Detection of Organophosphate Insecticide by an Immobilized-Enzyme System? Shelley Bhattacharya,* Carsten Alsen,* Hermann Kruse,§ and Peter Valentin* Department of Zoology, Visva-Bharati University, Santiniketan 731235,West Bengal, India; Abteilung fur Toxikologie der Universitat Kiel, D 2300 Kiel, Federal Republic of Germany; and Untersuchungsstelle fur Umwelttoxikologie des Landes Schleswig-Holstein, D 2300 Kiel, Federal Republic of Germany
Kinetic properties of acetylcholinesterase (AChE) covalently bound to poly(ma1einic anhydride) (PMA) were investigated in a Celite 545 (20-45 pm) column. The advantage of such a system was the constant rate of substrate hydrolysis for a continuous period of 12 h and the ability to detect low amounts of organophosphates in a water sample. The second-order rate constant (k2) for paraoxon at concentrations of M lies within the range of that found in the stirred suspension. However, a significant increase in the k2 value was noted at the lower concentrations of paraoxon ( and 10-lo M). Perfusion with a water sample revealed a typical organophosphate inhibition kinetics indicating the presence of M paraoxon equivalents in the water sample. Reactivation by obidoxime and additive inhibition by an internal standard of Daraoxon confirmed the contamination. Introduction In recent years immobilized enzymes have been shown to be of practical advantage (1-4). I t has been suggested that such water-insoluble enzyme derivatives could be successfully packed into columns, opening new possibilities of application (5-13). Studies dealing with practical applications of immobilized enzymes such as acetylcholinesterase are rather rare. Recently, for collection and detection of organophosphates in air and water, Goodson and Jacobs (14) comprehensively reported on the practical application of their gel-entrapped butyrylcholinesterase by electrochemical means in a continuous perfusion system. However, they have not dealt with all of the kinetic parameters in such a system. The present investigations were initiated by the availability of an immobilized acetylcholinesterase preparation. With regard to its stability and minimum change in the basic enzyme properties, the poly(ma1einic anhydride) acetylcholinesterase was found to be a favorable immobilized enzyme preparation. The aim was to develop a stable insoluble enzyme column, to evaluate the kinetic properties, and to utilize such a column for detection of acetylcholinesterase inhibitors in water. Materials and Methods Enzyme Immobilization. Acetylcholinesterase (E.C. 3.1.1.7, AChE, (Electrophorus electricus, 1000 units mg-l, Boehringer, Mannheim) in a 1:lOO mixture with human serum albumin (100%pure, Behring Werke, Marburg) was coupled with the copolymerizate of maleinic anhydride and butanediol divinyl ether (PMA vernetzt, Art. No. 10 272, Merck, Darmstadt) ( 4 ) with a slight modification in the pH (8.0) maintained during the coupling procedure (19), resulting in a 10-12.5% t Preliminary communications were presented at the Spring Meeting of the German Pharmacological Society a t Mainz, Federal Republic of Germany, March 1978, and at the Fourth International Congress of Pesticide Chemistry at Zurich, Switzerland, July 1978. * Address correspondence to this author at Visva-Bharati University. Abteilung fur Toxikologie der Universitat Kiel. 5 Untersuchungsstelle fur Umwelttoxikologie des Landes Schleswig-Holstein.
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Environmental Science & Technology
binding of the free enzyme to PMA. The preparation used in the present investigation had a bound protein content of 250 pg mL-l as measured by the modified Kjehldahl method (15) (for specific activity see Results). Enzymatic Activity in a Stirred Suspension. Enzyme activity in a stirred suspension was determined by means of the pH-stat technique under a nitrogen atmosphere (automatic microtitration device equipped with a 0.25-mL buret, Radiometer, Copenhagen). Experiments were carried out a t pH 7.8 and 30 OC with a reaction volume of 2 mL. The titrating agent was NaOH (3 mM) freshly prepared from Titrisol (Merck, Darmstadt). Preparation of the Column. Different aliquots of the PMA-AChE were brought into a reaction vessel containipg 250 mg of Celite 545 (20-45 pm, Serva, Heidelberg) suspended in 2.5 mL of 0.05 M phosphate buffer, pH 7.8, at 30 OC. After vigorous stirring for 30 min, the mixture was carefully poured into a double-jacketed glass column of 0.6-cm diameter maintained at 30°C. The outlet was closed by a D1 glass filter followed by 250 mg of sea sand (Merck, Darmstadt) in order to achieve the desired length of 1.75 cm which was found to be most suitable for further experiments with paraoxon.The total bed volume amounted to 0.5 mL, and the void volume as measured by cresol red was found to be 0.36 mL. Perfusion of Solutions. All solutions were perfused by means of a Varioperpex peristaltic pump (LKB Bromma) as described below. Studies of General Kinetic Properties. The enzyme column was equilibrated with 0.05 M phosphate buffer, pH 7.8, and a standard solution (referred to as the perfusion fluid) of the phosphate buffer with 30 mM NaCl and 4 mM MgCl2 for 10 and 15 min, respectively. Acetylthiocholine iodide (Boehringer, Mannheim) prepared in the perfusion fluid was perfused at varying initial substrate concentrations and flow rates. Acetylthiocholine iodide was used as the substrate since the enzyme activity was measured spectrophotometrically. Paraoxon (Mintacol, Bayer, Leverkusen) and carbaryl (Pestanal, Riedel de Haen, Seelze) added to the perfusion fluid were perfused at a rate of 0.8 mL min-l for defined incubation periods followed by perfusion of substrate (3 X M) plus the particular inhibitor, in order to simulate the reacting condition of a stirred suspension in the pH-stat method of determination of AChE activity. Experiments with Tap Water. When the substrate dissolved in tap water was perfused, the enzyme column showed a spontaneous loss in activity by 40% as compared to the standard conditions described above. Buffered tap water could not be used since there was a colloidal precipitation of calcium compounds. In order to reduce the loss in activity and to reach an ideal active condition for the column, 45 mM of NaCl and 6 mM of MgC12 were added to the tap water (referred to as water sample). The column was stable under this changed condition for an observed period of 6 h, which sufficed for the inhibition kinetics study. All perfusions were carried out at a flow rate of 0.8 mL min-l, and the order of perfusions was the same as in the inhibition experiments under buffered conditions. Reactivation experiments were done with obidoxime (Toxogonin, Merck,
0013-936X/81/0915-1352$01.25/0 @ 1981 American Chemical Society
Darmstadt). As an additional check for true organophosphate inhibition exhibited by the water sample, further experiments were conducted with paraoxon added to the water sample as an internal standard. Substrate ( 3 X M) or substrate plus obidoxime (1 X 10-4 M) were prepared in the water sample. Measurement of Enzyme Activity. Aliquots from the effluent collected after 10 min of substrate perfusion were measured spectrophotometrically (Shirlfadzu double-beam UV 200 spectrophotometer, Kyoto) at 405 nm for the thiocholine liberated, Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid), Boehringer, Mannheim) being used as the indicator. The amount of thiocholine produced was calculated from a standard curve, and enzyme activity was expressed in terms of micromoles of thiocholine produced per milliliter of perfusate in 10 min. To establish the calibration curve, we prepared fresh thiocholine in our laboratory. Acetylthiocholine iodide (500 mg) dissolved in 20 mL of distilled water was treated with 0.5 mL of concentrated HI (Merck, Darmstadt) at 70 "C for 2 h. After evaporation to dryness, it was crystallized in ethanol a t 4 "C. Crystallization was achieved within 3 h. After vacuum drying the crystallized sample was stored at 4 "C. Calculation of the Second-Order Rate Constant. The k2 value was calculated according to the following equation (16) k2
'J. acetylthiocholine hydrolysed 0.5
1.0
1.5 20 25
I.
1
2
3
4
6
5
7
10
9
8
11
12
f [mix mg-1 Figure 1. Extent of hydrolysis of acetylthiocholine (%) by PMAAChE-Celite column as a function of initial substrate concentration at different flow rates (mL min-l) depicted at each curve. 1/Sis expressed in mL (mg of substrate)-'. The amount of enzyme used in the column is 62.5 pg. Thioc ho line produced
25-
2 .o.
0.5.
i
= In 2/[to.dI)l 1.5-
where k2 denotes the bimolecular rate constant (M-l), t0.5 the time required for 50% inhibition, and (I) the inhibitor concentration. Results and Discussion Before the cdlumn experiments the specific activity of both free and bound enzymes was determined by means of the pH-stat method using acetylthiocholine iodide as a substrate. The data obtained were as follows: specific activity, 48 pmol min-l (mg of protein)-1 in both systems; K,, 2.00 X M for the free enzyme and 1.92 X M for the bound enzyme; k2,3.00 X 106 M-l min-1 for the free enzyme and 3.33 X lo5 M-l min-l for the bound enzyme. There was no significant difference in specific activity, K,, and k2 when acetylcholine iodide was used as the substrate in the stirred suspension, Preliminary experiments concerning proportionality between enzyme concentration and activity, temperature, and ionic requirements were conducted to obtain optimum conditions in the column. For the substrate acetylthiocholine the AChE activity is nearly pH independent (17);i.e., it displays a maximum activity over a broad range of pH. In the present investigation all experiments were done at a pH of 7.8, with 62.5 pg of bound enzyme in the column. Stability of t h e Column. At a definite flow rate and substrate concentration the column attains a steady state within 10 min and remains unchanged for an observed period of 12 h. It could also be stored and reused for -2 weeks with a negligible loss in activity. Effect of Flow Rate and Initial Substrate Concentration on Column Activity. Flow rate and initial substrate concentrations affecting the percentage hydrolysis of the substrate revealed an invdhe relationship which was more pronounced at comparatively higher substrate concentrations (Figure 1).Strictly inverse relationships were also observed in enzyme columns of trypsin (9) and ficin (IO). When t h e absolute amounts of thiocholine produced were plotted against the negative logarithm of substrate concentration, the usual bell-shaped curve was obtained (Figure 2), indicating an optimum concentration between 2 X 10-3 and 6X M. As compared to the stirred suspension, the range
1.0-
0.5
i
2
j
4 Ps
Figure 2. pS-aCtiJity curve of a PMA-AChE-Celite column with 62.5 pg of enzyme at different flow rates. Value at each curve denotes rate of perfusion (mL min-I).
is somewhat broadened in the column. This is probably due to the building up of a strong concentration gradient effecting a considerable difference in the macro- and microenvironmental substrate concentration. Determination of the Apparent K, ( K,'), Although Lilly et al. (IO) propose a modification of the integrated Michaelis-Menten equation for the determination of the apparent K , in an enzyme column, the same could not be applied to the present study because of the well-known substrate inhibition observed for AChE. Therefore, the LineweaverBurk plot was used to calculate the apparent K , of our column preparation (Figure 3). A t all flo$ rates studied there was always an increase (-10 fold) in the K,' as compared to that in the stirred suspension. A 4-5-fold increase in the K,' has been reported in enzyme columns (IO),and a 25-350-fold increase in artificial membranes (18).The parallel shift in this plot simulates an uncompetitive inhibition, although there is no inhibitor. Due to a longer residence time at the lower flow rate, a strong concentration gradient develops in the column because of enzyme reaction. As the substrate flows from above downwards, almost all of the substrate is hydrolyzed, and the enzyme molecules resident at the lower part of the column face no effective substrate concentration to react upon. In contrast, at higher flow rates the concentration gradient is decreased and more enzyme molecules become involved. These simultaneously occurring different phenomena, such as residence time and Volume 15, Number 11, November 1981 1353
.,
-V1 4-
log percentage activity
20 15 1.0 05
/
3-
2.5
0 1
2
3
4
5
6
7
0
incubation time ( h r ) -1000
1000
2000
3000
-1
LOO0
Flgure 4. Paraoxon inhibition kinetics at varying concentrations with 62.5pg of PMA-AChE and 0.8 mL min-l rate of perfusion.
S
Figure 3. Lineweaver-Burk plot to demonstrate the apparent increase in K,,, from higher to lower flow rates in the PMA-AChE-Celite column with 62.5 yg of enzyme. Flow rates are indicated at each curve. Apparent K, values were 10,3.3,2.5,1.7,and 1.1 M for flow rates of 0.5, 1.O, 1.5,2.0,and 2.5 mL min-I, respectively. decline of effective substrate concentration, cause conditions which do not obey the simple Michaelis-Menten kinetics any more. Theoretically, if the perfusion could be increased to a much higher level, one would perhaps have obtained an apparent K , closer to that in stirred suspensions but with a much lower l/2Vmax. Inhibition with Paraoxon, Carbaryl, and a Combination of Paraoxon and Carbaryl. A constant k z value was obtained for a concentration range of paraoxon around lo-’ M. However, an apparent increase in the k2 value was noted at lower concentrations of paraoxon (Table I and Figure 4), which is probably due to the continuous perfusion of the inhibitor through the column, a condition which is not provided for in stirred suspensions. Since paraoxon is an irreversible inhibitor, the active centers of the enzymes are progressively inactivated. The inhibitor concentration remaining constant throughout the incubation period, the chance of combination of the free active centers with the inhibitor is thus greatly enhanced in the column. As a result, the k 2 values are apparently magnified. Table I1 depicts the change in the degree of inhibition of the PMA-AChE in the column due to carbaryl, paraoxon, and a combination of carbaryl and paraoxon as compared to that in the stirred suspension. At all concentrations of paraoxon and carbaryl, there was a significant decrease in the rate of inhibition in the column. Mechanical or hydrophobic disturbances operating in a diffusion-limited system may account for this apparent change in inhibition kinetics in the column. Experiments with Tap Water. From the very beginning it was the aim of the present study to utilize such a column in the detection of organophosphates in water. Our experiments with tap water demonstrated typical organophosphate inhibition kinetics (Figure 5). Reactivation by obidoxime was 90-95%, indicating that the inhibitory effect originates from an organophosphate. Furthermore, an additive inhibition was observed when 1 X M paraoxon was added to the water sample as an internal standard. Since the specific organophosphate was not identified, from the time taken for 50% inhibition M paraoxon equivalents were calculated to be present in the water sample. Carbamates at concentration lower than 1 X 10-7 M showed a negligible effect in the column. Therefore, the inhibition demonstrated here by the 1354
Environmental Science & Technology
log percentage activity
1.0
2
1
3
4
incubation period ( h r l
Flgure 5. Inhibition kinetics demonstrated in the column by tap water (A)and tap water plus 1 X lob9 M paraoxon ( 0 )reactivation ; by obiM) after tap water (A)and tap water plus paraoxon doxime (1 X (0)inhibitions. Flow rate = 0.8 mL min-l; enzyme used in the column = 62.5yg Of PMA-AChE.
Table 1. k2 Values Observed for Varylng Paraoxon Concentrations with 62.5 pg of Enzyme In the Column and at a Perfusion Rate of 0.8 mL min-’ paraoxon concn, M
para ox on concn, ?A
105k2, M-I min-1
a x 10-7
2 x 10-8 2 x 10-9 2 x 10-10
1.54 1.54 1.50
4 x 10-7 2 x 10-7
105k2,
min-1
1 .ao 13.80 75.00
Table II. Time Required for 50% Inhibition and k2 Values at Different Concentrationsof Carbaryl, Paraoxon, and a Combination of Carbaryl plus Paraoxon in Stirred Suspensions and in Columns 105k2, M-’
10.59 mln
inhlbitor concn, M
stirred suspension
column
column
0.554 0.693
0.396 0.346
2.89 3.46
1.98 1.92
Carbaryl *
1 x 10-7 1 x 10-6 2 x 10-6
12.5 5
2 x 10-7 4 x 10-7
12 5
105
17.5 10
min-1
stirred suspension
0.66
Paraoxon
1X
17.5 9
M Carbaryl plus 2 X 15
M Paraoxon
5
To have a comparative bask for the determination of the k2 vaiues, we used 2.5 F g of bound enzyme both in the stirred suspension and in the column. In calculating the k2 values for carbaryl inhibition, we took into consideration reversibiiity of carbamylation.
water sample may be considered to be due to paraoxon equivalents alone. Reactivation by obidoxime to 90-95% also excludes the presence of organophosphates with a very quick aging time. In conclusion, the bound AChE-Celite-system proved to be quite a good tool for analytical purposes. Although there was significant alteration in the kinetic parameters, the stability of the column over long and continuous perfusion periods enables the detection of very low concentrations of paraoxon equivalents which is impossible in stirred suspension. Ohnesorge and Menzel(19) have also utilized the PMA-AChE in stirred suspension to quantify paraoxon equivalents in various water samples; since the investigators did not apply a continuous perfusion, they could demonstrate a marked inhibition only by enriching the water samples, by means of extraction. In comparison the PMA-AChE-Celite system in a column is a simplified analytical system for the detection of organophosphates or other acetylcholinesterase inhibitors in water. Acknowledgment
S.B. is grateful to Professor Dr. 0. Wassermann for the collaboration he arranged with Dr. C. Alsen. We thank Miss Marion Pauer for the excellent assistance rendered in preparing the figures. Literature Cited (1) Silman, J. H.; Katchalski, E. Annu. Rev. Biochem. 1966, 35, 873.
(2) Goldstein, L.; Katchalski, E. Fresenius 2. Anal. Chem. 1968,243, 375. (3) Brummer, W.; Hennrich, N.; Klochow, M.; Lang, H.; Orth. H. D. Eur. J. Biochem. 1972,25,129. (4) Alsen, C.; Bertram, U.; Gersteuer, T.; Ohnesorge, F. K. Biochim. Biophys. Acta 1975,377, 297. ( 5 ) Glubhofer, N.; Schleith, L. J. 2. Physiol. Chem. 1954,297,108. (6) Epstein, C. J.; Anfinsen, C. B. J. Biol. Chem. 1962,237,2175. (7) Riesel, E.; Katchalski, E. J. Biol. Chem. 1964,239,1521. (8) Fritz, H.; Schult, H.; Hutzel, M.; Wiedermann, M.; Werle, E. 2. Physiol. Chem. 1967,348,308. (9) Bar-Eli, A.; Katchalski, E. J. Biol. Chem. 1963,238,1690. (10) Lilly, M. D.; Hornby, W. E.; Crook,E. M. Biochem. J . 1966,100, 718. (11) Lilly, M. D.; Dunhill, P. Methods Enzymol. 1976,44,717. (12) Ngo, T. T.; Laidler, K. J. Biochim. Biophys. Acta 1975, 377, 316. (13) Ngo, T. T.; Laidler, K. J. Biochim. Biophys. Acta 1975, 377, 317. (14) Goodson, L. H.; Jacobs, W. B. Methods Enzymol. 1976, 44, 647. (15) Parnas, J. K. 2. Anal. Chem. 1938,114,261. (16) Aldridge, W. N.; Davison, A. N. Biochem. J. 1952,51,62. (17) Bergman, F.; Rimson, S.; Segal, R. Biochem. J . 1958,68,493. (18) Goldman, R.; Kedem, 0.;Katchalski, E. Biochemistry 1971,10, 165. (19) Ohnesorge, F. K.; Menzel, H., Department of Toxicology, University of Dusseldorf, West Germany, private communication.
Received for review September 29, 1980. Revised Manuscript Received May 29, 1981. Accepted July 6 , 1981. S.B. gratefully acknowledges the award of a DAAD Fellowship which enabled the present study in West Germany.
EnvironmentalAssessment of Industrial Discharges Based on Multiplicative Models M. Ross Leadbetter*t and W. Gene Tucker* U.S. Environmental Protection Agency, Industrial Environmental Research Laboratory, Research Triangle Park, North Carolina 277 11
The severity, S, of a substance in a discharge from an industrial source is defined as the ratio of substance concentration, either a t the source or a t some ambient point of interest, to a maximum specified “safe” concentration level. The source is considered “clean” unless S is expected to exceed unity on more than a given acceptably small proportion of time. Otherwise, it is “dirty”. The classification of a source as clean or dirty is made from (a) measurements of factors such as stack emission characteristics and (b) possible knowledge of the statistical properties of other factors (such as meteorology). Standard statistical decision techniques are used, with some novelty to take account of the forms of variation present (time fluctuations, measurement errors, etc.) and to best incorporate existing prior knowledge of the statistical parameters involved. Log normal distributional assumptions are used, coupled with multiplicative transport models in ambient cases.
I. Introduction A current approach to assessment of substances discharged from industrial processes ( 1 , 2 )is to compare the concentration, C, of the substance a t some point of interest (either at + Under Loan Agreement with University of North Carolina, Statistics Department, Chapel Hill, NC. t Chief, Special Studies Staff, Industrial Environmental Research Laboratory.
the point of discharge or at some downstream point) with some estimated “safe” concentration level, or standard, G. The severity of the source is then defined as the ratio S = C/G.
Suppose, for the moment, that there are no temporal (e.g., daily) fluctuations in a source’s discharge and ambient conditions. Then S has a constant value, and the source may be termed “clean” or “dirty” depending upon whether this value is less or greater than 1 (Le., C < G or C > G ) . The terms “clean” and “dirty” have no official standing, of course, but are conveniently brief and clear for our purposes here. S, of course, is unknown, but can be estimated from measurements of appropriate factors (e.g., emissions) giving some calculated estimate 3. The classification of the source as clean or dirty would be made on the basis of whether 9 was smaller or larger than some critical level, c (not to be confused with C denoting concentration). Since the measured factors (hence, also 9) are subject to measurement errors, it is intuitively plausible that, to have small probability of misclassifying dirty sources, c must be less than 1.Its precise value would be determined by standard statistical (hypothesis testing) techniques in such a way that the probability of misclassifying a dirty source is limited to some preassigned small value, p (e.g., 0.05). If the severity at the discharge point is of primary interest, 9 will be calculated directly from measurements of concentration at that point. On the other hand, when ambient severities are of concern, an appropriate transport (e.g., diffusion) model must be assumed in order to relate C and S to
This article not subject to US. Copyright. Published 1981 American Chemical Society
Volume 15, Number 11, November 1981
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