The determination of chlorsulfuron by the inhibition of acetolactate synthase
by Gary William Kagel
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Chemistry
Montana State University
© Copyright by Gary William Kagel (1987)
Abstract:
The enzyme acetolactate synthase (ALS, EC 4.1.3.18) is the site of action of the herbicide
chlorsulfuron (2-chloro-N-[(4-methoxy-6-methyl-l,3,5,-triazin2yl)aminocarbonyl]benzenesulfonamide). This enzyme was extracted from plant material and
immobilized in a cartridge packed with a silica based activated affinity media. The activity of the
immobilized enzyme was inhibited by nanomolar concentrations of chlorsulfuron.
A 60% recovery of radio-labeled chlorsulfuron from treated soil was demonstrated. Analytical
standards of chiorsulfuron recovered from soil inhibited immobilized ALS to a greater degree at higher
herbicide concentrations.
Results indicate this technique is capable of chlorsulfuron detection down to about 1 ppb (1.12 g/ha).
This technique, therefore, represents a method with the potential to be faster and more economical than
the present method which uses high performance liquid chromatography. THE DETERMINATION OF CHLORSULFURON BY THE INHIBITION OF
ACETOLACTATE SYNTHASE
by
Gary William Kagel
A thesis submitted in partial fulfillment
of the requirements for the degree
of
.
Master of Science
in
Chemistry
MONTANA STATE UNIVERSITY
Bozeman, Montana
July 1987
MAIN L>l,
tins
Cq^. <2/
ii
APPROVAL
of a thesis submitted by
Gary William Kagel
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
Approved for the Major Department
H e a d , Major Department
Date
Approved for the College of Graduate Studies
0
Date
Graduate Dean
ill
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of
the requirements for a master's degree at Montana State
University,
I agree that the Library shall make it available
to borrowers under rules of the Library.
Brief quotations
from this thesis are allowable without special permission,
provided that accurate acknowledgment of source is ma d e .
Permission for extensive quotation from or reproduction
of this thesis may be granted by my major professor, or in
his absence, by the Dean of Libraries when,
in the opinion
of either,
the proposed use of the material is for scholarly
purposes.
Any copying or use of the material in this thesis
for financial gain shall not be allowed without my written
permission.
This thesis is dedicated to Mr. and Mrs. Raymond Kagel
without whose boundless help, love, and understanding this
work could never have been completed.
V
ACKNOWLEDGEMENTS
.I thank my advisor, Dr. Pete Fay, for the help and
guidance he provided throughout this project.
I
also appreciate the assistance and encouragement
given by my committee, Wyn Jennings, Pat Callis, and Rich
Stout.
I would also thank my fellow graduate students Bill
NeiIsen, Tim Hanks, Edie Parsons, Mark Stannard, Eric
Gallandt, and Ed Davis for their help, support and
friendship.
Additional thanks to Joe DiTomaso for his help in
editing this thesis.
vi
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS -..................
v
TABLE OF CONTENTS ........................... vi
LIST OF TABLES............................ viii
LIST OF FIGURES .......................... .
ix
ABSTRACT..................
x
LITERATURE REVIEW ........................
I
Chapter
1
Introduction.................
Synthesis of Chlorsulfuron. . ..........
Persistence in S o i l ................ .. .
Mode of Action..........................
Acetolactate Synthase........ ' ........
Chlorsulfuron Determination . ..........
Enzymes as Analytical Reagents.. . . . .
Immobilized E n z y m e s ........ ■ .......... 11
2
EXTRACTION AND ASSAY OF ACETOLACTATE
SYNTHASE. . . . . . . . ..............
,
Abstract............................
Introduction..............................
Materials and Methods ..................
Results and Discussion....................
3
..I
2
3
4
4
7
9
13
13
13
14
21
IMMOBILIZED ACETOLACTATE SYNTHASE AS A
DETECTOR FOR CHLORSULFURON................ 23
Abstract..................
23
Introduction. ............................. 24
Materials and Methods . . . . . . . . . . .
24
Results and Discussion.................... 26
Conclusion. ............................... 28
vii
Chapter
4
Page
THE EXTRACTION AND DETERMINATION OF
Ch l o r s u l f u r o n f r o m s o i l .. ............... 30
Abstract............................... .
Introduction..........
Materials and Methods . . . . . ........
Results and Discussion....................
Conclusion........
BIBLIOGRAPHY.......... ................... 37
30
30
31
33
35
viii
LIST OF TABLES
Table
Page
1.
The ALS activity and protein concentration
for. 30 fractions collected from an anion
exchange HPLC c o l u m n ........ ........... 2 2
2.
Recovery data for the soil extraction and
enrichment of the extract for
chlorsulfuron.
................ ..
34
ix
LIST OF FIGURES
Figure
Page
1.
The structure of chlorsulfuron ............
2
2.
The biosynthetic pathways of valine, leucine,
and isoleucine ............................. 5
3.
Reaction pathway used to determine ALS
activity................................. 18
4.
Standard curve developed using standard
acetoin and a method designed by
Westerfeld, and.modified by Ray............ 19
5.
Standard curve developed using standard
serum albumen and the Bradford total
protein assay............................. 20
6.
Reaction of Hydropore-EP with a hydroxyl
moiety on the surface of a protein . . . .
26
7.
The stability of ALS in solution vs. that
of immobilized A L S ....................... 27
8.
The inhibition of immobilized ALS by
nanomolar concentrations of chlorsulfuron. 28
9.
The inhibition of immobilized ALS by soil
extracts .................... . . . . . .
10.
35
The inhibition of immobilized ALS by soil
extracts close to the detection limit. . . 36
X
ABSTRACT
The enzyme acetolactate synthase (A L S , EC 4.1.3.18) is
the site of action of the herbicide chiorsulfuron (2-chloroN-1 (4-methoxy-6-methyl-l,3,5,-triazin2yl)aminocarbonyl]benzenesulfonamide). This enzyme was
extracted from plant material and immobilized in a cartridge
packed with a silica based activated affinity media.
The
activity of the immobilized enzyme was inhibited by
nanomolar concentrations of chlorsulfuron.
A 60% recovery of radio-labeled chlorsulfuron from
treated soil was demonstrated.
Analytical standards of
chlorsulfuron recovered from soil inhibited immobilized ALS
to a greater degree at higher herbicide concentrations.
Results indicate this technique is capable of
chlorsulfuron detection down to about I ppb (1.12 g/ha).
This technique, therefore, represents a method with the
potential to be faster and more economical than the present
method which uses high performance liquid chromatography.
I
CHAPTER I
LITERATURE REVIEW
Introduction
Chlorsulfuron [2-chloro-^V- { (4-methoxy-6-methyl-l ,3,5triazin-2-yl) aminocarbonyI}benzenesulfonamide], (Figure I)
is a selective herbicide used for broadleaf weed control in
wheat ( Triticum aestivum L .) , oats (Avena sativa L .) , and
barley (Hordeum vulgare L.).(Levitt et a l ., 1981).
Chlorsulfuron, and other sulfonylurea herbicides, injure
sensitive weed species at extremely low rates of
preemergence and postemergence application (16 g/ha)
1982).
(Ray,
Wheat, is tolerant to chlorsulfuron as it rapidly
metabolizes the herbicide into nonphytotoxic compounds
(Sweetser et al.,
1982).
Chlorsulfuron represents a major
advancement in weed control technology as.it is highly
active at rates less than 0.5 g/ha, yet has shown little
injury to cereal crops and very low mammalian toxicity
(Levitt,
1978).
The primary site of chlorsulfuron activity is the
enzyme acetolactate synthase (ALS)
(EC 4.1.3.18).
ALS is
the first common enzyme in the biosynthetic pathway for the
amino acids valine,
leucine, and.isoleucine (Dailey and
2
Cronan, 1984).
Chaleff (1984) demonstrated that other
sulfonyl ureas are also potent inhibitors of ALS activity.
Cl
OCH3
CH3
Figure I.
The structure of chlorsulfuron
Synthesis of Chlorsulfuron
Chlorsulfuron is synthesized by adding an equivalent of
2-chlorobenzenesulfonyl isocyanate (Ulrich and Sayigh,
to a suspension of 2-amino-4-methoxy-6-acetonitrile.
1966)
After
stirring for 2 to 16 h at room temperature, the mixture is
filtered.
The precipitate is washed with ethyl ether to
yield chlorsulfuron which melts at 174-178 ° C .
Results of
nuclear magnetic resonance, infrared absorption spectra, and
elemental analyses of the product were consistent with the
proposed structure (Huffman and Schaefer, 1963).
The
compound is moderately soluble in methylene chloride,
less
soluble in acetone and acetonitrile, and slightly soluble in
3
hydrocarbon solvents.
25° c .
Its solubility in water is 125 ppm at
Chlorsulfuron is stable to sunlight when dry.
Hydrolysis occurs in distilled water with an average halflife of 4-8 weeks at pH 5.7-7.O and 20° C .
Hydrolysis is
accelerated to 1-2 days in solutions below pH 5.
Polar
organic solvents also promote hydrolysis (Hartley,
1983).
Persistence in Soil
Chlorsulfuron residues are degraded biologically by
soil microbes (Joshi et a l ., 1958), and chemically by acid
hydrolysis.
In some soils, however,
active levels for several yea r s .
they may remain at
Microbial degradation in
soil occurs more rapidly when moisture,
temperature, pH,
nitrogen and carbon content are optimal for microbial
growth.
Acid hydrolysis,
the major mechanism for breakdown
(Palm et al., 1980), proceeds more rapidly at high soil
temperature, high soil moisture and low soil pH.
Since
degradation rates are controlled by these soil variables,
persistence varies with time and site of application (Walker
and Brown,
1983).
There is a need for a suitable analysis
for chlorsulfuron in field soils.
The technique most
commonly used at present involves HPLC analysis
1982).
sample.
(Zahnow,
This method of analysis costs more than $200 per
4
Mode of Action
Chlorsulfuron selectively stops growth by inhibiting
cell division (Rost, 1984).
Ray (1984) and Chaleff (1984)
related this inhibition to ALS activity.
A correlation
between ALS inhibition and mitosis arrest was also
demonstrated by Rost and Reynolds
(1985).
They demonstrated
a reversal in chiorsulfuron-induced inhibition of cell
division by supplying isoleucine and valine to treated
'
plants, and Ray (1984) showed a reversal of chlorsulfuroninduced growth inhibition in pea root culture by isoleucine
and valine applications.
LaRossa and Schloss (1984) reported that another
sulfonyl urea herbicide, sulfometuron methyl,
is
bacteriostatic as a result of ALS inhibition.
These
herbicides are potent, selective inhibitors of ALS with
values as low as 18 nM for chlorsulfuron and 15 nM for
sulfometuron methyl.
Another promising new class of herbicides, the
imidazolinones, also inhibit ALS activity (Muhitch et a l .,
1987).
However, there is no apparent structural similarity
between the two herbicide classes.
Acetolactate Synthase
The biosynthesis of isoleucine and valine occurs
through parallel synthetic pathways-(Figure 2).
The first
step involves the ALS catalyzed synthesis of either
acetohydroxybutyrate or
-acetolactate from the condensation
5
of the active acetaldehyde from
respectively (Umbarger and Brown,
-ketobutyrate or pyruvate,
1958).
THREO NINE
THREONINE DEAMINASE
PYRUUflTE
ct-KETOBUTVRRTE
ACETOL ACT ATE SYNTHASE
cc-flCETOHYDROHY
BUTYRATE
I
I
I
cc-ACETOLACTATE
I
Zl
VALINE
LEUCINE
ISOLEUCINE
Figure 2.
Acetolactate synthase is the first common enzyme
in the parallel biosynthetic pathways of valine,
leucine, and isoleucine.
There are several isozymes of ALS in most species,and
all of those investigated apparently catalyze the formation
of both acetolactate and acetohydroxybutyrate (DeFelice et
a l ., 1985).
found,
ilvG,
1985).
In E. coli, six isozymes of ALS have been
four of which are encoded by a separate gene (iJvB,
ilvH,
iIvJ ), and have been described (Schloss et al.,
Only two of these have been purified (Grimminger and
6
Umbarger, 1979).
Later work revealed that the isozyme ALS-I
from E . coli, consists of two dissimilar polypeptides
(Eoyang and Silverman,
1984).
The larger subunit (60 kDa)
is presumably encoded by H v Q and the smaller subunit (9.5
kDa) has been designated as the ilvM product.
It has also
been determined that the stop and start codons of jJvG and
JJvM overlap.
Overlapping stop and start codons have also
been observed for several other genes whose gene products
are produced in equal amounts.
• Schloss et a l . (1985) developed an improved method for
the purification of ALS-II from Salmonella typhimurium and
showed that native acetolactate synthase has a molecular
weight of about 140 kDa and consists of two large and two
small subunits weighing about 59.3 and 9.7 k D a ,
respectively.
The contribution of the different subunits to enzyme
function has yet to be systematically examined.
isozyme is encoded by the IIvl (large) and H v R
The ALS-III
(small)
subunits (DeFelice et al., 1947), and is sensitive to
feedback inhibition by L-valine.
Some JJvH mutations lead
to production of a valine-resistant enzyme (Squires et al.,
1981), suggesting that the small subunit of ALS-III plays a
role in the valine sensitivity of that enzyme.
These
mutations also effect the specific activity of ALS-III, at
least for acetolactate formation.
ALS sensitive to end-
product inhibition by valine from E . coll strains W and K-12
7
s h o w sensitivity characteristics that are consistent with
the two-site model proposed to account for the regulation of
enzyme activity by specific small molecules (Bauerle et a l .,
1964).
Escherichia coli mutants which are missing one of
the two valine-sensitive ALS enzymes have major membrane
defects
(DeFelice et al., 1985).
ALS-I catalyzes the
reaction with acetolactate more efficiently than with
acetohydroxybutyrate, although the opposite effect is
measured for ALS-II (DeFelice et al., 1978).
This indicates
that the isozymes of ALS function in a complimentary
fashion.
Cara and De Felice (1979) separated these isozymes
by low pressure ion exchange chromatography.
al.
Grimminger et
(1979) purified ALS-I to near homogeneity and reported
that the isozyme consists of two subunits of approximately
60,000 molecular weight each with an isoelectric point of
4..9 and a pH optimum of 7.5.
Leavitt and Umbarger (1961)
reported that ALS requires thiamine pyrophosphate (TPP),
Mg++ , and flavin adenine dinucleotide (FAD)
Umbarger, 1964).
(Stormer and
This requirement for FAD is unusual for an
enzyme catalyzing a reaction in which no net oxidation or
reduction occurs.
Chlorsulfuron determination
Chlorsulfuron is persistent in so i l , therefore an
accurate soil assay method is needed.
Detection is
exceedingly difficult since the herbicide is applied to soil
at rates as low as 4 g/ha.
Phytotoxic residues can occur in
8
soil at concentrations' as low as 35 parts per trillion.
Computer software has been developed to model the
decomposition of chlorsulfuron in soil.
These programs take
into account the soil variables which affect rates of
degradation (Walker and Brown,
1964).
The method is not
widely used because it requires detailed soil histories.
Zahnow (1982;
1985)developed high performance liquid
chromatography (HPLC) methods for the determination of both
chlorsulfuron and sulfometuron methyl in soil using
selective photoconductivity detection (Walters, 1983).
He
was able to quantify chlorsulfuron at levels as low as 0.2
ppb.
Slates (1983) designed a similar HPLC method for
determining chlorsulfuron residues in grain, straw, and
green plants of cereals.
While these methods utilize the
selective conductivity detector,
they suffer from high
background levels of interference.
A sensitive enzyme-Iinked immunosorbent assay (ELISA)
has also been developed for chlorsulfuron with a detection
limit of 0.4 ppb.
This technique can quantify nanogram
levels of chlorsulfuron in crude soil extracts (Kelley et
a l ., 1985).
Although this has shown promise, more research
is needed to develop consistent antibodies.
Bioassays are also useful in detecting the presence of
low levels of chlorsulfuron in soil.
Nilsson (1981)
detected chlorsulfuron residues in soil treated at a rate of
10 g/ha 11 months before sampling.
In our research group we
9
have demonstrated a useful method of measuring chlorsulfuron
levels in soil by plant bioassay using five plant species
ranging in their sensitivity to the herbicide (Fay and
Fellows,
1987). Although bioassays can be very sensitive,
they are time consuming and difficult to quantify.
Enzymes as Analytical Reagents
Specific chemical substances often inhibit enzymes at
very low concentrations, and have been used as analytical
transducers
(Guilbault, 1969; Bowers, 1986).
It was
demonstrated that in vitro inhibition of cholinesterases
isolated from different animals could be useful for the
highly sensitive and selective assay of pesticides
(Guilbault, 1970).
Enzymes are normally very substrate-
specific due to the three dimensional nature of the binding
site.
This is advantageous because of the reduced
requirement for resolution of compounds from a matrix.
addition,
In
if the enzyme product is easily detected, an
enzymatic amplification is possible (Bowers, 1986).
A radiometric enzymic method was developed by Horvath
(1982) to measure organophosphprus and carbamate pesticide
residues in water.
By fluorometric assay of phosphatase,
Guilbault et a l . (1969) selectively determined bismuth,
beryllium, and selected pesticides.
Enzyme electrodes have
also been developed which directly determine the products
formed by the enzyme immobilized on or near the surface of
JL
10
the appropriate ion selective electrode.
This enables the
determination of substrates, cofactors, or inhibitors
(Guilbault and Montalvo,
1970).' More recently Haginaka et
a l . (1987) developed a postcolumn reactor consisting of
immobilized lactamase for the detection of HPLC-separated
lactamase inhibitors in human serum.
The major problems with enzymatic analyses is the
lability of the enzymes, and the high'cost associated with
obtaining large quantities of the enzymes.
One solution to
this problem is to immobilize the enzyme to a matrix which
permits reuse and/or increased stability (Guilbault,
In addition to added stability,
1970),
immobilization of enzymes is
more convenient and adaptable to automation (Fukui and
Tanaka,
1984).
Classically,
the term "immobilized enzyme"
has been used to describe an enzyme that has been chemically
or physically attached to a water-insoluble matrix,
polymerized into a water-insoluble gel, or entrapped within
a water-insoluble gel matrix or microcapsule (Zaborsky,
1973).
The sensitivity of ALS to the sulfonyl urea herbicides
at low concentrations may facilitate the use of this enzyme
as an analytical tool for the determination of chlorsulfuron
residues in soil.
Immobilized Enzymes
Immobilization of enzymes is attracting attention for
several reasons.
Possibly the most important area is the
11
biotechnology industry which needs new techniques for the
immobilization of enzymes, antibodies and other biological
materials.
These materials, once immobilized, are vastly
more amenable to analytical and preparative needs of
biological research.
An important attribute of immobilized enzymes is their
usefulness in flowing systems, where reactants are
constantly flushed.into the immobilized catalyst and
products are constantly flushed off, or where the enzyme
affinity for a particular molecule is utilized for
chromatography.
In addition to use as analytical
transducers, immobilized enzymes are presently used in
purification of useful compounds by stereospecific and/or
regiospecific interactions, selective treatment of specified
pollutants to solve environmental problems, and affinity
purification of bio-engineered products from crude ferments
and extracts.
A number of materials have been used to immobilize
enzymes including physical entrapment in crosslinked
polymers, gel matrices, microcapsules,
fibers (Menecke and Polakowski, 1981).
liposomes or hollowOther methods
include chemical attachment by covalent or ionic bonding of
enzyme to water-insoluble, functionalized polymers, and the
more recent use of macroporous silica covered with a
hydrophilic layer before functionalization (Alpert and
Regnier, 1979).
Enzymes are also immobilized by
12
lntermolecular crosslinking of enzyme with multifunctional,
low molecular weight reagent (Zaborsky, 1973).
13
CHAPTER 2
EXTRACTION AND ASSAY OF ACETOLACTATE SYNTHASE
Introduction
Acetolactate synthase is not commercially available
therefore it must be obtained by extraction from a suitable
plant source.
The source of the enzyme must be available in
large quantities and contain chlorsulfuron sensitive A L S .
Alfalfa sprouts were the plant source chosen because large
quantities can be grown in seven days in the dark which
eliminates chlorophyll interference.
Alfalfa is very
sensitive to chiorsulfuron (Burkhart, 1985) and actively
growing sprouts are a rich source of ALS because the enzyme
is required for cell division.
Materials and Methods
Alfalfa Sprouts.
Common alfalfa seed was obtained from
the Montana State Seed Testing Laboratory.
The seed was
surface sterilized in concentrated sulfuric acid for fifteen
minutes and rinsed quickly with sterile water to avoid
injury from heat accumulation from acid dilution.
Sterile
seeds (100 g) were placed on one-half of a 24 by 15 cm sheet
of germination paper.
The germination paper was moistened
and folded over to cover the seed.
Nine sheets containing
14
seeds were placed on a rack and covered with a second layer
of germination paper.
Each rack was wrapped with clean
polyethylene wrap and placed in a dark incubator at
approximately 20° C
The etiolated sprouts were harvested
after seven days and stored in sealed plastic bags at 5° C
for no more than one day before enzyme extractions.
Fresh
sprouts (10 g) were dried at 70° C for 3 days to determine
dry weight.
Reagents.
Total protein assay dye reagent was
purchased from BIO-RAD (Richmond, CA).
chemicals,
Biological
including acetoin (acetyl methyl carbinol),
cocarboxylase (thiamine pyrophosphate), dithiothreitol
(DTT), flavin adenine dinucleotide (FAD), sodium pyruvate,
creatine, and a-naphthol were obtained from Sigma (St.
Louis, MO).
All other reagents were of analytical grade.
Enzyme extraction.
The extraction procedure was
adapted from the method of Ray (1984).
Sprouts were
homogenized for 30 sec in a chilled 250 ml Waring blender
receptacle with one volume of ice-cold buffer containing:
0.1 M potassium phosphate,
I .0 mM sodium pyruvate,
10 uM
flavin adenine dinucleotide (FAD), 15% (v/v) glycerol, and 5
mM.dithiothreitol
(DTT).
The homogenate was filtered
through eight layers of cheesecloth.
The volume of the
filtrate was noted and phenylmethylsulfonyl fluoride (PMSF),
dissolved in a minimum of acetone, was added to a final
15
concentration ait 1.0 mM
PMSF.
The homogenization buffer
was initially adjusted to pH 8.5.
After homogenization the
pH decreased to the desired pH of 7.5 due to the release of
acidic compounds.
ALS substrate pyruvate, and the cofactor
FAD were included to stabilize ALS during extraction.
Glycerol was included to prevent enzyme denaturation by
forming strong hydrogen bonds to decrease the activity of
water.
Glycerol also minimized foaming which can cause
protein denaturation from surface tension effects (Scopes,
1984).
Dithiothreitol was necessary to maintain a reducing
environment to prevent oxidation of phenolic compounds which
could deactivate A L S , and to maintain the sulfhydryl groups
of ALS in a reduced state.
The protease inhibitor PMSF was
added to prevent ALS deactivation during homogenization.
The filtered homogenate was centrifuged at 25,000 g for 30
min at 4° C .
The pellet was discarded and the lipid layer
was removed from the surface.
The supernatant volume was
measured and chelex-treated ammonium sulfate was added
gradually to the chilled and constantly stirred solution to
obtain 20% saturation.
Since the addition of (NH4 )2SO4
acidifies the solution, one molar K0H was added dropwise to
maintain constant pH.
It was necessary to occasionally soak
the pH electrode in a 10% pepsin solution at pH 1-2 for 30
minutes to remove protein adsorbed to the glass electrode.
The solution was again centrifuged as described above, and
the precipitate discarded.
Additional ammonium sulfate was
16
added to.the supernatant to bring the solution to 60%
saturation.
Stirring was continued for at least 10 min to
allow complete equilibration between dissolved and
aggregated protein.
one h o u r .
The mixture was again centrifuged for
The resulting supernatant was discarded and the
precipitated protein dissolved over ice for 5 to 10 min in I
to 2 volumes of the precipitate with desalting buffer
containing:
20 mM phosphate (pH 7.5),
10 mM pyruvate, arid
0.05 mM MgCl2 . The protein solution was centrifuged at 500 g
for 5 min to remove denatured protein and desalted on a 300
mm by 22 mm glass column slurry packed with 15 g (dry w t ) of
Sephadex G- 2 5 .
The packed column was equilibrated with
desalting buffer for 3 h at room temperature prior to u s e .
Desalted protein was used immediately for assay, or quickly
frozen at -40° C .
Frozen protein was thawed over a 50° C
water bath with agitation for further use.
.Enzyme assay.
buffer containing:
ALS activity was measured in assay
20 mM phosphate (pH 7.5),
20 mM pyruvate,
0.5 mM thiamine pyrophosphate (TPP), 0.5 mM MgCl3 , and 10 uM
FAD.
One hundred ul of desalted enzyme solution, or
collected chromatographic fraction, was added to 400 ul of
assay buffer.
The solution was mixed and incubated at 30° C
in a water bath.
The reaction was stopped after 60 min by
adding 25 ul of 12 N sulfuric acid.
Acetolactate produced
by the enzyme was hydrolyzed to acetoin by heating in a
water bath at 60° C for 15 min..
The reaction steps are
17
illustrated in Figure 3.
Acetoin was quantitatively
measured by a modified version of the method developed by
Westerfield (1945).
Five ml of 0.5% creatine in water, and
0.5 ml of 5% alpha naphthol in 2.5 N NaOH were added in
rapid succession with mixing.
The alpha naphthol must be
mixed immediately prior to use as it rapidly oxidizes in
air.
The mixture was returned to the 60° C water bath for
15 min to complete color development.
Absorbance at 525 nm
was measured in a Varian series 634 dual beam
spectrophotometer (Varian Instrument Co., Walnut Creek, CA).
The reference cell contained either a reagent blank or a
blank containing substrate and ALS "killed" with 12 N H^SO^.
This latter blank eliminated interference of product formed
before the assay, or the quenching effect of protein on the
color development.
A standard curve for the acetoin assay
(Figure 4) was developed using known concentrations of
acetoin and plotting the absorbance of the assay solution
vs. the weight of acetoin.
Protein ass a y .
Protein concentrations were measured by
the method of Bradford (1976) with a prepared total protein
dye reagent containing 0.01% (w/v) Coomassie Brilliant Blue
G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid.
The binding of the dye to protein causes a shift in the
absorption maximum of the dye from 465 nm to the detection
wavelength of 595 nm.
Bovine serum albumin protein standard
was dissolved at specific concentrations in the ALS
18
desalting buffer described above.
Solution containing from
10 to 100 ug protein in a volume up to 100 ul was pipetted
into 10 ml test tubes.
The dye reagent concentrate was
diluted with four volumes of glass distilled water and
filtered slowly to prevent foaming, which can result in
particulate formation.
Five ml of diluted reagent were
added to each test tube and the contents mixed.
Absorbance
at 595 nm was measured after 2 min and before I h in 1.5 ml
cuvettes against a reagent blank prepared from 100 ul of the
same buffer as above and 5 ml of protein reagent.
A
standard curve was plotted as the weight of protein against
the corresponding absorbance.
Pyruvate
Acetolactate
o
oh
o
ALS
CH3--- C ----COO(-)
---- -
CH3--- C ---- C ----CH3
COO(-)
Acetoin
oh
H2SO4
Heat
Figure 3.
c h 3--- CH
o
----c ----CH3
To determine ALS activity, the enzyme is reacted
with pyruvate to form acetolactate which is
converted to the measured compound acetoin, by
acid hydrolysis.
19
0 75
-
0 50
-
0 25
-
0 05
-
.
.
.
.
Ug Acetoin
Figure 4.
1 00
-
0 75
-
0 50
-
0 25
-
0 05
-
.
.
.
.
.
Standard curve developed using standard acetoin
from a method designed by Westerfeld (1952), and
modified by Ray (1984).
jig Protein
Figure 5.
Standard curve developed using standard serum
albumin and the Bradford (1976) total protein
assay.
20
High performance liquid chromatography (HPLC).
In an
effort to further purify A L S , a Rainin gradient HPLC with a
Dynamax 300A AX (Rainin Instrument Co. Inc., Woburn, M A .)
silica-based polyethyleneimine (PEI) anion exchange
analytical
(4.6 mm ID x 22 cm) column was used.
The anion
exchange packing material is prepared by chemical
modification of the wide pore silica support to obtain a
uniform hydrophilic layer to completely cover the silica
surface.
An anion exchange ligand, PEI, is covalently
bonded to the hydrophilic layer at a specific ligand
density.
Polyethyleneimine is a weak anion exchange ligand
with a broad titration curve.
It carries primary,
secondary, and tertiary amine functional groups as the
ionizable species.
The charge density on a PEI matrix,
therefore, varies with the pH of the mobile phase.
The pH
necessary to maintain ALS activity was found to be useful
for separation with this packing material.
A gradient solvent system consisted of 5 mM potassium
phosphate (A) and 0.4 M K 2HPO4 at pH 7.5 (B).
The column
\
was equilibrated with A.
The enzyme was eluted in a
gradient from 0% to 100% B over 20 min.
Particulates were
removed by filtering enzyme samples to 0.22 urn with a
syringe filter.
The mobile phase also was filtered to 0.45
urn with a suction filtration apparatus.
The flow rate was
0.5 ml/min and the detection was made at a wavelength of 254
hm.
21
Results and Discussion
Extraction of 200g (fresh) or 23g (dry) of alfalfa
sprouts yielded 310 mg protein with a specific ALS activity
of. 8 nM/mg protein-hr.
Specific activity of the enzyme
extract was determined by standard curves developed for both
the ALS assay and the total protein assay (Figures 4 and 5).
Chromatograms of the desalted protein extract showed 12
to 15 partially resolved peaks.
Most of the peaks were
tightly grouped at the beginning of the chromatogram with
very little elution of proteins at high salt.
The
separation was therefore optimized by holding the gradient
at 0% B for 5 min,
linearly increasing B to 10% over 10 min,
and subsequently increasing to 50% B. by 10 min.
Following
development of this method on an analytical column, the
system was converted to a preparative mode with a 41.4 mm ID
column and a flow rate of 40 ml/min. . Eighty ml of the
protein extract was injected, and 40 ml fractions were
collected for 30 min.
ALS and protein assays were conducted in triplicate on
each fraction (Table I).
While Fraction 3 demonstrated ALS
activity, very little protein was detected.
Highest ALS
activity occured in fractions 17 through 21 which contained
large amounts of protein.
These results indicate that use
of HPLC can increase ALS purity by separating the enzyme .
from much of the coextracted protein.
22
Table I.
The ALS activity and protein concentration for 30
fractions collected from an anion exchange HPLC
column.
Fraction Number
total protein
(ug)
ALS activity
(ng product)
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
0.9
I .4
I .8
I .9
I .5
I .6
I .I
I .6
I .6
2 .I
2.0
2 .I
7.9
12.0
18.0
27.0
18.0
16.0
17.0
15.0
15.0
15.0
18.0
20.0
13.0
7.4
4.3
3.7
2.4
3.4
0.0
0.0
5.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
41.6
36.4
57 .I
18.2
15.6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
total
253
174
252
229
Injected sample
23
CHAPTER 3
IMMOBILIZED ACETOLACTATE SYNTHASE AS A DETECTOR FOR
CHLORSULFURON
Introduction
Chlorsulfuron is an important herbicide used to
control brbadleaf and selected grass weeds in small grain
crops (Levitt, 1978).
Common methods used to determine
chlorsulfuron levels in soil require high resolution
separation prior to detection due to a lack.of detector
selectivity (Jandera e t . a l ., 1987).
There are plant species grown in rotation with small
grains which are killed by soil residues of chlorsulfuron .
that are detectable only with costly instrumental analysis.
Acetolactate synthase, the site of action of chlorsulfuron,
could be used in vitro to measure very low levels of
chlorsulfuron, however ALS is labile and must be used.
o
'
immediately or frozen at -40 C to maintain activity.
The purpose of this study was to produce a stable form
of A L S , in order to develop a simple, inexpensive assay for
chlorsulfuron.
Materials and Methods
Activated Affinity Med i a .
from Rainin Inst. Co.
Hydropore-EP was obtained
(Woburn, M A ).
This material is
macroporous silica bonded to a hydrophilic spacer molecule
24
terminating in an active epoxide function.
high salt concentration (I M Phosphate)
At acid pH or
the epoxide function
spontaneously reacts with primary amines, sulfhydryl and
hydroxyl groups to form stable covalent bonds permanently
binding protein to the rigid particle.
The derivatized
affinity material provides a chemical environment which
binds yet permits active macromolecules such as .ALS to
maintain their biological activity..
Reagents.
Technical grade chlorsulfuron (94% purity),
a gift from E.I. duPont de Nemours Co.,
(Wilmington, DB) was
dissolved in a minimum of acetone and diluted further with
water or 20 mM potassium phosphate buffer (pH 7.5) to
desired concentrations. ' All other reagents were of
analytical grade.
Acetolactate synthase (ALS).. Acetolactate synthase was
extracted from alfalfa sprouts as described in Chapter 2.
The crude desalted extract (before HPLC) was used for
immobilization, as a high concentration of ALS was required.
Enzyme Immobilization.
The plastic cartridges used for
the enzyme reaction were filled with 0.4 g of dry
Ultraffinity-EP.
Cartridges were weighed to insure that
each contained the same amount of affinity material.
Eight ml of desalted enzyme extract at 15.5 mg
protein/ml was mixed with 2 ml of 4 M phosphate (pH 7.5) to
obtain the necessary salt concentration for optimum protein
25
binding to the epoxide.
Cartridges were filled with I M
phosphate until w e t , and the volume of phosphate required
was recorded.
One cartridge volume of ALS solution obtained
as described in Chapter 2 was loaded on each cartridge with
a syringe and left to react for 24 hr at 5 ° C .
Response to Chlorsulfuron.
Chlorsulfuron stock
solution (1.0 uM) was added at 0, 10, 20, or 40 nM
chiorsulfuron at constant dilution to the ALS assay buffer
described in Chapter 2.
Unbound protein was flushed from the cartridges with at
least 3 cartridge volumes of 0.1 M phosphate buffer at pH
7.5.
Each cartridge was loaded with one volume.of assay
buffer at the appropriate chlorsulfuron concentration.
enzyme reactors were sealed and incubated at 30 ° C .
The
After
40 min the reaction mixtures were flushed into 5 ml test
tubes and assayed for acetolactate as described in Chapter 2
Results and Discussion
One ml of crude protein extract solution was needed per
cartridge to achieve maximum and uniform loading.
It was
determined that approximately 6 mg of protein was bound to
the affinity material over the 24 hr period at 5° C .
Figure
6 illustrates the epoxide at the end of a spacer molecule
reacting with a hydroxyl group on the surface of a protein.
Immobilized ALS cartridges catalyzed the production of
7.6 ug of acetolactate per hour and maintained 95% of its
26
original activity after 14 days at 5 ° C .
Nonimmobilized
enzyme lost about 50% of its activity under similar
conditions (Figure 7).
IMMOBILIZATION RRArTTON
SPACER
SPACER
CH2
CH
CH2 + OHCH2— CH2---- PROTEIN
CH2— CH -C H 2- O CH2—CH-
PROTEIN
OH
Figure 6.
The immobilization of protein by Hydropore-EP is
the result of the epoxide reaction with hydroxyl,
sulfhydryl, or primary amino groups to form stable
covalent bonds.
ALS in solution catalyzed the production of 0.83 ug of
acetolactate per mg of protein, compared to 0.63 ug for
immobilized A L S .
The immobilized A L S , therefore, is 18%
less active than ALS in solution, however, this reduction in
activity is compensated for by increased stability.
The reduction in ALS activity as a result of
chiorsulfuron inhibition is illustrated in Figure 8.
Inhibition was linear from O to 40 nM chlorsulfuron.
27
Figure 7.
The rapid decrease in activity of ALS in solution
at 5 °C was prevented by immobilization.
4.00 I
3.67 -
3.33 -
3 .00 (Hg acetoin)
2.67-
CONCENTRATiCN OF CHL0RSULFUR0N (nM)
Figure 8.
Immobilized ALS was inhibited by nanomolar
concentrations of chlorsulfuron.
28
Conclusion
These results indicate that ALS immobilized on
activated affinity material maintains its activity and
stability is enhanced.
In addition the affinity material
can be packed dry into cartridges with Iuer connections and
derivatized with active enzyme, thus creating an immobilized
enzyme reactor which can be easily analyzed for the presence
of an inhibitor.
This system may be used to detect low
levels of chiorsulfuron in soil when combined with the
necessary cleanup and enrichment steps required for soil
extraction.
This method may have potential for detecting the
imidazolinone herbicides, also selective inhibitors of ALS
(Anderson et.'al., 1985).
The imidazolinones, however,
require much higher concentrations to inhibit ALS and are
used at much higher" rates than are sulfonylurea herbicides
(approximately 100 fold).
Therefore, the imidazolinones
would not interfere with chiorsulfuron determination unless
present at high levels.
29
CHAPTER 4
THE EXTRACTION AND DETERMINATION OF CHLORSULFURON FROM SOIL
Introduction
Current.methods for chiorsulfuron determination in soil
involve the use of plant bioassays, or.extensive sample
preparation and HPLC.
These methods are sensitive but both
expensive and time consuming.
Since detailed soil histories
are often lacking, it is difficult to predict whether
chlorsulfuron soil residues would be toxic to a sensitive
crop.
An economical chlorsulfuron soil assay combining the
sensitivity of a bioassay with the speed of an instumental
method would be of great val u e ;
The purpose of this study was to determine if ALS could
be used in vitro to detect nanomolar quantities of
chlorsulfuron in a soil extract.
Methods and Materials
Soil was obtained from the Montana State University
Plant Growth Center, and had no history of pesticide use.
Analysis indicated that the soil consisted of 47.2% sand,
18.8% clay, and 34.0% silt.
The soil pH was 8.5 and the EC
was 0.44 mmohs/cm.
The SPE disposable octadecyl (C^g ) columns were
purchased from J .T . Baker (PhiIlipsburg, New Jersey) and
30
Scintiverse-E scintillation cocktail was obtained from
Fisher Scientific (Fair Lawn, N J ).
Fifty g soil were weighed into each of four 250 ml
'
polypropylene centrifuge bottles.
Three samples were spiked
with 500 ul of water containing 0.125 uCi of triazine ring
labeled 14C-Chlorsulfuron.. The fourth sample was a blank
with only distilled water added..
Spiked soils were mixed well and dried.
One hundred ml
of aqueous 0.1 M NagCOg - 0.1 M NaHCOg (pH 10) was added to
each bottle.
The bottles were shaken vigorously for I hr,
centrifuged for 15 min at 5000 g, and decanted into 250 ml
erlenmeyer flasks.
A 500 ul aliquot of each replication was
added to 15 ml of scintillation cocktail and counted for 10
min.
A second 100 ml of the carbonate buffer was added to
each bottle.
The bottles were shaken again for one hour and
centrifuged as above.
Extracts were combined after an
aliquot of the second wash was taken for scintillation
counting.
Extracts were filtered and the filter papers were
counted by liquid scintillation (LS).
The .pH of the filtered extracts was adjusted, to 3.5 to
convert the anionic chlorsulfuron to the nonionic form.
cartridges were preconditioned with methanol.
SPE
The methanol
was flushed with water adjusted to pH 3.5 with H C l .
Soil
extracts were aspirated through the cartridges, and the
eluent was collected fdr LS counting.
The cartridges were
washed with several milliliters of pH 3.5 wat e r , and the
31
wash water was also collected for LS counting.
Air was
aspirated through the cartridges for 5 min to dry them.
Chlorsulfuron was eluted with three 500 ul aliquots of 100%
methanol and the eluent counted by L S .
The SPE packing was
removed and placed in a scintillation vial with 15 ml
cocktail to determine if any radioactivity remained in the
column.
A similar experiment using a ten-fold dilution of the
salt in the extracting buffer was used to determine if high
chlorsulfuron yield could be maintained with a corresponding
reduction in coextraction of organic matter.
Determination of chiorsulfuron in treated soils.
Soils
were treated at 0.1, I, 9, and 36 ppb chlorsulfuron.
Chlorsulfuron was extracted with 20 mM sodium carbonate at
pH 10 (as described above).
Following. SPE enrichment, the
extracts were evaporated to dryness, dissolved in 500 ul of
phosphate buffer (pH 7.5), and flushed onto immobilized ALS
cartridges.
The cartridges were washed with ALS assay
buffer and the cartridges assayed for the inhibition of ALS
activity (see Chapter 3).
Results and Discussion
Recovery data is shown in Table 2.
The first wash, and
the combined first and second wash contained 65 and 80% of
the radioactivity, respectively.
Only a slight loss of
radio-label occured after filtering indicating some
32
chlorsulfuron had adsorbed to soil particles.
When the pH
3.5 extract was aspirated through the SPE columns,
approximately 90% of the radio-label was retained on the C
I O
packing.
Only 1% of the labeled herbicide was lost from the
column with the pH 3.5 water wash.
About 95% of the bound
radio-label was recovered following elution with methanol.
A modified extraction using a ten-fold dilution of the
extractant resulted in less coextraction of interfering
compounds without loss of chlorsulfuron recovery.
The
modified extraction procedure and a solid phase extraction
of the crude extract proved to be satisfactory in removing .
the interfering compounds without excessive loss of
chlorsulfuron.
Table 2.
Chlorsulfuron recovery data following soil
extraction and extract enrichment.
Replication
A
C
Percent of spike
Sample
combined extracts
filter paper
SPE column
post SPE
water wash
methanol elution
B
86.7
I .3
0.5
8.4
0.6
65.9
76.0
0.6
0.9
11.4
I ;6
49.3
76.2
0.7
0.8
9.2
I .3
63 .I
The. response of immobilized ALS to soil extracts
33
treated with varying concentrations of chlorsulfuron
indicates that treated soils can be analyzed for
chlorsulfuron by the reduction in activity of immobilized
ALS (Figure 9).
To estimate the limit of sensitivity of
this assay, soils were treated at a lower range of
chlorsulfuron concentrations: 0.125, 0.25, 0.5, and 1.0 p p b .
In this case however, 100 g of soil was used.
are plotted in Figure 10.
The results
No statistical evaluation was
used to clearly distinguish detectable level of
chlorsulfuron as. only one sample was assayed at each
concentration. The data does suggest, however that it is
possible to detect chlorsulfuron in soil at I p p b .
Conclusion
. Chlorsulfuron can be efficiently extracted from a loam
soil using 20 mM sodium carbonate.
This extract was also
shown to be enriched in chlorsulfuron by a solid phase
extraction on octadecyl bonded phase silica columns.
Chlorsulfuron can be detected to about I ppm in soil by
exposing immobilized ALS to the extracts and noting the
inhibition of enzyme activity.
Although the current HPLC method with photoconductivity
detection has been shown to detect chlorsulfuron levels in
soil as low as 0.2 ppb, the method is costly and only allows
the elution of one sample at a time.
With the enzyme
inhibition assay, however, many samples may be analyzed in
tandom, at a much lower cost than the currently used
34
chromatographic method.
A detection limit of 1.0 ppb would
appear to be sufficient, as this level is below the
phytotoxic threshold for most the species used in rotation
with small grain crops.
6.00
CONCENTRATION OF CHLORSULFURON (ppb)
Figure 9.
Immobilized ALS was inhibited by extracts from
soil containing part per billion levels of
chlorsulfuron.
35
(Hg acetoin)
13.2
0.125
CONCENTRATION OF CHLORSULFURON(ppb)
Figure 10. Immobilized ALS can be used to detect as little
as 1.0 part per billion chlorsulfuron in soil.
36
BIBLIOGRAPHY
Agostiano, A., M. Caselli, and M . R . Rovenzano. 1985.
Analysis of pesticides and other organic pollutants by
preconcentration and chromatographic techniques. W a t e r ,
Air, Soil Pollut. 19:309-320.
Ahmad, I., and J . Hellebust. 1987. Glutamine synthetase
isoenzymes in the green soil alga Stichococcus
bacillaris Na e g . Plant Physiol. 83:259-261.
Anderson, P . C., and K . A. Hibberd. 1985. Evidence for the
interaction of an imidazolinone herbicide with leucine,
valine, and isoleucine metabolism. Weed S c i . 33:479483.
Anonymous. 1981."Glean". Technical Bulletin. E .I .
duPont de Nemours and Co., Inc., Biochem. Dep.,
Wilmington, DE 12 pages.
Baker, B . R . 1967. Interactions of enzymes and inhibitors.
J. Chem. Ed. 44:610-619.
Ballschmiter, K . 1983. Sample treatment techniques for
organic trace analysis. Pure A p p l . C h e m . 55:1943-1956.
Bauerle, R . H., M . Freundl ich, F.- C . Stormer, and H . E .
Umbarger. 1964. Control of isoleucine, valine and
leucine biosynthesis. II. Endproduct inhibition by
valine of acetohydroxyacid synthetase in Salmonella
typhimorium. Biochim. Biophys. Acta 92:142-149.
Bortslap, A. C., and M. Vernooy-Gerritsen. 1985. In-vivo
control of valine and leucine synthesis in the duckweed
Spirodela polyrhiza. Planta 164:129-134.
Bowers, L . D . 1986. Enzyme reaction detectors in HPLC.
In:"Reaction detection in liquid chromatography." Ed.
Ira S . Krull Marcel Dekker, Inc., N .Y . pp. 195-225.
Bradford, M . M . 1976. A rapid and sensitive method for
quantitation of microgram quantities of protein
utiltzing the principle of protein-dye binding.
Anal. Biochem. 72:248.
Burkhart, D . C. 1985. M.S. Thesis. The leaching, adsorption
and residual properties of chlorsulfuron. Montana State
University. 109 pages.
Chaleff, R . S . 1984. Acetolactate synthase is the site of
37
action of two sulfonylurea herbicides in higher plants.
Science 224:1443-1445.
Dailey, F . E., and J . E . Cronan. 1986. Acetohydroxy acid
synthase I, a required enzyme for isoleucine and valine
biosynthesis in Escherichia coli K-12 during growth on
acetate as the sole carbon source. J . Bacteriol.
165:453-460.
DeFelice, M., C. T . Lago, C . H . Squires, and J ..M . Calvo.
1985. Acetohydroxy acid synthase isoenzymes of
Escherichia coli K-12 and Salmonela typhimurium. A n n .
Microbiol. 133A:251-256.
DeFelice, M., J . Guardiola, B . Esposito, and M . Iaccarino.
1974. Structural genes for a newly recognized
acetolactate synthase in Escherichia coli K-12. J .
Bacteriol. 120:1068-1077.
DeFelice, M., and M. Levinthal. 1977. The acetohydroxy acid
synthase III isoenzyme of Escherichia coli K-12:
Regulation of synthesis by leucine. Biochem. Biophys.
R e s . Comm. 79:82-87.
DeFelice, M., T . Newman, and M . Levinthal. 1978. Regulation
of synthesis of the acetohydroxy acid synthase I
isoenzyme in Escherichia coli K-12. Biochim. Biophys.
Acta 541:1-8.
DeFelice, M., C . Squires, and M . Levinthal. 1978. A
comparitive study of the acetohydroxy acid synthase
isoenzymes of Escherichia coli K-12. Biochim. Biophys.
Acta 541:9-17.
Eoyang, L., and P . M. Silverman. 1984. Purification and
subunit composition of acetohydroxyacid synthase I from
Eschericha coli K-12. J . Bacteriology 157 :184-189.
Foley, M . E . 1985. Response differences of wheat (Triticum
aestivum) and barley (Hordeum vulgare) to
chlorsulfuron. Weed S c i . 34:17-21.
Grimminger, H., and H . E . Umbarger. 1979. Acetohydroxy acid
synthase I of Escherichia coli: Purification and
properties. J .. Bacteriol. 137:846-853.
Guilbault, G . G . 1966. Use of enzymes in analytical
chemistry. Anal. Ch e m . 38:527R-536R.
Guilbault,.G. G . 1968. Use of enzymes in analytical
chemistry. Anal. Ch e m . 40:459R-471R.
38
Guilbault, G . G . 1970. Enzymatic methods of analysis.
Pergamon Press, N . Y. 347 pages.
Guilbault, .G. G ., and J . D a s . 1970. Immobilization of
cholinesterase and urease. Anal. Biochem. 33:341-355.
Guilbault, G . G., D . N. Kramer, and P . L . Cannon, Jr. 1962.
Electrochemical determination of organophosphorous
compounds. Anal. Che m . 34 :1437-1439.
Guilbault, G . G., and J . G . Montalvo. 1969. A urea-specific
enzyme electrode. J . A m e r . Che m . S o c . 91 :2164-2165. _
Guilbault, G . G., and J . G . Montalvo. 1970. An enzyme
electrode for the substrate urea. J . A m e r . Chem. S o c .
92:2533-2538.
Guilbault, G . G., M. H . Sadar, S . S . Kuan, and D . Casey.
1970. Enzymatic methods of analysis: Trace analysis of
various pesticides with insect cholinesterases. A n a l .
Ch i m . Acta 52:75-82.
Guilbault, G . G., M. H . Sadar, S . Kuan, and D . Casey. 1970.
Effect of pesticides on liver cholinesterases from
rabbit, pigeon, chicken, sheep, and p i g . A n a l . Chim.
Acta 51:83-93.
Guilbault, G . G., M. H . Sadar, and M. Zimmer. 1969.
Analytical applications of the phosphatase enzyme
system: Determination of bismuth, beryllium and
pesticides. Anal. C h i m . Acta 44:361-367.
Guilbault, G . G., S . H . Sadar, and R . McQueen. 1969. A
fluorimetric enzymic method for the assay of mixtures
of organic acids. Anal. Chi m . Acta 45:1-12.
Hartley, D., and H . Kidd, Eds. 1983. The Agrochemicals
Handbook. Royal Society of Chemistry, Nottingham,
England. 422 pages.
Horvath, L., J . Volford, and T . Bursics. 1982. Application
of a radiometric enzymic method for monitoring
organophosphorus and carbamate insecticide residues in
water of the Danube river. Institute of Isotopes,
Hungarian Academy of Sciences, Budapest, Hungary.
Report No. IAEA-R-1793-F. 8 pages.
Huffman, K. R., and F . C . Schaefer.
J . O r g . C h e m . 28:1816-1821 .
1963. N-Cyanoimidates.
Hutchison, J . M., R . Shapiro, and P . B . Sweetser. 1984.
Metabolism of chlprsulfuron by tolerant broadleaves.
39
Pest. Biochem. Physiol.
22:243-247.
Jandera, P., J. Churacek, and P . Butzke. 1987. Liquid
chromatography of phenylurea herbicides and related
compounds on chemically bonded ion-exchange materials.
J . Chromatogr. 387:155-169.
Joshi, M . M., H . M . Brown, and J . A. Romesser. 1985.
Degridation of chiorsulfuron by soil microbes. Weed
S c i . 33:888-893.
Kelley, M . M., E . W. Zahnow, W. C . Petersen, and S . T . T o y .
1985. Chlorsulfuron determination in soil extracts by
enzyme immunoassay. J . A g r i c . Food C h e m . 33:962-965.
Krampitz, L . 0. 1948. Synthesis of alpha-acetolactic acid.
Arch. Biochem. 17:81-85.
Kunquan,. Y., and S . Hjerten. 1987. Gradient and isocratic
high-performance liquid chromatography of proteins on a
new agarose-based anion exchanger. J . Chromatogr.
385:87-98.
LaCara, F., and M . DeFelice. 1979. Chromatographic detection
of the acetohydroxy acid synthase isoenzymes of
Escherichia coli K-I2. Biochem. Biophys. Res.
Comm. 91:319-326.
LaRossa, R . A., and J . V. Schloss. 1984. The sufonylurea
herbicide sulfometuron methyl is an extremely potent
and selective inhibitor of Aeetolactate. synthase in
Salmomella typhimurium. J . B i o l . C h e m . 259 :8753-8757.
Leavitt, R . I., and H . E . Umbarger. 1961. Isoleucine and
valine metabolism in Escherichia coli. X . The enzymatic
formation of acetohydroxybutyrate. J . Biol. Chem.
236:2486-2491 .
Levitt, G . 1978 . U.S. Patent.
4 127 405 .
Levitt, G . 1983 . Sulfonylureas: New high potency herbicides
Pergamon, Oxford, UK pp. 243-250.
Levitt, G., H . L . Ploeg, R . C . Wdigel, Jr., and D . J .
Fitzgerald. 1981. 2-Chloro-N[(4-methoxy-6-methyl-l,3,5triazin-2-yl)aminocarbonyl]benzenesulfonamide, A new
herbicide. J . Agric. Food Ch e m . 29 :416-418.
Magee, P . T., and Robichoh-Szulmajster. 1968. The
regulation of isoleucine-valine biosynthesis in
Saccharomyces cerevisiae.
2. Identification and
characterization of mutants lacking the
40
acetohydroxyacid synthetase.
3:502-506.
European J . Biochem.
Manecke, G., and D . Polakowski. 1981. Some carriers for the
immobilization of enzymes based on derivatized
poly(vinyl alcohol) and on copolymers of methacrylates
with different spacer lengths. J . Chromatogr. 215:1324 .
McDonald, R . A., T . Satyanarayana, and J . G . Kaplan. 1973.
Biosynthesis of branched-chain amino acids in
Schizosaccharomyces pombe: Properties of acetohydroxy
acid synthetase. J . Bacteriol. 114:332-340.
Mendoza, C . E., P . J . Wales, H . A. McLeod, and W. P .
McKinley. 1968. Enzymatic detection of ten
organophosphorus pesticides and carbaryl on thinlayer chromatograms: An evaluation of indoxyl,
substituted indoxyl and I-naphthyl acetates as
substrates of esterases. Analyst 93:34-38.
Morishita, D . W.., D . C . Thill, D . G . Flom, T . C . Cambell,
and G . A. Lee. 1958. Method for bioassaying
chlorsulfuron in soil and wat e r . Weed S c i . 33:420-425.
Muhitch, M . J., D . L . Shaner, and M . A. Stidham. 1987.
Imidazolinones and acetohydroxyacid synthase from
higher plants. Plant Physiol. 83:451-456.
O'Sullivan, P . A. 1982. Response of various broad-leaved
w e e d , and tolerance of cereals, to soil and foliar
applications of DPX-4189. Can. J . Plant S c i . 62:715724.
Palm, H . L., J . L . Riggleman, and D . A. Allison. 1980.
Worldwide review of the niw cereal herbicide - DPX
4189. 1980 Pr o c . Brit. Crop P r o t . C o n f . Weeds, pp 1-5.
Ray, T . B . 1982. The mode of action of chlorsulfuron:. A new
herbicide for cereals. Pest. Biochem. Physiol. 17:10-17
Ray, T . B . 1984. Site of action of chlorsulfuron. Plant
Physiol. 75:827-831.
Ray, T . B . 11985. Sulfonylurea herbicides as inhibitors of
amino acid biosynthesis in plants. Trends Biochem. S c i .
11:180-183 .
Rost, T . L . 1984. The comparative cell cycle and metabolic
effects of chemical treatments on root tip meristems.
J . Plant Growth Reg u l . 3:51-63.
41
Rost, T . L., and T . Reynolds. 1985. Reversal of
chlorsulfuron-induced inhibition of mitotic entry by .
isoleucine and valine. Plant Physiol. 77:481-482.
Rubin, B., and J . E . Casida. 1985. R-25788 Effects on
chlorsulfuron injury and acetohydroxyacid synthase
activity. Need S c i . 33:462-468.
Schloss, J. V. 1984. Interaction of the herbicide
sulfometuron methyl with acetolactate synthase. In:
"Flavins and flavoproteins", Walter de Gruyter & Co.,
Berlin, p p . 737-740.
Schloss, J . V., D . E . VanDyk, J . F . Vasta, and R . M. Kutny.
1985. Purification and properties of SaJmonella
typhimurium acetolactate synthase isozyme II from
Escherichia coli HB101/pDU9. Biochem. 24:4952-4959.
Scopes, R. K. 1982. Protein purification. Springer-Verlag,
N . Y . 228 pages.
Shaner, D . L., P . C . Anderson, and.M, A. Stidham. 1984.
Imidazolinones: potent inhibitors of acetohydroxyacid
synthase. Plant Physiol. 76:545-546.
Shaner, D . L., and M . L. Reider. 1985. Physiological
responses of corn (zea mays) to AC 243,997 in
combination with valine, leucine, and Isoleucine. Pest.
Biochem. Physiol. 25:248-257.
Shaw, K . J., and C . M. Berg. 1980. Radioenzymatic assay for
the acetohydroxy acid synthase-catalyzed synthesis of
alpha-aceto-alpha-hydroxybutyrate. Anal. Biochem.
105:101-105.
Slates, R . V. 1983. Determination of chlorsulfuron residues
in grain, straw, and green plants of cerals by highperformance liquid chromatography. J . A g r i c . Food C h e m .
31:313-117 .
Squires, C . H., M . DeFelice, S . R . Wessler, and J . M . Cal v o .
1981. Physical characterization of the iJvHI operon of
Escherichia coli K-12. J . Bacteriol. 147:797-804.
Stormer, F . C., and H . E . Umbarger. 1964. The requirement
for flavine adenine dinucleotide in the formation of
acetolactate by Salmonella typhimurium extracts.
Biochem. Biophys. Res. Com. 17:587-592.
Sweetser, P . B., G . S . Schow, and J . M . Hutchison. 1982.
Metabolism of chlorsulfuron by plants: Biological basis
for selectivity of a new herbicide for cereals. Pest.
42
Biochem. Physiol.
17:18-23.
Swisher, B . A., and M . R. Weimer. 1986. Comparative
detoxification of chiorsulfuron in leaf disks and cell
cultures of two perennial weeds. Weed S c i . 34:507-512.
Tsui, P., and M . Freundlich. 1985. Starvation for i_ZvB
operon leader amino acids other than leucine or valine
does not increase acetohydroxy acid synthase activity
in Escherichia coli. J . Bacteriol. 162:1314-1316.
Ueda, T., and Y. Ishida. 1987. High-performance liquid
chromatographic separation of biological macromolecules
of new silica-based ion exchangers. J . Chromatogr.
386:273-282.
Ulrich, H., and A. A. R . Sayigh. 1966. Synthesis of
isocyanates and carbodiimides. Angew. C h e m . 5:704-712.
Umbarger, H . E., and B . Bro w n . 1957. Isoleucine and valine
metabolism in Escherichia coli. VII. A negative
feedback mechanism controlling isoleucine biosynthesis.
J. Biol. Ch e m . 233:415-420.
Umbarger, H . E., and B . Brown. 1958. Isoleucine and valine
metabolism in E. coli.. VIII. The formation of
acetolactate. J . Biol. C h e m . 233:1156-1160.
Walker, A., and A. Brown. 1983. Measurement and prediction
of chlorsulfuron persistence in soil. B u l l . Environ.
Contam. Toxicol. 30:365-372.
Westerfeld, W. W. 1945. A colorimetric determination of
blood acetoin. J . Biol. C h e m . 161:495-502.
Walters, S . M . 1983. Preliminary evaluation of highperformance liquid chromatography with
photoconductivity detection for the determination of
selected pesticides as potential food contaminants. J .
Chromatogr. 259 :227-242.
Wright, K . -S'., H . B . Halsall, and W. R . Heineman. 1986.
Digoxin homogeneous enzyme immunoassay using high
performance liquid chromatographic column switching
with amperometric detection. Anal. C h e m . 58:2996-2998.
Zaborsky, 0. 1973. "Immobilized enzymes.". CRC Press,
Cleveland, OH. p p . 1-3.
Zahnow, E . W. 1982. Analysis of the herbicide chlorsulfuron
in soil by liquid chromatography. J . A g r i c . Food C h e m .
43
30:854-857.
Zahnow, E . W. 1984. On the analysis of the herbicide
chlorsulfuron in soil by liquid chromatography. J.
A g ric. Food Ch e m . 32:953.
Zahnow, E . W. 1985. Analysis of the herbicide sulfometuron
methyl in fish and green plants by liquid
chromatography. J . Agric. Food Chem. 33:1206-1208.
Zweig; G., and T . E . Archer. 1958. Residue determination of
Sevin (I-naphthyl N-methylcarbamate) in wine by
cholinesterase inhibition and paper chromatography. J .
A g r i . Food Gh e m . 6:910-913.
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