EPA-R2-73-Z17
APRIL 1973 Environmental Protection Technology Series
Fluorescent Probes in the
Detection of Insecticides in Water
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
degradation from point and non-point sources of
pollution. This work provides the new or improved
technology required for the control and treatment
of pollution sources to meet environmental quality
standards.
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This report has been reviewed by the Environmental Pro-
tection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect
the view and policies of the Environmental Protection
Agency, nor does mention of trade names or commercial
products constitute endorsement of recommendation for
use.
ii
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ABSTRACT
Fluorescent probes are molecules whose spectral responses
are changed by their environment. A fluorescent probe
which is in equilibrium with the active site of cholin-
estrace enzymes will have emission spectra and quantum
yields corresponding to (a) the free probe and (b) the
probe-enzyme complex. Insecticides in water which com-
pete for the active site will displace the probe from
its complex and quench the fluorescence of the probe-
enzyme complex. The quenching effect will be related to
the concentration of the insecticide and the equilibrium
constant and quantum yield of the probe-enzyme complex.
The change in spectral response can be the basis of a new
analytical methodology for insecticides. The objectives
of the present research included synthesis of candidate
fluorescent probe molecules for cholinesterase enzymes
and evaluation of the feasibility of development of a new
analytical method for insecticides in water -
Active-site-directed, equilibrium fluorescent probes have
been synthesized and used in the development of the ana-
lytical system. Results with Dursban, Thiodan, and cer-
tain other insecticides are in the range of 1 x 10~7 M.
Insecticides which do not compete with, or displace the
probe from its complex are not detected. Experimental
parameters for design and synthesis of optimum probe
molecules were developed.
"This report was submitted in fulfillment of grant 16020
EAO under the sponsorship of the Environmental Protection
Agency."
iii
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CONTENTS
SECTION PAGE
*
I. Conclusions 1
II. Recommendations 2
III. Introduction 3
IV. Experimental 9
V. Discussion 19
VI. Acknowledgements 21
VII. References 39
VIII. Publications 41
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FIGURES
PAGE
Figure 1. Change in fluorescence of the TA-ChE
complex with Methyl Parathion 34
Figure 2. Change in fluorescence of the TA-ChE
complex with Dursban 35
Figure 3. Change in fluorescence of the TA-ChE
complex with Thiodan 36
Figure 4. Relative fluorescence effects with
Dursban 37
Figure 5. Relative fluorescence effect with
Thiodan 38
vi
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TABLES
Table I.
Table II.
Table III
Table IV.
Table V.
Table VI.
Table VII.
Table VIII,
Table IX.
Synthesis of Dansyl Phenolic
Sulfonamides
Synthesis of Dansyl Sulfonamides
Synthesis of Dansyl Sulfonates
from Phenols
Fluorescent Carbamates and Dns-F
Model Compounds
Enzyme Inhibition Studies on Bovine
Erythrocyte Acetylcholinesterase
(AChE) and Horse Serum Cholin-
estrase (ChE)
Aminoalkyl Dns Sulfonamides
(Anionic Site Probes)
Inhibition by Fluorescent Probes
Compounds which Compete for the
Probe-Enzyme Complex Using Probe
TA
PAGE
22
23,24,25
26
27
28
29
30
31,32
33
vii
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SECTION I
CONCLUSIONS
The application of fluorescent probe-enzyme systems to
detection of water pollution by insecticides represents
a new concept. It can be the basis for the development
of a new analytical methodology. The ultimate potentials
of the system were not approached because an optimum fluo-
rescent probe molecule was not synthesized during this
research. Such an optimum probe appears to be a realistic
potential from any extension of the present research.
Under presently available conditions, a series of phos-
phate insecticides can be detected in the range of 10"7M.
Sensitivity of the method with an optimum probe molecule
appears to be in the range of 10"^ molar. The new method-
ology has proven scientific and analytical feasibility.
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SECTION II
RECOMMENDATIONS
It is recommended that further exploratory research be
conducted with the aim of design and synthesis of an
optimum fluorescent probe molecule for the esteratic
site of cholinesterase enzymes. When such a probe mole-
cule becomes available, further research should be done
to determine the ultimate sensitivity and scope of the
analytical methodology.
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SECTION III
INTRODUCTION
Cholinesterase enzymes are irreversibly inhibited by
organophosphate and carbamate insecticides. For this
reason, many analyses for insecticides use these enzymes
as a part of the analytical method. There are two major
cholinesterases, acetylcholinesterase (AChE) and serum
Cholinesterase (ChE). These two enzymes are closely re-
lated in that both have an anionic site plus an esteratic
site; however, the enzymes are not identical. They differ
in their substrate preferences, and in other characteristics
Both of these enzymes were used in this research; however,
ChE is the more stable enzyme and, in addition, was avail-
able to us in a highly purified, stable state. This made
it possible for us to determine whether or not artifacts
would be introduced in any use of the commercial (impure)
enzyme. Although AChE has been purified, the process is
difficult and the pure enzyme is unstable. When the com-
mercial enzymes are used, it is possible that in any given
analytical method, one of these enzymes may have specific
advantages. Both were studied in much of the research,
but ChE was the enzyme of choice.
The inhibition of cholinesterases by organophosphate and
carbamate insecticides involves two steps. The first step
is the formation of an equilibrium complex, (the Michaelis
complex). The second step involves covalent bond formation
(phosphorylation or carbamylation) at the esteratic site.
These reactions are outlined in equations (1) and (2). The
enzyme is represented by E-OH. The insecticide is repre-
sented by I-X, in which X represents the alcoholic or
phenolic leaving group.
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Keq ,
Step 1 E-OH + I-X v N (E-OH ____ IX) (1)
Michaelis
Complex
Step 2 (E-OH ---- I-X) k2 E-O-I + HX (2)
inhibited
enzyme
= equilibrium constant
= reaction rate constant
This sequence of reactions is important because it is be-
lieved that the mechanism and mode of action of phosphate
and carbamate insecticides involves inhibition of cholin-
esterase enzymes, in vivo. They are also important because
any candidate analytical method which involves the enzyme
must be related to either the equilibrium in Step 1 or a
sequence of reactions involving Step 2.
Inhibition of the enzyme by phosphate or carbamate insecti-
cides has an overall reaction rate which is dependent on
both the equilibrium constant (K0q) in the first step and
the reaction rate constant (k£) in the second step. Various
organophosphate and carbamate insecticides often differ
markedly in the relative values of these two constants;
however, it is the esteratic site which is the ultimate
point of attack by these insecticides. For this reason, the
design for a fluorescent probe preferably includes complex
formation at the esteratic site. Complex formation at the
anionic site will allow detection of those insecticides
which complete for that site.
Many analytical methods for insecticides have been based
on the use of fluorogenic substrates. Such fluorogenic
substrates are designed to react with various hydrolytic
enzymes to produce products which are strongly fluorescent,
in a spectral range which is easily measured. This allows
measurement of the enzyme- fluorogenic substrate reaction
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rate in the presence and absence of insecticides. Un-
fortunately, measurement of rate constants is subject
to ill-defined catalytic and inhibition effects from
known or unknown impurities. For this reason, the use
of fluorogenic substrates has limitations as an ana-
lytical method for insecticides, particularly in water
pollution research. For these reasons, it was con-
sidered important to develop a new methodology which
could take advantage of the sensitivity of fluores-
cence measurements but which would not be dependent on
rate constant measurements. That new methodology is
the basis of the present research. It is discussed
herein.
Active-Site-Directed, Equilibrium
Fluorescent Probes
In 1967, this laboratory started a research program whose
objectives included the design and synthesis of new fluores-
cent molecules as tracer compounds. They were designed with
two potential objectives, (1) as a part of the development
of new analytical methods for insecticides and (2) for re-
search involving the mechanism and mode of action of insecti-
cides. The analytical methods research was specifically
limited to equilibria in enzyme systems to avoid the diffi-
culties inherent in measurement of comparative rate constants.
The objectives of the present research, included the syn-
thesis of active-site-directed, fluorescent equilibrium
inhibitors of cholinesterases. These fluorescent molecules
could compete with insecticides for the active site of the
enzyme. When such fluorescent molecules act as fluorescent
probes, changes in spectral responses occur when the probe
molecule is removed from the enzyme complex. Therefore, in
this new method, the change in spectral response of the
probe-enzyme complex could be related to the amount of in-
secticide present. The sensitivity of the method is dependent
on the equilibrium constant of the probe-enzyme complex, the
quantum yield of the complex and the degree of interference
(if any) of the fluorescence of the free probe. The se-
quence of reactions is as follows:
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When an insecticide in solution competes for the active
site of a suitable cholinesterase enzyme-fluorescent probe
complex, it will displace the probe from the probe-enzyme
complex. This displacement is reflected by a concomitant
change in the fluorescence. The analytical methodology
therefore involves measurement of change in fluorescence
which accompanies insecticide competition for the active
site of the enzyme. A suitable fluorescent probe is re-
quired. The research under this grant was, therefore,
involved in the design, synthesis, and testing of candi-
date fluorescent probe molecules, study of their spectral
responses with cholinesterase enzymes, and determination
of feasibility of the analytical method. Some of the
research has been published (2-8).
The equilibria involved are illustrated in equations (3)
and (4), where P designates an active-site-directed,
equilibrium fluorescent probe.
E-OH + P -—* (E-OH....P) probe-enzyme complex (3)
I-X
(E-OH I-X) insecticide-enzyme
complex (4)
The synthesis of fluorescent substrate analogs is discussed
by Himel and Chan (1). A fluorescent substrate analog is a
designed, fluorescent molecule which can act as an alterna-
tive substrate for the enzyme being used. Their synthesis
and application is a bioanalytical problem which involves
stereochemical, chemical, structural, solubility, binding
and spectroscopic factors.
Fluorescent probes can be excited directly by irradiation,
or indirectly by dipole-dipole energy transfer. This latter
is a form of sensitized fluorescence (1-2). In either case,
the first step in an analytical determination of insecti-
cides involves formation of the probe-enzyme equilibrium
complex. The dissociation constant of that complex and the
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presence or absence of dipole-dipole energy transfer con-
stitute design parameters which must be studied.
The interaction of chromophores in a fluorescent molecule
or its complex can be predicted in general but not speci-
fically. Isolation or interaction of chromophores is an
important design parameter. It determines the spectral
characteristics of the probe molecule.
The concept of active-site-direction was developed in the
early 1960's by several different laboratories (9). It
made possible the design of candidate fluorescent probes
having a significant measure of selectivity. That se-
lectivity stems from interaction of the probe molecule
at the active site of the enzyme rather than at some ill-
defined exo area. Interaction and formation of an equili-
brium complex at the active site is important because the
active-site is the major site of competition with organo-
phosphate and carbamate insecticides.
The importance of fluorescence techniques as research and
analytical tools lies in their wide scope and sensitivity.
That sensitivity can be orders of magnitude greater than
that available from absorption spectroscopy. A fluorescent
probe is a fluorescent molecule which is sensitive to its
environment. In general, a successful fluorescent probe
will have a strong pi-pi* transition, an excited state
which is more polar than the ground state and no dominant
n-pi* transitions. The latter are generally accompanied
by a low extinction coefficient of absorption, a low
quantum yield and show extensive intersystem crossing with
concomitant phosphorescence.
The development of the new analytical system required active
site directed, equilibrium fluorescent probes. Their de-
sign and synthesis involves one or more of the following
biochemical or spectroscopic parameters:
1. Stereochemical fit at the active site of the
enzyme.
2. A dissociation constant of the active-site
equilibrium complex in the range of 10~5 to
10-7M.
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3. An excitation minimum near 280 nm.
4. An excitation maximum near 330 nm or at
substantially longer wavelengths when
dipole-dipole interaction is not desired.
5. A quantum yield near zero for the free probe
in water.
6. A quantum yield of the probe-enzyme complex
of 0.5 or larger.
7. An emission maximum of the probe-enzyme
complex in the range of 500 nm or longer
wavelengths.
8
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SECTION IV
EXPERIMENTAL
Synthesis of Fluorescent Substrate Analogs as
Candidate Fluorescent Probes for
Cholinesterase Systems
No known fluorescent probes for cholinesterase enzymes were
available when this research was started. The first step
in the research program was the synthesis of a wide variety
of molecules. Design parameters for suitable fluorescent
probes were delineated.
The 5-dimethylaminonaphthalene-l-sulfonyl moiety (1) is
readily available and easily introduced into a variety of
structures. For this reason it was studied extensively
in this research. The NBD moiety (2) is a more recent
development in fluorescence research and has valuable
spectral characteristics.
N(CH3)2
(1)
(2)
9
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Candidate fluorescent probe molecules can be designed with
bridge systems which isolate the fluorescent moiety or
which allow electronic interaction (1-2). The spectral
results of isolation of chromophores and electronic inter-
action have been discussed in detail (1). The structural
factors are indicated in (3) and (4). In (3) the groups
A and B are separated by the bridge system X and Y, as
indicated, such that electronic interaction of A and B
takes place. This interaction is reflected in the com-
posite nature of the resulting spectra. In (4) however,
A and B moieties are separated by a methylene-type bridge
which serves to isolate A and B. In this case, only
dipole-dipole (sensitized fluorescence) can occur, and
the interaction is limited by distance and mutual orienta-
tion (1). In (3) the groups X and/or Y can include (a)
aromatic rings, (b) conjugated double bond systems
A-X-Y-B A-CH2-Y
(3) (4)
(c) atoms such as oxygen, sulfur, or nitrogen with lone-pair,
nonbonding electrons, or (d) -CO-, -SO-, -SO2~- In general,
the hydrogen atoms of the C^ group in (4) can be replaced
by any group which does not circumvent the isolation or one
or both can be replaced by alkyl groups.
Synthesis details have been published (2-6). Data are
collected in the following Tables. Synthesis data on an
extensive series of compounds are described. Many are new
compounds and all were synthesized to spectroscopic purity
requirements. All compounds were tested for photolytic
stability. Critical design parameters exist which determine
the utility of candidate fluorescent probe molecules syn-
thesized for use in the analytical system.
Reagents. Dansyl chloride (mp 68-70°C) was obtained from
Peninsular Chemresearch. Solvents were reagent grade for
synthesis and fluorescent grade for spectral studies.
Pyridine was stored over solid KOH prior to use. Tic film
used to monitor synthesis reactions was Eastman, fluorescent
and nonfluorescent types.
Analysis. Microanalytical data are by Midwest Micro Labora-
tories. Structures of new compounds were confirmed by ir
10
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(Beckman IR-10) and nmr (Varian HA-100). In each case the
spectra support the structure. We are indebted to Dr.
D. R. Leyden for the nmr spectra. All syntheses were moni-
tored by tic and by ir.
Synthesis of Dansyl Phenolic Sulfonamides. Pyridine Method,
Equimolar quantities (usually 0.004 mol) of the aminophenol
(or salt) and dansyl chloride (usually 1.1 g) were added
to 15 ml of dry pyridine. The reaction was kept under
nitrogen and stirred over a 24-hr period. Ether and water
were added and the ether layer was separated. The combined
ether extracts were washed with 1% HC1 and with water to
remove traces of pyridine, dried over drierite, filtered,
and ether was removed in vacuo. The reaction was monitored
by tic (benzene-acetic acid 75/25). Data on sulfonamides
are shown in Table II.
Synthesis of Dansyl Sulfonates from Phenols. The phenol
(0.02 mol) was added to dansylchloride (0.02 mol) in 300
ml of ether containing 50 ml of triethylamine (free of
dimethylamine)- The reaction was monitored with tic.
Reaction times averaged 4 days. The ether solution was
washed with water and then dried over drierite. The ether
was removed in vacuo. The product was recrystallized as
indicated. Data are given in Table III.
Synthesis of Fluorescent Carbamates. N-Methylcarbamates
were prepared by reaction of methylisocyanate with dansyl
phenolic sulfonamide (or oxime). Secondary sulfonamides
containing a phenolic OH can react to give the carbamate
or the urea carbamate. The former is formed in the absence
of catalyst in benzene or toluene solution. The latter is
formed when triethylamine is used in catalytic quantities.
Reaction times were 24 hr. Data are given in Table IV.
Data on model compounds are given in Table V. Enzyme in-
hibition data are given in Table VI.
Most dansyl derivatives exhibit useful fluorescent prop-
erties. Dansyl sulfonamides of reactive amines are readily
prepared such that binding properties from differences in
the sulfonamido substituent can be studied. Important re-
lationships between binding characteristics and structure
have been found in the series of compounds reported herein.
11
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The sulfonamido proton appears to have significant bio-
chemical importance in enzyme and protein binding. The
protolysis kinetics of the sulfonamido proton have been
studied (7).
In order to prepare fluorescent carbamate analogs, pure
sulfonamido phenols were required. The synthesis route was
from the bifunctional aminophenol, reactive at both amino
and phenolic groups. Prior to the development of tic
methods, specificity of reaction at the amino group was
generally assumed, or byproduct sulfonate was removed or
ignored. Most synthetic procedures in which dansyl chloride
was reacted with an aminophenol such as p-aminophenol gave
a mixture of phenolic sulfonamide (a) amino-sulfonate
(b) and sulfonate-sulf onamide (c). The synthesis routes
are simple but preparation of spectroscopic grade product
was complex. The pyridine method allowed production of
pure (a) directly, a unique and unexpected specificity of
reaction.
Dansyl sulfonates from a variety of phenols were found to
be reasonably chemically stable in water even at elevated
temperatures. All sulfonates tested were, however, photo-
lytically unstable to irradiation at the usual excitation
wavelength in the range of 340 nm. Dansyl chloride is un-
stable in all solvents tested, and its fluorescence is
quenched by the Cl to a major extent.
Dansyl fluoride is fluorescent in the same general range as
other dansyl derivatives. It is an inhibitor of AChE and
ChE. The inhibition of cho lines terase enzymes by sulfonyl
fluorides has been studied by Fahrney and Gold (10).
Excellent evidence from X-ray diffraction studies indicates
probably sulfonate formation at the active-site serine OH.
Irradiation of the inhibited enzyme would be expected to
give 5-dimethylaminonaphthalene-sulfonic acid (dans acid),
515
Photolytic stability is a critical design parameter for
fluorescent probes. p-Substituted phenolic sulfonamides
and their carbamate derivatives were less stable than the
meta isomers but had acceptable stability. tert-Sulfona-
mides para to a phenolic OH were grossly unstable.
12
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N-Aminoalkyl-dansylsulfonamide derivatives have been
studied as equilibrium probes for the active site of ChE.
Probes specific for the anionic site are listed in Table
VII. Their synthesis was as follows:
Reaction of dansyl chloride with a 3M excess of N,N-
dimethyl-l,3-diaminopropane in ether gave 5-dimethyl-
aminonaphthalene-l-sulfonamido-N,N-dimethyl-n-propyl
amine (compound 35, Table VII), referred to as TA in the
discussion. The aliphatic quaternary derivative (QA-
#36) was prepared by dissolving TA in ether containing
a 50 fold excess of methyl iodide. The quaternary deriva-
tive precipitated immediately. After four hours, the
product was removed by filtration and was recrystallized
from water, mp 201-202OC. The structure was verified by
infrared and NMR spectra. Purity was monitored by TLC.
Synthesis of N-methylacridinium iodide was a modification
of published data and was carried out of room temperature
by addition of acridine to a solution of methyl iodide in
dimethylformamide.
Fluorescence Measurements
Instrumentation. Emission, excitation, and absorption
spectra were obtained with a G. K. Turner "Spectro 210."
This instrument presents corrected emission and excitation
spectra. Samples for fluorescence measurements were tem-
perature controlled to 25±0.1° . pH measurements were
made at room temperature with a Radiometer Model 26 pH meter
standardized with NBS standard buffer solutions. Life-
time of fluorescence measurements were made using a TRW
nanosecond flash unit and decay computer coupled to a dual
gun oscilliscope. Appropriate filters (Corning) were used
to isolate excitation and emission wavelengths (10).
Chemicals and Solvents. Pure grade 5-dimethylaminonaph-
thalene-1-sulfonic acid (DnsOH) was from Pierce Chemical
Co. It was homogeneous on Eastman tic film, using benzene-
methanol (1:1), and was comparable to that of a highly
purified standard.
13
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Et^PP, p,p'-dichlorophenyl-2,2,2-trichloroethane, p,p'-di-
chlorophenyl-2,2-dichloroethylene, heptachlor, dieldrin,
and Thiodan were of analytical quality and gifts of the
Southeast Environmental Research Laboratory, Athens, Ga.
Maretin was a gift of the Chemagro Chemical Co. Solvents
and reagents were reagent grade or fluorescent quality.
All water was double distilled from glass. Buffers were
either Tris-HCl (0.05 M) or citrate-disodium hydrogen
phosphate (0.1 M). Any pH below or above the capacity of
these buffers was obtained with 0.1 N H2S04 or 0.1 N NaOH.
Lyophilized horse serum cholinesterase (ChE) (type IV),
bovine erythrocyte acetylcholinesterase (AChE) (type III),
electric eel AChE (type III), were purchased from Sigma.
Amounts of cholinesterase are given as milligrams of
enzyme preparation per milliliter. The bovine erythrocyte
AChE hydrolyzed 2.9 umoles of ACh per min per mg. The
electric eel AChE hydrolyzed 540 umoles of ACh per min
per mg of preparation. Horse serum ChE hydrolyzed 6.0
umoles of ACh per min per mg of preparation.
Purified ChE was prepared by the Method of Main (10) and
was used to determine the effect of enzyme purity on the
spectral responses of the probe-enzyme complex in the
presence of insecticides.
Methods. Fluorometric titrations were performed manually
with Hamilton microsyringes. Total volumes of all samples
for fluorometric measurements were 2.0 ml. Irradiation of
samples containing protein was limited to 5 min. or less
to avoid photooxidation of the tryptophan and histidine
residues in the protein. When mixing was necessary, cells
with fitted Teflon stoppers were used. Dissociation con-
stants (K0) were determined graphically after the methods of
Chen and Kernohan (11), Chen (12), and Jun et al. (8).
Cholinesterase activities were determined titrametrically
through a modified method of Nabb and Whitfield (13) using
0.005 N NaOH as the titrant. The titration was at pH
7.3 ChE solutions were made up in double-distilled water
without salt additives.
14
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The comparative methods of Parker and Rees (14) were
followed in quantum efficiency calculations. The equa-
tion used in calculating quantum efficiency has been given
by Fletcher (15). Quantum efficiencies of probe-enzyme
complexes were determined in aqueous systems. DnsOH was
used as the quantum yield reference standard with the
quantum yield taken as 0.36 in 0.1 m NaHCOs. All solu-
tions which did not contain protein were purged with
special purity nitrogen to retard oxygen quenching.
Fluorescence quality sample cells (Helima) 1.0 cm in
path length with Teflon stoppers were used. Where ab-
sorption was a problem, 0.3-cm microcells with special
cell holders were used (AMI-NCO). Peak areas were
integrated with a Hewlett Packard 3370A electronic
digital integrator interfaced with the spectrofluoro-
meter.
Enzyme Inhibition Data
In order to be useful, a candidate fluorescent probe
molecule must be an equilibrium inhibitor of the enzyme
active site. Inhibition data are given in Table VIII,
from pH Stat measurements.
Fluorescence Data with Insecticides
The fluorescent probe molecule TA
S02-NH -
N(CH3)2
15
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was studied in considerable detail. It is a competitive
inhibitor of ChE and is relatively specific for ChE.
Since it also reacts with phosphorylated enzyme to form
a complex, it is assumed that the probe molecule binds
(1) at the anionic site and (2) at an exo area adjacent
to the anionic site. Because it does not specifically
interfere with the esteratic site, phosphate and carbamate
insecticides which have a minimum bulk do not interfere
with the probe-enzyme complex. On the other hand, with
commercial enzyme, most chlorinated hydrocarbon insecti-
cides interfere with the probe-enzyme complex, presumably
at some exo area. This extension of the methodology was
unexpected and is limited to the commercial enzyme. It
is not present when the pure enzyme was used.
Quantitation of the decrease in fluorescence intensity of
the TA-ChE complex as a function of various insecticides
has been studied. The data are discussed in the following
section. Attempts to prepare a suitable fluorescent probe
specific for the esteratic site included the phenolic
sulfonamides below, its para isomer and their carbamate
esters.
In this series of compounds, both the meta and para isomers
are readily available by simple synthesis steps using the
selective pyridine method developed for this compound (6).
The fact that compound 3 is a non-competitive inhibitor of
the enzyme indicates that its major binding is adjacent to
16
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the esteratic site, thus a suitable ester derivative of
this compound should be studied. The carbamate ester of
the para isomer was obtained and found to have a limited
potential because of its relatively low inhibition of the
enzyme (cf. Table VI).
Analytical Data
The objective of this research was to synthesize one or
more fluorescent probe molecules which would be useful in
this new analytical methodology for insecticides and to
determine the range of sensitivity of the analytical
method. Those objectives were realized. Suitable fluores-
cent probe molecules can be synthesized and the analytical
method is feasible. Analytical sensitivity with several
insecticides is in the range of 10~^ M with the potential
for considerable increase in sensitivity when an optimum
probe molecule is made available. The design parameters
for an optimum probe molecule have been delineated by the
research.
A series of phosphate insecticides and other compounds of
interest were tested to determine whether they interferred
with the TA-ChE complex. The data are given in Table IX.
The fact that two organophosphates did not compete with
the probe-enzyme complex indicated that this probe was not
located at the esteratic site of the enzyme. It was later
determined that the binding of this probe was at (1) the
anionic site and (2) at a hydrophobic area adjacent to the
anionic site. Since none of the phosphate insecticides
which did give a positive response are known to bind at the
anionic site, it was assumed that their potential detection
by this analytical system was dependent on competition for
the hydrophobic area occupied by the probe molecule. No
carbamate type insecticides were found to respond to the
probe enzyme complex, however, the rather bulky cationic
carbamate molecule esserine did replace the probe, as evi-
dence by the expected change in fluorescence.
17
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When a series of chlorinated hydrocarbon insecticides were
tested, it was not expected that they would affect the
probe-enzyme complex. When the commercial enzyme was used,
a competitive replacement was found. The experimental re-
sults could be duplicated and after extensive research,
the effect was considered to be real and not an artifact.
Further study of the unexpected effect was made with
purified enzyme and here, it was found that chlorinated
hydrocarbons did not displace the probe from its enzyme
complex. The use of purified enzyme did not alter the
fundamental nature of the reaction of organophosphate in-
secticides with the probe-enzyme complex, hence the effect
of chlorinated hydrocarbons relates only to commercial
enzyme. It was not possible to determine the absolute
nature of the interaction of chlorinated hydrocarbons with
the probe-enzyme complex formed with commercial enzyme.
It was assumed that the enzyme system contained a protein
which was complexed near the active site which furnished
a hydrophobic area common to the chlorinated hydrocarbons
and the naphthalene moiety of the probe molecule.
In Figures 1-3 the change in fluorescence of the probe-
enzyme complex in the presence of methyl parathion, Dursban
and Thiodan is shown. The latter indicates the unexpected
effect of chlorinated hydrocarbons with the complex formed
with the commercial ChE enzyme. Figures 4 and 5, show a
family of curves and relate the effect of probe concentration
and insecticide concentration for Dursban and for Thiodan.
The latter indicates again the reproducibility of the com-
petitive effect with chlorinated hydrocarbons when the
commercial enzyme is used. Thiodan does not affect the
probe-enzyme complex formed from TA and the pure ChE enzyme.
18
-------
SECTION V
DISCUSSION
Choice of Enzyme
The requirement for analytical specificity means that the
enzyme for use in this research must have selective re-
action with organophosphate and carbamate insecticides.
For this reason, acetylcholinesterase (AChE) and serum
cholinesterase (ChE) were the only enzymes studied. ChE
was the enzyme of choice because of its ready availability
as commercial enzyme (about 1-5% enzyme) and the avail-
ability to us of pure, stable enzyme (10). The fact that
the inhibition of these two enzymes was not identical with
each probe molecule indicates that both enzymes may have
significant potential in any final study and method de-
velopment research. Relative inhibition data are given in
Table VIII for various probe molecules.
Synthesis of Candidate Probe Molecules
Synthesis and enzyme reaction data are given in Tables
I-VIII. Cholinesterases are unique enzymes in that they
have two substantially different active sites. The anionic
site is specific for aliphatic and quaternary amines and
apparently acts as a positioning site for the quaternary
moiety in acetylcholine. The esteratic site on the other
hand is a hydrolytic site and is the site which is phos-
phorylated and carbamylated during insecticide inhibition.
Utility as a fluorescent probe for this research was found
to be determined by the following criteria:
1. The candidate probe molecule must complex with the
enzyme with a K^ (dissociation constant) in the range
of ID"5 to 10"7M. This is a range related to that of
many insecticides and allows maximum change in fluores-
cence of the probe-enzyme complex when insecticide is
added.
19
-------
2. The candidate probe molecule must show competitive
inhibition of the enzyme indicating that it complexes
at the (a) anionic site, (b) the esteratic site, or
(c) both the esteratic and anionic sites.
3. An optimum probe molecule will complex at both the
esteratic and anionic sites or at the esteratic site
or at the esteratic site and an adjacent hydrophobic
area.
4. An optimum probe molecule will have a quantum yield in
water which is close to zero and a quantum yield of
the probe-enzyme complex which is in the range of 0.5.
5. The optimum fluorescence range for a probe-enzyme com-
plex is in the range of 500-600 nm.
6. The probe must be soluble in water in the range of
10~4 molar.
Near the end of the first year of this research program, the
probe molecule 5-dimethylaminonaphthalene-l-sulphonamido-N,
N,-dimethyl-n-propyl amine (compound 35, Table VII) was de-
veloped and found to be a promising probe molecule. The
structural formula is given in the experimental section. At
that time, it was noted that this probe molecule (given the
trivial designation TA) was specific for cholinesterase, and
did not react with AChE. After considerable study it was
determined that TA binds at the anionic site of ChE and at
an adjacent hydrophobic site. Other anionic sites probes
were also developed but the objective of development of a
probe suitable for the esteratic site was not obtained.
Relative solubility was found to be a critical factor and
substantially eliminates compound 20 which was an esteratic
site probe. Relative solubility also eliminates compound 9.
Water solubility in the range of 10"4 molar is a critical
parameter.
20
-------
SECTION VI
ACKNOWLEDGEMENT S
The research was carried out at the University of Georgia,
Department of Entomology, with Dr. R. T. Mayer and with
Dr. L. M. Chan. Synthesis research was carried out prin-
cipally by Mrs. Wissam Aboul-Saad and Miss Carol Lord.
21
-------
Table I. Synthesis of Dansyl Phenolic Sulfonamides
Yield Analysis
Compd and no. mp, C % Calcd % Found %
1 p-(5-Dimethylamino- 142-4 62 C 63.15 63.00
naphthalene-1- H 5.26 5.28
sulfonamido) phenol N 8.18 8.05
2 p-N-Methyl-(5-di- 114-5 45 C 64.04 63.70
methylamino- H 5.62 5.86
naphthalene-1- N 7.86 7.79
sulfonamido)-
phenol
3 m-(5-Dimethylamino- 165-6 50 C 63.15 63.27
naphthalene-1- H 5.26 5.41
sulfonamido) phenol N 8.18 8.16
R = 5-(dimethylaminonaphthalene)-l-sulfonyl (dansyl);
Rf - H or CH3.
R
22
-------
Table II. Synthesis of Dansyl Sulfonamides
Compd and no. mp, C
4 4-(5-Dimethylamino- 91-3
naphthalene-1-
sulfonamido) methyl-
ene-1,2-methylened i-
oxy benzene
5 2-(5-Dimethylamino- 235
naphthalene-1-
sulfonamido)
pyridine
6 3-(5-Dimethylamino- 194
naphthalene-1-
sulfonamido)
pyridine
7 N-(5-Dimethylamino- 76-7
naphthalene-1-
sulfonamidoethyl-
glycinate
8 m-(5-Dimethylamino- 129-30
naphthalene-1-
sulfonamido) benzo-
trifluoride
9 m-(5-Dimethylamino-
naphthalene-1-
sulfonamido) ethyl-
benzoate
10 p-(5-Dimethylamino- 218-20
naphthalene-1-
sulfonamido)
benzoic acid
Yield
50
Analysis
Calcd % Found %
50
50
50
70
68
75
C 62.31
H 5.21
N 7.28
C 62.39
H 5.20
N 12.84
C 62.39
H 5.20
N 12.84
C 57.14
H 5.95
N 8.33
C 57.87
H 4.31
N 7.10
C 63.29
H 5.53
N 7.03
C 61.60
H 4.86
N 7.56
62.37
5.41
7.18
62.27
5.31
12.54
62.39
5.22
12.89
56.77
5.90
8.23
57.93
4.43
7.04
63.39
5.56
7.14
61.63
4.74
7.43
23
-------
Table II. (continued)
Yield Analysis
Compd and no. mp, °C % Calcd % Found 9
11 m-(5-Dimethylamino- 222-3 80 C 61.62 61.50
naphthalene-1- H 4.86 4.94
sulfonamido) N 7.57 7.57
benzoic acid
12 5-Dimethylamino- 215 100
naphthalene-1-
sulfonamide
13 N-Methyl-5-di- 110-1 85 C 59.09 58.81
methylamino^ H 6.06 5.95
naphthalene-1- N 10.61 10.34
sulfonamide
14 N,N-Dimethyl-5-di- 100-2 85 C 60.43 60.53
methylamino- H 6.47 6.62
naphthalene-1- N 10.07 9.86
sulfonamide
15 (5-Dimethylamino- 141-2 78
naphthalene-1-
sulfonamido)
benzene
16 N-Methyl (5-di- 94-5 78 C 67.06 66.57
methylamino- H 5.88 5.94
naphthalene-1- N 8.24 8.18
sulfonamido)
benzene
17 N-Benzyl-5-dimethyl- 139
aminonaphthalene-1-
sulfonamide
24
-------
Table II. (continued)
Yield Analysis
Compd and no. mp, C % Calcd % Found %
18 8-(5-Dimethylamino- 152-4 58 C 66.84 66.61
naphthalene-1- H 5.04 5.17
sulfonamido) N 11.14 11.22
quinoline
19 8-(5-Dimethylamino- 207-8 73 C 64.86 64.87
naphthalene-1- H 5.16 5.21
sulfonamido)-6- N 10.32 10.39
methoxy quinoline
20 p-(5-Dimethylamino- 162-3 78 C 57.98 57.19
naphthalene-1- H 6.09 6.10
sulfonamido) benzyl N 5.88 5.90
phosphonate
25
-------
Table III. Synthesis of Dansyl Sulfonates
from Phenols RSO2-OR'
21 1,2-Dioxymethyl-
lene-4-(5-di-
methylamino-
naphthalene-
1-sulfonato)
benzene
22 5-Dimethylamino-
naphthalene-1-
(4-nitrophenyl)-
sulfonate
23 5-Dimethylamino-
naphthalene-1-
sulfonato)-3-
dimethylamino
benzene
24 4-(5-Dimethyl-
aminonaph-
thalene-1-
sulfonato)
acetophenone
25 4-(5-Dimethyl-
aminonaph-
thalene-1-
sulfonato)
acetophenone
oxime
Reac-
tion
time, Yield
°G days %
86-7 1
94
114
63
131-2
54
90-2
64
144-6
40
Analysis
Calcd %Found %
C 61.47 61.33
H 4.58 4.84
N 3.77 3.96
C 58.07
H 4.30
N 7.53
C 64.86
H 5.95
N 7.57
C 65.01
H 5.15
N 3.79
C 62.48
H 5.21
N 7.29
58.37
4.41
7.47
64.60
5.98
7.40
64.82
5.20
3.79
62.48
5.32
7.24
R = 5-(dimethylaminonaphthalene)-l-sulfonyl (dansyl);
R' = substituted phenols
26
-------
Table IV. Fluorescent Carbamates and Diis-F
0 Yield Analysis
Compd and no. mp, C % Calcd % Found %
26 p-(5-Dimethyl- 160-2 67 C 60.15 60.49
aminonaphthalene- H 5.26 5.55
1-sulfonamido) N 10.52 10.28
phenyl-N-methyl-
carbamate
27 p-N-(NT-Methyl- 159-61 97 C 57.89 57.82
carbamoyl)-N- H 5.26 5.47
(5-dimethyl- N 12.28 12.00
aminonaphthalene-
1-sulfonyl) phenyl-
N"-methyl carbamate
28 p-(5-Dimethylamino- 114-5 77 C 59.86 59.58
naphthalene-1- H 5.21 5.37
sulfonato) aceto- N 9.52 9.47
phenone (N-methyl-
carbamoyl) oxime
29 p-(5-Dimethylamino- 50 dec 80
naphthalene-1-N-
methylsulfonamido)
pheny1-N-methy1-
carbamate
30 S-Dimethylamino*- 50-2 95 C 56.92 57.49
naphthalene-1- H 4.74 4.90
sulfonyl fluoride N 5.53 5.53
27
-------
Table V. Model Compounds
Compd and no.
31 p-Benzensulfonamido-
phenol
32 p-Benzenesulfonamido-
pheny1-N-metby1-
carbamate
33 p-N-Methylbenzene
sulfonamidophenol
34 p-N-Methylbenzene
sulfonamidophenyl-
N-methylcarbamate
mp, °C
157-8
137-8
135-6
115-7
Yield
%
88
100
81
100
Analysis
Calcd %Found %
cf. Tingle and
Williams (1907)
C 54.90 54.87
H 4. 58 4. 58
N 9.15 8.93
C 59.32
H 4.94
N 5.32
C 56.25
H 5.00
N 8.75
59.48
4.98
5.23
56.31
5.09
8.81
28
-------
Table VI. Enzyme Inhibition Studies on Bovine Erythrocyte
Acetylcholinesterase (AChE) and Horse Serum
Cholinesterase (ChE)
Inhibition % of controls '
Compd. and no. 1 X 10~5M 5 X 10~5M 10 X 10~5M
AChE ChE AChE ChE AChE ChE
26 p-(5-Dimethylamino- 3-2 6±2 10-2 30±2 14±2 40-4
naphthalene-1-
sulfonamido)-phenyl-
N-methylcarbamate
27 p-N-(N'-methylcarba- 0.00 8-5 6±2 13±3 11-1 18-1
moyl)-N-(5-dimethy1-
aminonaphthalene-1-
sulfonyl) phenyl-N"-
methylcarbamate
28 p-(5-Dimethylaminonaph- 10±1 33±2 13-1 43-2 16-1 47±2
thalene-1-suIfonato)
acetophenone (N-methyl-
carbamoyl) oxime
30 5-Dimethylaminonaphtha- 9-3 23-1 18-2 33-3 24±2 35-2
lene-1-sulfonyl fluoride
Matacil (4-dimethyl- 79^2 30±4 58-3
amino-m-tolyl
methyIcarbamate)c
aAverage of three replicates. Average activity of con-
trol for AChE = 10.71 uM of acetylcholine hydrolyzed per hour,
and for ChE = 9.13 of acetylcholine hydrolyzed per hour.
cChemagro Crop., Kansas City, Mo.
29
-------
Table VII.
AminoaIky1-Dns-Sulfonamides
(Anionic Site Fluorescent Probes)
Compel and no.
35 5-Dimethylamino-
naphthalene-1-suIphon-
amido-N,N,-dimethyl-n-
propylamine
36 quaternary iodide of 35
mp.
119-20
201-2
C 60.80
H 7.46
N 12.50
C 45.28
H 5.87
N 8.80
60.43
7.23
12.27
45.16
5.86
8.73
37 N-Methylacridinium iodide
30
-------
Table VIII.
Enzyme Inhibition by Fluorescent Probes
Cone. Inhibition
Compound (M/l) AChE ChE
9, m-(5-Dimethylamino- 2 x 10"5 44
naphthalene-1-sulfon-
amido) ethylbenzoate 1 x 10 6
20 p-(5-Dimethylamino-
naphthalene-1-sulfon-
amido) benzyl diethyl _
phosphonate 1 x 10 46 21
3, m-(5-Dimethylamino- 1 x 10~4 48 41
naphthalene-1-suIfon-
amido) phenol 2 x 10~4 64 55
6, 3-(5-Dimethylamino-
naphthalene-1-sulfon- 4
amido) pyridine 1 x 10 28 2
quaternary of #6 1 x 10"4 34 2
8, m-(5-Dimethylamino-
naphthalene-1-sulfon- c
amido) benzotrifluoride 2 x 10 26 14
Dns-N-CH0COOH 1 x 10~5 45 0
H ^
11, m-(5-Dimethylamino-
naphthalene-1-sulfon- .
amido) benzoic acid 1 x 10 32 0
Dns-N-(CH2)2N-(CH3)3. I 1 x 10~4 3
2 x 10~5 37
Dns-N-(CH9)0N(CHo)o. I 1 x 10"4 4 37
H * 3 d 3
(#36)
Dns-N-(CH2)3-N(CH3)2 1 x 10~4 14
(#35) 2 x 10~5 47
31
-------
Compound
37, N-Methylacridinum
iodide
5, 2-(5-Dimethylamino-
naphthalene-1-sulfon-
amido) pyridine
7, N-(5-Dimethylamino-
naphthalene-1-sulfon-
amidoethylglycinate
5 x 10
-7
-6
1 x 10
2 x 10~6
2 x 10~5
1 x 10
-5
1 x 10
-5
Inhibition
AChE
58
75
100
ChE
58
85
11
32
-------
Table IX.
Compounds Which Compete for the Probe-Enzyme Complex
Using Probe TA
Insecticide or Compound Response
Naled +
Diazinon +
Pho rate +
Paroxon +
Methylparathion +
TEPP +
Guthion +
Ethion +
Dursban +
Malathion +
DDT +*
Heptachlor +*
Diedrin +*
Thiodan +*
DDE +*
Esserine +
Zectran
Vapona
*eommercial enzyme
33
-------
50
<" 40
ft
m
Z
ft
m 30
m 2 0
CA
-< 10
8
10
1 2
[METHYL PARATHiON]xio6 M
Figure 1
Change in fluorescence of the TA-ChE complex
with Methyl Parathion
34
-------
[DURSBAN] x io6 M
Figure 2
Change in fluorescence of the TA-ChE complex with Dursban
35
-------
2468
[THIODAN]X io6 M
10
Figure 3
Change in fluorescence of the TA-ChE complex with Thiodan
36
-------
ChE 0.1 mg/ml
X Excitation 330 nm
X Emission 515
1 2
[DURSBAN]X IO'M
Figure 4
Relative fluorescence effects with Dursban
37
-------
m40
Z
ChE 0.1 mg/ml
X Excitation 330 nm
X Emission 515
20
[TA] x 10~7M.
Q 2
A6
• 8
010
A 12
1 2 3
[THIODAN]xl06M
Figure 5
Relative fluorescence effects with Thiodan
38
-------
SECTION VII
REFERENCES
1. Himel, Chester M. and Chan, Lai-Man. Synthesis of
Fluorescent Substrate Analogs, Chapter VA in Concepts
in Biochemical Fluorescence. (Chen, R. and Edelhock,
H. Eds.) Dekker, New York. (in press).
2. Mayer, Richard T., and Himel, Chester M., Dynamics of
Fluorescent Probe-Cholinesterase Reactions, Biochemistry
11 2082 (1972).
3. Himel, Chester M. and Mayer, Richard T., Applications
of Fluorescence Spectroscopy to Fundamental Problems
in Insect Biochemistry, J. Georgia Entomol. Soc. 5
31 (1970).
4. Himel, Chester M. and Mayer, Richard T., 5-Dimethylamino-
naphthalene-1-sulfonic Acid (Dns Acid) as Quantum Yield
Standard, Anal. Chem. 42, 130 (1970).
5. Himel, Chester M. , Mayer, Richard T., and Cook, Larry L.,
Design of Active-Site-Directed Fluorescent Probes and
Their Reaction with Biopolymers, J. Polymer Sci. Pt. A-l
8 2219 (1970).
6. Himel, Chester M., Aboul-Saad, Wissam G., and Uk, Solang,
Fluorescent Analogs of Insecticides and Synergists.
Synthesis and Reactions of Active-Site-Directed Fluores-
cent Probes, J. Agr. Food Chem. 19 1175 (1971).
7. Whidby, J. F., Leyden, D. E., Himel, C. M., and Mayer,
R. T., A Nuclear Magnetic Resonance Study of the Pro-
tolysis Kinetics of 5-Dimethylaminonaphthalene-l-sulfonic
Acid and Its N-Methylsulfonamide, J. Phys. Chem. 75 4056
(1971).
8. Jun, H. W., Mayer, R. T., Himel, C. M., and Luzzi, L. A.,
Binding Study of p_-Hydroxybenzoic Acid Esters to Bovine
Serum Albumin by Fluorescent Probe Technique, J. Pharm.
Sci., 60 1821 (1971).
9. Fahrney, D. E. and Gold, A. M., J. Amer. Chem. Soc., 85
997 (1963).
39
-------
10. Main, A. R., Tarkane, E., Aull, J. L. and Soucie, W. G.,
J. Biol. Chem., 247 566 (1972).
11. Chen, R. F. and Kernohan, J. C., J. Biol. Chem., 242,
5813 (1967).
12. Chen, R. F. in Fluorescence Theory, Instrumentation
and Practice, (Guilbault, G. G., Ed.) Dekker, New York
(1967).
13. Nabb, D. P. and Whitefield, F., Arch. Environ. Health,
15, 147 (1967).
14. Parker, C. A. and Rees, W. T., Analyst 85, 587 (1960).
15. Fletcher, A. N., J. Mol. Spectrosc. 23 221 (1967).
40
-------
SECTION VIII
PUBLICATIONS
Mayer, Richard T., and Himel, Chester M., Dynamics of Fluores-
cent Probe-Cholinesterase Reactions, Biochemistry 11 2082
(1972).
Himel, Chester M. and Mayer, Richard T., Applications of
Fluorescence Spectroscopy to Fundamental Problems in Insect
Biochemistry, J. Georgia Entomol. Soc. J3 31 (1970).
Himel, Chester M. and Mayer, Richard T., 5-Dimethylamino-
naphthalene-1-sulfonic Acid (Dns Acid) as Quantum Yield
Standard, Anal. Chem. 42, 130 (1970).
Himel, Chester M., Mayer, Richard T., and Cook, Larry L.,
Design of Active-Site-Directed Fluorescent Probes and Their
Reaction with Biopolymers, J. Polymer Sci. Pt. A-l J3 2219
(1970).
Himel, Chester M. , Aboul-Saad, Wissam G., and Uk, Solang,
Fluorescent Analogs of Insecticides and Synergists. Syn-
thesis and Reactions of Active-Site-Directed Fluorescent
Probes, J. Agr. Food Chem. 19 1175 (1971).
Whidby, J. F., Leyden, D. E., Himel, C. M., and Mayer, R. T.,
A Nuclear Magnetic Resonance Study of the Protolysis Kinetics
of 5-Dimethylaminonaphthalene-l-sulfonic Acid and Its N-
Methylsulfonamide, J. Phys. Chem. 75 4056.
Jun, H. W., Mayer, R. T., Himel, C. M., and Luzzi, L. A.,
Binding Study of p-Hydroxybenzoic Acid Esters to Bovine
Serum Albumin by Fluorescent Probe Technique, J. Pharm. Sci.,
60 1821 (1971).
a U. S. GOVERNMENT PRINTING OFFICE : 1973—51
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
7. Re port No.
A Lces^ioE No
w
4. Title
Fluorescent Probes in the Detection of Insecticides in Water
". Airhor(s)
Chester M. Himel
9. Organization
Department of Entomology
University of Georgia
Athens, Georgia 30601
12. Sr-nsorir. Orgaxratioa Environmental Protection Agency
15. Supplementary Notes
Environmental Protection Agency Report
Number EPA-R2-73-217
5. Report Date
6.
8. Performing Orgat'izatioa
Report No.
10. Project No.
'/. -^ jiitract!Grant No.
16020 EAO
1 Type :>{ Report and
Period Covered
Research 6/1/70-5/31/7
16. Abstract
Objectives of this study included synthesis of candidate fluorescent probe molecules
for cholinesterase enzymes and evaluation of the feasibility of developing a new
analytical method for insecticides in water. This was accomplished. Results with
Dursban, Thioden and certain other insecticides are in the range of 1X10"7M. Insecticide;
which do not compete with, or displace the probe from its complex are not detected.
Experimental parameters for design and synthesis of optimum probe molecules were
developed.
17a. Descriptors
Fluorescent probes*, Insecticides* , Water
l~b. Identifiers
Analytical methodology*, Cholinerterase*, Enzymes* , Water*
J7c. COWRR Field & Group
18. Availability
IP. -Security Class,
^ Repot.)
W. Security C't ss,
(Page)
Abstractor H.P. Nicholson
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 2O24O
Institution EPA., Southeast Environmental Research Lab.
1O2 (REV JUNF 197!)
Athens, Georgia 30601
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