The milk portion of the diet had 0.16-0.46 mg Sr, with a n average of 0.29 mg. or 32.1% (Rehnberg et al., 1969). In the present study, analyses of the diets of children showed intakes of 0.792-2.43 mg Sr, with an average of 1.237 mg/kg (Murthy et al., 1972). T h e amount of Sr in the milk portion of the diet was calculated t o be 0.071-0.463 indicating that mg, with an average of 0.135 mg or 11.070, 1.102 mg or 89% of the S r ingested originated from food other than milk. In conflusion, these d a t a show wide seasonal a n d geographical variations in the trace element content of diets studied. Trace element content of water and air must also be considered to obtain a total environmental perspective.
Achnou,ledgrnent T h e authors are grateful to J. E. Campbell for his interest in the project; to Rebecca Martin for her help in preparing samples for atomic absorption analyses; and t o Helen Bachelor a n d R u t h Carver for statistical calculations.
Literature Cited Bancroft, T. A., “Topics in Intermediate Statistical Methods,” Vol. 1, pp 35-41, Iowa State Cniv. Press, Ames, Iowa, 1968. Bryant, F. J., Chamberlain, A . C., Spicer, G . S., Webb, M. S . W., Brit. Med. J . , 1371-5 (June 14, 1958). Duncan, D. B., Biometric,$, 11, 1-42 (1955). El-Gindy, M . M., Indian J . Agr. Sei , 29, 78-80 (1959). Engel, R. LV.. Price. N . O., Miller. R. F.. J . Y u t r , 92, 197-204 (1967. Feldman, C.. Jones, F. S., Tissue Analysis Laboratorv Report ( A n n . Prog Rept , July 31, 1964i, Health Phy. DiJ. ORNL 3697, 178-85 (19641. Glendennine. B. L.. Parrish. D. B.. Schrenk. \V. G.. A n a / . ( ‘ h e m . . 27, 1554-6 (1955) Golvkin, N A , KraLnora. L S , RI b Khoz 45,60-4 (1969) Gormican, A , , J . A m c r . Diet .4ss..‘56, 397-403 (1970). Harrison, G . E , , Raymond, LV, H. A , . Sutton. A , , Brit. .Wed J 589-92 (1960). Il’vitskii, N .A , . Asmaeva. Z . I.. Izi,. L’>s\h. l*ch Z a i . c d . I’i.iic./i Tekbnoi.. 2, 34-5 (1969). Jaulmes. P.. Hamelle. G . . A n n .Yutr A l i m e n t . . 25, B133-B203 (1971). Kent, \V,L.. McCance. R. A , , Bincheni. J , 35,877-83 (1941) Kleinbaum, H.. 2 Kindcrbeiik., 86,655-66 (1962). Kramer. C. Y.. Biometric,, 12, 307-10 (19561.
Leshchenko, P. D.. Kononko, L. N., Solomko, G . I . , Rudenko, A. K . , Vop. Piton., 31,81-5 (1972). Los, L. I., Pjatnickafa, L. K., ibid.,21,82-3 (1962). Malina, V . D.. Klyachko, Y. A.. Izt,, L(\,s.ch. I’ch. Zatvd , Pishch. Tehhnol., 4 , 32-3 (1969). Meranger. J. C., Smith, D. C..Can. J . Pub. Heaith. 63, 53-7 (1972). Murphy. E. b‘., Page, L.. Watt, B. K . . J. Amer. Diet. A S \ , , 5 8 , 115-7 (19711. Murthy. G. K . , Rhea, U . , J . Ilair? Sci.. 50,313-7 (1967). Murthy. G. K . . Rhea, U . , Peeler. J . T., ibid , pp 651-4. Murthy. G. K.. Rhea. U.. Peeler, J . T.. Ent,irnn. S c i . Techno/.. 5, 436-42 (1971). Murfhy, G . K . , Rhea, U. S . , Peeler, J. T., J . Dair) Sci., 55, 1666-74 (1972). Rehnberg, G . L., Strong. A. B., Porter, C. R.. Carter. hl. \V,,Enciron. Sci Tpchnoi.,3, 171-3 (1969). Schlettwein-Gsell, D.. Mommsen-Straub. S.. V. Sickel. I n t . Z. Vitarninfor.\ch.. 41, 429-37 (1971a). Schlettwein-Gsell, D., Mommsen-Straub, S., VI. Copper, ibid., pp 554-82 (197lbi. Schroeder. H. A.. Balassa. .J. J., Tipton. I . H.. J Chronic Dic , 15,51-65 (1962). Schroeder, H. A , . Nason. A . P., Tipton. I. H.. Balassa, J . J.. ibid.,19, 1007-64 (1966). Soman. S.D.. Panday. V . K . . Joseph. K . T., Raut, S.J.. Health Ph>S., 17,35-40 (1969). Stephanov, J . . Yaneva. S., Mladenova. S . . S h . Tr. ,Vaucii. I l s l e d . Khihicnen I n s t . , 12, 56-8 (1970). Tipton. I. H., Stewart. P. L.. Dickson, J., Heaitb Pb?.r.. 16, 45562 (1969). Tipton, I. H.. Stewart. P. L . , Martin, P. G., ibid.. 12, 1683-9 (1966). Tsvetkova. I. N., Gig. Sanit.. 34, 92-4 (1969). U.S. Public Health Serivce. “Radioassay Procedures for Environmental Samples.” 999-RH-27(1967). White. H . S.. J A m e r . Iliet. .4ss.. 55, 38 (1969). Yamagata, S . .,Vaturc, 83-4 (1962). Zook, E. G., Lehmann, ,J., J . Ass. Off. Anal. Chem., 48, 850-5 ( 1965i . Reccircd f o r roi,icri.A p r i l 2, 1973 A c c e p t c d .Augu\t 3. 19i.S Supplementary Material Available. Tables 11 and 111 will ap-
pear following these pages in the microfilm edition of this volume of the journal. Photocopies of the supplementary material from this paper only or microfiche (105 X 148 mm. 20X reduction, negative) containing all of the supplementary material for the papers in this issue may be obtained from the Journals Department. American Chemical Society. 1155 16th S t . , N.LV., \Vashington. D.C. 20036. Remit checks or money order for $3.00 for photocopy or $2.00 for microfiche, referring to code number ES&T-73-1042.
Detection and Estimation of Isopropyl Methylphosphonofluoridate and 0-Ethyl S-Diisopropylaminoethylmethylphosphonothioate in Seawater in Parts-per-TriIIion Level Harry 0 . Michel, Eric C. Gordon, and joseph Epstein’ D e f e n s e R e s e a r c h Branch, Edgewood Arsenal, Aberdeen Proving Ground, Md. 21 010
H
A procedure is described for the estimation of two very
potent anticholinesterase chemicals, viz., isopropyl methylphosphonofluoridate (GB) and 0-ethyl Sdiisopropylaminoethylmethylphosphonothioate (VX) in seawater in concentrations a t the parts-per-trillion level by an enzymatic technique. Kinetic constants for the reaction of other anticholinesterases with two sources of cholinesterase a n d for the reaction of the cholinesterases with several substrates are given. With these d a t a , the reader can select conditions for the development of procedures for estimating very low concentrations of these anticholinesterases in water. To whom correspondence should be addressed.
Some unserviceable munitions containing t h e toxic nerve gas GB (isopropyl methylphosphonofluoridate) or, in some cases, VX ( 0 - e t h y l S-diisopropylaminoethylmethylphosphonothioate) have, in the past, been disposed of by dumping into ocean waters. Eventually, these agents could find their way into the ocean. A knqwledge of the concentration, if any, of the nerve gases in the seawater in the vicinity of the disposed munitions can provide much information on the environmental impact of this method of disposal and other information relating t o such items as the ability of the munkions a n d their encasements t o withstand the pressures a t various ocean depths and the force of impact of collision of the containers with the ocean bottom. For this purpose, an extremely sensitive Volume 7, Number 11, November 1973
1045
technique was needed for the detection a n d estimation of G B a n d / o r VX concentrations in seawater. In this paper we give methods for detecting a n d estimating concentrations of G B a n d VX in seawater, singly or in combination, in t h e parts-per-trillion ( p p t ) g / m l ) level by the technique af enzyme inhibition.
Table I. Percent Inhibition of Enzyme by GB in Seawater at pH 7.2 and 25”C, Incubation Time of 20 Hr % inhibition Calculated
[GBI moles/ 1 X 10’
J’r in c ip le of Me tho d T h e determination of concentrations of organophosphorus compounds through their inhibition of cholinesterase enzymes has been in use for some time (Guilbault. 1970a). ’These enzymes, which catalyze t h e hydrolyses of various esters. are rapidly and irreversibly phosphonylated by toxic phosphorus esters. T h e resulting phosphonylated enzyme is catalytically inactive. T h e fraction of t h e original enzyme concentrat ion remaining active after incubation with the inhihitor will depend upon t h e time of contact between the inhihitor and t h e enzyme and the concentrations of’ t h e reactants. but t h e solution will always have a reduced catalytic activity. T h e activity. prior to and after incubation with the inhibitor, is determined from t h e rate of splitting o f a suitable enzyme substrate. ‘The decreased activity. under specified reaction conditions, is related to the inhibitor concentration. In this procedure, the activity of the cholinesterase was determined by t h e technique of Ellman et al. (1961). For the sequence of reactions involving a n inhibitor ( I ) , the enzyme ( E ) . a n d ester substrate (S) to form products (Pi. one can write t h e following equations: f:
0.64 1.28 1.92 2.56 3.21
- _ _ _ _ . _ _ ~ -
’
Eq 5 38.4 63.6 77.1 86.0 91.5
Eq 7
Found
9.2 17.3 24.9 31.7 38.0
7.6 17.0 23.0 27.5 32.0
If the rate of hydrolysis of t h e inhibitor is such that a significant portion of t h e inhibitor undergoes hydrolysis during t h e incubation period, then Equation 5 must he modified to reflect the effect of the changing inhibitor concentration on t h e rate of reaction with the enzyme. In such a case. the differential equation for the change in enzyme concentration with time becomes,
where k h is the first order hydrolysis rate and has the same t i m e units as k l . Integration a n d suitable mathematical manipulation leads to:
,
E + I - E 1
ES
I
If t h e time of incubation is made sufficiently long so t h a t e - Rh‘ < < < 1, t h e n Equation 6 reduces to Equation 7:
P
(3,
from which it can be determined t h a t the rate of formation or products will be (Guilbault, 1970b):
For t h e s a m e substrate a n d initial enzyme concentrations
At [Ill = 0, VI is t h e rate of formation of products using t h e initial concentration of enzyme, and Equation 5 may be written In V I = h,[I]?t
+
In
V1
(5)
T h u s , one can determine t h e concentration of inhibitor from a knowledge of t h e activities of t h e enzyme prior to a n d after incubation for a given length of time a n d t h e biomolecular rate constant for t h e reaction between inhibitor a n d enzyme. T h e equation is also useful in t h a t it enables one to select conditions for t h e inhibition reaction if a n approximate concentration of inhibitor is known. For example, if Vz is to be 50% of VI, a n d h l lO7M-1 min l , the product of [I] a n d t should he approximately 7 x 10 -“M.min. Moreover, a plot of In V I vs. I will be linear with a slope of - h ~ tand a n intercept equal to In Vz. S t a n d a r d curves can be constructed using this relationship. Bimolecular rate constants can be determined.
-
1046
Environmental Science & Technology
As in t h e case of Equation 5 , a plot of In V,/VI with concentration will be linear. T h e slope in this case is k l / k h .
Experirn e n tu 1 Materials and Equipment. Eel Cholinesterase. T h e enzyme (Source: Worthington Biochemical Corp.) is diluted in buffer so as to have a catalytic activity of 7 n mol/min/ ml vs. acetylthiocholine. T h i s is equivalent to about 10 x micromolar units/ml (as defined by Sigma Chemical Co.). Substrate. T h i s contains 0.002M acetylthiocholine, iodide (Source: Calbiochemi a n d 0.002M 5.5’dithiobis-2nitrobenzoic acid (Source: K&K Laboratories, Inc.). t h e latter neutralized with sodium hydroxide solution to pH 5.0. Buffer. 0.1M morpholinopropane sulfonic acid, 0.01M ethylenediaminetetraacetic acid and 0.1% gelatin, a d justed to p H 7.20. Determination of GB and VX. One-milliliter samples of seawater are mixed with 0.100-ml aliquots of eel cholinesterase in buffer. T h e samples are allowed to react for periods u p to 30 hr a t 25°C (see discussion on mixtures of VX a n d G R ) . T h e n 0.100 ml aliquots of substrate solution are mixed with t h e samples. After a further reaction period of exactly l hr, t h e absorbances of the samples are determined a t 412 n m . Samples of seawater containing known amounts nf GB a n d VX are allowed to react with the enzyme and substrate as described above. Blank s a m ples containing seawater, buffer, and substrate, but no enzyme. are incubated along with t h e other samples. Blanks are used to zero t h e spectrophotometer and unknown GI3 and VX values are calculated by interpolat inn from known concentrations.
Table I I. Representative Bimolecular Rate Constants of Some Organophosphorus Compounds with Cholinesterase from Various Sources at 25°C k 2 , M-' min-' Acetyl cholinesterase from Compound
Eel ChE
Acetyl ChE
Butyryl cholinesterase from horse serum
0
II
(CHJXHO-P-F
iGB)
I
7 x 107
1 . 4 x 107
1 . 7 x 107
1.7 X l o 8
6.0x 107 =
2.1 x 107
2.0 x 108Q
3 . 6 x 107
3.3 x 108Q
1 . 6 x 107
CHI
0
II
iCH ),CCHO-P-F
I CH
I
CH, 0
I1 I CH
CHCHCH CHO-P-F AH
&H
\I
H,C /CH---CH \CH.-CH.
H
Q
O
II
,C-0-P-F
4 x 108
I
CH O
0
II
II
C,H,O-P-0-P-C,H,
I
(TEPP)
I
OC H
2.1
x 106
2 . 0 x 107
OC,H
0 tCH 1 CHO-P-F
II
(DFP)
2 x 104c
I OCHtCH,)
4 . 8 x 1040
1.5 x 1 0 7 ~
1 . 4 x 105 b
2.5 x 1 0 7 b
0
II I
tCH ) CHO-P-L
tDAP1
OCH(CH 0
1
/CH(CH
II
CH,-P-X
)i
H,S
I
CCH
iVX) 'CHt
3
x 107
2.3 X lo6
CH,)
0
II I
C H 0-P-SC OC H
H,K / C H
C'
(Amiton)
H
2.5 X lo6
3.0 x 107b
a Boter, H. L., Ooms. A. J. J., Rec. Trav. Chim., 85, 21-30 (1966). Ooms. A. J. J., PhD Thesis, University of Leiden, Holland, The Netherlands. Jandorf, B. J., J . Agri. Food Chem., 4, 853-8 ( 1956).
Determination of VX. A 25-ml sample of seawater is brought to p H 10 with sodium hydroxide solution, then extracted twice with 5-ml portions of methylene chloride. T h e combined methylene chloride extracts are acidified with 0.1 ml of 0.1N hydrochloric acid a n d evaporated to dryness a t room temperature under a stream of nitrogen. T h e residue is dissolved in water. An aliquot of t h e aqueous solution is treated as under the determination of GB a n d VX.
Discussion General. For determination of concentrations of nerve gases in t h e parts-per-trillion level ( 10-lO-lO-llM), several criteria for the reaction between inhibitor a n d enzyme, and enzyme and substrate must be m e t . First, the reaction rate constant for the inhibitor and enzyme must be of the order of 1OiM-I m i n - l if t h e t i m e of incubation is to be not unreasonably lengthy. Second, since first-order kinetics of enzyme inhibition is required, t h e inhibitor concentration, therefore, must be of the order of 10-I2M, a n d , a t this and even lower concentrations, t h e enzyme must have a relatively high activity toward its substrate. T h e turnover number should be 105-106m i n - I . Use of Equations 5 , 6 , and 7. In t h e case of VX, which has a first-order rate constant of hydrolysis a t p H 7.2" and
25°C of 2.5 x 10-5 min-1, no serious error is introduced by t h e use of Equation 5 for even long ( u p to 30 hr) incubation periods. However, with GB, whose rate of hydrolysis in seawater adjusted to p H 7.2 is 4.12 X min-I (Epstein, 1970), Equation 6 or 7 m u s t be used. A 20-hr incubation of enzyme with GB in water is sufficiently long so t h a t one can use Equation 7. T a b l e I shows the expected loss in activity of eel cholinesterase in contact with seawater containing different concentrations of GB for 20 hr a t p H 7.2, 25"C, using Equations 5 a n d 7; t h e inhibition determined experimentally using GB in solutions 0.46M in NaCl a n d 0.053M in M g S 0 4 under t h e same conditions is also shown. For the calculated values, k l was taken as 6.4 X 10i.W1 m i n - l (Table 111). Specificity. From Equation 5 we see t h a t two anticholinesterase materials, differing widely in their hl values, can give t h e same rate of formation of psoducts if their concentrations are such t h a t h,[I]1 = hl'[I]2. Table I1 lists some bimolecular rate constants of some organophosphorus compounds with different sources of cholinesterase a t 25°C. If eel cholinesterase were used as a source of enzyme, the same degree of inhibition of the enzyme would be achieved after a given time of incubation of the enzyme with GB or T E P P if t h e concentration of T E P P were 35 times t h a t of GB. I t is highly unlikely t h a t a n Volume 7, Numbe;ll, November 1973
1047
organophosphorus compound, other t h a n G B or .VX, will be found in seawater in areas of d u m p e d munitions in concentrations sufficiently high to cause a n interference. Nevertheless, in t h e event t h a t this very unlikely possibility did occur, a partial solution to this problem can be obtained if identical test conditions are employed using two sources of enzyme, for example. eel cholinesterase a n d horse serum cholinesterase. Using Equation 5, we c a n determine t h e ratio of the bimolecular rate constants for t h e reactions of eel cholinesterase and of horse serum cholinesterase with t h e anticholinesterase. As a general rule, t h e ratio of reactivities of organophosphonates such as t h e nerve gases with eel and horse serum cholinesterases will be >1, whereas the opposite is usually true for the organophosphates used in pesticides. If t h e necessary information can be obtained, a n a p proximate value of t h e bimolecular rate constant for t h e reaction between a n unknown organophosphate a n d t h e enzyme is useful for class identification of t h e anticholinesterase. Thus. if eel or red cell cholinesterase is inhibited irreversibly and if a hl value of l O 7 - l O S M - 1 m i n - 1 is obtained. it can be assumed t h a t t h e inhibitor is a n organophosphonate rather t h a n a n organophosphate. A precise value of t h e biomolecular rate constant for t h e reaction between a cholinesterase a n d a n organophosphorus compound is a powerful tool for t h e identification of a particular organophosphorus ester. However, t h e rate constant obtained in t h e reaction between G B a n d eel cholinesterase varies significantly with such factors as ionic strength (Table 1111, p H ( T a b l e IV), a n d temperature (Table V ) . For identification purposes, t h e determination of t h e bimolecular rate constant of a n unknown must be made under exactly the s a m e experimental conditions (e.g., p H , temperature, ionic strength, buffer constituents) as those Table I l l . Inhibition of Eel Cholinesterase by GB; Effect of Sodium Chloride Concentration (pH 7.2, f = 25"C, 0.0091M MOPS,' 0.01% gelatin) NaCI. M
kl X lO-'M-'
0 0.082 0.163 0.25 0.334 0.416
3.1 4.0 4.8 5.4 6.1 6.4
min-I
a Morphilinopropane sulfonic acid
Table I V . Inhibition of Eel Cholinesterase by GB; Variation in Rate with Change in pH (f = 25"C, 0.33M KCI, 0.01 % gelatin) PH
kl X
5.0 6.0 7.0 8.0 9.0 10.0
M - ' min-'
0.68 3.4 6.8 7.1 6.7 4.2
Table V. Inhibition of Eel Cholinesterase by GB, Effect of Temperature (pH 7.2, 0.0091M MOPS,' 0.01% gelatin) Temp, " C
kl X
15 25 38 a
Morphilinopropane sulfonic acid
1048
Environmental Science 8 Technology
M-'min-'
2.0 3.06 4.3
Table VI. Half-life of GB in Water at 25"C, pH 7.2, in Presence of Mg2+ and EDTA [Mg2+1
0 03 0 02 0.02 0.02
[EDTA]
0.02 0.03 0.10
t i 2.
mln
120 180 460 ca 1100
used in determining the constant for a n organophosphorus ester of known structure. VX, because it contains a n amine function, is particularly suitable for extraction from seawater into organic solvents a n d re-extraction into a n aqueous phase for reaction with t h e enzyme. T h i s property of VX is important both to the specificity a n d sensitivity aspects of t h e analysis. VX is extracted quantitatively with methylene chloride from seawater adjusted to p H 10. T h e distribution coefficient of VX a t p H 10 between methylene chloride a n d water has been determined to be greater t h a n 5O:l. T h u s , two 5-ml extractions of 25 ml of t h e seawater extracts more t h a n 99% of t h e VX from t h e seawater. Addition of a few drops of hydrochloric acid solution a n d evaporation of t h e organic solvent leave a residue t h a t is t h e n dissolved in 1 ml of water. In addition to separation of VX from a n y of a host of organophosphorus anticholinesterases, this procedure also has t h e following advantages: T h e extracted VX is now in a relatively pure form, a n d a bimolecular rate constant with t h e enzyme can be determined under precisely specified conditions a n d can be compared with t h a t of a n authentic sample for identification purposes; a n d t h e VX concentration in t h e 1-ml s a m ple is twenty-five times t h a t in t h e seawater sample; a more sensitive analysis is t h u s possible or t h e time of incubation can be materially reduced. Finally, advantage can be taken of t h e vastly different rates of hydrolysis of VX a n d G B in seawater to achieve a degree of specificity. G B has a half-life of fewer t h a n 10 sec a t p H 10 in seawater, whereas the VX half-life a t this p H is approximately 14 hr. G B in seawater, adjusted to p H 10 with a few drops of 0.1M N a O H solution, will have been more t h a n 99.9% hydrolyzed after 2 min. T h e degree of inhibition given by samples prior to a n d after hydrolysis can serve to help identify t h e anticholinesterase responsible for the activity. M e t h o d s for M i n i m i z i n g Loss of S e n s i t i v i t y D u e to Hydrolysis of GB. If the hydrolysis rate of G B were one order of magnitude lower t h a n it is, a solution of G B 6.32 X 10-12M in concentration would inhibit 32% of t h e enzyme after a 20-hr contact period. T h i s may be compared with t h e inhibition of 38% which assumes a negligible rate of hydrolysis. T h e hydrolysis rate of G B can be reduced by ( a ) lowering t h e p H of t h e reaction medium or (b) addition of chelating materials to sequester t h e activity of t h e magnesium ion. In connection with ( a ) , lowering t h e p H to 6.2 reduces t h e hydrolysis rate constant to 4.3 x 10-4 min-1. However, there is a lowering also of t h e bimolecular rate constant of t h e G B a n d eel cholinesterase reaction from approximately 7 x 1 o 7 ~ - Im i n - I to 3.5 x 1 0 7 ~ - 1 m i n - I . Although there is some gain in t h e percentage inhibition by lowering t h e p H , it is not large as indicated by t h e above calculation. A concentration of 6.32 x 10-12M G B incubated with eel cholinesterase for 20 hr at 25°C a t p H 6.2 would yield a percentage inhibition of 19%. In connection with ( b ) , t h e high hydrolysis rate of G B in seawater has been attributed to t h e presence of M g 2 + (Epstein, 1970; Epstein a n d Mosher, 1968). Addition of
Table V I I . Enzymic Activities of Eel and Horse Serum Cholinesterases with Various Substrates at 25°C Eel ChE Substraten
Acetylcholine ( 1 ) Acetylthiocholine (2) Butyrylcholine ( 1 ) Phenylacetate (2) p N 0 ~phenylacetate (2) 2,6-diCI indophenolacetate (2) lndoxylacetate (2)
Vmax,
meq/min
1.63X 1.45 x 10-4 ... 1.44x 10-4 2.18 x 10-6 6.5 x 10-7 3.6 x 10-5
Km
3 x 10-4 1.3x 10-4 ...
1.8x 5.2 x 7.0 x 2.3 x
10-3 10-4 10-5 10-3
HS ChE k 3 > min-
’
6.7 x 105 6.0 x 105 ...
5.9 x 9.0 x 2.7 x 1.5x
105 103 103 105
Vmnx,
meq/mln
1.4x 10-4 1.71 x 10-4 4.7 x 10-4 3.2 x 10-4 1.2 x 10-4 7.5 x 10-6 8.2 x 10-5
Km
1.85x 10-3 6.6 X 1.6x 10-3 5.7 x 10-3 4.0 x 10-3 1.3x 10-4 6.7x 10-4
k 3 1 bm
u-
1.33 x 1.63x 4.48 x 3.05 x 1.14x 4.14 X 7.81 x
1
104 104 104 104 104
IO’ 103
‘Two reaction media were used: the first, designated by ( 1 ) . was 0 . 3 3 M KCI and 0.01% gelatin at pH 7 . 4 for pH stat measurements and the second, designated by ( 2 ) . was 0.01M MOPS, O.OlM MgS04, 0.01% gelatin. and 0.001M EDTA. k3 = V,,,/Eo; Eo (enzyme concentration) for eel ChE with acetylcholine = 2.43 X l O - ’ O . for HS ChE with phenylacetate = 1.05 X 10-*M.
ethylene diaminetetraaceticacid ( E D T A ) lowers the hydrolysis rate of G B (Table VI) in MgZ+-containingwaters. However, t h e bimolecular rate constant of t h e reaction between G B a n d eel cholinesterase in t h e presence of 0.10M E D T A is about one half of t h a t in t h e absence of E D T A . T h e percent enzyme inhibited, therefore, is of t h e s a m e order as t h a t obtained by lowering t h e p H to 6.2. S a m p l i n g of W a t e r . Because of the relatively high rate of hydrolysis of G B in seawater, samples taken for analysis should be adjusted to p H ca. 5.0, where the hydrolysis rate is at a minimum (Epstein, 1970), a n d stored at t e m peratures as close to 0°C as possible. T h e addition of a p proximately 1 ml of 0.1N HC1 to 50 ml of seawater will bring t h e sample into t h e correct p H range. M i x t u r e s of VX a n d GB i n S e a w a t e r . T h e bimolecular rate constants of VX and G B with eel cholinesterase in seawater medium a t p H 7.2 and 25°C are different [ h l ( V X ) = 1.6 X 107M-l m i n - l ., h 1 ( ~ =.6.4 ~ ) x 107~-1 m i n - l ] , but approximately the same concentrations (calculated as p p t ) will give the same degree of inhibition if t h e incubation time is approximately 30 hr. T h u s , a s a m ple 6.5 p p t of either VX or G B or combinations of each totaling 6.5 ppt will give a n inhibition of approximately 50% of t h e enzyme after 30 hr of incubation. If both VX and G B are suspected, then one sample can be run as indicated in t h e procedure using a time of incubation of 30 hr. A second sample, extracted and reextracted, can be a n a lyzed for VX concentration, keeping in mind that the rate constant for the reaction between VX and eel cholinesterase will now be different from t h a t in the seawater (Table 11). T h e concentration due to G B in the first sample can be calculated from the total concentration of GB and VX less the concentration of VX found in the extracted s a m ple. It h a s been our experience t h a t t h e activity can be reproducibly determined to k3%. We consider a 10% change in activity as sufficient. For a 30-hr incubation. then, as little as 1 ppt of G B or VX can be determined. E x t r a c t i o n . Extraction of t h e seawater sample for VX will also extract G B to some extent, although the likelihood t h a t G B in any significant amount will remain after p H adjustment to 10 is extremely small because of the high rate of hydrolysis of G B at this p H . T h e distribution coefficients of G B between several organic solvents and water have been determined (Rosenthal et al.. 1956). T h e distribution coefficient for G B between methylene chloride and water is approximately 17. S u b s t r a t e s for U s e w i t h Cholinesterases. T h e method described herein for analysis of G B and VX requires t h a t t h e agents, at very low concentrations-i.e.. lO-llMreact sufficiently rapidly with very dilute concentrations of the enzyme so that a n appreciable inactivation of t h e enzyme takes place in a not too long time period and t h e enzyme concentration remaining after incubation-i.e..
> K,, Equation 8 reduces to:
1.’
=
fi,E,,e-’, h 1
(9)
Under these conditions, the rate of formation of products is maximum (V,,,), independent of the substrate concentration and dependent only upon the concentration of the enzyme remaining after its reaction with the inhibitor. K,, a n d h3 for a n u m Table VI1 gives the values of V,,,, ber of substrates determined under particular conditions for eel a n d horse serum cholinesterase. T h e use of acetylthiocholine-dithiobisnitrobenzoatesystem for assaying cholinesterase activity has been discussed in detail (Ellman, 1961). When the d a t a of Table VI1 are used, the rate of formation of products for concentration of enzyme 1012M, calculated from Equation 8 is 0.36 mol/l. min. Reaction of t h e formed thiocholine with 5,5’-dithiobis-2-nitrobenzoicacid (already contained in the substrate solution) immediately produces t h e highly colored p-nitrothiophenolate anion, which has a molar extinction coefficient of 1.36 X l o 4 at 412 n m . After 1 hr, it would be expected that the absorptivity of a solution containing the enzyme-substrate mixture would be ca. 0.3 unit when viewed through a 1-cm cell. An examination of t h e d a t a i’n Table VI1 will quickly reveal that other substrates can be used.
Literature Cited Boter. H . L.. Ooms. A. J . .J.. K e c Trac. Chim.. 85, 21-30 (1966). Ellman. G . L.. Courtney, K . D.. Andres, \-., J r . . Featherstone. R 11..Hiochem. Pharmacoi.. 7.88-95 (1961). Epstein. .J.. Science, 170, 1396-7 (1970). Epstein, .J.. Mosher, I$-.A , . J . Ph\,s. Chem.. 72, 622-5 (1968). Guilbault. G . G . “Enzymatic Methods of Analysis,” pp 11-12, Pergamon. New York. N.Y., 1970a. Guilbaulr. G . G . “Enzymatic Methods of Analysis.” pp 217-24. Pereamon. New York. K Y . ,1970b. .Jand&f. B. J . . J . Agr Food (’hem., 1,853-8 (1956). Ooms. A. .J. .J.. PhD Thesis. I-niversity of Leiden. Holland. The Netherlands. 1961. Rosenthal. R. LV.. Proper. R.. Epstein. J . . J . Ph\s (‘hem.. 60, 1596-7 (1956). ReccivPd f o r ret,ieii April .5, 1973 Accepted Augusr9. 197.9 Volume 7,Number 11, November 1973
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