Chem. Res. Toxicol. 2011, 24, 135–143
135
Inhibition with Spontaneous Reactivation of Carboxyl Esterases by Organophosphorus Compounds: Paraoxon as a Model Jorge Este´vez,* Adolfo Garcı´a-Pe´rez, Jose´ Barril, and Eugenio Vilanova Unidad de Toxicologı´a y Seguridad Quı´mica, Instituto de Bioingenierı´a, UniVersidad Miguel Herna´ndez, Elche (Alicante), Spain ReceiVed October 7, 2010
In this work kinetic data were obtained for different paraoxon concentrations incubated with chicken serum and the soluble fraction of chicken peripheral nerve. A kinetic model equation was deduced by assuming a multienzymatic system with three different simultaneously occurring molecular phenomena: (1) inhibition; (2) simultaneous spontaneous reactivation; (3) “ongoing” inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied to analyze the experimental data versus the concentration of the inhibitor and the preincubation time in an inhibition experiment. The best-fitting model in the soluble fraction of chicken peripheral nerve was compatible with a resistant component (22%) and with two sensitive enzymatic entities (37 and 41%). The corresponding secondorder rate constants of inhibition (ki ) 1.8 × 10-3 and 5.1 × 10-3 nM-1 min-1, respectively) and the spontaneous reactivation constants (kr ) 0.428 and 0.011 min-1, respectively) were estimated. The bestfitting model in chicken serum was compatible with a resistant component (5.6%) and with two sensitive enzymatic entities (22.1 and 72.3%). The corresponding second-order rate constants of inhibition (ki ) 5.8 × 10-2 and 2.0 × 10-3 nM-1 min-1, respectively) and the spontaneous reactivation constants (kr ) 0.0044 and 0.0091 min-1, respectively) were estimated. These parameters were similar to those observed in spontaneous reactivation experiments with preinhibited paraoxon samples. The consistency of the results of all the experiments is considered an internal validation of the methodology. The results are also consistent with a significant ongoing inhibition. The proportion of enzymatic components shown in this work by the inhibition and reactivation of paraoxon is similar to that previously observed in inhibition experiments with mipafox in both tissues, demonstrating that this kinetic approach provides consistent results in complex enzymatic systems. The high sensitivity (at nanomolar concentrations) of these esterases suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation. 1. Introduction The acute and delayed neurotoxicity of organophosphorus compounds (OPs) is mediated by the inhibition of esterases such as acetylcholinesterase or neuropathy target esterase (NTE) by a covalent organophosphorylating reaction (reaction i in Figure 1). It is usually considered irreversible and yields an inactive phosphorylated protein, thus producing a time-progressive inhibition. This reaction is similar to that of the first catalytic reaction step of the carboxyl-ester substrate, which yields the intermediate acyl-enzyme that is quickly deacylated by hydrolysis to release the acidic product. In contrast, the phosphorylenzyme is not normally reactivated because the dephosphorylating reaction (reaction r in Figure 1) does not occur, or is very weak, and so the enzyme is practically inhibited irreversibly. In some cases, the phosphoryl-enzyme can undergo a dealkylating reaction called “aging” (reaction a in Figure 1), which is not significant under the experimental conditions of the examples with paraoxon provided in this paper because all of the activity is recovered overnight after removal of the inhibitor by ultrafiltration. In some cases, the dephosphorylation reaction occurred at a detectable rate; therefore, spontaneous reactivation should be considered in the kinetic data analysis. In vitro experiments involve preincubating the enzyme preparation with an inhibitor concentration (I) during inhibition * Author to whom correspondence should be addressed (e-mail:
[email protected].
times (t) and then incubating with a substrate during the enzyme-substrate reaction time (ts) to measure residual enzyme activity (E) (Figure 2). After incubation with the soluble fraction of chicken peripheral nerve, most soluble phenyl valerate esterase (PVase) activity was inhibited at a low concentration (nanomolar levels) in assays in which the substrate was added to a preincubated mixture of tissue with the non-neuropathic organophosphorus compound (OP) paraoxon (O,O′-diethyl p-nitrophenyl phosphate): residual activity should include the so-called soluble neuropathy target esterase (S-NTE) which, by definition, is considered resistant to the permanent and progressive (covalent) inhibition by paraoxon. However, paraoxon was proposed to be a potent reversible inhibitor of S-NTE (2), thus creating the need to introduce profound methodological changes as paraoxon is used in the standard NTE assay to inhibit only nonrelevant esterases (10). Therefore, the need exists to understand the kinetic behavior of most sensitive soluble PVases in the presence of paraoxon. The model equations and approaches for analyzing the kinetic behavior of multienzymatic systems in the presence of an inhibitor, with or without spontaneous reactivation, have been recently reviewed (7). If more than one enzyme is present in the preparation or a partial spontaneous reactivation by dephosphorylation occurs, a complex kinetic model needs to be applied
10.1021/tx100346c 2011 American Chemical Society Published on Web 12/14/2010
136
Este´Vez et al.
Chem. Res. Toxicol., Vol. 24, No. 1, 2011
Figure 1. Reaction of inhibition (i), reactivation (r), and aging (a) of esterases by organophosphorus compounds. This scheme assumes that the formation of a Michaelis-type intermediate complex in the inhibition reaction does not significantly affect the kinetics of the reaction because the low solubility of the organophosphorus compounds does not allow the concentrations to cause significant saturation (1).
Figure 2. Typical time course in an inhibition experiment. Enzyme preparation is treated with an inhibitor (paraoxon) (first vertical arrow) and is preincubated during the “inhibition time” (ti) (continuous horizontal line). Then a substrate is added (PV) (second vertical arrow) and incubated during the “substrate time” (ts) (dashed horizontal line) to allow the substrate-enzyme reaction to measure the residual active enzyme (mark of the inhibition). Finally, the reaction with the substrate is stopped (third vertical arrow) by adding SDS, and a color reagent is added before absorbance is measured. The absorbance values are referred to as the absorbance of the enzyme preparation without an inhibitor. The addition of a substrate usually stops inhibition due to dilution and competitive substrate protection.
for an accurate calculation of inhibitory potencies and to discriminate between the different esterases encountered in the preparation. A proposed model equation can only be accepted if consistent results are obtained when different inhibitor concentrations are assayed as follows: the best-fitting model is the same with the same number of components, the same kinetic constants, and the same amplitude (proportion of the different components) (7). Nonetheless, when the preparation includes enzymatic components of different sensitivities, the best-fitting model equation and, therefore, the number of discriminated components and their estimated kinetic constants and deduced amplitude values may apparently differ for the various inhibitor concentrations tested. This has been overcome by using an approach with several tiers and by analyzing data with a three-dimensional model equation that has been successfully applied to the analysis of peripheral nerve soluble PVases inhibition by mipafox (N,N′di-isopropyl phosphorodiamidefluoridate) (6, 7). The spontaneous reactivation of paraoxon-inhibited esterases has been observed in both the chicken peripheral nerve soluble fraction and chicken serum (2, 3, 9). The precise analysis of the combined spontaneous reactivation and inhibition kinetics of multienzymatic systems has always been hindered not only by the systems’ complex mathematical performance but also by difficulty in interpretation of the results. Additional complexity involves the medium containing a highly sensitive component the inhibition of which may continue during the substrate reaction (“ongoing inhibition”) (7, 8). Here a kinetic model has been used, which includes the inhibition process and a spontaneous reactivation in a complex multienzymatic system in which ongoing inhibition occurs in the most sensitive component. The model is used to analyze the chicken peripheral nerve soluble fraction and the serum esterases inhibited by paraoxon. This work continues the development of methods for simultaneously characterizing interactions of organophosphate neurotoxins with biological samples that potentially contain numerous sites, that is, enzymes, that are susceptible to inhibition. Enumeration of sensitive components and the associated inhibi-
tion and reactivation kinetics are determined, in this case, for paraoxon reactions with chicken serum and the soluble fraction of chicken peripheral nerve by applying the considerations and kinetic models described by Este´vez and Vilanova (7). A useful commercial software which allows a 3D fit by applying userdefined equations has been used to implement kinetic models with the data. Even though this paper does not deal directly with organophophorus-induced delayed polyneuropathy (OPIDP) and does not study neuropathy target esterase (NTE), chicken tissues were chosen as the source of biological material and phenyl valerate (PV) as the substrate because there are extensive studies into chicken PVases (brain, spinal cord, peripheral nerve, liver, ...) using paraoxon to eliminate (inhibit) nonrelevant esterases for the induction of OPIDP because it is neither an inducer of such neurotoxicity nor an inhibitor of NTE (2, 3, 6, 8). The model equations are used by means of the 3D fit according to the best kinetic model that can explain the different kinetic phenomena observed, and these are employed to (1) discriminate PVase activity components in the soluble fraction of peripheral nerve searching for permanently inhibited (nonspontaneously reactivated) PVases, which should be discarded as neurotoxicity targets; (2) confirm that the use of complex equations by means of a 3D fit according to the best kinetic model is both applicable and useful in other enzymatic systems such as PVases of chicken serum; and (3) compare the kinetic components discriminated within chicken serum PVases with those in peripheral nerve soluble PVases as the former have been suggested to mirror the response to inhibitors of the latter (9).
Materials and Methods 2.1. Chemicals. O,O′-Diethyl p-nitrophenyl phosphate (paraoxon, D9286, total impurities e 10%) was obtained from SigmaAldrich S.A. (Madrid, Spain), and phenylvalerate was purchased from Lark Enterprise (Webster, MA). A stock solution of 10 mM paraoxon was prepared in acetone and dissolved in 50 mM TrisHCl buffer (pH 8.0) containing 1 mM EDTA immediately before the kinetic assays; the final acetone concentration was 0); (2) component 1 is the most sensitive, so k1 > k2 and k2 > k3. (3) The following complementary restriction was also applied: E10 + E20 + E30 ) 100%. A 3D fitting (percent of phenylvalerate esterase activity versus t and I) was done with the data described in Figure 3. The results are provided in Table 1 (line A), and the deduced 3D surface is plotted in Figure 5. The system allows more than one solution, although this depends on the initial value in the interactive computing estimation. The results were accepted only if the reactivation constants were of the same order of magnitude as in the reactivation experiment, which is shown later. The consistency of the results improved if a correction factor for the ongoing inhibition during the substrate reaction was included in the most sensitive component. The I50(30 min) values for each component (Ei) were obtained by approximation and by applying the equation
% activity (Ei) ) [(kr · 100)/(ki · I + kr)] +
[(ki·I·100)/(ki·I + kr)]
· e-(ki·I+kr)·30 (5)
140
Este´Vez et al.
Chem. Res. Toxicol., Vol. 24, No. 1, 2011
for the previously estimated kinetic constants and by then carrying out successive iterations with different I values in an electronic spreadsheet to obtain the I value for the percent of activity equal to 50 ( 0.1%. Spontaneous Reactivation Constants Obtained from Dilution Experiments. The data reported by Barril et al. (3) in an inhibition experiment with peripheral nerve soluble PVase activity with paraoxon, followed by spontaneous reactivation after dilution, were used to estimate the reactivation and inhibition kinetic constants. In this experiment, samples were inhibited with 10 nM paraoxon to be then diluted to around 0.5 nM paraoxon. The recovery level was similar to the activity expected if inhibition with 0.5 nM paraoxon was applied in the inhibition experiment shown in Figure 3. The reactions to be considered are as follows: kr
E - P 98 E + P
The differential equations associated with these reactions are (7)
variation of E as a function of t: dE/dt ) -a · E + kr · EP variation of EP as a function of t: dEP/dt ) a · E - kr · EP When the initial conditions were t ) 0 min, E(t ) 0) ) E0 and EP(t ) 0) ) EP0, the solution of this system of equations is as follows:
E ) (EP0 + E0)·kr/(k · I + kr) + [E0 - (EP0 + E0)·kr/(k · I + kr)] · e-(k·I+kr)·t Mathematical models were applied and corresponded to one, two, or more inhibited enzymes, which spontaneously reactivated. The initial concentrations of the active enzymes and the residual concentration of the inhibitor were considered significant. The corresponding model described by Este´vez and Vilanova (7) was applied as follows:
E ) A · k1r /(k1 · I + k1r) + B · k2r/(k2I + k2r) +
kr
EP 98 E + P
(6)
[E20 - B · k2r /(k2I + k2r)] · e-(k2·I+k2r)·t + R where A ) E1P0 + E10 ) total concentration of E1 and B ) E2P0 + E20 ) total concentration of E2. The following restrictions were applied: A + B + R ) 100% and k1 > 1. The restriction k1 > 1 nM-1 min-1 forces the estimate of the secondorder rate kinetic constant of the most sensitive component. This mathematical model corresponds to two sensitive components capable of spontaneously reactivating, and a resistant component (R). This R component is probably the equivalent to the low sensitive component E3 detected in the inhibition experiment. The system estimated it as a resistant component because the inhibitor concentration was lower to that needed to cause significant inhibition. The resulting kinetic parameters and I5030 values are shown in Table 1, line B.
(III)
and the resulting kinetic equation for this reaction (7) is
E ) ET - EP0 · e-kr·t
(7)
where ET ) EP0 + E0 ) total enzyme concentration ) 100%. The mathematical models corresponding to the one, two, or more inhibited enzymes that had spontaneously reactivated were used. The best fit according to the F test was
E ) (E0 + EP0) - EP0 · e-kr·t + R
ki
E + PX 98 E - P + X
[E10 - A · k1r/(k1 · I + k1r)] · e-(k1·I+k1r)·t +
Study of the Reactivation Progress Experiment after Removal of the Inhibitor by Ultrafiltration. The enzyme was inhibited and the inhibitor was thoroughly removed after several ultrafiltration steps (see Materials and Methods). The inhibitor concentration was practically I ) 0, then a ) 0. The diagram showing this process is as follows:
(8)
where kr is the reactivation constant, E0 is the proportion (amplitude) of the initial active enzymatic component, EP0 is the proportion (amplitude) of the initial inhibited enzymatic component, and R is the enzymatic fraction resistant to inhibition. The following constraint was applied: E0 + EP0 + R ) 100%. The deduced kinetic parameter values are shown in Table 1, line C, and the curve is plotted in Figure 4. The inhibited nonultrafiltered controls showed 17% PVase activity. Consistency between the Kinetic Behavior in Inhibition Experiments and in Reactivation Experiments. The consistency of the estimated parameters was checked by comparing the results of the inhibition experiments with those obtained with the reactivation experiments. The kinetic model for these situations was the same, but the starting conditions differed. Therefore, different mathematical equations were applied. The comparison made is presented in Table 1, lines A-C. 3.4. Kinetic Inhibition Model with a Simultaneous Spontaneous Reactivation Process in Chicken Serum. Analysis of the Data from the Inhibition Experiment Considering Simultaneous Reactivation. The data reported in Garcı´a-Pe´rez et al. (9), that is, in the inhibition experiment of chicken serum soluble PVase activity with paraoxon, were used to estimate the kinetic constants. Spontaneous reactivation and ongoing inhibition were considered. The profile of the curves in the time-progressive inhibition (parallel lines for a long time) suggests that spontaneous reactivation occurred simultaneously. Extrapolating the data in this experiment to the preincubation zero time did not converge to 100%. This indicates that the ongoing inhibition during the substrate incubation is apparently significant under the assayed conditions. Therefore, the ongoing inhibition during substrate incubation needs to be considered. The kinetic inhibition model with simultaneous spontaneous reactivation was applied to the inhibition data by considering model equations with one, two, or three exponential components with or without a constant component and by also taking into account the factor for the ongoing inhibition effect in the most sensitive component. The F test was applied. The best-fitting model consisted of three enzymatic entities, two of which were inhibited and spontaneously reactivated, whereas one was permanently inhibited. This mathematical model is
Paraoxon Model of Inhibition
Chem. Res. Toxicol., Vol. 24, No. 1, 2011 141
E ) [e-ka · I] · {[(kr1 · E10)/(k1 · I + kr1)] + [(k1 · I · E10)/(k1 · I + kr1)] · e-(k1·I+kr1)·t} + [(kr2 · E20)/(k2 · I + kr2)] +
(9) -(k2·I+kr2)·t
[(k2 · I · E20)/(k2 · I + kr2)] · e
+
-k3·I·t
E30 · e
where enzymatic components E1 and E2 were inhibited and spontaneously reactivated and enzymatic component E3 was only inhibited. This is similar to eq 4, except that the third component is only inhibited. A 3D plotting (Figure 6) was used by fitting the inhibition data described by Garcı´a-Pe´rez et al. (9). Only the results obtaining reactivation constants of the same order of magnitude as the reactivation constants obtained in the reactivation experiment shown later were accepted. The consistency of the results improved if a correction factor for the ongoing inhibition during the substrate reaction was included in the most sensitive component. The results are shown in Table 2, expt A. Analysis of the Data from the Reactivation Progress Experiment after Removal of the Inhibitor by Ultrafiltration. The data reported by Garcı´a-Pe´rez et al. (9), in a reactivation experiment of chicken serum soluble PVase activity after removal of the inhibitor by ultrafiltration, were used to estimate the reactivation kinetic constants. This situation is described in reaction III. The model applied assumed that the most sensitive component (E1) was totally preinhibited, that the second (E2) was partly preinhibited, and that there was a resistant component (R)
E ) EP10 - EP10 ekr1·t + (E20 + EP20) - EP20 ekr2·t + R where kr1 and kr2 are the reactivation constants of enzymatic components E1 and E2, respectively, E20 is the proportion (amplitude) of the initial active enzymatic component E2, EP10 and EP20 are the proportion (amplitude) of the initial inhibited enzymatic components E1 and E2, respectively, and R is the enzymatic fraction resistant to inhibition. The following constraints were applied: (1) EP10 + E20 + EP20 + R ) 100% and (2) E20 + EP20 ) 72.3 by considering the estimated value of E2 in the inhibition experiment (Table 2, line A). R was fixed to a value of 7% of the residual activity of the inhibited but not ultrafiltered controls (9). The constraints involve fixing the parameters estimated in the previous inhibition experiments. The resulting kinetic parameters values are shown in Table 2, line B, and are similar to those in the inhibition experiment.
Discussion Evidence of Spontaneous Reactivation. The spontaneous reactivation experiments in a soluble fraction of the peripheral nerve and in chicken serum not only reveal that inhibited soluble PVase activity is able to totally reactivate but that reactivation is also time-progressive (see Figure 4). The spontaneous reactivation phenomenon of the preinhibited carboxyl esterases taking place with different inhibitors has been described in other works. A 40% reactivation has been observed in the samples preinhibited with stereoisomer P(-) of soman in NTE studies in hen brains after 18 h at 37 °C (15). A spontaneous reactivation (around 90%) after 20 h in hen brains that had been NTE and cholinesterase preinhibited with some phosphoramidates (17, 18) has also been described. Moreover, the spontaneous reactivation
Figure 6. Representation of the inhibition kinetics of chicken serum PVases by paraoxon. The inhibitory surface obtained by fitting the 3D model equation to the data of inhibition of paraxon in chicken serum is shown in Garcı´a-Pe´rez et al. (9). The surface reflects the results of the best model according to the F test. It corresponds to a model with three enzymatic components, two of which were inhibited and reactivated simultaneously, whereas the other was permanently inhibited. Further restriction was seen as the kinetic constants had to be of the same order of magnitude as those obtained in the reactivation assays shown in Table 2.
of carboxyl esterases preinhibited with paraoxon has been described in a particulate fraction of a hen brain (4) and in a soluble fraction of the peripheral nerve in chicken (3). Kinetic Behavior of Peripheral Nerve Soluble PVases with Paraoxon. Paraoxon has been used to discriminate nonneuropathic carboxyl esterases as it is neither an inducer of OPIDP nor an inhibitor of NTE. Knowledge of the kinetic behavior of the soluble PVases of the peripheral nerve with paraoxon is necessary to learn the reversibility of long-term inhibition and to discriminate the non-neuropathic carboxyl esterases in soluble fractions of peripheral nerve. The model with the mathematical equations of a simple kinetic model (E + I f EI) does not prove to be an appropriate data fit in the time-progressive inhibition curves in a soluble fraction of peripheral nerve. Another kinetic model is required when inhibition and spontaneous reactivation are considered to occur simultaneously (E + I f EI f E + P) and when the model equations are carried out according to Estevez and Vilanova (7). A 3D fit is the best tool to fit the equations to the data in this complex model (6). The reactivation constant (kr) as a result of the reactivation experiment after ultrafiltration is similar to the kr obtained from the second sensitive component in the reactivation experiment after dilution. Only one sensitive component is obtained in the reactivation experiment after ultrafiltration in a soluble fraction of peripheral nerve. Another sensitive component may have also been spontaneously reactivated during the ultrafiltration and washing steps because the inhibited but nonultrafiletred controls show 17% PVase activity for the soluble fraction of peripheral nerve, whereas the PVase activity is higher at the zero reactivation time. On the other hand, a control experiment after ultrafiltration shows reactivation close to 100% of activity at overnight, suggesting that the aging effect may be considered negligible under these experimental conditions and is, therefore, not considered in the model equations.
142
Este´Vez et al.
Chem. Res. Toxicol., Vol. 24, No. 1, 2011
Table 2. Kinetic Constants (ki) and Proportions of Components Obtained from the Different Inhibition and/or Reactivation Experiments with Paraoxon in Chicken Seruma expt b
A B
E1 (%)
k1 (nM-1 min-1)
k1r (min-1)
0.0584
0.0044 0.0021
22.1 20.7
I5030 (nM)
E2 (%)
0.43
72.3 (19.4 + 52.9)c (fixed E20 + E2I0 ) 72.3)
k2 (nM-1 min-1)
k2r (min-1)
0.0020
0.0091 0.0112
I5030 (nM)
E3 (%)
13.7
5.6 7 (fixed)
k3 (nM-1 min-1)
I5030 (nM)
4.3 × 10-6
5373
a I50 values were calculated from the kinetic constants for each component. (A) Experiment for time-progressive inhibition with eight different inhibitors concentrations; (B) reactivation after ultrafiltration with additional restrictions. The R2 coefficients were 0.9869 (expt A) and 0.9983 (expt B). b Apparent first-order ongoing inhibition constant (ka′) ) 0.0025 nM-1. c This value corresponds to E20 + EI20 ) E2 (the free initial enzyme plus the initial inhibited enzyme) of component 2.
Table 3. Sensitivity of the Different Components Discriminated by Inhibition with Mipafox and Paraoxona PXN component
sensitivity
peripheral nerve soluble esterases I (36.6 -37%) +++ II (41-47.8%) ++ III (15.6-22%) + serum esterases I (21-22%) +++ II (72-75%) ++ III (5-5.6%) +
MPX
I50 (nM)
% b
0.24-0.26 6-12 740
I50 (nM)
% c
37 41 22
++ +++ -
69-71 11-12 -
22.1 72.3 5.6
++ +++ +
>100 3.6-4 .1000
d 0.43 13.7 >2000
sensitivity
36.6 47.8 15.6 e 21 74 5
a I50 values are indicated (for 30 min) with the proportion of the component given in %. b From the data in this paper. c From the 3D fitting in the paper of Estevez et al. (6). d The results obtained in this paper from the time-progressive inhibition data in the paper of Garcia-Perez et al. (9). e From the experiment with a fixed inhibition time in the paper of Garcia-Perez et al. (9): +++, very high; ++, high; +, low; -, resistant.
The 3D fit enables all of the data in the same fit to be included simultaneously. The amplitudes (proportion) of the obtained components by 3D fitting in inhibition data are similar to those obtained in the inhibition experiments with mipafox (Table 3). The outcomes indicate that the PVases with paraoxon behave in the same way as three different enzymatic entities. Table 1 shows how both the inhibition constants (and the corresponding I5030 values) and the reactivation constants are consistent with those obtained in the reactivation experiments after either dilution or ultrafiltration. These observations may be considered an internal validation of the applied kinetic model and the 3D fit. It is concluded that a soluble fraction of peripheral nerve contains two paraoxon-sensitive components of 37 and 41% (I50) 0.24 and 6 nM, respectively, for 30 min) and that 22% of total activity is resistant to the highest tested concentration (200 nM). The same number and proportion of components are observed in the same tissue by inhibition with mipafox (Table 3). However, the relative sensitivity of the time-progressive inhibition for mipafox and paraoxon differs (Table 3). The first component is the most sensitive for paraoxon, whereas the second component is the most sensitive component for mipafox. In any case, both components are very sensitive enzymes (I50 on the order of nanomolar) if compared with other esterases (i.e., neuropathy target esterases bound to brain membranes), with I50 for 30 min on the order of micromolar (5, 11, 14, 19). Finally, the consistency of the results obtained in the experiments done with paraoxon and mipafox in both tissues may be considered an internal validation of the strategies to characterize kinetic behavior. Model Equation with Spontaneous Reactivation and a 3D Fit Applied to the Data from Chicken Serum PVases with Paraoxon. The data from the chicken serum PVases (9) were reviewed to corroborate the applicability of the obtained equations from the different kinetic mechanisms and the 3D fit. No kinetic inhibition constants were provided in the cited work.
A similar consistency of the results, as in peripheral nerve soluble PVases with paraoxon, is obtained. The reactivation constants (kr1 and kr2), as a result of the reactivation experiment after ultrafiltration, are similar to those obtained in the inhibition experiment. In addition, three components have been obtained with proportions similar to those in the inhibition experiment with mipafox at a fixed time (Table 3) (8). However, the relative sensitivities of the time-progressive inhibitions for mipafox and paraoxon differ (Table 3). The first most sensitive component (E1, 21-22%) is the most sensitive for paraoxon, whereas the second most sensitive component (E2, 72-75%) is the most sensitive component for mipafox. In any case, both components are very sensitive enzymes (I50 on the order of nanomolar). With both inhibitors, a minor resistant (I50 > 1000 nM) component of about 5-6% was also observed. The consistency of the results obtained in the experiments with paraoxon and mipafox may be considered an internal validation. Thus, it is confirmed that using complex equations by means of a 3D fit in accordance with the best kinetic model is applicable and useful in other kinetic systems such as the PVases of chicken serum. Toxicological Meaning and Applications for Measuring NTE. To discriminate and measure neuropathy target esterases (NTE), those esterases sensitive to paraoxon (an organophosphate that does not induce neuropathy) are excluded as targets of the neurotoxic process (10, 13, 16). This work proves that the soluble PVases of the peripheral nerve can be progressively inhibited by paraoxon with time, but not permanently for it to be spontaneously reactivated. The soluble PVase activity is able to reactivate about 65% of total activity when it is preinhibited with 40 µM paraoxon and when residual PVase activity is
