&EPA
United States
Environmental Protection
Agency
Industrial Environmental Researcn EPA-600 2-80-083
Laboratory May 1980
Cincinnati OH 45268
Research and Development
Alternate Enzymes for
Use in Cholinesterase
Antagonist Monitors
("CAM's")
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental 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.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-80-083
May 1980
ALTERNATE ENZYMES FOR USE IN CHOLINESTERASE ANTAGONIST MONITORS ("CAM'S")
by
Louis H. Goodson
Vicki J. Appleman
Midwest Research Institute
Kansas City, Missouri. 64110
Contract No. 68-03-0299
Project Officer
John E. Brugger
Oil and Hazardous Materials Spills Branch
Industrial Environmental Research Laboratory-Cincinnati
Edison, New Jersey 08817
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Industrial Environmental Research
Laboratory-Cincinnati, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection Agency,
nor does mention of trade names of commercial products constitute endorse-
ment or recommendation for use.
ii
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FOREWORD
When energy and material resources are extracted, processed, converted,
and used, the related pollutional impacts on our environment and even on our
health often require that new and increasingly efficient pollution control
methods be used. The Industrial Environmental Research Laboratory-Cincinnati
(lERL-Ci) assists in developing and demonstrating new and improved methodolo-
gies that will meet these needs both efficiently and economically.
This report describes a search for alternate enzymes to be used in the
Cholinesterase Antagonist Monitor (CAM) systems. When operated with cholines-
terase, the CAM instruments work well for the detection of organophosphate
and carbamate pesticides on a real-time basis, but they cannot detect com-
pounds that do not inhibit Cholinesterase. In the present investigation,
a group of commercially available enzymes was investigated to determine poten-
tial usefulness in the CAM instruments for the detection of non-cholinesterase-
inhibiting, toxic materials in water, such as chlorinated hydrocarbons, aryl
phosphates, phenols, and heavy metals. Thus, CAM systems, designed for mon-
itoring of water supplies and municipal and industrial effluents, can be used
to alert responsible personnel to a spill of a wide range of toxic and hazar-
dous materials and can be used as a means of tracing the course of these
materials in waterways. Information on this subject beyond that supplied
here may be obtained by contacting the Oil and Hazardous Materials Spills
Branch, lERL-Ci, USEPA, Edison, New Jersey 08817.
David G. Stephan
Director
Industrial Environmental Research Laboratory
Cincinnati
iii
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ABSTRACT
The CAM series of instruments ("CAM" is an acronym for "cholinesterase
antagonist monitor") was previously constructed to automatically monitor the
levels of organophosphate and carbamate pesticides in water. In these real-
time monitoring devices an immobilized enzyme, cholinesterase, is inactivated
in the presence of organophosphate and carbamate pesticides. The residual
activity of the enzyme, related to the level of inhibitor, is determined by
measuring the electrochemical response of the system to a substrate readily
hydrolyzed by the enzyme.
It was expected that the CAM system could be used to detect hazardous
materials other than organophosphate and carbamate pesticides if enzymes that
were inhibited by other toxic and hazardous materials of interest could be
found. Therefore, a preliminary study was conducted to determine how the
CAM system could be adapted to respond to toxic materials, such as chlorinated
hydrocarbons, phenols, aryl phosphates, cyanides, and heavy metals in water.
The approach consisted of a search for enzymes that: (1) could be used in
the CAM systems in place of immobilized cholinesterase, (2) would be inhibited
by low levels of one or more of the toxic substances of interest, and (3)
were detectable by electrochemical means. In this search, more than 20 en-
zymes were evaluated to see whether they met the following requirements: (1)
were commercially available at moderate cost, (2) were sufficiently stable
for study, (3) required no expensive cofactors, and (4) used substrates that
were inexpensive and changed redox potentials as a result of enzyme action.
Laboratory Investigations of the following five enzymes were made: al-
kaline phosphatase (E^ coli), carboxylesterase (hog liver), glucose oxidase
(Aspergillus m'ger), carbonic anhydrase (bovine erythrocyte), and hexokinase
(yeast).Inhibition studies of these enzymes showed that glucose oxidase,
carboxylesterase, and alkaline phosphatase were not significantly inhibited
(>15%) by 10" M solutions of the toxic test materials. Carbonic anhydrase
was significantly inhibited by 10 M solutions of methoxychlor, DDT, toxa-
phene, pentachlorophenol, cyanide ion, and cadmium and mercury compounds.
Hexokinase was significantly inhibited by 10 M or 10 M solutions of chlor-
dane, DDT, heptachlor, aldrih, lindane, methoxychlor, toxaphene, mercuric
ion, and tri-o-cresyl phosphate.
IV
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Hexokinase and carbonic anhydrase are potentially useful in a CAM-type
instrument for detection of selected hazardous materials. However, neither
enzyme has yet been adapted to the CAM system.
This report was submitted in fulfillment of Contract No. 68-03-0299,
Task No. 3, by Midwest Research Institute under the sponsorship of the U.S.
Environmental Protection Agency. This report covers the period 3 November
1976 to 3 July 1977 and work was completed as of 18 November 1977.
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CONTENTS
Foreword i i i
Abstract i v
Figures and Tables viii
Abbreviations ix
Acknowledgment x
1. Introducti on 1
2. Conclusions 2
3. Recommendations 3
4. Experimental Approach 4
5. Methods of Enzyme Analysis 9
6. Enzyme Inhibition Studies 10
7. CAM Instrument Adaptation 16
References 26
Appendices
A. Fact sheet for candidate enzymes 28
B. Methods of enzyme inhibition analysis 58
C. Abstracts of previous CAM-Instrument reports 68
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FIGURES
Number Page
1 Scope of enzyme studies 8
2 Carbonic anhydrase-catalyzed reactions investigated for potential
use in monitoring devices 17
3 Apparatus for electrochemical "beaker studies." 18
4 Prototype apparatus for determination of carbonic anhydrase
activity 24
TABLES
Number Page
1 Hazardous Materi al s Selected for Study 5
2 Inhibition of Glucose Oxidase.. 11
3 Inhibition of Alkaline Phosphatase 12
4 Inhibition of Carbonic Anhydrase 13
5 Inhibition of Hexokinase 15
6 Examination of p-Nitrophenylthioacetate and p-Nitrothiophenol
with Platinum-Wire Electrodes 20
7 Results of Electrometric Assay of Carbonic Anhydrase Activity 23
vlii
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ABBREVIATIONS
ADP --Adenosine diphosphate
ATP —Adenosine triphosphate
BCA —Bovine carbonic anhydrase
CAM --Cholinesterase Antagonist Monitor, general term for those instru-
ments using Cholinesterase in an electrochemical cell for detection
and monitoring of Cholinesterase antagonists
CAM-1 --Cholinesterase Antagonist Monitor, automatically detects and moni-
tors water supplies for Cholinesterase inhibitors
CAM-4 --Battery-operated, portable version of CAM-1
DDO —2,2-Bis(p-chlorophenyl)-l,l-dichloroethane
DDT —2,2-Bis(p-chlorophenyl)-!,!,1-trichloroethane
GTP --Guanosine triphosphate
NAD --Oxidized form of the co-factor, nicotinamide adenine dinucleotide
(also called diphosphopyridine nucleotide)
NADH --Reduced form of NAD (see above)
p-NPA --p-Nitrophenyl acetate
p-NPTA --p-Nitrophenyl thioacetate
p-NTP —p-Nitrothiophenol
2,4,5-T --2,4,5-Trichlorophenoxyacetic acid
TOCP --Tri-o-cresyl phosphate
Tris --Tris(hydroxymethyl)-aminomethane partially neutralized with HC1;
a buffer
TTP --Thymidine triphosphate
UTP --Uridine triphosphate
ix
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ACKNOWLEDGMENTS
The work upon which this publication is based was performed pursuant
to the earlier Contract No. 68-01-0038 and the current Contract No. 68-03-
0299 with the Environmental Protection Agency; this report describes the work
on Task III of the current contract. Task I of this contract evaluated the
response of the Cholinesterase Antagonist Monitor Model No. 1 (CAM-1) to a
series of commercially available pesticides, including both organophosphates
and carbamates. Task II covered the design, fabrication, and evaluation of
a portable version of CAM-1, which has been designated as CAM-4. Task III
was devoted to an investigation of alternate enzyme systems for use in CAM-
1 and CAM-4. Work on all tasks has now been completed.
The authors wish to thank Mr. William B. Jacobs, Dr. Donald R. Sellers,
Dr. Patrick E. Guire, Mr. Charles Barker, and Mr. Brian Cage, for their tech-
nical assistance. We also wish to thank Dr. Thomas Hoover of EPA's Southeast
Water Quality Laboratory, Athens, Georgia, and Dr. John E. Brugger and Ms.
Allison Tepper of EPA's Industrial Environmental Research Laboratory-Cincin-
nati, Edison, New Jersey, for their technical assistance and encouragement.
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SECTION 1
INTRODUCTION
As the manufacture and use of industrial and agricultural chemicals has
accelerated, the danger of pollution of natural waterways has increased.
This contract was performed pursuant to the Environmental Protection Agency's
interest in the development of instrumental methods for the detection and
continuous monitoring of hazardous materials spills.
The Cholinesterase Antagonist Monitor (CAM-1) was developed by Midwest
Research Institute on an earlier EPA Contract (No. 68-01-0038). The instru-
ment is capable of monitoring water for the presence of organophosphate and
carbamate pesticides on a real-time basis. Detailed information regarding
the theory of operation and the physical components of CAM-1 can be found
in EPA Report No. R2-72-010, August 1972.
Task 1 of the present contract, entitled "Evaluation of a Warning Device
for Organophosphate Hazardous Material Spills," delineated the sensitivity
of the CAM-1 instrument to various pesticides. During Task II, entitled "CAM-
4, A Portable Warning Device for Organophosphate Hazardous Material Spills,"
a portable version of the CAM—designated CAM-4--was developed and tested.
The practical worth of the CAM system was thereby extended to include in-the-
field monitoring. ^
This publication is a report on Task III, "Alternate Enzymes for Use
in CAM Instruments." Presently, the detection capability of the CAM instru-
ments is limited to toxic materials that inhibit the enzyme Cholinesterase.
The objective of Task III was to examine other enzyme systems for potential
use in CAM to detect additional toxic materials.
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SECTION 2
CONCLUSIONS
The present report is concerned with the investigation of a number of
enzyme systems to determine their potential usefulness in the CAM instrument
for the detection and monitoring of toxic substances in the aquatic environ-
ment. In enzyme-inhibition studies, measurements were made of the decrease
in enzyme activity resulting from incubating candidate enzymes with dilute
solutions of chlorinated hydrocarbons, phenols, aryl phosphates, cyanide ion,
and heavy metal ions. As a result of these studies, the following conclusions
can be drawn:
1. Carbonic anhydrase is inhibited by 5 x 10 M solutions of several
chlorinated hydrocarbons and inorganic substances, such as pentachlorophenol,
cyanide ion, and mercuric ion. Although carbonic anhydrase has not yet been
adapted to the CAM system, such adaptation may be both practical and useful.
2. Hexokinase is inhibited by low levels (10~4 to 10~5 M) of DDT, chlor-
dane, lindane, and toxaphene. It is also inhibited by mercuric ion and by
tri-o-cresyl phosphate. Therefore, hexokinase is considered to be a potential
sensor for these toxic substances and should be considered for adaptation
to a real-time monitoring system.
3. Glucose oxidase, carboxylesterase, and alkaline phosphatase are not
promising for use in CAM as sensors for the battery of toxic substances tested.
4. Although the aim of this study was to adapt new enzymes for use in
the electrochemical detection system of the CAM instrument, immobilized horse
serum cholinesterase and eel cholinesterase remain—at this time—the only
enzymes that have been us.ed successfully.
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SECTION 3
RECOMMENDATIONS
On the basis of the preliminary investigation of "Alternate Enzymes for
Use in Cholinesterase Antagonist Monitors," it is clear that little is known
about the possible application of hundreds of enzymes to problems in the
detection and monitoring of toxic and hazardous materials. However, this
field appears promising. Therefore, the following recommendations are ger-
mane:
1. Studies on the potential use of carbonic anhydrase and hexokinase
for the detection and monitoring of toxic substances—especially chlorinated
hydrocarbons—in water should be continued.
2. A larger number of available enzymes should be screened to determine
their potential sensitivity to toxic substances of interest, including chlori-
nated hydrocarbon pesticides, phenols, solvents, aryl phosphates such as tri-
o-cresyl phosphate, and heavy metals.
3. Studies of the inhibition of oxidative enzymes should include the
co-immobilization of these enzymes with the NAD-NADH system to demonstrate
the practicality of using oxidative enzymes for the detection of toxic materi-
als in water or air. These enzymes are especially important in the metabolism
of living systems and should be particularly sensitive to highly toxic sub-
stances.
4. One or more promising enzymes should be selected and adapted for
use in a CAM-like instrument for monitoring water or air on a real-time basis.
5. New breadboard enzyme-monitoring systems should be evaluated to
determine their sensitivity to toxic materials of interest and their suscep-
tibility to interferences in the environments in which they would be used.
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SECTION 4
EXPERIMENTAL APPROACH
The framework of Task III permitted the examination of any of the 200
to 300 commercially available enzymes for detection of hazardous materials.
As this was such a broad area for investigation, it was immediately apparent
that arbitrary but logical restrictions on the selection of enzymes and toxic
and hazardous materials for laboratory study were needed. Criteria for candi-
date enzymes and toxic materials of interest were outlined early in the pro-
gram in order to ensure acquisition of the most useful information during
the limited time available. A scheme for laboratory examination of candidate
monitoring enzymes evolved. The following paragraphs describe the selection
of toxic materials and candidate enzymes and the scheme for laboratory testing.
TOXIC MATERIALS OF INTEREST IN WATER MONITORING
It is recognized that virtually every substance has the potential for
presenting some hazard to the public health or welfare depending upon its
physical properties, its toxicity, and the quantity, time, and place of its
discharge. As only a finite number of hazardous compounds could be tested
in vitro with each enzyme in the laboratory, it was necessary to carefully
determine which toxic substances were of greatest environmental concern and
therefore could be considered as target compounds for extending the detection
capabilities of the CAM instrument.~ A review of EPA criteria for designation
of hazardous substances was made. ' '
EPA regulations (40 CFR 116) place designated hazardous substances into
five categories (X,A,B,C,D) on the basis of reportable quantities (1,10,100,
1000,5000 Ib, respectively). The determination of minimum reportable quanti-
ties is based on the chemical and toxicological properties of the substance
of concern. Many of these hazardous substances can be placed in six chemical
classes of interest to the present study. Materials in two classes, organo-
phosphates and carbamates, are detectable by the CAM systems when operated
with cholinesterase. Therefore, materials for this study were largely se-
lected from four other classes: (1) chlorinated hydrocarbons, (2) cadmium
compounds, (3) cyanide compounds, and (4) mercury compounds. In addition,
several other hazardous materials were also examined. Table 1 lists materials
tested and, where applicable, identifies the appropriate EPA "reportable
quantity (RQ)" hazardous category.
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TABLE 1. HAZARDOUS MATERIALS SELECTED FOR STUDY
Material
EPA
Category
Material
EPA
Category
Chlorinated hydrocarbons;
Aldrin X
Chlordane X
ODD X
DDT X
Endosulfan X
Endrin X
Heptachlor X
Lindane X
Methoxychlor X
Pentachlorophenol A
Toxaphene X
Trichlorophenol X
2,4,5-T acids B
2,4,5-T esters B
Cyanide compounds;
Potassium cyanide A
Sodium cyanide A
Mercury compounds:
Mercuric acetate
Mercuric chloride
Aryl phosphates;
Phenylphosphate, disodium
salt
Tri-o-cresyl phosphate
Tricresylphosphate
(mixed cresols)
Cadmium compounds;
Cadmium chloride
Others;
Lead acetate
Potassium permanganate
Strontium acetate
D
B
SELECTION OF CANDIDATE ENZYMES
Prior to beginning laboratory work it was necessary to decide which en-
zymes showed greatest promise for detection of the target compounds and for
adaptation to CAM. Twenty enzymes were considered for laboratory study be-
cause: (1) the literature indicated that they were inhibited by the toxic
substances of interest and (2) the enzymes yield sulfhydryl groups upon reac-
tion with substrates, making them more easily adaptable to the CAM system
(the utility of cholinesterase in the CAM system depends on the enzymatic
production of sulfhydryl groups). The following 20 enzymes by no means in-
clude all potentially useful monitoring enzymes but provided the starting
point for this work:
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Glucose oxidase (1.1.3.4)
Xanthine oxidase (1.2.3.2)
Pyruvate dehydrogenase (1.2.4.1)
Succinic dehydrogenase (1.3.99.1)
Glutathione reductase (1.6.4.2)
Thiol oxidase (1.8.3.2)
Glutathione dehydrogenase (1.8.5.1)
Cytochrome oxidase (1.9.3.1)
Catalase (1.11.1.6)
Hexokinase (2.7.1.1)
Carboxylesterase (3.1.1.1)
Arylesterase (3.1.1.2)
Lipase (3.1.1.3)
Acetyl[esterase (3.1.1.6)
Alkaline phosphatase (3.1.3.1)
Acid phosphatase (3.1.3.2)
Urease (3.5.1.5)
ATPase (3.6.1.3)
Carbonic anhydrase (4.2.1.1)
S-Alkyl cysteine lyase (4.4.1.6)
Information pertinent to the selection of enzymes for laboratory testing
was assembled for each of the 20 candidate enzymes in the form of fact sheets.
The fact sheets for all candidate enzymes are included in this report as
Appendix A.
The relative merits of the various enzymes, based on the following enzyme
selection criteria, were considered by a panel of five MRI biochemists:
1. Commercial availability and cost: Use of enzymes not commercially
available is precluded. Although cost may be minimized by enzyme immobiliza-
tion, which permits reuse, it should not be so high as to prohibit eventual
routine use of the immobilized enzyme in a CAM-like water monitor.
2. Stability: Extremely labile enzymes are difficult to handle and
have a low likelihood of succesful use, therefore, enzymes for laboratory
study should be reasonably stable.
3. Specific activity: Specific activity is an expression of the catalyt-
ic efficiency of the enzyme. Enzymes with high specific activity are desired
since they are expected to have the greatest bioamplification factor and to
be the most sensitive to pollutants.
4. Substrate specificity: Enzymes with low substrate specificity are
versatile and should be easily adaptable to the CAM instruments.
5. Cofactor requirements: A requirement by an enzyme for cofactors,
such as NAD-NADH, imposes an additional burden. Regeneration or reuse of
expensive cofactors presents complications that cannot be satisfactorily stud-
ied within the scope of the current contract. Therefore, enzymes not requir-
ing cofactors are preferred.
6. Response to inhibitors: The candidate enzyme should be inhibited
by low concentrations of the hazardous materials of interest in water monitor-
ing. Literature reports of inhibition are considered, when available.
Enzyme Commission numbers for enzyme identification.
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7. Electrochemical detection: The enzyme should produce or destroy
a substance that is easily oxidized or reduced electrochemically. Indirectly,
the enzyme may produce a product that, through reaction with another material,
becomes either eas.ily oxidizable or reducible.
A point system for judging the relative merits of candidate enzymes was
devised. In each of the following areas, a maximum of three points was award-
ed: (1) reported inhibition by pollutants of interest, (2) expected adapta-
bility to the CAM instrument, and (3) other factors such as cost and cofactor
requirements (1-5 above).
As a result of discussions and a review of the fact sheets, the five
panel members mutually agreed on the following point assignments:
Enzyme Points
X
Carbonic anhydrase (4.2.1.1) 8
Succinic dehydrogenase (1.3.99.1) 7
Glucose oxidase (1.1.3.4) 6-1/2
ATPase (3.6.1.3) 6
Alkaline phosphatase (3.1.3.1) 6
Hexokinase (2.7.1.1) 6
Glutathione reductase (1.6.4.2) 5-1/2
Cytochrome oxidase (1.9.3.1) 4-1/2
Laboratory studies began with carbonic anhydrase and the other enzymes
were then tested in order of point ranking as time permitted. Carboxylester-
ase was subsequently included for study.
ORGANIZATION OF LABORATORY STUDIES
Laboratory studies proceeded through the following three stages: (1)
methods of enzyme analysis, (2) in vitro inhibition studies, and (3) CAM-in-
strument adaptation. When a problem was encountered during the investigation
of a particular enzyme, the likelihood of its solution within a reasonable
time and its anticipated effect on the final usefulness of the enzyme were
evaluated. Based on these considerations, a decision was made whether to
continue laboratory work with the enzyme. This decision-making process was
necessary in order to ensure that preliminary data were obtained and an under-
standing of potential problems was developed for several enzymes within the
time and effort limitations of this contract. Figure 1 illustrates the scope
of the laboratory work for each enzyme studied.
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CD
Tasks
Literature search
Selection of assay method
Purchase of enzyme and reagents
Modification of assay method for
enzyme inhibition assay
Enzyme test with battery of
toxic substances
Determination of sensitivity
of enzyme to specific inhibitors
Investigation of alternate means to
measure enzyme activity electro-
chemically
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SECTION 5
METHODS OF ENZYME ANALYSIS
A literature review of assay methods was conducted for each enzyme se-
lected for study. Although many methods were available, spectrophotometric
methods of analysis were preferred, as the equipment was available and pro-
cedures were simple and readily adaptable to inhibition studies. Once a
defined method had been chosen, enzymes and other reagents were ordered from
commercial suppliers. As noted previously, commercial availability of enzymes
was a prerequisite. Because no supplier of succinic dehydrogenase (the second
enzyme chosen for study) was found, it was immediately eliminated from this
work.
Published methods of enzyme analysis are often designed solely for the
detection of active enzyme in assay mixtures. When used for this purpose,
excess quantities of substrate may be used. However, for inhibition testing,
standard concentrations of enzyme and substrate are used and the effect of
given quantities of toxic materials on the activity assay are measured. The
standard concentrations of enzyme and substrate are critical to the detection
of inhibition. For example, excess substrate may "protect" the enzyme from
inhibition when the inhibitor concentration is low in comparison to the sub-
strate concentration.
4
The Worthington Enzyme Manual was often used as the initial source of
assay methods. These methods were modified prior to testing for inhibition
by toxic chemicals in order to ensure that maximum sensitivity was obtained.
Curves of enzyme activity versus substrate concentration and of enzyme activi-
ty versus enzyme concentration were generated. The reactant concentrations
selected were: (1) the substrate concentration (no excess substrate) allowing
maximum activity and (2) the smallest assayable concentration of enzyme.
The method of adding the test compound (water soluble and organic solu-
ble) to the reaction mixture was specified for each screening test. When
organic solvents were used, the effect of the solvent on the enzyme reaction
was taken into account because organic solvents, even at less than 10% by
volume, can inhibit enzyme activity. When the amount of inhibition resulting
from solvent effects was small, the "enzyme control" containing the organic
solvent and no inhibitor represented 100% activity. Appendix B contains the
revised methods of enzyme inhibition analysis used in this work and references
the original sources.
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SECTION 6
ENZYME INHIBITION STUDIES
The effect of known concentrations of toxic chemicals on enzyme activity
was measured in vitro. The purpose of the measurements was to screen a number
of chemicals against a number of enzymes to find those enzymes that were
sensitive to low concentrations of specific toxic chemicals. In this way,
the expected usefulness of an enzyme for water monitoring could be anticipated
before any effort was given to CAM-adaptation.
GLUCOSE OXIDASE/ALKALINE PHOSPHATASE
The results of inhibition studies of glucose oxidase and alkaline phos-
phatase are presented in Tables 2 and 3. With the exception of a slight
inhibition of alkaline phosphatase by mercury compounds, these enzymes were
not significantly (>15%) inhibited by any of the target materials. Thus,
glucose oxidase and alkaline phosphatase do not appear to be useful for moni-
toring the toxic chemicals tested.
CARBOXYLESTERASE
The toxic arylphosphate, tri-o-cresyl phosphate (TOCP), has been receiv-
ing increased attention in environmental studies conducted by EPA. It has
been reported that certain carboxylesterases (E.G. 3.1.1.1, sometimes called
aliesterase or tributyrinase) are sensitive to inhibition by TOCP. In light
of this report and because it is believed that a capability for detecting
TOCP would be valuable, a comparative study of the inhibition of hog liver
carboxylesterase and eel cholinesterase by TOCP was conducted. Additional
toxic materials were not screened.
In studies of the sensitivities of hog liver carboxylesterase (Sigma
Chemical Co. No. E-3128) and eel cholinesterase to TOCP, it was demonstrated
that both enzymes were rather insensitive to TOCP, i.e., a 10 M solution
of TOCP produced no more than 15% inhibition of carboxylesterase or eel cho-
linesterase.
CARBONIC ANHYDRASE
Table 4 presents the results of inhibition studies of carbonic anhydrase.
Significant inhibition pi5%) occurred at 10 M and 10 M by cyanide-con-
taining and mercury compounds, respectively. These tpxicant+|evels suggest
that carbonic anhydrase would detect low levels of CN~and Hg in water.
10
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TABLE 2. INHIBITION OF GLUCOSE OXIDASE
Chemical
class
Organophosphate
pesticides
Chlorinated
hydrocarbons
Cyanide compounds
Cadmium compounds
Mercury compounds
Aryl phosphates
Test compound Concentration Inhibition
ppnF (%)
Dichlorwos (DDVP)
Dursban
Ethion
Naled
Parathion
Aldrin
Chlordane
DDD
DDT
Endosulfan
Endrin
Heptachlor
Lindane
Methoxychlor
Pentachl orophenol
2,4,5-T acid
2,4,5-T butyl ester
2,4,5-Trichlorophenol
Potassium cyanide
Sodium cyanide
Cadmium chloride
Mercuric acetate
Mercuric chloride
Phenyl phosphate,
di sodium salt
Tri-o-cresyl phosphate
24
35
39
38
26
37
41
32
36
41
38
37
29
38
27
26
31
20
7
5
23
32
27
20
37
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
29
29
N.S.
N.S.
a/ ppm (w/v) = fig/ml. All solutions contained the test compound at levels
of 10 M. (Some solutions were hazy indicating that the solubility of
the inhibitor in 10% aqueous acetonitrile was exceeded.)
b/ "N.S." means that no significant inhibition (i.e.,>15%) was detected.
11
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TABLE 3. INHIBITION OF ALKALINE PHOSPHATASE
Chemical
class
Organophosphates
Carbamates
Chlorinated
hydrocarbons
Cyanide compounds
Mercury compounds
Aryl phosphates
Others
Test compound Concentration, Inhibitior
ppnF' (%)
DDVP R
Dursban*
Endosulfan
Ethion
Naled
Par at hi on
Carbofuran
Sevin
Aldrin
Chlordane
DDD
DDT
Heptachlor
Lindane
Methoxychlor
Pentachlorophenol
2,4,5-T acid
2,4,5-T ester
Toxaphene
2,4,5-Trichlorophenol
Potassium cyanide
Sodium cyanide
Mercuric acetate
Mercuric chloride
Phenyl phosphate,
di sodium salt
Tri-o-cresyl phosphate
Lead acetate
Potassium permanganate
Strontium acetate
24
35
41
39
38
26
22
20
37
41
32
36
37
29
38
27
26
31
41
20
7
5
32
27
20
37
38
16
22
N.S*/
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
a/ All solutions contained the test compound at concentrations of 10 M.
(Some solutions were hazy indicating that the solubility of the inhibitor
in 10% acetonitrile was exceeded).
b/ "N.S." means that no significant inhibition (i.e.,>15%) was detected.
12
-------
TABLE 4. INHIBITION OF CARBONIC ANHYDRASE
% Inhibition at several concentrations—
Test compound Grade purity 10~4 M 5xlO"5 M 10"5 M 10"6 M
DDT relatives:
DDT
DDT
Methoxychlor
h/
Anal, std.-'
100%
Anal. std.
97%
Commercial
90%
34
28
40
29
28
25
11 —
5 —
55 —
Chlorophenoxy compounds:
2,4,5,-T
acids
2,4,5-T
butyl esters
Anal. std.
99%
Anal. std.
100%
+6
21
9
26
7 —
13 —
Aldrin-toxaphene group (toxaphene family);
Toxaphene Anal. std. 44 39 3 —
100%'
Highly halogenated aromatics;
Pentachlorophenol Commercial 70 72 12 —
34%
2,4,5-Tri- Anal. std. 35 21 8 —
chlorophenol 98%
Cyanide compounds:
Potassium cyanide Reagent 100 — 100 46
Sodium cyanide Reagent 100 — 79 21
Mercury compounds;
Mercuric chloride
Cadmium compounds:
Cadmium chloride
Reagent
Reagent
100 —
21 —
55 0
a/ Percentage depression in the activity of a standard enzyme solution.
b/ Anal. std. - Analytical standards received from EPA Pesticide and Toxic
Substances Effects Laboratory, Research Triangle Park, North Carolina.
13
-------
All chlorinated hydrocarbons tested, except 2,4,5-T acid, inhibited car-
bonic anhydrase at 5 x 10 M. Pentachlorophenol, toxaphene, andrDDT ranked
first, second, and third, respectively. At concentrations of 10" M, none
of these pesticides inhibited the enzyme by 15%.
Inhibition studies with several highly toxic substances have indicated
the expected utility of a monitoring device based on carbonic anhydrase.
An expanded capability for detection of hazardous materials could result from
the adaptation of carbonic anhydrase to the CAM instrument.
HEXOKINASE
Compounds that inhibited hexokinase by 90% in vitro at 3 x 10 M in-
cluded DDT, mercuric salts, chlordane, lindane, methoxychor, and toxaphene.
As seen in Table 5, 50% inhibition was_caused by exposure to aldrin and to
endosulfan at concentrations of 3 x 10" M and to tri-o-cresyl phosphate at
a concentration of 10 M.
During tests with hexokinase, an increase in sensitivity was achieved
by adding the substrate, glucose, after a 5-minute incubation of hexokinase
and test compound. Additional manipulations might further increase the sensi-
tivity of this enzyme to toxic chemicals.
Adaptation of hexokinase to an automatic monitoring device is promising
but was not pursued during these studies.
14
-------
TABLE 5. INHIBITION OF HEXOKINASE
% Inhibition at several concentrations-
Test compound 3xlO"4 M 10~4 M 10"5 M
Chlorinated hydrocarbons:
Aldrin
Chlordane
ODD
DDT
Endosulfan
Heptachlor
Lindane
Methoxychlor
Pentachlorophenol
Toxaphene
Trichlorophenol
2,4,5-T acid
Organophosphates:
DDVP
Dursban
Naled
Parathion
Carbamates:
Furadan
Inorganic Ions:
Strontium
Cadmium
Cyanide
Mercury
Aryl Phosphates:
Phenlyphosphate,
disodium salt
Tri-o-cresyl phosphate
Tricresylphosphate
(mixed cresols)
55 17 . . —
92 22, 6(£7 —
— . /
93 40 61-7
54 59 T-T .
— 40 4(Wy
100 49 55^7
100 26 —
0 — T-T
100 52. IQO^7-^7
26 5,68^7 —
24 — —
23 — —
17 — —
22 — —
— 12 —
15 — —
0 — —
— 38 —
— 6 — h/
95 77 63s-7
13 — —
50-90 3.4 —
— 29 —
a/ Percent inhibition was calculated by assigning 100% activity
ane control containing no
b/ Value was obtained using
in this report. Glucose
cubated for 5 min. This
tion. c
test compound.
a modification of the test procedure
is added after inhibitor and enzyme
procedure increases the sensitivity
to a di ox-
given
are in-
to inhibi-
c/ Concentration was 2x10 M.
15
-------
SECTION 7
CAM INSTRUMENT ADAPTATION
The goal of instrument adaptation experiments was to discover a way to
incorporate alternate enzymes into an existing automatic monitoring device.
This investigation was primarily restricted to methods that could make use
of the electrochemical detection system of the present CAM-type instruments.
Use of this approach required the selection of substrates that are catalyzed
to produce sulfhydryl groups (e.g., thiocholine esters are hydrolyzed by
cholinesterase to yield sulfhydryl groups, which are detected by CAM). Non-
electrochemical cell methods for generating electrical signals (pH) indicative
of enzyme activity were also considered. Carbonic anhydrase was the only
enzyme for which these adaptation experiments were performed.
CARBONIC ANHYDRASE: ADAPTATION TO CAM
Three reactions catalyzed by carbonic anhydrase were studied for poten-
tial use in an automatic monitor. These reactions are illustrated in Fig-
ure 2.
Hydrolysis of p-Nitrophenyl Acetate
Hydrolysis of p-nitrophenyl acetate (p-NPA), Figure 2a, is the basis
for the spectrophotometric assay of carbonic anhydrase activity (Appendix B-
1) used in the inhibition studies. The ability to distinguish p-NPA from
its enzyme-catalyzed products is a requirement for the successful use of the
hydrolysis reaction of p-NPA in CAM.
A series of electrochemical studies was conducted in beakers with plati-
num-wire electrodes similar to those used in the CAM'S electrochemical cell
(Figure 3). These studies were designed to determine the electrical proper-
ties of substrate and of substrate-enzyme solutions under various electrical
conditions. Voltages between platinum electrodes in a test solution were
measured while the resistance, in series with the battery and electrodes,
was decreased stepwise. Test liquids were a p-NPA solution (4 x 10 M in
Tris buffer/10% acetonitrile,pH 7.4) and a p-NPA solution also containing
2 x 10 M bovine carbonic anhydrase (BCA). The results of the studies demon-
strated that for the resistances between 10 megohms and 15000 ohms: (1) high
voltage values (0.8 to 2.0 v) were obtained and (2) electrochemical cell
voltages for p-NPA solutions with and without BCA were indistinguishable.
The following explanations for these results are proposed: (1) each solution
16
-------
2a.
0=C-CH,
I ^
0
NO,
carbonic
anhydrase
NO,
NO,
0
+ HO-C-CH,
OH
p-Nitrophenyl
acetate
p-Nitrophenolate p-Nitrophenol
an ion
0=C-CH-
2b.
NO,
carbonic
*
anhydrase'
SH
0
+ HO-C-CH-
NO,
p-Nitrophenylthioacetate
p-Nitrothiophenol
2c. C0
carbonic
anhdrase
H2C03
-> HCO,
Figure 2. Carbonic anhydrase-catalyzed reactions investigated for potential use in monitoring devices.
-------
9V
Battery
Electrometer
Measuring Voltage
Resistance
Substitution
Box
Platinum-Wire Electrode
Test Solution
Magnetic Stirrer
Figure 3. Apparatus for electrochemical "beaker studies". Cell potential
measurements were made at fixed resistance values.
18
-------
behaves only as substrate because of insufficient product formation, (2) each
solution behaves only as a product because of spontaneous hydrolysis of the
substrate, and (3) the substrate, p-NPA, and the reaction product, p-nitro-
phenol, cannot be distinguished electrochemically under the experimental
conditions used.
In "beaker studies," solutions of substrate and product were examined
separately under identical conditions of temperature, buffer, and pH. Fluctua-
tions in voltage readings resulting from electrode conditioning were taken
into account by repeating tests until stable and characteristic readings were
obtained at each resistance. The results indicated that p-NPA could not be
distinguished from p-nitrophenol electrochemically, using platinum-wire elec-
trodes; therefore, p-NPA was abandoned as a substrate.
Hydrolysis of p-Nitrophenylthioacetate
Theoretically, the sulfur analogue of p-NPA, i.e.,p-nitrophenylthioace-
tate (p-NPTA), was very promising for use with carbonic anhydrase in CAM,
although the use of p-NPTA as a carbonic anhydrase substrate had not been
previously reported. Because it was theorized that the hydrolysis product,
p-nitrothiophenol (p-NTP), Figure 2b, would lend itself to electrochemical
detection due to the presence of a free sulfhydryl group, further studies
were conducted.
Enzyme-catalyzed hydrolysis of p-NPTA in solution was examined spectropho-
tometrically and electrochemically, as follows:
Spectrophotometric Assay—
Upon standing, solutions of p-NPTA were observed to yellow with time.
The yellow color presumably indicated the formation of the hydrolysis product.
Clear, colorless solutions of p-NPTA were prepared by dissolving the material
in acetonitrile, which was added to cold diethylmalonate buffer (pH 7.2)
and kept at 4 C. Scans (350 to 850 nm) of the clear p-NPTA solutions and
of the yellow, hydrolyzed material showed that Spectrophotometric observations
of the reaction could be made at 410 nm.
The analysis was performed by preparing clear solutions of p-NPTA as
described above. A known amount of carbonic anhydrase was placed in the
sample cuvette of a Beckman Model 25 spectrophotometer. Buffered substrate
was added to both reference and sample cuvettes and the 00,, Q recorded for
5 min. In this way, spontaneous hydrolysis was automatically subtracted from
the sample and enzymatically-catalyzed product formation was observed.
Assays done in this manner revealed a greater activity/min in the sample
cuvette in all instances. Further proof of actual carbonic anhydrase activity
towards p-NPTA was that the moles of substrate hydrolyzed per minute decreas-
ed as enzyme concentration in the sample was reduced ten-fold. Detailed
studies to characterize and optimize this reaction were not conducted.
19
-------
Electrochemical Assay-
Cold solutions of p-NPTA and p-NTP (5 x 10"4 M) in 0.01 M diethylmalonate
buffer-10% acetonitrile were prepared. Measurements obtained with platinum-
wire electrodes at fixed resistance values are shown in Table 6. Lower vol-
tages were consistently observed with solutions of p-NTP. Therefore, it
appeared that the hydrolysis product produced the low voltages characteristic
of sulfhydryl groups, thus allowing differentiation between substrate and
product in the CAM instruments.
Hydrolysis of p-NPTA was further tested in a breadboard CAM system, which
operates on the same water-substrate-current cycle as CAM and uses the same
platinum electrodes. A starch pad with no enzyme was placed between the elec-
trodes and baselines were determined with the test solutions. Water was
pumped through the pad during the sample cycle. The two compounds, p-NPTA
and p-NTP, were distinguishable in CAM at 5 x 10 M. Baselines with p-NPTA
were ca. 750 mv, while p-NTP produced baselines of ca. 250 mv. In principle,
this electrochemical difference should be quite satisfactory for monitoring
the activity of an enzyme and its subsequent inhibition. Thus, the use of
p-NPTA and carbonic anhydrase in the CAM instrument remains theoretically
possible. However, there are several problems associated with adaptation
of this enzyme system for use in a real-time monitoring device. The problems,
yet to be resolved, are as follows:
1. p-NPTA is not very soluble in aqueous solutions. Mixed solvents
must be used; however, some organic solvents inhibit enzyme activity.
2. The substrate, p-NPTA, hydrolyzes spontaneously in a 10% organic-
aqueous solution at room temperature. A method for handling the p-NPTA so
that it remains intact prior to enzymatic hydrolysis must be devised.
TABLE 6. EXAMINATION OF p-NITROPHENYLTHIOACETATE AND
p-NITROTHIOPHENOL WITH PLATINUM-WIRE ELECTRODES
Resistance at
substitution box,
megohms
10.0
6.8
4.7
3.3
2.2
1.5
1.0
0.68
0.47
Current, pA
0.87
1.3
1.8
2.6
3.9
5.7
8.4
12.2
17.3
Voltage
with
p-NPTA, v
1.1
1.15
1.2
1.27
1.37
1.45
1.52
1.6
1.68
Voltage
with
p-NTP, v
0.31
0.33
0.35
0.38
0.44
0.50
0.59
0.70
0.85
Voltage
difference,
mv
790
820
850
890
930
950
930
900
830
20
-------
3. The specific activity of carbonic anhydrase towards p-NPTA is very
low. A way must be found to generate sufficient quantities of product for
detection during a sampling cycle of reasonable length.
4. An immobilized carbonic anhydrase product with sufficient activity
towards p-NPTA must be prepared to allow read-out in the electrochemical cell
of CAM. Preliminary studies of three methods for immobilization of carbonic
anhydrase were conducted: (1) starch-gel entrapment on open-pore polyurethane
foam, (2) photoimmobilization on a solid support, and (3) hydrophobic adsorp-
tion to phenoxyacetyl cellulose.
Starch-gel entrapment—Carbonic anhydrase was substituted for cholines-
terase (ChE) in the procedure for preparing the ChE pads presently used in
CAM instruments. ' The maximum weight of carbonic anhydrase allowing con-
venient handling was used and no attempt was made to modify the ChE procedure
to better accommodate carbonic anhydrase. These carbonic anhydrase pads did
not produce the low baseline voltage expected of an active enzyme pad in the
CAM system.
Photoimmobilization--Photoimmobilization with stepwise bifunctional
reagents (e.g., 4-azido-2-nitrophenyl-y-aminobutyryl N-oxysuccinimide) for
covalent bigd|ng of enzymes to a variety of solid supports has been success-
fully used. ' Enzymes have been coupled to matrices such as paper, cotton,
nylon cloth, and polyurethane with good Retention of activity. Carbonic
anhydrase was photoimmobilized on a 1 cm cellulose sponge in an attempt to
make a product with greater absolute activity and less enzyme wash-out.
Activity on the sponge after photolysis was not assayed spectrophotometric-
ally. The product was qualitatively evaluated by testing it in CAM. Sub-
strate was passed through the sponge and into the electrochemical cell.
Again, the low voltage characteristic of the active enzyme was not seen.
Hydrophobic adsorption--A new product from Regis Chemical Company,
11,12,13
Enzorb-A (phenoxyacetyl cellulose), is available for immobilization of enzymes
and other proteins by means of hydrophobic adsorption. Immobilization can
be accomplished by passing a solution of enzyme or other proteins through
an Enzorb-A column. This material reportedly binds 0.3 to 0.5% of its own
weight in protein, often with near 100% retention of enzyme activity. This
relatively simple procedure seemed promising for the immobilization of car-
bonic anhydrase. It was envisioned that such an enzyme column could be placed
in the continuous flow system of CAM in front of the electrochemical cell.
Enzorb-A (2 ghwas carried through the batch preparation given in the
technical bulletin using 10 mg of carbonic anhydrase. Alphacel (Nutritional
Biochemical Corporation), a cellulose product with no phenoxyacetyl groups,
served as the control material. All filtrates and washes from Enzorb-A and
Alphacel were analyzed for carbonic anhydrase activity spectrophotometrically.
Activity unaccounted for (representing enzyme remaining on the column) was
calculated by difference. The Enzorb-A retained 2.9 mg carbonic anhydrase,
while 0.08 mg remained on the Alphacel control column.
21
-------
Although the data indicated binding of the enzyme protein, the column
was not immediately usable with the electrochemical cell as an indicator of
enzyme activity. Several suggestions for modifications in column preparation
and techniques for operating the column in CAM remain to be explored.
In summary, the inability to produce carbonic anhydrase products usable
in CAM by starch-gel entrapment, photochemical immobilization, and hydrophobic
column preparations during this report period was not unexpected. These
results should also be viewed in light of the fact that carbonic anhydrase
has a low affinity for the substrate, p-NPTA, usable in CAM, in contrast to
the high affinity of cholinesterase for choline esters. In the cholinesterase
system, an enzyme preparation containing relatively little enzyme can convert
sufficient substrate to be detectable by CAM, but this is not the case with
carbonic anhydrase. It is anticipated that greater quantities of active
enzyme, longer substrate incubation times, or higher incubation temperatures
will be required for use of carbonic anhydrase in CAM. The preliminary ex-
periments performed under this contract have provided a preview of possible
adaptation techniques and an understanding of the potential problems to be
solved.
Hydrolysis of Carbon Dioxide
Figure 2c illustrates the naturally occurring, "physiological" reaction
of carbonic anhydrase. The specific activity of carbonic anhydrase towards
the substrate, C02, is much greater than that towards either p-NPA or p-NPTA.
With the device snown in Figure 4, the use of C02 as substrate and of carbonic
anhydrase as enzyme was tested. Development of this apparatus was an attempt
to instrument the Wilbur-Anderson electrometric assay of carbonic anhydrase
activity (Appendix B-2), which measures the time required for a saturated
C02 solution to lower the pH of a 0.02 M Tris-HCl buffer from 8.3 to 6.3 at
0 C. It was believed that a pH measurement of buffer solution (with or with-
out carbonic anhydrase) might produce an electrical signal indicative of the
presence or absence of enzyme activity and could be usable in a continuous
monitor for carbonic anhydrase inhibitors.
A series of experiments was designed to establish a set of operating
parameters under which time differences in the pH drop for tests with and
without carbonic anhydrase could be observed (note that the COp-hydrolysis
reaction proceeds in the absence of enzyme). Initial tests in which C0? and
buffer solutions entered a T-tube connector and flowed directly to a pH elec-
trode produced very "noisy" pH readings. Incorporation of a short coiled
tube between the T-tube connector and pH electrode did not correct the noise
problem. However, using a mixing vessel or a large T-connector with an in-
ternal stirring bar (Figure 4), smooth recordings of pH with time were gen-
erated. The following parameters were varied while pH tracings were made:
1. Flow rates of C02 and pH 8.0 buffer (thereby changing ratios of the
two reactants and the time interval between mixing and pH measurement).
4°C).
2. Mixing vessels (thereby changing time delay prior to pH measurement),
3. Temperature (reactants were mixed at room temperature (25°C) or at
22
-------
In this series of experiments, a time difference in pH drop with and
without carbonic anhydrase was not seen. The probable reason for this result
was that the lag time between contact of reactants and measurement of pH was
too long. r
To ascertain which time intervals between mixing of reactants and pH
measurements might be required jo a flow-through system, the electrometric
carbgmc anhydrase assay method *»* was evaluated at room temperature and
at 4 C. The results are shown in Table 7. The time required for the pH to
fall from 8.3 to 6.3 was shown to be temperature dependent. At 4°C, the "time
window" was 27 sec, while at 25°C, it was 6 sec. In both cases, the concen-
tration of enzyme was 10 mg per 10 ml of a buffer/C09 mixture. (Notice that
the windows were shortened when the enzyme concentration was reduced ten-fold).
In order to detect enzyme activity on a continuous basis, as in the CAM
system, measurements must be made in the time window bracketed by the blank
and test times. Therefore, careful consideration of design parameters, in-
cluding a provision for refrigeration, would be required to develop a carbonic
anhydrase monitor such as that tested.
In conclusion, the preceeding discussions demonstrate the feasibility
of adapting a carbonic anhydrase inhibition-based system for use in the CAM
instrument to monitor chlorinated hydrocarbons and cyanide-containing and
mercury-containing compounds. The following systems appear most promising
for the development of a carbonic anhydrase inhibition-based water monitor:
TABLE 7. RESULTS OF ELECTROMETRIC ASSAY OF CARBONIC ANHYDRASE ACTIVITY
Temperature,
L
4
25
25l/
Carbonic anhydrase
in reaction, mg
Blank
0.01
0.001
Blank
0.01
0.001
Blank
0.01
Test time,-
sec
30.0
3.0
20.0
8.0
2.0
6.0
15.0
2.0
Time,-/
sec
27.0
10.0
6.0
2.0
13.0
I/ Time required for pH drop from 8.3 to 6.3; average of several tests
2J Blank time minus test time="time window"
3/ A 3-ml saturated COoSolution was used instead of a 4-ml solution
23
-------
ro
Mixing Vessel:
Buffer
To pH Meter
Stir Bar
Magnetic
Stirring
Plate
Magnetic
Stirring
Plate
Buffer
To pH
Electrode
-j v~ y
pH 8.0 pH 8.0 Buffer/
Carbonic Anhydrase
_T-tube Connector
(See Insert)
Mixing Vessel
(See Insert)
Drip Tip
Glass pH Electrode
Flow-through
pH Assembly
oj1"1^
VX^ ^x/
OO
OO
pH Meter
Strip-chart Recorder
Figure 4. Prototype apparatus for determination of carbonic anhydrase activity.
-------
1. Hydrolysis of p-nitrophenylthioacetate (p-NPTA): p-NPTA is hydrol-
yzed in the presence of carbonic anhydrase to form p-nitrothiophenol. As
in the present CAM system—using cholinesterase—enzyme inhibition by toxic
materials can be detected electrochemically by monitoring production of the
-SH group in the hydrolysis product. Although the detection system would
remain the same, several problems, such as low specific activity for p-NPTA
hydrolysis and development of a method of enzyme immobilization, must be
solved before a carbonic anhydrase /p-NPTA system could be adapted to the
CAM instrument.
2. Hydrolysis of carbon dioxide (C02): In the presence of carbonic
anhydrase, p-NPTA is rapidly hydrolyzed in solution to form hydrogen and
bicarbonate ions, causing a pH change. The presence of toxic, enzyme-inhibit-
ing materials in water will affect the residual enzyme activity, which can
be determined by monitoring the pH. Utilization of a carbonic anhydrase/CO^
system would require major changes in the detection system of the CAM instru-
ment as presently configured. However, instrumentation of the Wilbur-Anderson
assay method previously described appears to be a practicable detection al-
ternative for use in an automatic water monitor such as CAM.
25
-------
REFERENCES
1. Federal Register, Environmental Protection Agency, "Designation and
Determination of Removability of Hazardous Substances from Water," Pro-
posed Rules, Vol. 39, No. 164, Part IV, Thursday, August 22, 1974.
2. Federal Register, Environmental Protection Agency, "Hazardous Substances
Designation, Removability, Harmful Quantities and Penalty Rates," Vol.
40, No. 250, Part IV, December 30, 1975.
3. Federal Register, Environmental Protection Agency, "Hazardous Substances
Definitions, Designations, Revocation of Regulations, Proposed Expansion
of Criteria of Designation and Proposed Determination of Reportable
Quantities," Vol. 40, No. 116, Part IV, February 16, 1979.
4. Decker, L. A. (ed.), Worthington Enzyme Manual. Worthington Biochemical
Corporation, Freehold, New Jersey (1977).
5. EPA Final Report, "Assessment of the Need for the Characterization of
and the Impact Resulting from Limitations on Aryl Phosphates," Contract
No. 68-01-4313, MRI Project No. 4309-L.
6. Drisch, K., "Carboxylic Ester Hydrolases," in Vol. V, The Enzymes (P.O.
Boyer, ed.), Academic Press, New York, (1971) p. 63.
7. Goodson, L. H. and W. B. Jacobs. "Rapid Detection System for Organo-
phosphates and Carbamate Insecticides in Water." EPA-R2-72-010, U.S.
Environmental Protection Agency, Washington, DC, (1972) p. 66.
8. Goodson, L. H., W. B. Jacobs, and A. W. Davis. "An Immobilized Choiin-
esterase Product for Use in the Rapid Detection of Enzyme Inhibititors
in Air and Water." Anal. Biochem.. j>l (2): 362-367 (1973).
9. Guire, P. "Photochemical Immobilization of Enzymes and Other Biochem-
ical s," Chapter 21 of Methods in Enzymology. Vol. 44 (Klaus Mosback,
ed.). Academic Press (1974).
10. Guire, P. "Enzyme Immobilization With a Thermochemical-Photochemical
Bifunctional Agent." U.S. Patent No. 3,959,078, May 1976 (assigned to
Midwest Research Institute).
26
-------
11. Technical Product Bulletin. Regis Chemical Company, 8210 N. Austin
Avenue, Morton Grove, Illinois 60053.
12. Worthy, W. "New Support Simplifies Enzyme Immobilization." Chem. Eng.
News (1977), pp. 23-24.
13. Butler, S. G. "Enzyme Immobilization by Adsorption on Hydrophobic Deriva-
tives of Cellulose and Other Hydrophobic Materials." Arch. Biochem.
Biophys., 171:645-650 (1975a).
14. Wilbur, K. M. and N. G. Anderson. "Electrometric and Colorimetric Deter-
mination of Carbonic Anhydrase." J. Biol. Chem., 176:147 (1948).
27
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APPENDIX A
FACT SHEET FOR CANDIDATE ENZYMES
A-l: GLUCOSE OXIDASE (1.1.3.4)
Reaction Catalyzed: Glucose + H20 + 02 >H202 + gluconic acid
Source: Worthington Cost: $45.00/100 mg (130 units/mg)
(Aspergillus niger)
Stability: Stable 6-12 months at 4°C
Specific Activity: 80 lU/mg
Substrate Specificity: Relative rates - D-glucose=100, D-mannose=20,
2-deoxy-D-glucose=20, other hexoses=negligible
Cofactor Requirements: None
Inhibitors: Ag+, Hg++, Cu++, H202, F" (halides F~»C1~, Br")
Electrochemical Detection: H202 + RSH—»R-S-S-R
1. The formation of the reaction product, hLOp, could be monitored
electrochemically. A sulfhydryl group could be fed into the system and,
in the presence of active enzyme, H202 would oxidize the sulfhydryl
compound and produce characteristic low voltages, as seen in the absence
of enzymes in the cholinesterase-based system. When the enzyme is in-
hibited, HpOp production would decrease and the lower rate of sulfhydryl
oxidation would produce an increase in the cell voltage.
2. Yellow Springs Instrument Model 25 Oxidase Meter could be used for
direct electrochemical detection.
3. The reaction could be coupled with glutathione peroxidase (1.11.1.9)
and the loss of -SH monitored.
References: Auses, J. P., S. L. Cook, and «J. T. Maloy. "Chemiluminescent
Enzyme Method for Glucose." Anal. Chem.. 47(2):244-249 (1975).
28
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Bostick, D. T. and D. M. Hercules. "Quantitative Determination
of Blood Glucose Using Enzyme Induced Chemiluminescence of
Luminol." Anal. Chem., 47(3):447-452 (1975).
Bouine, J. C., M. T. Atallah, and H. 0. Hultin. "Parameters
in the Construction of an Immobilized Dual Enzyme Catalyst."
Biotechnol. Bioenq.. 18:179-187 (1976).
Clark, L. C. Jr., and C. Lyons. "Electrode Systems for Continuous
Monitoring in Cardiovascular Surgery." Annals N.Y. Acad. Sci..
102(29):29-45 (1962).
Duke, F. R., N. Weibel, D. S. Page, V. G. Bulgrin, and J. Luthy.
"The Glucose Oxidase Mechanism Enzyme Activation by Substrate."
J. Amer. Chem. Soc., 91:14 (1969).
Finelli, V. N., et al. "Effects of Metal-Binding Fractions of
Tobacco Smoke on In Vitro Activity of Enzymes." Arch. Environ.
Health, 25:97-100 (1972).
Greenfield, P. F. "Inactivation of Immobilized Glucose Oxidase
by Hydrogen Peroxide." Anal. Biochem., 65(1-2):109-124 (1975).
Greenfield, P. F. and R. L. Laurence. "Characterization of
Glucose Oxidase and Catalase on Inorganic Supports." J. Food
Sci., 40:906-910 (1975).
Guilbault, G. G. and G. L. Lubrano. "An Enzyme Electrode for
the Amperometric Determination of Glucose." Anal. Chim. Acta,
64: 439-455 (1973). "~
Horvath, C., B. A. Solomon, and J. M. Engasser. "Measurement
of Radial Transport in Slug Flow Using Enzyme Tubes." Bio-
technol. Bioeng.. 12(4):431-439 (1973).
Horvath, C. and B. A. Solomon. "Open Tubular Heterogeneous
Enzyme Reactors: Preparation and Kinetic Behavior." Biotech-
nol. Bioeng.. 14:885-914 (1972).
Kilburn, D. M. and P. M. Taylor. "Effect of Sulfhydryl Reagents
on Glucose Determination by the Glucose Oxidase Method." Anal.
Biochem.. 27:555-558 (1969).
Lahoda, E. 0. and C. C. Liu. "Electrochemical Evaluation of
Glucose Oxidase Immobilized by Different Methods." Biotechnol.
Bioeng., 17:413-422 (1975).
Leon, L. P., S. Narayanan, R. Dellenbaclr, and C. Horvath. "Im-
mobilized Glucose Oxidase Used in the Continuous Flow Determin-
ation of Serum Glucose." Clin. Chem.. 22(7):1017-1023 (1976).
29
-------
Llenado, R. A. and G. A. Rechnitz. "Ion-Electrode Based Auto-
analysis System for Enzymes." Anal. Chem., 45(6):826-833
(1973).
Llenado, L. and 6. A. Rechnitz. "Ion-Electrode Based Automatic
Glucose Analysis System." Anal. Chem., 45(13):2165-2170 (1973).
Loren, E. C. Jr., and F. J. Burger. "Trace Determination of Metal
Ion Inhibitors of the Glucose-Glucose Oxidase System." Mikro-
chim. Acta. (Wien), 538-545 (1968).
Mell, L. D. and 0. L. Maloy. "Amperometric Response Enhancement
of the Immobilized Glucose Oxidase Enzyme Electrode." Anal.
Chem., 48(11):1597-1601 (1976).
Mell, L. D. and J. L. Maloy. "A Model for the Amperometric
Enzyme Electrode Obtained through Digital Simulation and Ap-
plied to the Immobilized Glucose Oxidase System." Anal. Chem.,
47(2): 299-307 (1975).
Messing, R. A. "Communications to the Editor." Biotechnol.
Bioeng. 16:525-529 (1974).
Fran-Minki, C. and G. Brown. "Construction and Study of Elec-
trodes Using Cross-Linked Enzymes." Anal. Chem., 47(8):1359-
1364 (1975).
Nagy, G., L. H. Von Stoys, and G. G. Guilbault. "Enzyme Elec-
trode for Glucose Based on an Iodide Membrane Sensor." Anal.
Chim. Acta. 66:443-455 (1973).
Nakamura, S. and Y. Ogura. "Mode of Inhibition of Glucose Oxi-
dase by Metal Ions." J. Biochem.. 64(4):439-447 (1968).
Ramachandran, K. B. and D. D. Perlmutter. "Effects of Immobiliza-
tion on the Kinetics of Enzyme-Catalyzed Reactions. I. Glucose
Oxidase in a Recirculation Reactor System." Biotechnol. Bio-
eng., .18:669-684 (1976).
Rogers, M. 0. and K. G. Brandt. "Interaction of Halide Ions
with Aspergillus niger Glucose Oxidase." Biochem., 10(25):
4630-4635 (1971).
Rogers, M. J. and K. G. Brandt. "Multiple Inhibition Analysis
of Aspergillus niger Glucose Oxidase by D-Glucal and Halide
Ions." Biochem.. 10(25):4636-4637 (1971).
Toren, E. C. "Trace Determination of Metal Ion Inhibitors of
the Glucose-Glucose Oxidase System." Mikrochim. Ichnoanal.
Acta, 3:538-545 (1968).
30
-------
Weibel, M. K. and C. Dodge. "Biochemical Fuel Cells, Demonstra-
tion of an Obligatory Pathway Involving an External Circuit
for the Enzymatically Catalyzed Aerobic Oxidation of Glucose."
Arch. Biochem. Biophys.. 169:146-151 (1975).
Weibel, M. K. and H. J. Bright. "The Glucose Oxidase Mechanism."
J. Biol. Chem.. 246(9):2734-2744 (1971).
Weibel, M. K. and H. J. Bright. "Insolubilized Enzymes, Kinetic
Behavior of Glucose Oxidase Bound to Porous Glass Particles."
Biochem. J., 124:801-807 (1971).
Weibel, M. K., W. Dritschilo, H. G. Bright, and A. E. Humphrey.
"Immobilized Enzymes: A Prototype Apparatus for Oxidase En-
zymes in Chemical Analysis Utilizing Covalently Bound Glucose
Oxidase." Anal. Biochem., 52:402-414 (1973).
Williams III, D. C., G. F. Huff, and W. R. Seitz. "Evaluation
of Peroxyoxalate Chemiluminescence for Determination of Enzyme
Generated Peroxide." Anal. Chem.. 48(7):1003-1006 (1976).
31
-------
A-2: XANTHINE OXIDASE (1.2.3.2)
Reaction Catalyzed: Xanthine + H20 + 02 > urate + H202
Source: Gallard-Schlesinger Cost: $40.00/100 mg (0.5 lU/mg)
(buttermilk)
Stability: Ammonium sulfate suspensions of the enzyme are stable for weeks
when refrigerated and for several days at room.temperature.
Specific Activity: 1.35 lU/mg
Substrate Specificity: Exhibits low specificity and attacks a number of
aldehydes, purines, pteridines, pyrimidines, azo-
purines, and other heterocyclic compounds. Sub-
strates: xanthine, acetaldehyde, benzaldehyde
Cofactor Requirements: None
Inhibitors: Quinazolines, benzodiarone, cyanide ion, alkylsalicylic acids
Electrochemical Detection: Detect H?02 directly or monitor its reaction
with RSH (See Appendix A-l Glucose Oxidase;
Electrochemical Detection of H^Op).
References: Massey, V., et al. "On the Mechanism of Inactivation of Xanthine
Oxidase by Cyanide." J. Biol. Chem.. 245:6595-6598 (1970).
McCoubrey, A., et al. "Inhibition of Enzymes by Alkylsalicylic
Acids." J. Pharm. Pharmacol., 22:333-337 (1970).
Wildbrett, G., et al. "Influence of Organic Insecticides on
Enzymes. Influence of the Structure of Phosphoric and Thiono-
phosphoric Acid Esters on Their Inhibitory Action in Relation
to Milk Xanthine Dehydrase In Vitro." Z. Naturforsch, 22:307-
312 (1967).
Marcolongo, R., et al. "The Inhibition of Xanthine Oxidase Activ-
ity by Benzodiarone." Clin. Chim. Acta.. 41;91-94 (1972).
32
-------
A-3: PYRUVATE DEHYDROGENASE (1.2.4.1)
Reaction: Pyruvate + oxidized > 6-S-acetyl-lipoate + C09
Catalyzed lipoate i
CH- S-CH-(CH?),-COOH CH~COS-CH-(CH9).COOH + CO-
I
-------
A-4: SUCCINATE DEHYDR06ENASE (1.3.99.1)
Reaction Catalyzed: Succinate + acceptor = fumarate + reduced acceptor
Source: Sigma Chemical Co., No. S-3630 (bacterial) Cost: Inquire
*
Stability: N.A.
Specific Activity: 55 lU/mg (bovine heart; succinate)
5.5 lU/mg (Micrococcus lactilyticus)
Substrate Specificity: The bovine heart enzyme oxidizes succinate, L-methyl-
succinate, L-ethylsuccinate, L-chlorosuccinate, and
D- and L-malate.
Cofactor Requirements: None. PMS (phenazine methosulfate) is the best elec-
tron acceptor. FP (ferriflavoprotein) is another
acceptor.
D
Inhibitors: Mirex, DDT, toxaphene, endrin, dieldrin, Guthion , manganese
Electrochemical Detection:
1. Investigate the possibility of directly monitoring the oxidation-
reduction of the electron acceptor.
2. The reduced acceptor could possibly be oxidized by R-S-S-R to liber-
ate the electroactive R-SH group.
References: Bernath, P. and T. P. Singer. "Succinic Dehydrogenase," Methods
in Enzymology, Vol. 5. Academic Press, New York (1964), pp.597-
614.
Davis, K. A. and Y. Hatefi. "Succinate Dehydrogenase. I. Puri-
fication, Molecular Properties and Substructure." Biochem.,
10(13):2509-2516 (1971).
Hanstein, W. B., K. A. Davis, M. A. Ghalambor, and Y. Hatefi.
"Succinate Dehydrogenase. II. Enzymatic Properties." Bio-
chem.. 10(13):2517-2524 (1971).
Heidker, J. C., et al.R "Inhibition of Mitochondrial Electron
Transport by Guthion , Some Related Insecticides and Degrada-
tive Products." Bull. Environ. Contam. Toxicol., 8:141-146
(1972).
McPhail, L. C. and C. C. Cunningham. "The Role of Protein and
Lipids in Stabilizing the Activity of Bovine Heart Succinate
Dehydrogenase." Biochem., 14(6):1122-1130 (1975).
"N.A." - Information not available.
34
-------
Seth, P.K., et al. "In Vitro Inhibition of Succinic Dehydro-
genase by Manganese and its Reversal by Chelating Agents."
Environ. Physiol. Biochem.. £(4):176-180 (1974).
Singer, T. P. "Determination of the Activity of Succinate, NADH,
Choline, and Glycerophosphate Dehydrogenases." Methods of
Biochem. Anal., 22:123-175 (1974).
Singer, T. P., E. B. Kearney, and W. C. Kenney. "Succinic De-
hydrogenase." Advances in Enzymology. 37^189-272 (1973).
Yarbrough, J. D. and G. B. Moffett. "The Effects of DDT, Toxa-
phene, and Dieldrin on Succinic Dehydrogenase Activity in Insec-
ticide-Resistant and Susceptible Gambusia Affinia." J. Agric.
Food Chem.. 20(3):558-560 (1972).
Yarbrough, J. D. and M. R. Wells. "Vertebrate Insecticide Re-
sistance: The In Vitro Endrin Effect on Succinic Dehydrogenase
Activity in Endrin-Resistant and Susceptible Mosquitofish."
Bull. Environ. Contam. Toxicol., 6(2)-.171-176 (1971).
Yarbrough, J. D. and F. M. McCorkle. "The In Vitro Effects of
Mirex on Succinic Dehydrogenase Activity in Gambusia Affinis
and Lepomis Cyanellus." Bull. Environ. Contam. Toxicol., 1:1(4):
364-370 (1974).
Zanetti, G., Y. M. Galante, P. Arosio, and P. Cerletti. "Inter-
actions of Succinate Dehydrogenase With Cyanide." Biochim.
Biophys. Acta.. 32^:41-53 (1973).
Zeijlemaker, W. P., D. V. Dervartanian, C. Veeger, and E. C.
Slater. "Studies on Succinate Dehydrogenase. IV. Kinetics
of the Overall Reaction Catalyzed by Preparations of the Puri-
fied Enzyme." Biochem. Biophys. Acta.. 178:213-224 (1969).
35
-------
A-5: 6LUTATHIONE REDUCTASE (1.6.4.2)
Reaction Catalyzed: Reduced . oxidized >NAD(P) + 2 glutathione
NAD(P) glutathione
Source: Sigma Chemical Co., No. G-4751, (yeast) Cost: $6.30/mg (100 units/mg)
Sigma Chemical Co., No. G-6004 (wheat germ) $25.00/g (100 units/g)
Stability: N.A.*
Specific Activity: Yeast=64.4, human erythrocyte=61, rat liver=839
Substrate Specificity: Specif*i'c for NAD(P)
Cofactor Requirement: Reduced NAD(P)
Inhibitors: 5-Nitro furaldehyde and nitrobenzene derivatives, mercury
Electrochemical Detection: Detection of -SH, as with cholinesterase-based
system.
References: Kosmidevs, S. "Glutathione Level in the Blood in Experimental
Poisoning with Metallic Mercury." Med. Pracy.. 16_: 206-210
(1965).
Mykkanen, H.M. "Effect of Mercury on Erythrocyte Glutathione
Reductase Activity." Bull. Environ. Contam. Toxicol., 12(1):
10-16 (1974).
Pekkanen, T. J. "The Effect of Experimental Methyl Mercury
Poisoning on the Activity of the TPNH-Specific Glutathione
Reductase of Rat Brain Liver." Acta Vet. Scand., 13:14-19
(1972).
Yawate, Y., et al. "Red Cell Glutathione Reductase: Mechanism
of Action of Inhibitors." Biochem. Biophys. Acta, 321:72-83
(1973).
"N.A." - Information not available.
36
-------
A-6: THIOL OXIDASE (1.8.3.2)
Reaction Catalyzed: 4 R:CR''SH + 02 »2 R:CR'-S'S-CR':R + 2H20
Source: Bacteria, yeast, liver, heart, muscle
Stability: N.A.*
Specific Activity: 19.8 lU/mg (thiophenol)
Substrate Specificity: The following substrates were oxidized: thiophenol,
catechol, diethyldithiocarbamate, methylmercaptoimi-
dazole, and resorcinol.
Cofactor Requirements: None
Inhibitors: lodoacetic acid, iodoacetamide, N-ethylmaleimide, p-chloromer-
curibenzoate, bromoacetyl-CoA, arsenic trioxide
Electrochemical Detection:
1. Detection of -SH, as with cholinesterase-based system.
2. Use of an oxygen detector.
References: Barman, T. E. Enzyme Handbook. Vol. 1. Springer-Verlag, New
York (1969), p. 220.
Boyer, P. D. (ed.). The Enzymes. 3rd Ed., Vol. 7. Academic
Press, New York (1972), p. 391.
Dixon, M. and E. C. Webb. Enzymes. 2nd Ed. Academic Press, New
York (1964).
"N.A." - Information not available.
37
-------
A-7: GLUTATHIONE DEHYDROGENASE (1.8.5.1)
Reaction Catalyzed: 2 Glutathione + dehydro- —) oxidized + ascorbate
ascorbate glutathione
*
Source: N.A.
Stability: N.A.
Specific Activity: N.A.
Substrate Specificity: Dehydro-D-araboascorbate and 1,2,3-triketocyclopro-
pane are reduced, but at lower rates than dehydroas-
corbate.
Cofactor Requirements: None
Inhibitors: Unknown
Electrochemical Detection: Detection of -SH, as with present cholinester-
ase-based system.
References: Dixon, M. and E. C. Webb. Enzymes, 2nd Ed. Academic Press, New
York (1964), p. 374.
"N.A." - Information not available.
38
-------
A-8: CYTOCHROME OXIDASE (1.9.3.1)
Reaction Catalyzed: 4 Ferrocytochrome c + 02 = 4 Ferricytochrome c + 2H?0
Source: Gallard-Schlesinger (beef heart) Cost: $15/vial (25 U/vial)
Substrate Cost: $50/100 mg (Sigma from concuda)
$8.00/100 mg (Sigma from equine heart)
Stability: N.A.*
Specific Activity: 6.7 lU/mg (Pseudomonas)
Substrate Specificity: Cytochrome c from various sources. Relative rates-
P. aeruginosa=100. baker's yeast=4.5, beef=0.5
Cofactor Requirements: None
Inhibitor's: Cyanide, sulfide, azide, fluoride, hydroxylamine, metyrapone
Electrochemical Detection:
1. Investigate the possibility of directly monitoring the oxidation-
reduction of ferrocytochrome c.
2. Use of an oxygen detector.
References: VanBuuren, K. J. "Binding of Cyanide to Cytochrome aa." Biochem.
Biophys. Acta. 256:243-257 (1971).
Harmon, H. J., et al. "Inhibition of Cytochrome c Oxidase by
Hydrophobic Metal Chelators." Biochim. Biophys. Acta. 368(1):
125-129 (1974).
Muijsers, A. 0. "Biochemical and Biophysical Studies on Cyto-
chrome Oxidase. IV. Reaction with Fluoride." Biochim. Bio-
phys. Acta. 333:430-438 (1974).
Nicholls, P. "Biochemical and Biophysical Studies on Cytochrome
aa 3.8. Effect of Cyanide on the Catalytic Activity." Bio-
chim. Biophys. Acta. 275_: 279-287 (1972).
Nicholls, P. "Inhibition of Cytochrome c Oxidase by Sulphide."
Biochem. Soc. Trans.. _3(2):316-319 (1975).
Roots, I. and A. 6. Hildebrandt. "Non-Competitive and Competi-
tive Inhibition of Mixed Function Oxidase in Rat Liver Micro-
somes by Metyrapone." Naunyn-Schmiedeberg's, Arch. Pharmacol.,
227:27-38(1973).
*
"N.A." - Information not available.
39
-------
Yoshikawa, S. "The Reactions Between Cytochrome Oxidase and
Its Inhibitors." J. Japan Biochem. Soc.. 46(11):967-983 (1974).
Yoshikawa, S. "The Inhibition Mechanism of the Cytochrome Oxidase
Reaction IV. The Nature of the Kinetically Inactive Complex
of Cytochrome Oxidase with Cyanide." J. Biochem. (Tokyo),
73:637-645 (1973).
Yoshikawa, S. "Classification of Inhibitors Based on Modes of
Action." JL Biochem. (Tokyo), 71:859-872 (1972).
Yoshikawa, S. "The Inhibition Mechanism of the Cytochrome Oxi-
dase Reaction. Inhibition by Hydroxylamine." J. Biochem.
(Tokyo), 68:145-156 (1970).
40
-------
A-9: CATALASE (1.11.1.6)
Reaction Catalyzed: 2H202 »2H20 + 02
Source: Worthington (bovine liver) Cost: $27.00/100 ml (3,000 fl/ml)
Worthington (human erythrocyte) $45.00/2 ml (160,000 U/ml)
Stability: All preparations stable 6-12 months at 5°C. Do not freeze.
Specific Activity: N.A.
Substrate Specificity: Alkyl hydrogen peroxides can replace H?0? as sub-
strate. Ethanol, formate, nitrous acia, and thiol
compounds can replace the second H?0? as hydrogen
donor. ^ *
Cofactor Requirements: None
ii
Inhibitors: Cu , ascorbate, sodium azide, formaldehyde, hydrazine, pyrazole
Electrochemical Detection:
1. Detection of Hp02 directly or indirectly. (See Appendix A-l, Glucose
Oxidase, Electrochemical Detection.)
2. Use of an oxygen detector.
References: Ceniotti, 6., et al. "Anti-Catalase Activity of Phenylenedi-
amines In Vitro and In Vivo." Enzymologia. 30:290-298 (1966).
Feinstein, R. N., et al. "Effect of Compounds Related to Amino
Triazole on Blood and Liver Catalase IV." ANL-7535, US AEC
Argonne Nat. Lab., 96 (1968).
Feytmans, E., et al. "Effect of Pyrazole on Rat Liver Catalase."
Biochem. Pharmacol.. 23:1293-1305 (1968).
Thurman, R. G., et al. "Inhibition of Catalase in Perfused Rat
Liver by Sodium Azide." Ann. Acad. Sci.. 168_: 348-353 (1969).
"N.A." - Information not available.
41
-------
A-10: HEXOKINASE (2.7.1.1)
Reaction Catalyzed: ATP + D-hexose = ADP + D-hexose-6-phosphate
Source: Aldrich (yeast) Cost: $13.50/10 mg (2,500 U/10 mg)
Stability: N.A.
Specific Activity: 800 lU/mg (glucose)
Substrate Specificity: Relative rates - D-glucose=100, D-fructose=180,
D-mannose=80, D-galactose=0.2, D-glucosatnine=70
Cofactor Requirements: ATP
Inhibitors: Mg , disulfides, Ca , Al , chlorinated pesticides, diiso-
propylf1uorophosphate
Electrochemical Detection:
1. Couple with G-6-P dehydrogenase and NAP peroxidase (1.11.1.1), meas-
uring HpOp loss.
2. Couple with G-6-P dehydrogenase and glutathione reductase, measuring
-SH.
References: Barnard, E. A. "Hexokinases from Yeast." Methods in Enzymology,
42:6-20 (1975).
Bigl, V., et al. "Some Properties of Soluble and Solubilized
Particle-Bound Hexokinase." J. Neurochem.. 18_:721-727 (1971).
Bowers, L. D., et al. "An Immobilized-Enzyme Flow-Enthalpimetric
Analyzer: Application to Glucose Determination by Direct
Phosphorylation Catalyzed by Hexokinase." Clin. Chem.. 22_(9):
1427-1433 (1975).
Britton, H. G., et al. "The Order of Addition of Substrates
to Yeast Hexokinase." Biochem. J.. 128:104P (1972).
Coburn, H. 0. and J. J. Carrol. "Improved Manual and Automated
Colorimetric Determination of Serum Glucose with Use of Hex-
okinase and Glucose-6-Phosphate Dehydrogenase." Clin. Chem.,
1£:127 (1973).
Colowick, S. P. "The Hexokinases." in Vol. IX, The Enzymes (P.
D. Boyer, H. Lardy, and K. Myback, eds.). Academic Press,
New York (1973), p. 1.
"N.A." - Information not available.
42
-------
DeAlaniz, M. 0., et al. "Comparative Studies Regarding Different
Ways of Expression of Enzymatic Activity." Enzymol., 38:85-
88 (1970). —* —
Derechin, M., Y. M. Rustum, and E. A. Barnard. "Dissociation
of Yeast Hexokinase Under the Influence of Substrates." Bio-
chem.. 11:1793 (1972).
Genin, M.S., Y. F. Romanov, and V. Andreev.,(Russian) "Antagon-
istic Nature of the Effect of Mg and Ca Ions on Hexokinase
Activity." Biokhimiya. 37(4):732-735 (1972); Chem. Abstr..
77, 149037q(l972).
Domagk, 6. F. "Diisopropylfluorophosphate." Physio!. Chem.
348:381-384 (1967). '
Harrison, W. H., et al. "Aluminum Inhibition of Hexokinase."
Lancet. 2:277 (1972).
Jones, J. G., et al. "Essential and Nonessential Thiols of Yeast
Hexokinase: Reactions with lodoacetate and lodoacetamide."
Biochem., 14(11): 2396-2403 (1975).
Jones, J. G. "Active-Site Thiol Groups of Yeast Hexokinase."
Biochem J.. 115_:41P (1969).
Kanda, F., et al. "Competitive Inhibition of Hexokinase Iso-
enzymes by Mercurials." J. Biochem. (Tokyo), 79_(3): 543-548
(1975).
Morris, D. L., et al. "The Preparation of Nylon-Tube-Supported
Hexokinase and Glucose 6-Phosphate Dehydrogenase and the Use
of the Coimmobilized Enzymes in the Automated Determination
of Glucose." Biochem. J.. 147(3):593-603 (1975).
Murakami, K., et al. "Difference Between Hexokinase Isoenzymes
in Sensitivity to Sulfhydryl Inhibitor." J. Biochem. (Tokyo),
74:321-341 (1973).
Otieno. S., et al. "Essential Thiols of Yeast Hexokinase Alkyla-
tion by a Substrate-Like Reagent." Biochem.. 14(11):2403-2410
(1975).
Purich, D. L., et al. "The Hexokinases: Kinetic, Physical,
and Regulatory Properties." Adv. Enzym. Related Areas of Mol.
Biol., 39:249-326 (1973).
Ricard, J., et al. "The Theory of Alternative Substrates in
Enzyme Kinetics and Its Application to Yeast Hexokinase."
Eur. J. Biochem.. 31:14-24 (1972).
43
-------
Roustan, C., et al. "Yeast Hexokinase: Interaction with Sub-
strates and Analogs Studied by Difference Spectrophotometry."
Eur. J. Biochem.. 44:353-358 (1974).
Sadar, M*. H., et al. "A Specific Method for the Assay of Select
Chlorinated Pesticides." J. Agric. Food Chetn., 19:357-359
(1971).
Wright, W. R., et al. "Glucose Assay Systems: Evaluation of
a Colorimetric Hexokinase Procedure." din. Chem., 17:1010-
1015 (1971).
-------
A-ll: CARBOXYLESTERASE (3.1.1.1)
Reaction catalyzed: R-COO-R1 +
R-COOH + R'OH
Source: Sigma Chemical Co. and Boehringer Manheim Co.
Stability: N.A.**
Specific Activity: One unit will hydrolyze 1.0 u mole per minute of ethyl
butyrate to butyric acid and ethanol (pH 8.0, 25 C).
Substrate Specificity: Wide specificity for carboxylic esters; especially
aliphatic carboxylic esters. p-Nitrophenyl acetate.
Cofactor requirements: None.
Inhibitors: Complete inhibition with DFP.
References: Boursnell, J. C. and E. C. Webb. Nature, 164:875 (1949).
Dixon, M. and E. C. Webb. Enzymes, 2nd Ed. Academic Press,
New York (1964), p. 218.
Muggins, C. and J. Sapides. "Chromogenic Substrates IV. Acyl
Esters of p-Nitrophenol as Substrates for the Colorimetric
Determination of Esterase." J. Biol. Chem.. 170: 467 (1947).
**Synonyms: Carboxylic ester hydrolase, tributyrinase, hog liver esterase.
"N.A." - Information not available.
45
-------
A-12: ARYLESTERASE (3.1.1.2)
Reaction Catalyzed: Phenylacetate + H,,0 = phenol + acetate
Source: Sheep serum
Stability: N.A.*
Specific Activity: 1.2 lU/mg (paraoxon)
Substrate Specificity: Relative rates - paraoxon=100, p-nitrophenyl acetate=
1200, DFP=40, tabun=180
Cofactor Requirements: None
Inhibitors: N.A.
Electrochemical Detection: Use a thiophenol acetate as substrate and moni-
tor the appearance of -SH.
References: Barman, T. E., Enzyme Handbook, Vol. II. Springer-Verlag, New
York (1969), p. 502.
"N.A." - Information not available.
46
-------
A-13: LIPASE (3.1.1.3)
Reaction Catalyzed: Triglyceride + H20 = diglyceride + fatty acid ion
Source: Sigma Chemical Co., No. L-3001 (wheat germ) Cost: $4.50/g (7 U/mg)
Stability: Highly purified, homogenous preparations are extremely labile.
Worthington's two preparations are stable 1 year refrigerated
and dry.
Specific Activity: N.A.
Substrate Specificity: Tri-, di- and mono-glycerides are attacked in de-
creasing order of activity.
Cofactor Requirements: None. May require emulsifiers for full activity.
Calcium is required as an activator.
Inhibitors: Phosphate, magnesium, ATP, ascorbic acid, paraoxon, alcohols,
sulfonyl halides, phenoxybenzamine, organophosphates
Electrochemical Detection: Use of a pH electrode.
References: Jacks, T. <3. "Phosphate Inhibition." J. Am. Oil Chem. Soc.,
51:112-113 (1974).
Kryson, J. L. "Paraoxon Inhibition of an Insect Egg Lipase."
Biochim. Biophys. Acta, 239:349-352 (1971).
Mates, A. "Inhibition by Alcohols." Lipids. 8:549-552 (1973).
Maylie, M. F. "Action of Organophosphates and Sulfonyl Halides
on Porcine Pancreatic Lipase." Biochim. Biophys. Acta, 276:
162-175 (1972).
Santhanam, K. "Studies on the Mechanism of Inhibition of Li-
polysis by Phenoxybenzamine." Life Sci. (II), 10:437-442
(1971).
Tsai, S. C. "Inactivation with ATP, Magnesium and Ascorbic
Acid." J. Biol. Chem., 248:5278-5281 (1973).
"N.A." - Information not available.
47
-------
A-14: ACETYLESTERASE (3.1.1.6)
Reaction Catalyzed: Acetic ester + H^O = alcohol + acetate
Source: Hog kidney, oranges
Stability: N.A.
Substrate Specificity: Greatest esterase activity toward esters of acetic
acid. Hog kidney: n-propylacetate, n-propylchloro-
acetate, mono-, di-, and tri-acetin, and n-nitro-
phenyl-acetate. Orange: methyl acetate, mono-,
di-, and tri-acetin, ethyl acetate, and acetylcholine.
Cofactor Requirements: None
Inhibitors: N.A.
Electrochemical Detection: Use a thiophenol acetate as substrate and moni-
tor the appearance of -SH.
References: Barman, T. E. Enzyme Handbook, Vol. II. Springer-Verlag, New
York (1969), p. 506.
"N.A." - Information not available.
48
-------
A-15: ALKALINE PHOSPHATASE (3.1.3.1)
Reaction Catalyzed: Orthophosphoric monoester + H20 = alcohol + orthophosphate
Source: Aldrich (chicken intestine) Cost: $36/g (0.9-2.2 U/mg)
Aldrich (E. coli) $29/5 mg (20-30 U/mg)
Stability: Lyophilized preparation stable 1-2 years when stored at 5°C.
Specific Activity: 24.8 lU/mg (E.coli), 350 ID/rng (calf intestine)
Substrate Specificity: p-Nitrophenyl phosphate is often used as the sub-
strate. Catalyzes a variety of phosphate esters
including esters of primary and secondary alcohols,
sugar alcohols, cyclic alcohols, and phenols
Cofactor Requirements: None
Inhibitors: Orthophosphate, L-tryptophan, L-phenylalanine, L-cysteine,
urea, NAD, Mg, strontium, cyanide, Cd, Al, Be, periodate, per-
manganate, reference to insecticides
Electrochemical Detection: A thiophosphate ester, if a suitable substrate,
may make detection of -SH possible as with the
present cholinesterase-based system.
References: Bamberger, C., J. Botbol, and R. L. Cabrini. "Inhibition of
Alkaline Phosphatase by Beryllium and Aluminum." Arch. Bio-
chem.Biophys., L23:195-200 (1968).
Belfield, A. and D. M. Goldberg. "Comparison of Sodium/3-Glyc-
erophosphate and Disodium Phenyl Phosphate as Inhibitors of
Alkaline Phosphatase in Determination of 5'-Nucleotidase Activ-
ity of Human Serum." Clin. Biochem., 3_:105-110 (1970).
Kshirsagar, S. 6. "The Effect of Stable Strontium on the Alka-
line Phosphatase Activity of Rat Tissues - In Vitro Studies."
Biochem. Pharmacol., 24:13-20 (1975).
Landoit, R. and E. E. Gutwein. "Alpha-Particle Autoradiography
of Alkaline Phosphatase Inhibition by Cadmium." Internat.
J. Appl. Rad. Iso.. 22:127-128 (1971).
Moss, D. W. "Influence of Metal Ions on the Orthophosphate Ac-
tivities of Alkaline Phosphatase." Biochem. J., 11(2) (1964).
Naqui, S. N. H. and S. A. Qureshi. "Studies of In Vivo Effect
of Varying Doses of Insecticides on the Phosphomonoesterases
of the Desert Locust (Schislocerca gregaria)." Folia Biologi-
ca., 17:409-419 (1969).
49
-------
Ohlsson, J. T. and I. B. Wilson. "The Inhibition of Alkaline
Phosphatase by Periodate and Permanganate." Biochim. Biophys.
Acta, 350:48-53 (1974).
Thandolt, R. "Alpha Particle Autoradiography of Alkaline Phos-
phatase Inhibition by Cadmium." Int. J. Appl. Rad. Iso., 22:
127-128 (1971). ~~
Thorling, E. B and M. S. Niazy. "Inhibition of Leucocyte Alkaline
Phosphatase by Cyanide In Vivo." Acta Med. Scand.. 194:271-
276 (1973).
50
-------
A-16: ACID PHOSPHATASE (3.1.3.2)
Reaction Catalyzed: Orthophosphoric monoester + hLO = alcohol + orthophosphate
Source: Aldrich (wheat germ) Cost: $16.00/g (0.15-0.3 U/mg)
Stability: Stable for years when maintained refrigerated as a dry powder.
Specific Activity: 93.3 lU/mg (E. freundei), 1,650 lU/mg (human)
Substrate Specificity: Broad esterase activity
Cofactor Requirements: None
Inhibitors: Heparin, formaldehyde, citrate, transition metals, reference
to insecticides
Electrochemical Detection: A thiophosphate ester, if a suitable substrate,
may make detection of -SH possible, as with
the present cholinesterase-based system.
References: Naqui, S. N. "Studies on In Vivo Effect of Varying Doses of
Insecticides of the Phosphomonoesterases of the Desert Locust."
Folia Biol., 17:409-419 (1969).
Van Etten, R. L. Letter - "Transition Metal Ion Inhibition of
Enzyme Catalyzed Phosphate Ester Displacement Reactions."
J. Am. Chem. Soc.. 96(21):6782-6785 (1974).
51
-------
A-17: UREASE (3.5.1.5)
Reaction Catalyzed: Urea + H20 = C02 + 2NH3
Source: Aldrich (jack bean meal) Cost: $15.00/mg (100 U/mg)
Aldrich (Bacillus sp.)
Stability: Purified urease is not very stable. Bacterial urease may be
stabilized by impurities and is stable 6-12 months at 4 C.
Specific Activity: 2,130 lU/mg (jack bean), 2,430 lU/mg (Bacillus sp.)
Substrate Specificity: Absolute substrate specificity
Cofactor Requirements: None
Inhibitors: Heavy metal ions, ammonia, metal ions
Electrochemical Detection: Adapt an ammonia-specific electrode to CAM-1.
References: Hughes, R. B. "Inhibition of Urease by Metal Ions." Enzymologia,
36:332-334 (1969).
Toren, E. C. Jr. "Trace Determination of Metal Ion Inhibitors
of the Urea-Urease System by a pH Stat Kinetic-Method." Mikro-
chim Acta. 5:1049-1058 (1968).
52
-------
A-18: ATPase (3.6.1.3)
Reaction Catalyzed: ATP + H£0 = ADP + orthophosphate
Source: Sigma Chemical Co. (pork brain) Cost: $8.00/5 U
Substrate Cost: ATP $40/10 g
Stability: N.A.*
Specific Activity: 114 lU/mg (beef heart with DNP), 24 lU/mg (skeletal
muscle myosin)
Substrate Specificity: Relative rates - ATP=100, TTP=125, GTP=75, UTP=2.0
Cofactor Requirements: Mg++ required for activity
Inhibitors: Aldrin, dieldrin, DDT, KeponeR, toxaphene, polychlorinated
biphenyls, Plictran
Electrochemical Detection:
1. Couple the reaction to an easily monitored reaction such as DPN-»DPNH.
2. Couple the reaction with isocitrate dehydrogenase (1.1.1.42) and
glutathione reductase (1.6.4.2) and measure -SH.
3. Couple the reaction with isocitrate dehydrogenase (1.1.1.42) and
carbonic anhydrase (4.2.1.1) and measure H .
References: Davis, P. W., et al. "Organochlorine Insecticide, Herbicide
and Polychlorinated Biphenyl (PCB) Inhibition of NaK-ATPase
in Rainbow Trout." Bull. Environ. Contam. Toxicol.. 8:69-72
(1972).
Desaiah, D., et al. "Inhibition of Fish Brain ATPases by Aldrin-
transdiol, Aldrin, Dieldrin and Photodieldrin." Biochem. Bio-
phys. Res. Commun.. 64(1):13-19 (1975).
Desaiah, D., et al. "Inhibition of Spider Mite ATPase by Plic-
tran and Three Organochlorine Acaricides." Life Sci., 13:1693-
1703 (1973). ~~~
Desaiah, D., et al. "Toxaphene Inhibition of ATPase Activity
in Catfish, Ictalurus Punctatus, Tissues." Bull. Environ.
Contam. Tcxicol., 13(2):238-244 (1975).
Desaiah, D., et al. "Inhibition of ATPase Activity in Channel
Catfish Brain by Kepone and its Reduction Product." Bull.
Environ. Contam. Toxicol., 13(2):153-158 (1975).
"N.A." - Information not available.
53
-------
Hexum, T. D. "Studies on the Reaction Catalyzed by Transport
(Na, K) Adenosine Triphosphatase. I. Effects of Divalent
Metals." Biochem. Pharmacol.. 23(24):3441-3447 (1974).
Koch, R. B., et al. "Polychlorinated Biphenyls: Effect of Long-
Term Exposure on ATPase Activity in Fish." Bull. Environ.
Contam. Toxicol.. _7:87-92 (1972).
Schneider, R. P. "Mechanism of Inhibition of Rat Brain ATPase
by DDT » Biochem. Pharmacol.. 24(9):939-946 (1975).
Tabakoff, B. "Inhibition of Sodium, Potassium, and Magnesium
Activated ATPases by Acetaldehyde and "Biogem'c" Aldehydes."
Res. Comm. Chem. Pathol. Pharmacol.. 7:621-624 (1974).
54
-------
A-19: CARBONIC ANHYDRASE (4.2.1.1)
Reaction Catalyzed: HgCOg (or H+ + HC03") = C02 + hLO
Source: Aldrich (beef blood) Cost: $10.00/50 mg (3,000-4,000 U/rag)
Stability: Lyophilized enzyme shows no loss of activity when stored refrig-
erated for 1 year.
Specific Activity: N.A.*
Substrate Specificity: Hydration of aldehydes: acetaldehyde, 2-, 3-, and
4- pyridine aldehydes, CH3CH?CHO, (CHo)? CHCHO.Es-
terase activity: p-nitrophenyl acetate and p-nitro-
phenyl thioacetate
Cofactor Requirements: None
Inhibitors: Methyl mercuric chloride, phenylmercuric chloride, lead ni-
trate, silver nitrate, dieldrin,. DDT, chlordane, 2,4,5-T,
cyanide, azide, heavy metal ions, cyanotnercurials
Electrochemical Detection: Possibility of detection of -SH if esterase
activity can be directed to a thioester.
References: Christenson, 6. M. "Effects of Selected Water Toxicants on the
In Vitro Activity of Fish Carbonic Anhydrase." Chem. Biol.
Interact., 13(2):181-192 (1976).
Hague, R. "Binding of the Chlorinated Hydrocarbon bis(p-Chloro-
phenyl) Acetic Acid with the Enzyme Carbonic Anhydrase." Bui 1.
Environ. Contam. Toxicol.. 14_(01): 143-146 (1975).
Maguire, J. "Carbonic Anhydrase Inhibition." Bull. Environ.
Contam., 13(5):625-629 (1975).
Maren, T. H. "Inhibition by Anions of Human Red Cell Carbonic
Anhydrase B: Physiological and Biochemical Applications."
Science, 191(4226):469-472 (1976).
Meiller, D. S. "Enzymatic Basis of DDE Induced Eggshell Thinning
in a Sensitive Bird." Nature. 259(5539):122-124 (1976).
Kandell, M. and A. G. Gonnall. "Some Characteristics of Human,
Bovine and Horse Carbonic Anhydrases Revealed by Inactivation
Studies." J. Biol. Chem.. 245(9):2444-2450 (1970).
Pocker, Y., M. W. Beug, and V. R. Ainardi. "Coprecipitation
of Carbonic Anhydrase by l,l-bis(p-Chlorophenyl)-2,2,2,-Tri-
chlorethane, l,l-bis(p-Chlorophenyl)-2,2-Dichloroethylene,
5: and Dieldrin." Biochemistry, 10(8)-.1390-1396 (1971).
"N.A." - Information not available.
55
-------
Pocker, Y. and J. E. Meany. "The Catalytic Versatility of Ery-
throcyte Carbonic Anhydrase. I. Kinetic Studies of the Enzyme-
Catalyzed Hydration of Acetaldehyde." Biochemistry. 4(11):2535-
2540 (1965).
Pocker, Y. and J. E. Meany. "The Catalytic Versatility of Car-
bonic Anhydrase. II. Kinetic Studies of the Enzyme-Catalyzed
Hydration of Pyridine Acetaldehyde." Biochemistry, 6(1):239-
246 (1965).
Pocker, Y. and J. E. Meany. "Catalytic Versatility of Erythro-
cyte Carbonic Anhydrase. The Enzyme-Catalyzed Hydration of
Nitrophenyl Acetate." J. Am. Chem. Soc.. 87:809 (1965).
Wilbur, K. M. and N. 6. Anderson. "Electrometric and Colori-
metric Determination of Carbonic Anhydrase." J. Biol. Chem.,
176:147 (1948).
56
-------
A-20: S-ALKYL CYSTEINE LYASE (4.4.1.6)
Reaction Catalyzed: S-methyl-L-Cysteine = pyruvate + NH3 + methyl mercaptan
Source: Pseudomonas cruciviae
Stability: N.A.*
Specific Activity: Variously substituted cysteines and cysteine sulfoxides
Cofactor Requirements: None
Inhibitors: N.A.
Electrochemical Detection: Detection of -SH, as with cholinesterase-based
system.
References: Barman, T. E. Enzyme Handbook, Vol. II. Springer-Verlag, New
York (1969) p. 812.
"N.A." - Information not available.
57
-------
APPENDIX B
METHODS OF ENZYME INHIBITION ANALYSIS
B-l CARBONIC ANHYDRASE INHIBITION ASSAY METHOD*
Reaction Catalyzed
carbonic
anhydrase
0
CH3-COH
p-nitrophenylacetate
p-nitro-
phenolate am" on
p-nitrophenol
acetic
acid
Reagents
Carbonic anhydrase (4.2.1.1): BCA (bovine erythrocyte) Sigma Chemical
Co. No. C-7500, Lot No. 86C-8125. 0.15 mg/50 ml buffer.
p-Nitrophenylacetate (NPA): Sigma Chemical Co. No. N-9126, Lot. No.
44C-1530.
For organic inhibitors: 4 x 10" _M NPA (366 mg/5 ml acetonitrile)
For inorganic inhibitors: 4 x 10 M NPA (36.6 mg/5 ml acetonitrile)
Tris: 0.009 M, brought to a final ionic strength of 0.09 with NaCl and
adjusted to pH 7.5 with concentrated HC1
Pocker, Y. and J. T. Stone. "The Catalytic Versatility of Erythrocyte
Carbonic Anhydrase. III. Kinetic Studies of the Enzyme-Catalyzed Hy-
drolysis of p-Nitrophenol Acetate." Biochemistry, £(3):668-678 (1967),
58
-------
Reaction Conditions (in cuvette)
BCA: 10"7 M
p-NPA: 2 x 10"3 M
Acetonitrile: 10%
Buffer: 0.01 M; pH 7.2, diethylmalonate
Procedure
Inorganic Inhibitors--
1. Place 2.7 ml buffered enzyme in the cuvette.
2. Add 0.03 ml aqueous inhibitor and mix.
3. Layer 0.3 ml 0.04 M p-NPA in acetonitrile on top of the aqueous phase
and mix.
4. Read the OD.QQ for 2 min.
Organic Inhibitors--
1. Place 2.7 ml buffered enzyme in the cuvette.
2. Layer 0.3 ml acetonitrile solution of inhibitor on top of the aqueous
phase.
3. To this add 0.03 ml 0.4 M p-NPA standard and mix.
4. Read the OD,g0 for 2 min.
59
-------
B-2 CARBONIC ANHYDRASE ASSAY, ELECTROMETRIC METHOD*
Reaction Catalyzed
C02 + H20—»H2 C0|—?H+ + HC03"
Reagents
Lyophilized enzyme powder: Sigma Chemical Co. No. C-7500, dissolve
in ice cold water (0.1 mg/ml). Store in ice bath. Immediately prior
to use, dilute to a concentration of approximately 0.01 mg/ml in ice
cold water.
Carbon dioxide-saturated water: Bubble C02 gas through 200 ml ice cold
water for 30 min prior to assay. During saturation process, store water
at 0 to 4 C in an ice bath.
Tris-HCI buffer: 0.02 M, pH 8.0. Store in an ice bath at 0° to 4°C
before and during use.
Procedure
Blank determination--
1. Add 6.0 ml of chilled 0.02 M Tris-HCI buffer (pH 8.0) to a 10-ml beaker.
2. Maintain temperature at 0° to 4°C and record pH.
3. Withdraw4 ml of chilled C02~saturated water and add to Tris buffer.
4. Immediately start a stop watch and record the time (T ) required for
the pH to drop from 8.3 to 6.3.
Enzyme Determination—
1. Add 6.0 ml of chilled 0.02 M Tris-HCI buffer (pH 8.0) to a 10-ml beaker.
2. Maintain temperature at 0° to 4°C and record pH.
3. Add 0.1 ml of freshly diluted enzyme.
4. Quickly add 4 ml of C02-saturated water and record the time (T) required
for the pH to drop from 8.3 to 6.3.
Wilbur, K. M. and N. G. Anderson. "Electrometric and Colorimetric Deter-
mination of Carbonic Anhydrase," J.Biol. Chem., 176, 147 (1948).
Decker, L. A. (Ed.) Worthington Enzyme Manual. Worthington Biochemical
Corporation, Freehold, New Jersey (1977), pp. 280-281.
60
-------
Calculation
2(T0-T)
Umts/mg = (j) (mg enzyme in reaction mixture)
T = time without enzyme (blank)
T = time with enzyme (test)
61
-------
B-3 GLUCOSE OXIDASE INHIBITION ASSAY METHOD*
Reaction Catalyzed
D-glucose + 0- — > D-glucono-S-lactone
H900 + o-dianisidine perox1dase > oxidized o-dianisidine
i * (460 nm)
Reagents
Glucose oxidase (1.1.3.4): Type V(Aspergil1us Niger). Sigma Chemical
Co. Lot No. 17C-02141. 5mg/ml (1385 units/ml). For a standard solution,
make a 1:40 dilution of the enzyme, 0.125 mg/ml buffer.
18% Glucose: Allow mutarotation to come to equilibrium by standing
overnight.
Potassium phosphate buffer: 0.1 M (pH 6.0)
Peroxidase: Dissolve peroxidase at a concentration of 200 jjg/ml in glass
distilled water.
1% o-Dianisidine: Note o-dianisidine has been reported to be carcin-
ogenic in the solid form.
Dianisidine-buffer mixture: Prepare by dissolving 0.1 ml of 1% o-di-
anisidine in 12 ml of 0.1 M potassium phosphate buffer (pH 6.0).
Procedure
1. Add 0.990 ml of inhibitor solution (in 0.1 M phosphate buffer, pH 6.0/
10% acetonitrile) to a test tube.
2. Add 0.010 ml of standard enzyme solution and incubate at room temperature
for 4 min.
3. Set the spectrophotometer at 460 nm and 25°C. Pipette into cuvette as
follows:
Sample Reference
Dianisidine-buffer mixture (pH 6'.0)...2.5 ml 2.6 ml
18% Glucose ........................... 0.3 ml 0.3 ml
Peroxidase ............................ 0.1 ml 0.1 ml
Test solution of glucose oxidase
inhibitor ........................... 0.1 ml —
Decker, L. A. Wgrthington Enzyme Manual. Worthington Biochemical Cor-
poration, Freehold, New Jersey (1977), pp. 37-38.
62
-------
4. Read the OD.go at room temperature for 4 min.
Calculation
_
Umts/mg = (11.3) ( mg enzyme/ml reaction mixture)
63
-------
B-4 ALKALINE PHOSPHATASE INHIBIION ASSAY METHOD*
Reaction Catalyzed
p-nitrophenylphosphate >p-nitrophenol + HoPO,
(410 nm)
Reagents
Alkaline phosphatase (3.1.3.1): Type III-S (Escherichia coli), Sigma
Chemical Co. No. P-4377, Lot No. 32C-6030 (144 units/ml).For a standard
solution, dilute to 3.7 1 enzyme/250 ml buffer.
p-Nitrophenylphosphate (p-NPP): 0.05 M
Tris-HCl: 1.5 M (pH 8.0)
Reaction Conditions (in the cuvette)
Q
Alkaline phosphatase: 2 x 10 M
p-NPP: 0.5 mM
Inhibitor: 10"4 M
Acetonitrile: 10%
Procedure
1. Set the Beckman 25 recording spectrophotometer at 410 nm.
2. Add, as follows, to the sample and reference cuvettes:
Sample Reference
Buffer — 2.7 ml
Buffered enzyme 2.7 ml —
10 M inhibitor in acetonitrile..0.3 ml 0.3 ml
3. Mix the contents and incubate 2 min at room temperature.
4. Add 0.03 ml p-NTP to each cuvette and record the OD.,Q for 2 min.
Decker, A. L. (Ed.) Worthington Enzyme Manual. Worthington Biochemical
Corporation, Freehold, New Jersey (1977), pp. 138-140.
64
-------
B-5 CARBOXYLESTERASE INHIBITION ASSAY METHOD*
Reaction Catalyzed
carboxvlesterase
410 nm
CH3-C-OH
p-nitrophenylacetate
Reagents
p-nitro-
phenolate anion
p-nitrophenol
acetic
acid
CarboxyTesterase (3.1.1.1): Type I (porcine liver), Sigma Chemical Co.
NO. E-3128, Lot No. 106C-8010 (0.065 units/ml).
p-Nitrophenylacetate: 3.5 x 10
Tris: 0.02 (pH 8.0)
Procedure
1. Set the spectrophotometer at 410 nm.
2. To an aliquot of standard enzyme, add a small volume of methanolic TOCP
(calculated to result in the desired test concentration).
3. Incubate at room temperature.
4. Add 3.0 ml of buffered p-NPA to the sample and reference cuvettes.
5. Add 50 ul of the esterase - TOCP (from Step 2) to the sample cuvette.
6. Record the OD41Q for 3 min.
*Decker, L. A. Horthington Enzyme Manual. Worthington Biochemical Cor-
poration, Freehold, New Jersey (19//), pp. 280-281.
Sadar, M. H. and 6. G. Guilbault. "A Specific Method for the Assay of
Select Chlorinated Pesticides" J. Agr. Food Chem.. 19(2):357359.
65
-------
B-6 HEXOKINASE INHIBITION ASSAY METHOD*
Reaction Catalyzed
D-glucose + ATP hexokini*se> glucose-6-phosphate + ADP
glucose- + NAD r' > «1 uconate-6-phosphate J- NADH
D-phospnate (340 nm)
Reagents
Hexokinase: 0.0146 units/ml
Glucose: 1.86 mM (in buffer)
Tris-HCl: 0.5 M (pH 8.0), with 13.3 mM/MgCl2
ADP: 16.5 mM (in buffer)
NAD: 6.8 mM (in buffer)
Glucose-6-phosphate dehydrogenase: 0.97 units/ml
Procedure
1. Set the Beckman 25 recording spectrophotometer at 340 nm (UV source),
2. Add, as follows, to the sample and reference cuvetts and mix:
Sample Reference
Tris-MgCl? buffer .................. 2.28 ml 2.28 ml
Glucose c ......................... 0.50ml 0.50ml
ATP ................................ 0.10 ml 0.10 ml
NAD ................................ 0.10 ml 0.10 ml
3. Add and mix either a or b:
Sample Reference
a. Organic inhibitor in dioxane..0.1 ml 0.1 ml
b. Inorganic inhibitor/buffer.... 0.1 ml 0.1 ml
66
-------
4. Add and mix:
Sample Reference
6-6-PDase 0.02 ml 0
Hexokinase 0.02 ml 0
5. Read at OD for 3 min.
67
-------
APPENDIX C
ABSTRACTS OF PREVIOUS CAM-INSTRUMENT REPORTS
C-l CAM-4, A PORTABLE DEVICE FOR OR6ANOPHOSPHATE HAZARDOUS MATERIAL SPILLS
(ENVIRONMENTAL PROTECTION TECHNOLOGY SERIES: FINAL REPORT ON TASK II,
CONTRACT NO. 68-03-0299)
Previously, an instrument designated as CAM-1 ("CAM" is an acronym for
"cholinesterase antagonist monitor") was constructed to continuously monitor
the levels of organophosphate and carbamate pesticides in water (ponds, streams,
plant outfalls, etc). CAM-1 is a sophisticated research instrument that can-
not be conveniently used in the field or from a boat because of its non-rug-
gedized construction. To meet the requirements for an equally sensitive but
portable system, CAM-4 was developed. CAM-4 is a field version of CAM-1 that
will operate continuously in the field from a 12-v DC power supply for eight
hours, or from 110-v AC. The present report describes the design, fabrica-
tion, and evaluation of CAM-4.
Operation of the CAM instruments is based on inactivation of the enzyme
cholinesterase by organophosphate and carbamate pesticides. The extent of
inactivation, which is proportional to the amount of inhibitor present, is
determined by measuring the response of the system to a substrate readily
hydrolyzed by the enzyme.
The conventional method for detecting organophosphates and carbamates
in water requires the addition of cholinesterase, a buffer, and an enzyme-
hydrolyzable substrate, e.g., butyrylthiocholine iodide, to a water sample,
followed by spectrophotometric determination of the residual substrate concen-
tration. The spectrophotomeric method is disadvantageous because: (a) a
significant quantity of costly enzyme is needed for each test, and (b) the
system is not easily adpated to continuous use since the optical windows
become dirtied by the flow of contaminated water. It is necessary to correct
this problem by comparing the response of the contaminated sample, plus rea-
gents, to that obtained with a contaminated sample alone. However, even with
this correction, frequent cleaning is necessary. The CAM systems eliminate
these problems. Entrapping a given amount of enzyme in starch gel on the
surface of open-pore polyurethane foam makes repeated use possible over ex-
tended periods of time. Residual activity of the enzyme is measured elec-
trochemical ly.
In the CAM system, residual activity is determined during a sampling
cycle by the level of substrate hydrolysis product present in the electro-
chemical cell. Presence of a cholinesterase inhibitor reduces the rate of
68
-------
substrate hydrolysis (thiol formation) and produces an increase in cell vol-
tage. The magnitude of the increase above the enzyme pad potential is a
function of the residual enzyme activity; that is, the voltage rises as the
enzyme is inhibited. A voltage increase from one sampling cycle to the next,
above a designated alarm threshold, is used to trigger an "alarm" indicating
the presence of cholinesterase inhibitors.
Development of a portable system necessitated elimination of the follow-
ing convenience features from CAM-1: the computer logic circuits, automatic
pad changer, digital voltmeter, strip chart recorder, and audible alarm compon-
ents. CAM-4 contains an inverter to .transform DC to AC current and a digital
printer to record cell voltage. An operator must read the digital printout
to determine when there is an "alarm" condition and when it is necessary to
replace the enzyme pad.
CAM-4 possesses detection and monitoring capabilities equal to those
of CAM-1. The sensitivity of CAM-4 to subtoxic levels of DDVP, Systox , Fura-
dan , malathion, Sevin , and other pesticides in water is comparable to the
sensitivity of CAM-1 for the same pesticide solutions.
This report was submitted in fulfillment of Contract No. 68-03-0299,
Task No. 2, by Midwest Research Institute under the sponsorship of the U.S.
Environmental Protection Agency. This report covers the period from 3 April
1975 to 3 May 1976 and work was completed as of 3 July 1976.
69
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C-2 EVALUATION OF "CAM-1", A WARNING DEVICE FOR OR6ANOPHOSPHATE HAZARDOUS
MATERIAL SPILLS (ENVIRONMENTAL PROTECTION TECHNOLOGY SERIES EPA-600/77-
219, NOVEMBER 1977; FINAL REPORT ON TASK I, CONTRACT NO. 68-03-0299)
The Cholinesterase Antagonist Monitor (CAM-1) has been operated with
water containing a variety of pollutants including organophosphates, carbam-
ates, chlorinated hydrocarbons, and various other economic poisons, and its
sensitivity to many of these materials has been measured. With few exceptions
only the organophosphates and carbamates are detectable with CAM-1. One of
these exceptions is zinc, at 10 ppm, which inactivates cholinesterase and
behaves in CAM-1 like the organophosphates. Another compound detectable under
certain conditions is the reversible cholinesterase antagonist, tributyl amine
hydrochloride; it is detectable for only one or possibly two cycles when a
sudden increase in the concentration of the reversible inhibitor occurs.
The non-reversible enzyme inhibitors, on the other hand, produce repeated
voltage increases until the enzyme in CAM-1 is completely inactivated. CAM-
1 is recommended only for the detection of non-reversible inhibitors.
Correlation of the sensitivity of CAM-1 with the chemical structures
of a group of organophosphate pesticides has shown that CAM-1 is generally
0
more sensitive for the phosphate ( 0-P=0) compounds than for the phosphoro-
0 0 p
thioate ( 0-£=S) or the phosphorodithioate ( S-P=S) compounds, even though
0 0
the animal toxicities of these different types of compounds may be very close.
Operation of CAM-1 in simulated sea water (3% NaCl) changes the voltages
registered on the digital voltmeter, but it does not change the sensitivity
of CAM-1 for compounds like DDVP; thus, CAM-1 is suitable for the detection
of cholinesterase inhibitors in either sea or brackish waters. CAM-1 has
much promise for monitoring of water supplies and plant effluents for cholin-
esterase inhibitors, but it is so new that it should be investigated under
the conditions of intended usage prior to putting it into regular service.
70
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C-3 RAPID DETECTION SYSTEM FOR ORGANOPHOSPHATES AND CARBAMATE INSECTICIDES
IN WATER (ENVIRONMENTAL PROTECTION TECHNOLOGY SERIES EPA-R2-010, AUGUST
1972; FINAL REPORT, CONTRACT NO. 68-01-0038)
An apparatus for the detection and monitoring of water supplies for haz-
ardous spills of organophosphate and carbamate insecticides has now been de-
signed and fabricated. The new unit is called the Cholinesterase Antagonist
Monitor, CAM-1, because it produces an alarm in 3 min when toxic or subtoxic
levels of Cholinesterase antagonists are present in water. Response of this
apparatus to subtoxic levels of azodrin, Sevin , dimetilan, malathion, para-
thion, and DDVP has already been demonstrated. CAM-1 uses immobilized Cholin-
esterase for the collection of Cholinesterase inhibitors from the water sup-
plies. The activity of the immobilized Cholinesterase is determined automati-
cally in an electrochemical cell by passing a substrate solution over the
enzyme at regular time periods. A minicomputer is used to automate the de-
tection process and to signal an alarm when there is a rapid loss of enzyme
activity—a situation that occurs in the presence of organophosphate and
carbamate insecticides in the water sampled.
This report was submitted in fulfillment of Project No. 15090-GLU, Con-
tract No. 68-01-0038, under sponsorship of the Water Quality Office, Environ-
mental Protection Agency.
71
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-80-083
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
ALTERNATE ENZYMES FOR USE
MONITORS ("CAM'S")
5. REPORT DATE
IN CHOLINESTERASE ANTAGONIST
May 1980 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
L.H. Goodson
V.J. Appleman
8. PERFORMING ORGANIZATION REPORT NO,
MRI 3820-B, Final Report
Task III
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, Missouri 64110
10. PROGRAM ELEMENT NO.
1BB610 Project 00202
11. CONTRACT/GRANT NO.
68-03-0299
12. SPONSORING AGENCY NAME AND ADDRESS
Industrial Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
ninr.innati. OH 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Report 11/76-9/77
14. SPONSORING AGENCY CODE
EPA/600/12
15. SUPPLEMENTARY NOTES
This report describes Task III of Contract No. 68-03-0299
16. ABSTRACT
The Cholinesterase Antagonist Monitors ("CAM'S") normally use cholinesterase as the
sensor in the detection of organophosphate and carbamate pesticides. The present in-
vestigation has been concerned with a search for alternate enzymes that could be used
in the CAM system and that would enable it to detect a variety of other types of
environmentally important toxic chemicals including chlorinated hydrocarbons, phenols,
aryl phosphates, cyanide, heavy metals, etc. Five enzymes including alkaline phos-
phatase, carboxyl esterase, glucose oxidase, carbonic anhydrase, and hexokinase have
been incubated with dilute solutions of toxic chemicals and the degree of enzyme in-
hibition measured. Two of the enzymes, hexokinase and carbonic anhydrase, were in-
hibited by low levels of toxic test compounds. It is concluded that these and
similar enzymes are of potential value in the detection and monitoring of toxic
substances in water.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. cos AT I Field/Group
Enzyme inhibitors
Monitors
Enzymes
Chemical agent detection
Water pollution
Glucose oxidase; carboxyl
esterase; alkaline phos-
phatase; carbonic anhy-
drase; hexokinase; toxic
substances; chlorinated
hydrocarbon, aryl phospha
cyanide, and heavy metal
6A
13B
e, phenol,
etection
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
72
ft U.S. GOVERNMENT POINTING OFFICE: 1980-657-146/5671
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