Fluorometric System Employing immobilized Cho Iineste rase f o r Assaying Anticholinesterase Compounds GEORGE G. GUILBAULT and DAVID N. KRAMER U. S. Army Chemical laboratories, Edgewood Arsenal, Md.
b To develop a fluorometric system for the assay of anticholinesterase compounds using an immobilized (insolubilized) cholinesterase, new fluorogenic substrates were investigated: the acetyl and butyryl esters of 1 and 2-naphthol. The esters are nonfluorescent compounds, hydrolyzed b y cholinesterase to highly fluorescent materials. The rate of change in the fluorescence of the solution due to production of 1-naphthol (Aex = 3 3 0 mp, ,A, = 460 to 470 mp) and 2-naphthol (Aex = 3 2 0 mp, ,A, = 410 mp) is measured and correlated with enzyme activity. Using immobilized cholinesterase (prepared b y insolubilization of cholinesterase in a starch gel on a polyurethane foam pad) and 2-naphthyl acetate as substrate, a continuous fluorometric system was developed for the assay of anticholinesterase compounds in air and water. As long as the enzyme i s active, a fluorescence is produced, due to the hydrolysis of the ester to 2-naphthol. Upon inhibition, the fluorescence drops to a level approaching zero. By employing batch methods with 2-naphthyl acetate as substrate, 0.00050 to 0.10 unit per ml. of soluble horse serum cholinesterase and 0.001 0 to 1 .OO unit per ml. of acylase may b e determined with standard deviations of 11.0 and l.2yo, respectively. Using 2-naphthyl phosphate, acid phosphatase in concentrations of 0.00050 to 0.1 00 unit per ml. may b e determined with a deviation of 1 1 . 2 % .
-
A
procedure for the rapid determination of cholinesterase (6) is based on the enzymic hydrolysis of two nonfluorescent substrates, resorufin butyrate and indoxyl acetate, to the highly fluorescent compounds, resorufin and indoxyl. In attempting to automate this procedure into an apparatus for the assay of anticholinesterase compounds in the atmosphere, several difficulties were encountered. The resorufin butyrate, although stable for ordinary laboratory use, was not stable enough for use in a continuous device. The indoxyl formed upon enzymic hydrolysis of indoxyl acetate is not air-oxidized under ordinary conditions, but, in the presence of FLUOROMETRIC
large volumes of air sampled in a continuous device, it was very rapidly oxidized to the nonfluorescent indigo blue. For some time 1- and 2-naphthyl esters have been used as colorimetric substrates for esterases. Seligman (14) and Kramer and coworkers (9) utilized the myristate and laurate esters as substrates for pancreatic lipase, and Seaman and Winell (IS) prepared the acetate ester of 1-naphthol for the histochemical localization of esterases of the prostate gland. Leisnert (IO)used the 1- and 2naphthyl acetates for detecting esterases of lymph nodes, Tashian and coworkers (15) for the estimation of carboxylic esterase, and Bernsohn et al. (3) for determination of brain esterase. dugustinson (1) proposed the qualitative differentiation of aryl esterase from cholinesterase with 1- and 2-naphthyl acetate. Hercules and Rogers (8) have shown that 1- and 2-naphthol are fluorescent, and Moss (12) proposed the fluorometric determination of alkaline phosphatase at pH 10 using 1- and Z-naphthy1 phosphate as substrates, but gave no data. Since it was believed that 1and %naphthol are fluorescent only in the ionic form a t pH's greater than 10 (16), no fluorometric methods were developed using the esters of 1- and 2naphthol for acid phosphatase or other enzymes. This report shows that the molecular forms of 1- and 2-naphthol are very fluorescent, whereas the esters are nonfluorescent. Fluorometric methods are described for the direct, rapid, and sensitive estimation of Cholinesterase, acid phosphatase, and other enzymes. 'c'sing 2-naphthyl acetate as substrate, a continuous fluorometric system was developed for the assay of anticholinesterase compounds, employing cholinesterase entrapped (insolubilized) in a starch gel matrix on a polyurethane foam pad. EXPERIMENTAL
Reagents. ENZYMES. Cholinesterase, horse serum (Arinour Industrial Chemical Co.), specific activity 1.90 units. One unit represents 1 pmole of acetylcholine hydrolyzed per milligram of enzyme per minute. Acetylcholinesterase (bovine erythrocytes and eel), lipase (porcine pancreas
and steapsin), a-, p-, and ychymotrypsin, acid phosphatase (activity 1.0 unit), and acylase were as described (5, 6). Immobilized (insolubilized) cholinesterase. The immobilization of cholinesterase in a starch gel was originally described by Vasta, Usdin, and Aldrich (17'). , Because of limitations in air and liquid flow, this preparation was found unsuitable for use in a continuous system. Instead the enzyme was immobilized by entrapment in a starch gel matrix on a polyurethane foam pad (2) (Scottfoam, Scott Paper Co., Philadelphia). These pads, 10 mm. in diameter and 6 mm. thick, were cut using a very sharp cork bore. The activity of the enzyme in these pads was analyzed electrochemically (2) and found to be about 1 unit per pad. SUBSTRATES.1- and 2-Xaphthyl acetate were obtained from the Sigma Chemical Co. The butyrates were prepared by conventional procedures (5, 6). 2-Saphthyl acid phosphate, sodium salt, was obtained from the Calbiochemical Co., Los dngeles. A stock 10-ZM solution was prepared in triply distilled water. The synthesis of 9,lO-diacetoxyanthracene, 1,5,9,10-tetraacetoxyanthracene, and 2-acetamido-9,lO-diacetoxyanthracene (Table I, compounds I, 11, and 111) was accomplished by the reductive acetylation of the corresponding 9,10-anthraquinone, 1,&dihydroxyanthraquinone, and 2-aminoanthraquinone (Eastman Chemical Co. Rochester, N. Y ,), using acetic anhydride-pyridine (3 to 1)and excess zinc dust. The mixture was cast into ice water and the resulting solid was collected, dried, and recrystallized from ethanol. 9-Acetoxyanthracene (Table I, IV) was prepared in the conventional manner, using acetic anhydride and pyridine, from anthrone, and was similarly recrystallized from ethanol. 4-(p-Bromoanilino)1,2-diacetoxynaphthalene (Table I, V) was obtained by reaction of l-naphthoquinone-4-sulfonic acid, sodium salt, with p-bromoaniline in 50 ml. of glacial acetic acid containing 10 ml. of water for 1 hour a t room temperature with efficient stirring. The mixture was diluted with 10 volumes of water to obtain the 4-(p-bromoanilino)-2-naphthoquinone (Table I, VI1 d). An identical procedure was employed to produce compounds VI1 a, b, and e, as well as the precursor for VI. I n the case of VI, 2,6-dibromo-p-aminophenol was employed in place of the VOL 37, NO. 13, DECEMBER 1965
1675
and a Corning filter 72786 (F). At right angles to the pad was placed the exit slit to the DK-2, and the emission a t 410 mp was automatically recorded. A solution of 3 X 10-4M 2-naphthyl acetate in Elving buffer, pH 7.4, was passed over the pads a t a rate of 0.5 ml. per minute, using a Holter parastaltic liquid pump; or air can be sampled over the enzyme a t a rate of 1 liter per minute using a Brailsford blower (Model TD-1, Brailsford and Co., Inc., Milton Point, Rye, N. Y.) a t a differential pressure of 2 inches of Hg and an air flow rate of 1 liter per minute with up to 1 ml. of liquid per minute. As long as the cholinesterase is active, the 2-naphthyl acetate (111) is hydrolyzed to the highly fluorescent 2-naphthol, and a high fluorescence is recorded (Figure 2). When the air or water becomes contaminated with an anticholinesterase compound, such
FILTER
FLASK
Figure 1. Experimental cence apparatus
PROCEDURES
Determination of Cholinesterase. To 2.80 ml. of MacIlvaine buffer, p H 7.40, is added 0.10 ml. of 10-*M 2naphthyl acetate. The spectrofluorometer is set to wavelengths of 320 and 410 mp, and adjusted to read zero. At zero time, 0.1 ml. of a solution of the enzyme to be assayed (containing 0.00090 to 0.50 unit of cholinesterase
fluores-
aniline derivative. The 2-naphthoquinone derivatives, thus obtained, were recrystallized from 95% ethanol. Reductive acylation of 4-(4’-bromoani1ino)- and 4-(4’-hydroxy-3’,5’-dibromoanilino)naphthoquinone,using acetic anhydride-pyridine in the presence of excess zinc dust yielded the corresponding 4-(4’-bromoanilino)-l ,Z-diacetoxynaphthalene (Table I, V) and 4-(4’acetoxy -3‘,5’, - dibromoanilono) - 1,2-diacetoxynaphthalene (Table I, VI). These compounds were recrystallized from ethanol. The analysis and melting points are given in Table I. MACILVAINE B U F F E ~A . MacIlvaine buffer of constant 0.10M ionic strength, pH 7.40, was prepared by dissolving the appropriate amounts of disodium hydrogen phthalate, citric acid, and potassium chloride in triply distilled water (4). Apparatus for Analysis of Enzymes. An Aminco-Bowman spectrophotofluorometer (SPF) was used for all assay of soluble enzymes. It was equipped with a xenon lamp, an optical unit for proper control of the fluorescence excitation and emission wavelengths, a Beckman linear recorder, and a thermoelectric cooler to maintain constant temperatures. The excitation and emission wavelengths used were 330 and 460 mp for 1-naphthol and 320 and 410 mp for 2-naphthol. The instrument was calibrated using a number of fluorescence standards as recommended by White and Argauer (18). Enzyme System. X continuous fluorometric system for the assay of anticholinesterase compounds was designed and constructed using the fluorescence attachment to the DK-2 spectrophotometer. A special glass tube was prepared (Figure 1) which fits into the fluorescence attachment to the DK-2. This tube has a lower constriction, 10 mm. wide and 30 mm. high, into which were stacked four enzyme pads containing immobilized cholinesterase. The wavelength of 320 mp was employed for excitation, using a Sylvania 360 BL 4W lamp (L)
1676
as an insecticide, the esteratic activity is blocked, no fluorescence is produced, and the fluorescence drops to a low value. A drop in the base line fluorescence indicates the presence of a contaminant, and the rate of fall of the fluorescence with time provides a semiquantitative estimation of the concentration of inhibitor; if the identity of this compound is known.
ANALYTICAL CHEMISTRY
Table 1.
Esters Prepared as
(All fluorescence readings
Wavelength, mp ExcitaFluorescence, tion Emission units/M
Compound
Observations
0COCH,
I
HSCOCO
OCOCHB
* OCOCH,
NCOCH3 385
430
9 X 106
Slow spontaneous and ensymic hydrolysis
380
410
1
x
Slow spontaneous rate; rate of hydrolysis by ChE and chymotrypsin moderete
OCOCHS
rn
0COCH,
Iv
106
or 0.0030 to 3.0 units of acylase) is added, and the change in the fluorescence of the solution, due to production of 2-naphthol, is then recorded us. time, usually for 2 minutes:
The slope of this curve, A F / A t , is then recorded, and from calibration plots of AF/At us. enzyme concentration, the activity of enzyme can be calculated.
- $J
0-COR
ChE
II
I R
= CHs(l-K-Ac)
1-Naphthol
= CaHT (1-N-Bu)
R
3
CHs(2-N-Ac)
2-Naphthol
= C~H~(~-N-BU)
IV
I11
Substrates for Esterases in pH 7.4 Elving buffer) Compounds
RESULTS A N D DISCUSSION
Wavelength, m r ExcitaFluorescence, tion Emission units/M
Observations
OCOCH3
*OcoCh N H
x
340
460
290
340
2.5 X
490
510
0
1
104
Q
Slow spontaneous and enzymic hydrolysis. Hydrolysis products nonfluorescent
Br
v
OCOCH,
"
Br
0 /
VI 0
6
N O c o c H 3
R VI1
lo3
Fluorescent only in EtOH; hydrolysis product nonfluorescent. M.D. 1245' C. Calcd. foFC2iH,?OeBrlN: C, 48.0; H, 3.09. Found: C, 47.7; H, 3.0
Br
6COCHa
Determination of Phosphatase. If acid phosphatase is to be assayed, 0.1 ml. of 10-2M 2-naphthyl acid phosphate is added, and a t zero time, 0.1 ml. of the unknown acid phosphatase to be assayed (containing 0.0015 to 0.15 unit) is added to the substrate as above. Operation of Enzyme System. The enzyme pads are placed in the glass tube gently, the Holter liquid pump and Brailsford blower are connected as shown in Figure 1, and the assembly is placed in the fluorescence attachment to the DK-2. Substrate is then pumped over the pads a t a rate of 0.5 ml. per minute until a n equilibrium fluorescence is reached (usually 10 to 15 minutes). The system can then be run continuously for up to 12 hours, sampling either air or water. A steady fluorescence will be maintained until an anticholinesterase material is encountered. At this point (see Figure 2) the fluorescence drops, and the presence of such a material is usually indicated in less than a minute.
Esters nonfluorescent ; hydrolysis product fluorescent, reading = 1 x lo4. Slow enzymic hy-
Naphthyl Esters as Substrates. Since the indoxyl and resorufin esters proved to be useless in a continuous system for the analysis of anticholinesterase compounds, a search was made for other stable esters t h a t might yield fluorescent compounds upon hydrolysis. The esters of 1- and 2naphthol have been used as substrates for esterases (1, 3, 9, 10, 13-15), and since 1- and 2-naphthol are known to be fluorescent (8), they were investigated. Udenfriend (16) reports, however, that the molecular forms of these compounds (present at pH < 9) are nonfluorescent, and that only the ionic forms (pH > 10) are fluorescent. This, if true, would seriously limit any use of these substrates fluorometrically in enzymology, since the rate of spontaneous hydrolysis of most esters is very fast a t pH > 9. Moss ( 1 1 , l a ) proposed the use of 1- and 2-naphthyl phosphate as substrates for alkaline phosphatase a t a pH of 10, but offered no details on substrate stability, accuracy, etc. The method proposed was not direct, but required a 15-minute reaction time, before the fluorescence of the naphthol was measured. Hercules and Rogers (8) have shown that the molecular forms of 1- and 2-naphthol are fluorescent, although not as fluorescent as the ionic species. Using the A,, of 2-naphthol as 426 mp and the excitation as 313 mp, they showed that the fluorescence of this compound is constant a t pH 7 to 8 a t about 6001, of the fluorescence a t p H 10. To resolve these discrepancies in the literature, the fluorescence of 1- and 2naphthol was carefully studied a t various pH's in triply distilled water. The pure naphthols were obtained by hydrolyzing the purified acetate with base. The p H was then adjusted to the desired VOL. 37, NO. 13, DECEMBER 1965
1677
level with acid or base, and all fluorescence wavelengths recorded were corrected as described above. The values of the fluorescence are reported as the “fluorescence coefficient,” defined as the fluorescence reading, obtained a t zero sensitivity setting on the .4miqcoBowman SPF, in units on the photomultiplier divided by the concentration in moles per liter. Average values were obtained using several concentrations of pure material. The absolute value of the fluorescence coefficient can be related from instrument to instrument by comparison to the fluorescence coefficient of a standard which has excitation and emission wavelengths close to those of the material under investigation. I n this case, quinine sulfate in 0.1M sulfuric acid with A,, of 350 mp and A,, of 450 mp was used. The constancy of this term from one spectrofluorometer to another is illustrated by the observation that the fluorescence coefficient of quinine sulfate obtained on the author’s spectrofluorometer (10 years old) of 1.40 X 106 is almost identical to that obtained on two new instruments, one a t Midwest Research Institute in Kansas City (1.39 X lo6) and the other at these laboratories (1.38 X 106). The fluorescence coefficients (FC) and wavelengths of maximum excitation and emission of the molecular and ionic forms of 1- and 2-naphthol are illustrated below:
60
50 ln
t
1 z
i
40
z V W Ln V E
30
0 73 2
U
20
10
I 0
=
pH 1-8 330 mp; A,, = 460-470 mp FC = 1.0 x 104
Figure 2.
=
p H 1-8 310-32Omp; , A, = 350 mp (1.6 X lo5) ,A, = 420 mp (1.2 X lo5)
Although the fluorescence of 1naphthol is about 5 times greater a t p H > 10 than at 8, the fluorescence of 2naphthol is about the same, provided the emission at 350 mp is used instead of 420 mp. To obtain maximum separation of wavelengths (as is necessary in a continuous system employing filters), a n emission wavelength of 420 mp was used. The fluorescence coefficients obtained for 1- and %naphthol may be compared to values of 1.6 X IO7 and 1.0 X IO5 for resorufin and indoxyl. Thus, 2-naphthol is as fluorescent as indoxyl, but one hundredth as fluorescent as resorufin. The rates of hydrolysis of the various naphthol esters by various enzymes are 1678
a
ANALYTICAL CHEMISTRY
I
I
I
I
I
I
I
15
16
17
18
19
20
21
I
22
Typical operation and response curve for fluorescence apparatus
given in Table 11. From aspects of stability and enzyme activity, 2-naphthyl acetate appears to be the preferred substrate for horse serum cholinesterase. The Michaelis constant, K,, for the naphthyl esters is given in Table 111, with the value for indoxyl acetate given for comparison.
P-
a pH >IO ,A,
= 370 mp; ,A, = 460-470 mp FC = 5.4 x 104
OH
,A,
I
10
TIME, MINUTES
OH
,A,
I
5
0-
A,.
p H 10 = 370 mu
The rate of increase in fluorescence, AP per minute, was found to be measurable a t 2-naphthyl acetate concentrations ranging from 10-2 to as low as 5 X lO-’iCf. Using the optimum substrate concentration of 3.4 X 10-4M in Elving buffer, pH 7.40, concentrations of cholinesterase of 0.000308 to 0.123 unit may be determined with a standard deviation of 1 1 . 0 (Table IV). In addition to horse serum cholinesterase, the hydrolysis of 2-naphthyl acetate is catalyzed by acylase, lipase, and phosphatase (Table 11). With 2-naphthyl acetate, 0.0010 to 1.0 unit of acylase was determined with a deviation of i1.2%, and acid phosphatase in concentrations
of from 0.001 to 0.100 unit per ml. can be determined by measuring the rate of production of 2-naphthol from 2-naphthyl phosphate (Table IV). Experimentally, it was found that the optimum fluorescence and rate of hydrolysis of 2-naphthyl acetate, AF per minute, were obtained using 3.4 X 10-4M substrate in Elving buffer, pH 7.4. The variation of these parameters as a function of p H and type of buffer is given in Table V. Other Esters. Since the naphthols are fluorescent, the corresponding anthrols should be even more highly fluorescent. Hence a series of anthrols and substituted naphthols was prepared, as possible substrates for cholinesterase (Table I). It was discovered, however, t h a t a n exactly opposite situation exists in the case of the anthrols; the esters are highly fluorescent, and the hydroxy compound formed upon enzymic hydrolysis is nonfluorescent. This promised to be an excellent possibility for incorporation into an automatic enzyme system, since a nonfluorescent compound would be produced continuously, provided cholinesterase is active; then, upon inhibition by anticholinesterase compounds, a fluorescence would be produced (exactly opposite to Figure 2). However, none of the esters prepared was hydrolyzed a t an appreciable rate by cholinesterase. Compound IV in Table I was the most promising, but its rate of hydrolysis was too low for an analytical system. The esters of a number of naphthyl derivatives were prepared for testing as substrates (Table I, compounds V to VII). Compounds V and VI were fluorescent, had nonfluorescent hydrolysis products, but were hydrolyzed very slowly by
cholinesterase. All the derivatives prepared of naphthoquinone were nonfluorescent (Table I, compound VII, R = H, CHI, OCHI, and Br), but had fluorescent hydrolysis products. Very little difference in fluorescence was observed with varying R, all values being approximately 1 X lo4. When R equaled Br, the hydrolysis product of VI1 possessed very little fluorescence (fluorescence coefficient = 1 X lo3). Fluorescence Enzyme System. T h e use of the starch gel method of Vasta and Usdin (17) for the immobilization of cholinesterase was unsuitable, because of limitations of air and liquid flow. The use of a polyurethane foam pad for a support of the enzyme [immobilized using Connaught starch and glycerol as described (2)] permitted the simultaneous flow of up to 1.0 mi. of substrate and 1 liter per minute of air through the pads a t a differential pressure of less than 2 inches of Hg. I n addition, there was sufficient contact of air and liquid to enzyme to ensure a speedy reaction. To test the loss of enzyme from the pads, substrate was passed over the pads a t various flow rates for up to 24 hours. At 0.5 ml. per minute, about 15% of the enzyme is washed off the pads in the first 25 ml. of effluent, but very little enzyme is lost thereafter. After 15 hours, there is significant activity in the enzyme pads, as indicated by a high fluorescence reading. Larger substrate flow rates will cause more enzyme to be washed off the pads, as expected. -it a flow of 5 ml. per minute, all the enzyme is mashed off in about 4 hours; a t 10 ml. per minute, the enzyme is lost in 1 hour. Up to 1 ml. of liquid per minute could be passed over the enzyme without appreciable loss of activity in 12 hours. When water was passed over the enzyme for analysis a t a flow rate of 0.5 ml. per minute, the substrate flow rate was also 0.5 ml. per minute. When water was sampled, a substrate flow rate of 1.0 ml. per minute could be used, but generally 0.5 ml. per minute iq sufficient to cause rapid changes in AF per minute when inhibitor if encountered (Figure 2). A solution of 3 X 10-~Jf2-naphthyl acetate in Elving buffer, pH 7.4, was
Table II. Enzymic Hydrolysis of Naphthyl Esters [Ester concentration = 3.4 x 10-4M in Elving buffer, pH 7.4. Activity and source of enzymes as described (S)] AFlminute, fluorescence units/min. Enzyme a-NAca a-NBu* p-NAcc P-NBud Horse serum cholinesterase 0.95 1.18 1.53 1.15 Bovine erythrocyte cholinesterase 0 0 0.275 0 Acylase 0.30 0.48 1 15 1.40 0 0 0.25 0 a-,p-, and 7-Chymotrypsin Porcine pancreas lipase 0.45 1.3 1.89 2.75 Steapsin lipase 0.20 1.3 0.70 2 0 0.15 0 1.03 0 Phosphatase Spontaneous 0.02 0.08 0.02 0.025 a 1-Naphthyl acetate. 6 1-Naphthyl butyrate. 2-Naphthyl acetate. 2-Naphthyl butyrate.
Table 111. Substrate 1-Naphthyl acetate 1-Naphthyl butyrate 2-Naphthyl acetate 2-Naphthyl butyrate 2-Naphthyl phosphate Indoxyl acetate
Cholinesterase, unit/ml. Present Found
Acylase, units/ml. Present Found
0.000308 0.000617 0.00123 0.00617 0.0617
0.00100 0.00500 0.0100 0.0500 0.100
0.123
1.oo
Enzyme Horse serum ChE Horse serum ChE Horse serum ChE Horse serum ChE Acid phosphatase Horse serum ChE
used in all analyses with the enzyme system, since these were found to be the optimum conditions for enzyme analysis and for maximum change in AF per minute. The substrate was stable for use in the continuous system for the 12hour operation time. The base line did not deviate appreciably during this period. Fresh solutions should be prepared from the stock solution (10-231) daily; the stock solution is stable for a t least 2 weeks when kept refrigerated. Many organophosphorus insecticides are potent inhibitors of horse serum cholinesterase. Sarin (isopropyl methylphosphonofluoridate) and Systox { thiolate isomer = o,o-diethyl S-[2(ethylthio)ethyl] phosphorothiolate ] are two insecticides that are good in vitro inhibitors of cholinesterase (Y), and hence should be determinable in the system described. Figure 2 was a typical response curve obtained from 1 pg. of Sarin or 10 pg. of Systox(thio1ate isomer)
Table IV.
0.000310 0.000617 0.00121 0.00614 0.0619 0.121
Michaelis Constants for Fluorogenic Substrates
0.00101 0.00508 0.0100 0.0498 0.0995 1.015
K, 1.5 x 10-4 3.0 x 10-4 1.8 x 10-4 2.6 x 10-4 1.7 x 10-4 3.4 x 10-4
contained in 1 ml. of contaminated water or 1 liter of polluted air. Within 1 minute one is able to detect the presence of these compounds (the fluorescence drops from 60 units on the DK-2 to 40 units in 1 minute). The system can not quantitatively indicate the exact concentration of inhibitor, only the presence of such materials. If the identity of the anticholinesterase compound is known, its approximate concentration can be estimated. For example, 100 pg. of Systox per ml. of water gave essentially the same response as 10 p g . per ml. (Table VI); with 1 pg, per ml. of Systox, the response time was twice that of 10 pg. (the fluorescence dropped from 60 to 40 scale units in 2 minutes). The fluorescence system was extremely useful for continuous monitoring in a laboratory, and after 12 hours of operation gave a good response when exposed to water contaminated with inhibitor. The system has several
Determination of Enzymes
Acid phosphatase, unit/ml. Present Found 0.000500
0.00100
O.OO500 0.0100 0.0250 0.0500
0.000508 0.00101 0.00498 0.00995 0.0250 0.0508
ChE
f0.6 0.0 -1.6 -0.5 f0.3 -1.6 Std. dev., yo k1.0
Rel. error, yo Acylase +1.0 $1.6 0.0 -0.4 -0.5 f1.5 f1.2
VOL. 37, NO. 13, DECEMBER 1965
AP +1.6 +l.O -0.4 -0.5 0.0 +1.6 k1.2
1679
Table V. Effect of p H on Hydrolysis of 2-Naphthyl Acetate 2-Kaphthyl acetate = 3.4 x 1 0 - 4 ~ ; ChE = 0.0625 unit/ml.
Fluorescence (max.), AF’/min. units
PH 6.0, phosphate 6.76, Elving 7.0, Elving 7.4, Elving 7 . 4 , Tris 7 . 9 , Elving 8.0, Tris
0.60 1.42 1.50 1.80 0.62 1.80 0.70
4.7 7.0 8.0 10.0 4.6 10.0 6.0
of cholinesterase, and hence should prove useful in studies of air and water pollution. B y using enzyme systems, other than cholinesterase, in an immobilized form in this system, continuous assay of other compounds that are potent enzyme inhibitors should be possible. ACKNOWLEDGMENT
The authors thank P. Beli and L. Strauche for their help in setting up the fluorescence system. LITERATURE CITED
limitations: It mill not respond to insecticide chemicals which are not in vitro inhibitors of cholinesterase, such as the phosphorothionate compounds, Compounds such as malathion or parathion are detectable in larger concentrations. Any fluorescence contamination in the water or air that has excitation and emission wavelengths a t a region similar to 2-naphthol will interfere, since a level of fluorescence will remain, despite the inhibition of cholinesterase. The proposed system should be applicable to the detection of any inhibitor
(1) Augustinson, K., Acta Chem. Scand. 16, 240 (1962). (2) Bauman, E. K., Goodson, L. H.,
Guilbault, G. G.. Kramer. D. N.. ANAL.CHEM.37. 1378 (1965): (3; Bernsohn, J., l t a/., J: Sezhochem. 10,
483 (1963). (4) Elving, P., Olson, E. C., J . Am. Chem. SOC.79, 2697 (1957). (5) Guilbault, G. G., Kramer, D. N., ANAL.CHEM.36. 409 (1964). ( 7 j Guilbauft, G.‘ G.,-’Kramer, D. N., Cannon, P. L., Ibzd., 34, 1437 (1962). (8) Hercules, D. RI., Rogers, L. B., Ibzd., 30, 96 (1958). (9) Kramer, S. P.. et al., Arch. Biochem. Biophys. 102, 1 (1963).‘ (10) Leisnert, K., Arch. Pathol. Anat. Physiol. 335, 491 (1962).
Table VI. Detection of Insecticides Using Enzyme System
Inhibitor concn., pg.a - AF/min. * Systox, 1.0 10 Systox, 10.0 20 Systox, 20.0 20 Parathion, 10.0 10 Parathion, 100 18 Per ml. of solution added. * Scale units on DK-2 per minute.
(11) Moss, D. UT.,Biochem. J . 76, 32 (1960). (12) Moss, D. W., Clin. Chim. Acta 5 , 283 (1960). (13) Seaman. A.. Winell. RI.. Acta Histochem. 8 , 381 (1959). (14) Seligman, A,, Ravin, H., Arch. Biochem. Biophys. 42, 337 (1953). (15) Tashian, R., et al., Biochem. Biophys. Res. Commun. 14, 256 (1964). (16) Udenfriend, S., “Fluorescence Assay in Biology and Medicine,” pp. 26, 29, Academic Press. New York. 1962. (17) Vasta, B., Usdin, V., Aldrich, F., Final Report, Contract DA 18-108AMC-828, Llelpar, Inc., Falls Church, TTa., 1964. (18) White, C., Argauer, R. J., AKAL. CHEY.36, 368 (1964). I
,
RECEIVEDfor review May 3, 1965. Accepted September 15, 1965. Division of Analytical Chemistry, 149th Meeting, ACS, Detroit, Rlich., Bpril 1965.
Spectrochemical Analysis of Oxygen-Free EIectroIyticaIIy Pure Copper by a Globule Arc Procedure WILLIAM E. PUBLICOVER United States Metals Refining Division, American Metal Climax, lnc., Carteret, N . 1.
b A globule arc procedure for use with the emission spectrograph detects phosphorus, zinc, cadmium, and mercury in oxygen-free copper a t levels less than 0.5 p.p.m.; provides for analysis of 2 9 other elements with the same exposure, and uses an easily adaptable sampling method. Involved in the development is the design of a special electrode and a method of preparing dilute standards wiih concentrations as low as 0.1 p.p.m.
T
of oxygen-free copper for the manufacture of electronic components and other critical applications requires that the zinc, cadmium, and mercury contents shall be less than 1 p.p.m. and the phosphorus less than 3 p p.m. Various investigators have developed spectrochemical procedures for the analysis of a total of 16 impurities in electrolytically pure copper and copper alloys. Leichtle (5), Smith (9), and Fromes and Schatz (4) have reHE USE
1680
ANALYTICAL CHEMISTRY
ported on d.c. arc procedures. Young (10) and Schatz (8) have described methods using spark and multiple source excitation. Deal (2) describes a spectrochemical procedure limited to the determination of lead in oxygen-free copper. Sone of these procedures report analytical sensitivity limits for zinc, cadmium, and phosphorus below the level of 10 1i.p.m. and no references are made t o the determination of mercury. Further studies of elemental sensitivities using point-to-point or point-to-plane procedures have established ultimate detection limits of 10 p.p.m. for phosphorus, 5 p.p.m. for zinc, and 2 p.p.m. for cadmium. Although early studies by hlilbourn (6) demonstrate the potential of high sensitivities using a globule arc technique, spectrographers, in general, have come to prefer the use of solid copper electrodes for a point-to-point d.c. arc method. Principal objections to the globule arc procedure were the lack of excitation stability, impurity contamina-
tion from impure graphite electrodes and high background levels due to the addition of the carbon spectrum. These objections have diverted many spectrographers from consideration of the globule arc method and have encouraged a general acceptance of the d.c. arc point-to-point procedure. Further studies of the d.c. globule arc by Price (7) covered the determination of 10 elements in copper but the sensitivities achieved were, in all cases, no better than commonly found with conventional point-to-point systems and, in some instances, were less sensitive. The belief that the globule arc procedure had not been utilized to its full potential has led to the development of the procedure to be described. This procedure provides for the analysis of zinc, cadmium, mercury, and phosphorus a t concentration levels less than 1 p.p.m. and, in addition, provides for the analysis of 25 other elements a t levels of 1 p.p.m. or less. The method incorporates a form of spectrochemical
