalocyanine (+300 mV vs. Ag/AgCI) [36] electrodes were used for this
141
Enzimatic inhibition in analytical applications
(A)
./'
OR'
Enz.-Ser-OH + HO-P,
o" OR"
,/"
inhibition
OR'
+ H 20
Enz.-Ser-O-P,
)
II
o
OR"
(B)
o
/I
2(RO}-P--{}-Enzyme
+
2-PAM
Phosphorylated enzyme
1
lL ~
+I
(;
N
N
W
"O-P-(ORJz
+
Enzyme-OH
CH 3
Phosphorylated oxime
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 epoxy
resin, 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.lA
current [38] or the reduction current ofthiocholine-mercury compound was measured
at -550 mV vs. SCE at mercury film-covered silver electrode [39].
Another possibility is to use acetyI- or butyrylcholine:
Acety1come
h 1· + H 20 Acetylcholinesterase~ Ch 0 l'me +" CH'3C'100H
Choline formed as a product of the above reaction can be then oxidised with the
oxygen from the solution in the presence of another enzyme, choline oxidase:
.
Cholme + 20 2
Choline oxidase
•
.
Betame + H20 2
142
T. Krawczy,iski vel Krawczyk
The last reaction can be monitored due to detection of hydrogen peroxide on Pt
electrode at +600-+700 mV vs. Ag/AgCI [14,16,29,40,41] or with commercial H 2 0 2
detector [42,43]. Also the measurement of the oxygen consumption can be carried
out 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 with
formation 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 dehydrogenase
catalysing the oxidation of propionaldehyde by nicotine adenine dinucleotide (NAD):
. ldeye
h d + NAD+
ProplOna
Aldehyde dehydrogenase
~
ProplOlllC
. . aCl.d + NADH
NADH formed was re-oxidised by feerricyanide in the presence of another enzyme
diaphorase:
NADH + Fe(CN)~- Diaphoras~ NAD+ + Fe(CN)t
and oxidation current of ferrocyanide to ferricyanide was then monitored at +81 mV
vs. Ag/AgCI [30]. Also inhibition of tyrosinase catalysing oxidation of various
substrates (catechol, -dopamine, L-DOPA, epinephrine [46] or Catechol Violet [47])
to quinone with consecutive quinone oxidation current monitoring on glassy-carbon
polypyrrole-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 changing
the pH of tested solution (and hence - the potential) during the course of enzymatic
reaction (also inhibited in the presence of pesticide). This change can be monitored
with various pH electrodes like glass electrode [13,18,22,26,48-51], metal oxide pH
electrodes (e.g. Pd/PdO or Ir/h0 2 [21,39]) and pH ISFETs [23-25]. Also a commercial enzyme field effect transistor (ENFET) with immobilised urease was used for
this purpose [28] as CO2 and NH 3 , both pH changing species, are formed during the
hydrolysis of urea. Despite pH-sensitive sensors also change of concentration of
acetylcholine being the substrate of enzymatic reaction catalysed by acetylcholinesterase was measured with commercial (Corning Model 476200) liquid-membrane
electrode sensitive to acetylcholine [52]. Interesting measurement of change of redox
couple Fe(CN)~-/Fe(CN)~- potential was applied in the method elaborated by Ivanitskii 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
2TChH+ + 2Fe(CN)~-_
143
TCh-TCh 2+ + 2H+ + 2Fe(CN)t
so the decrease of acetylcholinesterase activity due to pesticide caused less decrease
of the potential after addition of substrate compared to the same situation when there
was no inhibitor in the solution.
Spectroscopic detection
Spectroscopic methods like spectrophotometry VIS or fluorimetry are more
seldom used than electrochemical ones described in previous sub-paragraphs. The
most 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 is
measured at A= 405 nm [7,16,17,53-55]. Another possibility of spectrophotometric
determination of pesticides based on the inhibition of cholinesterase activity is the
measurement of coloured species formed by a-naphthol with p-nitrobenzenediazonium fluoroborate [10,12] in the broad range of wave-lengths from 440 to 560 nm
depending on pH. In this case a-naphthol is a product of hydrolysis of a-naphthyl
acetate catalysed by acetylcholinesterase. Inhibition of cholinesterases can be also
utilised 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 organochlorine
pesticides) [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 methods
based on enzyme inhibition
The analytical parameters of enzyme determination methods based on enzyme
inhibition like linearity range, accuracy or precision depend mainly on the pesticide
determined and the method of detection and it is difficult to quote all the data for
every pesticide and method (not taking into account the enzyme used as more than
90% papers cited in this review are based on inhibition of cholinesterases). Some
imagination about this variety one can obtain from the data for the most often
determined organophosphorus pesticide paraoxon. For this model pesticide range of
concentration determined was e.g. from 2 x 10- 10 to 1 X 10-7 mol 1-1 [7], from 1 x
10-9 to 1 X 10-6 mol I-I [22] and from 9 x 10-9 to 5 X 10-7 mol 1-1 [12] with
potentiometric, amperometric or spectrophotometric detection, respectively. From
the 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. The
precision of the determination of paraoxon was better than 2% (RSD) both for
amperometric [43] or spectrophotometric [10,12] detection.
T. Krawczynski vel Krawczyk
144
The most frequently reported analytical parameter of the method is the detection
limit, hence it seems to be the most representative parameter for the comparison of
different detection techniques used for the determination of pesticides by enzyme
inhibition. 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 for
carbamate and organochlorine pesticides. In the second Table one from the chemical
warfare agents group (sarin) is also reported.
Table 1. Application of esterases inhibition for the determination of organophosphorus pesticides *)
Pesticide
Paraoxon
Detection method
Amperometry
Potentiometry
Spectrophotometry
Enzyme
Limit of detection,
molr l
Reference
AChE
AChE or BChE
BChE
AChE
BChE
AChE
BChE
1 x 10-10-1 X 10-7
5 x 10-9-3 X 10-7
3 x lO-1O~l X 10-8
1 x 10-9-4 x 10-8
7 x 10-7-1.5 X 10-6
2 x 10-10-5 X 10-5
2 x 10-7
19,21,29,30,35,37,41
32,40,44
33,37,42,43
18,22,52
27,49
7,10,12,54
55
Potentiometry
AChE
BChE
AChE
9 x 10-9-1 X 10-7
1.2 x 10-9
2 x 10-7-1 X 10-6
Parathion
Amperometry
Potentiometry
Fluorimetry
AChE
AChE
BChE
3.5 x 10-9-2.5 x 10-7
6 x 10-7
9 x 10-9-5 X lO- R
40,44
18,26
9
Malathion
Amperometry
AChE or BChE
BChE
AChE
2 x 10-7-2.5 X 10-6
2 x lO- R
1 x 10-10-1.5 X 10-9
32,44
43
18,22
AChE
AChE
AChE or BChE
1 x 10-9-1 X 10-6
1 x 10-6
1 x 10-7
14,31
/23
24
Dichlorvos
Amperometry
Potentiometry
Trichlorphon
Amperometry
Potentiometry
15,35
33
11,18,25,26
*)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, parathion
methyl and ethyl, phosphamide, systox, tetram.
Table 2. Determination of other pesticides based on enzyme inhibition
Pesticide
Detection method
Enzyme
Aldicarb
Amperometry
Potentiometry
AChE
AChE
Carbaryl
Amperometry
AChE
AChE or BChE
BChE
Limit of detection,
(moll- l )
1x
lO- R-2 X
4 x 10-7
10-7
1.3 x 10-10-1 x 10-7
1 x 10-9-1 X 10-6
1.1 x lO- R
Reference
30,41
26
19,21,35,37
32,34
33
145
Enzimatic inhibition in analytical applications
Table 2 (continued)
Potentiometry
Spectrophotometry
Carbofuran
Amperometry
Potentiometry
AChE
BChE
AChE
2.5 x lO--7
2 x lO-5
2 x lO-R
AChE
BChE
BChE
Urease
9 x lO-9-2.5 x lO-R
1 x lO-7
4 x lO-6
4 x lO-1O
57
56
Lipase
1 x lO-7
57
Lipase
3 x lO-6
6
BChE
BChE
0.01 ngml- 1
2 x 10-4 IJ.g ml- 1
53
38
Fluorimetry
Lipase
Alkaline
phosphatase
Heptachlor
Fluorimetry
Sarin
Fluorimetry
Spectrophotometry
Amperometry
8,16-18,35,37
37
49
28
0.8IJ.g ml- 1
51J.g ml- 1
Aldrin
Lindane
18
49
17
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 from
3 min [35] to 1 h [42] depending on the concentration of paraoxon. However, in the
case of organophosphorus pesticides this time can be considerably longer if the
reactivation procedure is carried out (e.g. even 6 h [19,21]).
There are also few papers describing the determination of pesticides or paralysing
gases in real samples, e.g. in tap [7], drinking [16] or synthetic sea [18] water,_ in
natural waters [43], in lake water and soil extracts [44], in lagoon water and kiwi
fruits [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 considerable
interest in analytical chemistry. Enzymes are frequently inhibited by low concentration of metals, so enzymatic methods are commonly used for the determination of
such inhibitors. From the point of view of economy and ease of handling, conventional enzymatic procedures can be dramatically improved by using immobilised
enzymes and flow methods. The sensitivity and selectivity of enzymatic methods
together with simplicity, speed and ease of automation make these methods highly
promising for the use in environmental protection. Detection of enzyme inhibitors
can be performed in a very sensitive way, since the interaction of a single inhibitor
molecule with an enzyme can result in a large reduction of the enzyme activity, the
enzyme 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 is
monitoring 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 with
sulphydryl groups of the enzyme which contributes to non-stabilisation of the
enzyme-substrate complex.
The inhibition of urease was the most frequently studied from the point of view
of 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, cobalt
and manganese [63], zinc and chromium(VI) [65]. The electrochemical detection was
applied in examination of other enzymes inhibition by heavy metals, e.g. glucose
oxidase (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 electrochemical
methods were used less frequently than e.g. spectrophotometric one. In this method
of 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 and
Fe(HI) [65] also other enzymes were used. Alkaline phosphatase inhibition was tested
for 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 ofHg
using ~-fructofuranosidase (invertase) [78]. In the case of this enzyme also the
change of optical rotation was the measure of enzyme activity and its inhibition by
Ag [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 the
components of their active centres. After removing of metal ion, e.g. by treating with
strongly complexing agent, the obtained apo-enzyme form is inactive. However, its
activity increases rapidly after introducing traces of metal ions and this increase was
also 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 are
mostly employed as they catalyse redox reactions where redox species (e.g. H 2 0 2)
are formed. These products of enzymatic reaction can be monitored amperometrically. Potential inhibitors like heavy metals decrease the formation of redox product
and hence amperometric signal decreases proportionally to the concentration of
inhibitor. 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 presence
of choline oxidase the final product of enzymatic reaction is hydrogen peroxide
formed due to the oxidation of liberated choline. The mixture of these enzymes was
immobilised on nylon net at the surface of commercial amperometric H 2 0 2 detector
forming biosensor [43]. The inhibition of BChE by Hg, Pb and Cd was examined
and 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. In
another biosensor, where BChE was immobilised in nitro-cellulose membrane, the
decrease of amperometric signal of the oxidation of hydrolysis product thiocholine
due to heavy metals was utilised for the determination of Cu and Pb in the range from
1 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 or
0.02 moll- 1 hydroxylamine solutions. The enzyme was also inhibited by Tl(I), Cd
and Bi. Also inhibition effect of glucose oxidase immobilised on nylon membrane at
the Pt electrode surface was investigated for 16,metal ions [68]. Only Cu, Hg and Ag
exhibited significant inhibition. The enzyme electrode could be reactivated by
EDTA, the reactivation being most effective for Cu. The ability to restore the enzyme
activity following Cu inhibition, and the linear response of the detector between 2.5
x 10-4 and 5 x 10-3 moll- 1 indicated a prospect for the use of a flow system for
determining this enzyme inhibitor. In another work glucose oxidase was immobilised
in poly aniline conducting polymer [69], so this detector could be considered as
chemically modified electrode. The inhibition by 15 metal ions including also heavy
metals like Zn, Cd, Cr(HI), Cu, Mn, Fe(H), Co, Ni and AI, was investigated. Only Cu
inhibited the enzyme and Mn in low concentration exhibited an activation effect. It
was also demonstrated that another enzyme from oxidase group, horseradish peroxidase was inhibited by some metal ions. The enzyme was immobilised in carbon paste
and the inhibition effect of Ni, Co and Mn was eliminated by EDTA being also the
component of electrode matrix [70]. Cu was found also to inhibit oxalate oxidase
interfering the determination of oxalate [71]. The inhibition effect was removed by
EDTA. Compagnone et al. [85] have tested amperometric biosensors with 15
enzymes from oxidases group for the determination of 12 metal ions. The best results
were obtained for Cd with D-amino acid oxidase (range 5-20 ppm, detection limit 2
ppm), 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 limit
1 ppm), Se(IV) with glutathione oxidase in the same range and with detection limit
of 0.5 ppm and for V(V) with glutathione oxidase (determination in the range 0.3-2
ppm). Hg compounds were also determined due to inhibition of invertase [86] with
indirect amperometric detection of hydrogen peroxide formed after oxidation of
glucose in the presence of glucose oxidase on Pt electrode at +650 mV vs. Ag/AgCl.
As glucose was formed during the reaction:
Sucrose + H2 0
Invertase
I
D-Glucose + D-Fructose
148
T. KrawcZy'lski vel Krawczyk
the decrease of the amperometric signal was observed in the presence of invertase
inhibitor. The method with enzyme immobilised on nylon net at the electrode surface
was applied for the determination of Hg compounds, MetHg and EtHg in the range
1-50 ppb. The biosensor used was reactivated by dipping in 10 mmoll- I cysteine
solution for 10 min. In contrast to free enzyme in solution, its immobilisation
removed interferences of Ag.
Potentiometric detection with immobilised enzymes
Also potentiometric biosensors with immobilised enzymes (mainly urease) were
tested for the inhibition effect of metal ions. Urease catalyses hydrolysis of urea
forming 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 for
monitoring the analytical signal. However, in the earliest analytical works, in which
the inhibition of urease was the base for heavy metals determination [63], the enzyme
in 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. Picogram
to nanogram amounts of these metals were determined in water solution with relative
error < ± 20%. Also Winquist eta!' [61] used urease in solution or immobilised on
test plates containing dry reagent strips with all necessary chemicals for the very
sensitive evaluation of Hg content down to 5 nmol 1-1. In their work an ammonia
gas-sensitive iridium thin metal oxide semiconductor detector was used. Zurn and
Muller [15] used photolithographically patterned enzyme membranes with urease
immobilised onto the pH-sensitive gate area of a miniaturised transducer (ISFET) for
the detection of Cu based on enzyme inhibition. Such a biosensor was able to detect
Cu(ll) in water in the ppm-range without preconcentration. The inhibition of urease
immobilised in enzyme reactor was also applied to the determination of Hg in the
range up to 7 nmol [59]. Ammonia formed in enzymatic reaction was detected by an
ammonia gas electrode. The sample volumes were 5 or 25 ml and the total Hg
concentrations 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 for
determination of free as well as complex Hg(ll) ions. Only silver and copper
interfered. Also potentiometric detection (pH electrode) of acetic acid formed during
hydrolysis of acetylcholine catalysed by acetylcholinesterase was applied for the
determination of Cu and Hg down to 1 mg 1-1 due to inhibition effect [51]. The
enzyme was immobilised on acetate cellulose membrane. Similar results were
presented for acid phosphatase [51].
Other detection methods
In the mentioned above methods, based on other than electrochemical detection
techniques, mainly immobilised enzymes were used for the determination of metal
ions based on inhibition. Metal inhibition studies of urease immobilised on controlled
pore 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 from
the biocatalytic hydrolysis of urea was compared before and after exposure to the
metal ion solutions [58]. The strongest inhibition by Hg, Ag, Cu, Zn, Pb, Cr(III) and
Co was 'observed for the urease immobilised with cyanuric chloride- and glutaraldehyde-activated support, however, only for Hg the calibration is given in the range
from 1 x 10-7 to Ix 10-3 moll-I. The biosensor was reactivated with 0.75 mol I-I
diethylenetriaminepentaacetic acid (DTPA) (pH 6). Urease immobilised on a
polymer support (VA Epoxy) in the reactor was also inhibited by Cli [66]. Ammonia
concentration formed in enzymatic reaction was measured photometrically with
indophenol method. The elaborated method was applied for the Cu speciation in
drinking and surface water samples. A solution of EDTA was able to reactivate the
inhibited enzyme. The use of urease in solution with the same indophenol spectrophotometric detection enabled the determination of Hg and other heavy metals in
aqueous soil extracts [64] (Hg down to 10 ppb and Cu down to 5 ppb) and in surface
water, ground water and waste water samples [65] (Hg down to 0.05 ppb). Also
inhibition of urease immobilised in column enzyme thermistor [81] as well as on
SAW/impedance enzyme resonator [82] was applied for the determination of Hg
down to 1 x 10-9 mol I-I and 1 x 10-7 m'oll- l , respectively. The last l11ethod was
successfully applied to the determination of Hg in waste water and the results agreed
well with those obtained by AASmethod.
Fluorimetric determination of Hg based on the inhibition of enzymatic activity
of urease immobilised on CPG in a flow-injection configuration was described by
Bryce et ai. [60]. The linear determination range was between 0.5 and 100 ppb and
the sampling frequency was 6 h- l . The immobilised enzyme reactor was regenerated
by L-cysteine injected between samples. Cu was found to interfere strongly~. The same
conditions, i.e. acid urease immobilised on CPG in column reactor and flow-injection
technique but with thermometric detection, were applied for the determination of Cu
in 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 urease
to Cu than urease (from jack beans) commonly used for metal sensing, regeneration
of enzyme did not require any metal chelating agent, no decrease in enzyme activity
is observed because of irreversible inhibition and of performing intermittent monitoring of Cu using a thermistor device is possible. Screening method for trace Hg
analysis using flow-injection with urease inhibition and a fluorescence detection was
described by Narinesingh et ai. [62]. In their method a urease in flowing solution was
used. Calibration curves were found to be linear up to 22 ppb and the detection limit
of 0.2 ppb could be achieved. Hg was determined in soil samples with the results well
comparable with AAS cold vapour technique.
The spectrophotometric studies of alkaline phosphatase inhibition were based on
the determination of p-nitrophenol at A = 400-405 nm formed in the catalytic
decomposition ofp-nitrophenyl phosphate substrate [73,87]. The method was applied
for the determination of Be in a flow-injection system with enzyme immobilised on
CPG in the range from 1.5 x 10-5 to 1.5 x 10-4 mol I-I as well as for the determination
of other metal ions (Cu, Zn, Cd, AI, Fe(III), Ni and Co) [87], or Be and Zn in the
18-90 ng and 0.6-6 g range, respectively [73]. In the case of fluorimetric study of
T. Krawczynski vel Krawczyk
150
inhibition of alkaline phosphatase by metal ions, umbelliferone phosphate was used
as a substrate of enzymatic reaction [56]. The inhibition by Be and Bi (without any
incubation) allowed the determination of these metals in the range 12-130 ppb with
mean error ±1.59'0 and 3-63 ppm with mean error ±1.3%, respectively.
Oxidation of o-dianisidine with hydrogen peroxide catalysed by horseradish
peroxidase, inhibited by Hg, was used for the determination of this metal down to 3
x 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 phosphatase
was 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-Lg
ml- I. The same reaction, i.e. oxidation of o-dianisidine by HzO z was used for the
spectrophotometric measurement of glucose oxidase inhibition by metal ions.In this
case hydrogen peroxide oxidising o-dianisidine was formed as a product of catalytic
oxidation of glucose in the presence of glucose oxidase. The method was used for
the determination of Ag in the range from 2 x 10-6 to 10-5 mol 1-1 [75]. Also Hg in
the 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 in
the presence of xanthine oxidase was studied spectrophotometrically [74]. Utilisation
of the linear relationship between relative enzyme activity and inhibitor concentration
allowed sensitive and selective determination of Ag and Hg (10-9-10-8 mol 1-1 ), and
of 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 to
a-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. Zn
activated 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 in
Table 3.
Table 3. Determination of metals based on enzyme inhibition
Metal
determined
Mercury
Enzyme inhibited
Urease
Invertase
BChE
Xanthine oxidase
Peroxidase
Glucose oxidase
Copper
Urease
Detection method
Limit of detection,
mol 1-1
Reference
Potentiometry
Spectrophotometry
Fluorimetry
SAW resonator
Spectrophotometry
Polarimetry
Amperometry
Spectrophotometry
Spectrophotometry
Amperometry
5 x 10-9-2 X 10-7
2.5 x W-IO-1 x lO-R
2.5 x 10-9-1 x lO-R
1 x 10-7
lxlO- 1l
2 x lO-R
3 x 10-5
1 x lO-R
5 x 10-6
1 x 10-5
59,61,63
64,65,69
60,62
82
71
79
43
74
72
68
Spectrphotometry
Potentiometry
Thermometrv
1.5 x 10-7-1 x lO- R
2 x 10-6-3 X 10-6
1 x 10-6
64-66
15,63
67
151
Enzimatic inhibition in analytical applications
Table 3 (continued)
Silver
BChE
Alkaline phosphatase
Xanthine oxidase
Glucose oxidase
Amperometry
Spectrophotometry
Spectrophotometry
Amperometry
1 x 10-9
1.5 x 10-5
1 x 10-7
5 x 10-5
Invertase
Spectrophotometry
Polarimetry
Fluorimetry
Potentiometry
Amperometry
Spectrophotometry
8 x 1O-R-2 X 10-7
1 x 10-7
9 x 10-10
2 x 10-8
1 x 10-4
2 x 10-6
Urease
Glucose oxidase
39
87
74
68
~
72,79
80
62
63
68
75
Lead
BChE
Peroxidase
Alkaline phosphatase
Urease
Amperometry
Spectrophotometry
Spectrophotometry
Potentiometry
5 x 10-7-1 X 10-5
5 x lO-R
3 x 10-9
2 x 10-6
39,43
72
72
63
Zinc
Alkaline phosphatase
Peroxidase
Urease
Spectrophotometry
Spectrophotometry
Spectrophotometry
1 x 10-6-1 X 10-5
2 x 10-6
8 x 10-4
73,87
72
65.
Cadmium
BChE
Alkaline phosphatase
Peroxidase
Amperometry
Spectrophotometry
Spectrophotometry
5 x 10-5-6 X 10-5
9 x 10-6-1 X 10-4
5 x 10-6
39,43
72,87
72
Beryllium
Alkaline phosphatase
Fluorimetry
Spectrophotometry
1.3 x 10-6
3 x 10-7-1.5 X 10-5
56
73,87
Bismuth
Alkaline phosphatase
BChE
Spectrophotometry
Fluorimetry
Amperometry
1 x 10-6
1.4 x 10-5
1 x 10-5
73
56
39
Nickel
Urease
Alkaline phosphatase
Potentiometry
Spectrophotometry
2 x 10-6
2 x 10-6
63
87
Cobalt
Urease
Alkaline phosphatase
Potentiometry
Spectrophotometry
2 x 10-6
1.5 x 10-5
63
87
Spectrophotometry
Spectrophotometry
1.8 x 10-4
3 x 10-7
65
74
Chromium Urease
Xanthine oxidase
V(V), Au(III) and Tl(I) as well as Fe(III), AI, Mg, Ca and Ba were also determined by inhibition of xanthine
oxidase 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 to
the analysis of cyanide. Cyanide expresses its toxicity by binding to the terminal
component in the electron transport chain in the mitochondria, cytochrome oxidase.
It blocks an intermolecular electron transfer thus stopping terminal electron transport
T. Krawczynski vel Krawczyk
152
to oxygen. Cytochrome oxidase inhibition by cyanide is known to be uncompetitive
toward 02'
As toxicity of cyanide manifests itself in inhibition of cytochrome oxidase in
living organisms, it was obvious to use the inhibition of this enzyme for the
determination of cyanide. In this determination amperometric detection (see Fig. 3
for the principle of the method) of reduction current of cytochrome c on carbon paste
electrode at -150 mV vs. Ag/AgCI [88] or decrease of oxygen consumption with
Clark electrode [89] were utilised. In the first method cyanide could be determined
in 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 was
applied 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 and
that of cytochrome oxidase restraining metabolic respiration is shown in Figure 4.
The amperometric detection was based on the measurement of reduction current of
ferricyanide used as mediator instead of cytochrome c in the case of cytochrome
oxidase:
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 with
detection limit below 5 x 10-5 moll-I. The binding of cyanide to tyrosinase was also
reversible. In another method of determination of cyanide due to tyrosinase inhibition
with amperometric detection the reduction current of o-dichinone at -200 mV vs.
SCE was measured (Fig. 5). The biosensor used consisted of glassy-carbon electrode
with tyrosinase immobilised in polypyrrole layer [46]. The determination of cyanide
up to 1 X 10-6 mol I-I with very low detection limit of 2 x 10-8 mol I-I was possible
making this device particularly interesting for monitoring cyanide, e.g. in drinking
water. Also amperometric biosensor with horseradish peroxidase immobilised on
glassy-carbon disc of rotating platinum and glassy-carbon ring-disc electrode was
used for the determination of cyanide in ppb range [91]. The principle of the method
is shown in Fig. 6. In this method the reduction current of ferrocenium ions on Pt
ring at 0 V vs. SCE was measured. Indirect determination of traces of cyanide was
Graphite
r - •. particle=t-(cyt. c).)~(cyt.
e-/
Ccyt.
C)red
\icyt.
- - . • _. - - - . - - . . -
- . . -,
a-CU.l". ~(cyt. a.-cu"I.,!,,(H:P
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]
153
Enzimatic inhibition in analytical applications
D(ox)
0''''1
~ Cyt c (redlXCyt OX
~,~
Cyte
10XI
(OX)X
lip (sl
Oz
Cyl Ox (...,
r
!
eN
pPO
M (red)X
M (ox)
(OXIX
lip.
lb'
~
PPO (red)
T
Figure 4. Comparison of the terminal sequence of electron transport in aerobic respiration chain (a) with
amperometric 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
- ..._ ...._ ...-..~. . ._"-r-'~...;l........Jl,_ _
quinone
-..... -
200 mVvs SeE
catechol
phenol or catechol + Oz
Figure 5. Schematic description of the electroenzymatic cycle for the detection of phenol or catechol as
substrate 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 to
complexation) of invertase [92]. In the presence of 2 X 10-8 mol I-I Hg cyanide from
the 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 1-1
Ag with accuracy ±3 %.
T. Krawczynski vel Krawczyk
154
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 HRP
inhibition [91]. Fe(cph - ferrocene (electron mediator)
Fluoride
Fluoride content is a parameter that should be controlled in waters occurring there
from phosphate fertilisers manufacture, the aluminium and steel industries, oil wells
and effluents from atomic energy plants. Fluoride may also be added to natural water
supplies 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 of
enzyme 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 method
allowed the determination of fluoride in the linear calibration range from 8 x 10-7 to
8 X 10-6 mol 1-1 with limit of detection of 1.6 x 10~6 mol 1-1 in water [95], whereas
with 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 following
reactions:
Glucose 6-phosphate + H 20
Glucose + O2
Acid phosphatase
~
Glucose oxidase
•
2-
Glucose + HP04
Gluconolactone + H 20 2
155
Enzimatic inhibition in analytical applications
An amperometric measurement of oxygen consumption with Clark electrode was
applied 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% (variation
coefficient for n = 12 at 2 x 10-3 mol 1-1 ). Non-competitive inhibition mechanism
was postulated.
Another enzyme used for the determination of fluoride was urease [97]. In this
method CO 2 formed during enzymatic reaction was detected potentiometrically with
gas sensitive electrode. Fluoride in the range 3 x 10-4-1 X 10-2 mol 1-1 could be
determined.
Other species
The methods mentioned above were also applied for the determination of other
than cyanide or fluoride species. Gaseous hydrogen sulphide was determined up to
40 ppm with limit of detection 1 ppm due to inhibition of cytochrome oxidase with
amperometric detection [89]. Sulphide was also determined due to decrease of
inhibition 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 the
determination of iodide in the range from 1 X 10-7 to 7 X 10-7 mol 1-1 [92] and the
inhibition of tyrosinase was used for the determination of chlorophenols in the range
from 4 x 10-7 to 2 X 10-6 mol 1-1 [46]. Inhibition of acid phosphatase by phosphate
was applied for their determination in the range up to 2 X 10-4 mol 1-1 with limit of
detection 2.5 X 10-5 mol 1-1 by method described for fluoride [96].
Organic-phase biosensors suitable for monitoring low levels of enzyme inhibitors
in non-aqueous media, were described by Wang et ai. [98]. The inhibition of
tyrosinase or horseradish peroxidase by thiourea, benzoic acid, diethyldithiocarbamate, hydroxylammonium sulphate and mercaptoethanol was exploited for highly
sensitive amperometric measurements in organic media. Fast on-line monitoring of
various inhibitors was illustrated in a flow-injection operation, with a detector based
on enzyme inhibition and an acetonitrile carrier solution.
Limits of detection of methods elaborated for the determination of some from the
above mentioned species are reported in Table 4.
Table 4. Determination of other enzyme inhibitors
Inhibitor
determined
Enzyme inhibited
Detection method
Cyanide
Tyrosinase
Cytochrome oxidase
Peroxidase
Invertase
Amperometry
Amperometry
Amperometry
Polarimetry
Fluoride
Liver esterase
(lipase)
Spectrophotometry
AChE
Urease
Acid phosphatase
Potentiometry
Potentiometry
Potentiometry
Amperometry
Limit of detection,
molr 1
2 x 1O-x-5 X 10-5
4 x 10-7-5 X 10-7
ppb
2 x lO- x
Reference
46,90
88,89
91
92
95
2 x 1O-x-5 X 10-5
1.6 x 10-5
1 x 10-4
1 xlO-4
93,94
22
97
96
T. Krawczynski vel Krawczyk
156
Table 4 (continued)
Hydrogen
sulphide
Iodine
Phosphate
Cytochrome oxidase
Amperometry
3 x 10-5
89
Invertase
Polarimetry
1 x 10-7
92
Polarimetry
10-7
92
Invertase
Acid phoshatase
Amperometry
3x
2.5
X
10-5
96
Acknowledgement
The author wish to greatly appreciate the help (~f PT(~f M. Trojanowicz in critical review
manuscript.
This work was performed underfinQncial support (~f BST-562/9197 Grant.
(~f
the
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3. Zollner H., Handbook ofEnzyme Inhibitors, VCH Verlagsgesellschaft, Weinheim, 1989.
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Received August 1997
Accepted January 1998