United States
Environmental Protection
Agency
Robert S. Kerr Environmental Research ^
Laboratory
Ada OK 74820
Research and Development
EPA-600/S2-83-021 May 1983
<&ER& Project Summary
Biochemical Analyses for
Detection and Assessment of
Pollution in the Subsurface
Environment
Jenq C. Chang, Andrea B. Arquitt, Rosalee Merz, Elizabeth R. Doyel,
Phyllis T. Norton, Laura B. Frazier, Jerri Z. Jackson, JoAnn J. Webster,
Jeffrey L. Howard, Otis C. Dermer, and Franklin R. Leach
Selected biochemical analysis tech-
niques were investigated for potential
use in detecting and assessing pollution
of subsurface environments.
Procedures for determining protein,
nucleic acids, organic phosphate,
lipopolysaccharides, and various
coenzymes and enzyme systems were
evaluated. These procedures were
modified and adapted for application to
environmental samples, and sensitiv-
ities were determined in terms of
numbers of Escherichia co//cells which
could be detected.
Standard spectrophotometric and
fluorimetric methods for protein, DNA,
RNA, and organic phosphates lacked
sufficient sensitivity for successful!
application to subsurface environ-
mental samples. Methods for coen-
zymes and enzymes which employed
enzymatic cycling procedures could be
made highly sensitive but required use
of very sophisticated and difficult micro-
procedures. Two highly promising
procedures were the Limulus
amebocyte lysate test, which embodies
a built-in amplification, since
lipopolysaccharide activates an enzyme
which then catalyzes the reaction to be
measured, and the bioluminescence or
chemiluminescence procedures. These
methods are currently applicable to
many environmental samples, and
it should be possible to significantly
increase their sensitivity, reliability, and
applicability by further study.
Disclaimer — Although the research
described in this project summary has
been funded wholly or in part by the
United States Environmental
Protection Agency through grant
number R-804613 to Oklahoma State
University - Oklahoma Agricultural
Experiment Station, it has not been
subjected to the Agency's required peer
and policy review and therefore does
not necessarily reflect the views of the
Agency and no official endorsement
should be inferred.
This Project Summary was developed
by EPA's Robert S. Keff Environmental
Research Laboratory, Ada. OK. to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Basic to technological advancements
in any scientific area are sensitive,
accurate, and facile analytical techniques.
The past several decades of extensive
methods development research have
supported an astounding advance in
biochemical research and in molecular
biology. The present studies selected
biochemical analysis techniques
resulting from this developmental
research to determine their applicability
for detection and assessment of pollution
in the subsurface environment.
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First the reproducibility of results
available from published procedures was
established, and modifications necessary
to improve on the ease and reproducibility
of the assays were made.
Second, the sensitivities of the pro-
cedures were assessed for both
minimum quantities of biochemicals and
minimum numbers of cells of Escherichia
coli which could be detected under the
assay conditions.
Third, procedures which showed
particular promise, or a high degree of
sensitivity, including enzymatic, cycling,
and chemiluminescent assays, were
studied in detail. Where possible, the
assay conditions were optimized.
Fourth, the most promising assay,
firefly luciferase for ATP, was studied
extensively. Commercially available
reagents and equipment were evaluated.
Each component of the reaction mixture
was examined for its essentiality and the
proper concentration to be used. Less
extensive work was done on the Limulus
amebocyte lysate test for lipopoly-
saccharides.
Finally, selected assays were applied to
authentic environmental samples.
Standard Biochemical Tests
Standard biochemical tests on protein,
DNA and RNA were evaluated:
Protein—Lowry; dye binding with
Coomassie blue and bromosulfalein;
and o-phthalaldehyde without and
with hydrolysis.
DNA—Diphenylamine; diaminoben-
zoic both spectrophotometric and
fluorimetric; and fluorimetric using
ethidium bromide or DAI.
RNA—Orcinol and ethidium bromide;
Organic phosphate—Phosphomol-
ybdate and with extraction.
Several independent analyses were
repeated often enough to establish both
Table 1. Standard Biochemical Determination Methods and Their Sensitivities
statistical confidence and operator
experience. Since the assays measured a
particular substance in a culture of
Escherichia coli cells, the sensitivity of
the assay could be expressed as the
amount of a given substance required,
and as the number off. co//'cells required
to give the minimum detectable
response. Table 1 presents ranges and
sensitivities for these assays and makes
note of complications encountered.
While each of these assays was
satisfactory within its recognized
inherent limits and biochemically
understandable limitations, none was of
sufficient sensitivity to be applicable to
the sparse populations of organisms
likely to be present in subsurface
environmental samples.
Enzyme Assays
In part, the goal of this research was to
increase and/or optimize the sensitivity
of assays by using normally available
laboratory equipment. Since enzymes act
# E. coli
'ubstance Method or Principle
'rotein Lowry
Dye binding
First Author
Lowry
McKnight
Specific Methods
Folin phenol, biuret
Coomassie blue
Range*
10 • 80 ug
0.5 - 10 ug
cellsf
1O>
Complication Remarks
Phenol, tyrosine interfere
Not all proteins bind dyes to the
same extent ™
Fluorescent
Ftadioisotopic
McGuire
Kutchai
Butcher
Schutz
Bromosulfalein
Without Hydrolysis
With Hydrolysis
0.5 - 10/jg
1 -25 /jg
1.5 - 12ng(Ala)
W
Labeling amino terminal 0.08 - 2.5 fjg
2x10* Only amino groups
— 6 ng of protein; background is
the problem
— Reproducibility poor
DNA
RNA
Organic
Phosphate
Burton
DAB A
Fluorescent
....
Orcinol
Ethidium bromide
Molybdoantimontyl-
phosphoric acid
....
Abraham
Setaro
Cattolico
Various
Kapuscinski
Ceriotti
Going
Diphenylamine
Spectrophotometric
Fluorimetric
Ethidium bromide
DAPI
Fluorimetric
Direct
Extraction
5 - 50 ug
25 - 750 ug
0.1 - 1.6 ug
0.02 - 0.16 pg
1 -16 ng
1 - 50 ug
5 - 20 ng
40 - 640 ng
0.2 - 6.4 ng
6 x W A Deoxyribose determination
10a Not as sensitive, but one can
run spectrophotometric or
106 fluorimetric assay depending on
the DNA content of the unknown
6x10* Also reacts with RNA
3 x 10*
10s For pentose analysis
1 06 Also reacts with DNA
10s
10* 6 fgA 77VE. coli cell
"Range found in this laboratory to yield linear and accurate results.
tr/»e number of Escherichia coli cells required to yield the minimum amount of substance that is determined by particular methods.
2
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catalytically, they produce an amplified
effect; that is, several hundred, thousand
or so molecules of product for each
molecule of enzyme. For example, the
fluorimetric limit of detection of NADH is
60 ng and that of alcohol dehydrogenase
as a protein is also 60 ng. If the alcohol
dehydrogenase is used to reduce NAD+to
NADH which occurs at a rate of 1000
molecules of NADH produced per minute
of incubation by each molecule of alcohol
dehydrogenase, a 10-minute incubation
would yield a 10,000-fold increase in the
sensitivity of measuring alcohol
dehydrogenase over just a protein
determination.
Further increases in sensitivity can be
achieved by extended incubations;
however, care must be taken because of
inactivations and inhibitions. The
additional sensitivity given by incubation
assays as compared to the normal contin-
uous assays was determined. Table 2
presents some of these results and also
lists the minimum number of E, coli cells
detectable by that enzyme assay.
None of these direct or incubation
assays for enzymes was of sufficient
sensitivity for the expected subsurface
environmental samples.
Coupled Enzymes and
Enzymatic Cycling
When the products and reactants of a
reaction are not easily determined,
sometimes it is possible to utilize one of
the products in a succeeding reaction
catalyzed by another enzyme.
The following scheme differentiates
between these two processes.
Table 2. Sensitivities of Enzyme Assays as Bioindicators in Terms of E. coli Cell Number
Coupled:
E'AUX
1 I *>2
E.lnd
PI 1
1 ' «-"3
SIC, \ 1
1 + P2
3 "^ "4
. 2 + P3 + P4
Cycling:
E,
SI C
PI 1
SI 1
P4- P
S' i p
1 + PS
2 + ^3
In coupling, a product of the first reaction
is used as a substrate for the second
reaction producing two more products.
Usually one of these latter products is
easured. For enzymatic cycling, the
product of the first reaction which is used
Enzyme E. C. tf
Lactate Dehydrogenase
1.1.1.27
Alkaline Phosphatase
3.1.3.1
Catalase
1.11.1.6
A deny late Kinase
2.7.4.3
Assay Method
Continuous
Incubation
Continuous
Incubation (1 h)
Incubation (24 h)
Continuous
Incubation
Continuous
Incubation
Limit of
Detection
0.1 pg
1.0 ng
20 ng
1.0 ng
0.09 ng
0.2 vg
1.0 ng
3.0 ng
0.1 ng
Minimum Cell
Concentration
(mr^l
1 x /0«
8.5 x 706
1 x 70'
4 x 10s
as a substrate for the second is converted
to a substrate for the first. Thus the cyclic
conversion 81—• PI—- S, allows
production of much greater amounts of
the products.
With the coupled assay for ATP, a linear
range of 0.2 to 20 nmol was obtained. The
enzymatic cycling procedure for meas-
uring ATP yielded an effective range of
0.1 pmol to 10 nmol. Thus, the increase in
sensitivity obtainable with enzymatic
cycling procedures is clear. Table 3
summarizes some of the sensitivities
obtained using coupled and cycling
assays.
Bioluminescent and
Chemiluminescent Assays
Several symposia, conferences, and
reports devoted to bioluminescence and
chemiluminescence, and the commercial
development of reagents and instrumen-
tation, have brought these methods to the
forefront. Their sensitivity is greater than
most of the methods (except cycling)
discussed above. In fact they can be used
even after cycling reactions to determine
the final product.
The following scheme shows .the
reactions involved in bioluminescence
and chemiluminescence.
Bioluminescence
Firefly
LH2 + Lu + MgATP
AMP + MgPP
Lu - LH2 - AMP + 02
CO2 + OL + Light
= Lu - LH2 -
•LU + AMP +
Bacterial
BLu -t- FMNH2 + 02 - BLu-
FMNHOOH
BLu + FMNHOOH + RCHO —BLu +
RCOOH + FMN 4- H20 + Light
It is also possible to use NAD(P)H : FMN
oxidoreductase
H++FMN -NAD'+FMNHj
which allows any NADH yielding'
reaction or couple to be assayed.
Table 3. Sensitivities of Coupled and Cycling Assays
Bioindicator
Pyridine Nucleotides
Adenosine Triphosphate
Assay Method
Cycling
NADP +
NAD +
Coupled Enzyme
Cycling
Minimum Cell
Limit of Concentration
Detection (mr^)
37.0 pg
3 ng
WO ng-'
100 pg
Sx 10*
5x10*
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Chemiluminescence
Luminol
coo-
+ N2 + 3 H20 + Light
coo-
The properties of most of the commer-
cially available firefly luciferases were
compared. Experiments revealed that
Tricine buffer yielded a conformation of
firefly luciferase which was especially
reactive. The various assay conditions
were studied and the assay was
optimized. Table 4 lists a few of the
factors tested and shows the increase in
sensitivity. Table 5 shows an example of
the assay requirements for one of the
commercial preparations. Table 6 shows
a comparison of ATP determination by the
firefly luciferase (bioluminescence) and
enzymatic cycling procedures. As
indicated, a sensitivity of 50 fg of ATP was
achieved by the bioluminescence method
using commercial reagents and instru-
mentation.
Using bacterial luciferase, as little as
0.25 ng of FMN could be detected. With
the luminol assay, it was possible to
measure 2 pg of iron porphyrins which
corresponded to 10 £. coli cells. The
bacterial luciferase system in the form of
dried bacterial cells was tested as an
indicator as described in the Microtox
procedure.
Cascaded Reactions
The response of the Limulus
amebocyte lysate to lipopolysaccharides
from gram-negative bacteria is formation
of a gel. Since this reaction series is
similar to that in blood coagulation, and
the lipopolysaccharide activates an
enzyme or factor which acts catalytically,
the assay is very sensitive. As little as 100
fg of lipopolysaccharide, and as few as
10 £. coli cells, were detected by this
method.
Environmental Samples
Many of the assays were applied to
spring water samples, soil samples, and
core materials. The bioluminescence and
cascaded reaction assays were
particularly promising, but interfering
substances found in the soil and core
materials require further research to
optimize the application of these
biochemical determinations to the
difficult environmental solid samples.
Table 5. Requirements for the Firefly
Luciferase Assay*
Omissions
None
- 5 mM MgSQ*
- 0.5 mM EDTA
- 50 /jig Luciferin (LH^
- 0.5 mM DTT
- 50 tig Bovine serum
albumin (BSA)
Additions
None
MgSOt
LH2
DTT
BSA
MgSOt + LH2
MgSO* + LH2 + DTT
Light Units
(1 ngATP)
20.6
0
12.6
5.2
13.6
20.0
0.1
3.7
0.5
1.1
0.3
13.8
13.6
*This experiment done with Firelight enzyme.
Table 4. Improvements in the Firefly Luciferase Assay
Factor
Reaction vessel
Reaction volume
Luciferin
Buffer
Additives
Enzyme
Light effect on glass reaction
vessel (Pico-Lite)
Fold
Change Increase
/7/-1CC
05m!
None
w n 9 ml
^" Complete
purified
maintained
2
5
2
3
10
4
9 (deer, in okg.J
D
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Table 6. Comparison of Enzymatic Cycling and Firefly Lucit'erase Determinations of A TP
Parameter
Range*
Sensitivity"
Cosff
Productivity
Inhibitors
Equipment
Technical competence
required
Turnaround time
Specificity
Assay
Firefly Luciferase
0.2 pmol - 100 pmol
0. 1 fmol (50 fgl
6C/assay
25/hr or 200/8-hr day
Metal ions, Pol'
Photometer
Technicians
30 min
ATP only
Enzymatic Cycling
0.3 pmol - 10 pmol
0. 1 pmol (SO pg)
9.5C/assay
96/5-hr or 192/8-hr day
None encountered to date
Fluorometer
Enzymologist
5hr
ATP. NADH, NADPH
"Useful range of A TP which can routinely be measured.
"Smallest amount of A TP detected by the assay.
tBased on 1979 prices, when the experiment was done.
Jenq C. Chang, Andrea B. Arquitt. Rosalee Merz, Elizabeth R. Doyel, Phyllis T.
Norton, Laura B. Frazier, Jerri2. Jackson, JoAnnJ. Webster, JeffreyL Howard,
Otis C. Dermer, and Franklin R. Leach are with Oklahoma State University,
Stillwater. OK 74078
William J. Dunlap is the EPA Project Officer (see below).
The complete report, entitled "Biochemical Analyses for Detection and Assess-
ment of Pollution in the Subsurface Environment," (Order No. PB83-182 303;
Cost: $14.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
P.O.Box 1198
Ada. OK 74820
U. S. GOVERNMENT PRINTING OFFICE: 1983/659-095/1947
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Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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Fees Paid
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Penalty for Private Use $300
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