Wood, J. M. 1974. Biological cycles for toxic elements in the environ-
ment. Science 183, p 1049.

Yamada, T. 1968. Uptake of phenylmercuric acetate through the root
of rice and distribution of mercury in the rice plant. Nippon

Nogei Kaishi 42 (7) pp 435-439.


Introduction
Conclusions
Materials and Methods
Results and Discussion

References

CONTENTS

iii


iy
o

=
o
=
=
wi = 15
ve
z§
=> 0
ox
orf
z3
=
zo?
=
or
=
—
= 0
55 60 65 10 15 8.0
pH

Figure 2. The in vitro methylation of mercuric nitrate as a function of pH.

that the cofactors plus methionine are involved in an enzymatic reaction
forming S-adenosyl-methionine which, when formed, reacts with the mercuric
nitrate enzymatically to form methylmercury. The latter possibility is
the more plausible because the specific cofactors, the concentrations of
the cofactors used, and the optimum pH for methylmercury formation are
extremely similar to those which Cantoni (1955 and 1957) found to be
required for the enzymatic synthesis of S-adenosyl-methionine.

The acetone protein precipitation procedure was used to partially
purify the enzyme system forming methylmercury. Each protein fraction from
the acetone precipitation procedure was incubated with methionine, mercuric
nitrate and the various cofactors for 4 hours at pH 7.5. After the
incubation period, each fraction was analyzed for methylmercury. The proteins
precipitated between 70% and 92% acetone addition contained the enzyme system
which forms methylmercury.

Because of the possibility of S-adenosyl-methionine being the methyl-
donating compound, S-adenosyl-methionine (Sigma Chemical Company) was used
as a substrate along with mercuric nitrate. Proteins precipitated between
70% and 92% acetone were divided into two equal volumes. One was incubated
with methionine and mercuric nitrate, and the other was incubated with S-adenosyl-
methionine and mercuric nitrate, The final concentrations of methionine and
S-adenosyl-methionine were 1_x 10 3m, and the final concentration of mercuric
nitrate in each was 1.1 x 107M. ‘The incubation period was 4 hours at 30° C.
Each incubation mixture was analyzed for methylmercury, S-adenosyl-methionine
as substrate produced a relative concentration of 25 for methylmercury, while
methionine as substrate produced a relative concentration of 1, barely
distinguishable from background.


that the transfer of the methyl group from methylcobalamin to mercury
could be accomplished non-enzymatically in mild reducing conditions.

Methylcobalamin is not the only biological compound capable of
donating a methyl group in biological reactions. Cantoni (1955, 1957,
and 1975) and Mudd (1973) have examined S-adenosyl-methionine as a
methyl-donating compound and have found that it effectively methylates
amino acids, proteins, and carbohydrates in biological reactions.

In a previous study (Gay, 1975) I reported the formation of
methylmercury in Pisum sativum under several conditions; i.e., when peas
were sprayed with a solution of mercuric nitrate, when peas were grown
in soil containing mercuric nitrate or phenylmercuric acetate and when
pea tissues were infiltrated and incubated in solutions of mercuric
nitrate or phenylmercuric acetate. It was noted that very low concentra-
tions of methylmercury were found and that younger peas produced more
methylmercury than did older peas.

The purpose of this study was to investigate the mechanism of the
methylation of mercury compounds in peas and to determine whether methyl-
mercury is the end product in this pathway.

CONCLUSIONS

In vitro methylation of inorganic mercury has been accomplished by
an enzyme system isolated from Pisum sativum. The methyl-donating
compound was found to be S-adenosyl-methionine.

Formation of methylmercury in the peas is viewed as an intermediate
step in the biochemical pathway of mercury in peas.

MATERIALS AND METHODS

The common dwarf garden pea, Pisum sativum cultivar Little Marvel,
was used in all investigations. The seeds were planted in vermiculite
and harvested 13 to 14 days after planting.

The infiltration and incubation procedure and analytical methodologies
are discussed in detail elsewhere (Gay, 1975). The selected tissues
are placed in a mercury solution and infiltrated under partial vacuum for
15 minutes. The tissues are then incubated at normal pressure for various
periods of time in complete darkness.

A Hewlett-Packard gas chromatograph equipped with a nickel-63
linearizing electron capture detector is used to detect the presence
and quantities of methylmercury in the samples.

Partial purification of enzyme systems was accomplished with an
acetone protein precipitation procedure. The amount of acetone added to
obtain the various fractions is expressed as the percent of acetone in
the supernatant solution. Spectral grade acetone was used in all cases.

2


INTRODUCTION

The continuing release of mercury to the biosphere from natural
as well as manmade sources warrants the study of plant exposures to
mercury. Data on the effects of mercury pollution, the uptake and
distribution of mercury, the transformation and identification of mercury
species and the possibility of cycling of mercury in and through plants
are vitally needed to complete the environmental assessment of this
pollutant.

Many investigators, Aarkrog and Lippert (1971), Alvarez (1974),
Barker (1972), Gerdes et al. (1974), John (1972), Lee et al. (1972),
Smart (1964), Smith (1972), Tkachuk and Kuzina (1972), Van Loon (1974a
and 1974b), and Yamada (1968), have reported the uptake and distribution
of mercury in a variety of plant species.

Recently, the loss of metals from plants has been investigated.
Siegel et al. (1974) reported on the loss of a volatile form of
mercury from plants. The plants were harvested and the mercury content
of fractions from the total number of plants harvested was determined.
It was found that fractions of plants analyzed immediately after
harvesting contained more mercury than plant fractions analyzed at later
times. The suspected volatile form of mercury was found to be very
soluble in hexane, less soluble in methanol and least soluble in water.
This volatile mercury compound is not methylmercury or dimethylmercury.

Beauford et al. (1975) have obtained evidence that higher plants
may contribute heavy metals naturally to the atmosphere. Peas, broad
beans, and pine seedlings were grown in solutions of radioactive zinc,
lead, or copper. A small fraction of the radioisotopes absorbed by the
roots was released from the leaves, The metal release from the leaves
was not found to be quantitatively associated with water loss. However,
there was a requirement that the plants be continuously fed with the
radioisotope, Removal of the plants from the radioactive solutions
resulted in a complete cessation of radioisotope loss. All three metals
were released from the plants but not in equivalent amounts per unit of
tissue,

The methylation of mercury by aquatic organisms has been shown by
Jengen and Jernelov (1969), Langley (1973), and Bisogni and Lawrence
(1973). Wood et al. (1968) showed the methylation of mercury by
methanogenic bacteria. Imura et al. (1972) showed the methylation of
mercury by a liver homogenate from tuna. The role of methylmercury in
these investigations has not been clearly elucidated.

Various investigators, Imura et al. (1971), Ingraham (1964), Wood
et al. (1968), Wood (1974), and Bertilsson and Neujahr (1971) have used
methylcobalamin as a methyl-donating compound. Wood et al. (1968) found


Methylmercury was formed when 6 milliliters of a supernatant
solution, obtained by grinding 1 gram of tissue per 5 milliliters of
phosphate buffer, was incubated for 4 hours at 30 C, in the dark with
magnesium, manganese, ATP, methionine and mercury in the following final
concentrations: 4 x 10° "M magnesium and manganese, 1 x 10 *M ATP, 2.5 x
10°"M methionine, and 5.5 x 10 °M mercuric nitrate.

12 ry
. a
S
=-
23° °
—
2
a |
Sz
oe 61
zs
34
«fz e
wo —_>+"——~e
Zeit
ze
=
=

0 4 — —+ —— —f

0 5 10 5 20

TIME IN HOURS

Figure 1. Time course for the formation of methylmercury in the
infiltration and incubation procedure.

This same incubation mixture was used to determine the optimum pH of
the enzyme system which forms methylmercury. An equal weight of tissues
from peas grown for 14 days in the greenhouse was used for each of five pH
values being tested. The tissues were ground in a specific pH buffer and
the various cofactors were dissolved in the same buffer. After a 4-hour
incubation of each at 30° C, the incubation mixture was analyzed for methyl-

mercury. Maximum formation of methylmercury under this set of conditions
occurred at pH 7.5 (Figure 2).

Two possibilities exist for the mechanism involved in the enzymatic,
Ain vitro, methylation of mercuric nitrate with methionine and the various
cofactors used. The methylation reaction may be straightforward, utilizing
methionine as the methyl-donating compound but requiring the cofactors to
complete the enzyme system methylating mercury. The other possibility is

4


EPA-600/3-76-082
July 1976 Ecological Research Series



REFERENCES

Aarkrog, A., and Lippert, J. 1971. Direct contamination of barley with
Stor, 59¢e, 58¢o, S5zn, 203yg, and 2}9Pb, Radiation Botany 11 (6)
pp 462-472,

Alvarez, R. 1974, Sub-microgram per gram concentration of mercury in
orchard leaves determined by isotope dilution and spark-source
mass spectrometry, Anal. Chim. Acta 73, pp 33-38.

Barker, W. G. 1972. Toxicity levels of mercury, lead, copper, and
zinc in tissue culture systems of cauliflower, lettuce, potato, and

carrot, Can, J. Botany 50, pp 973-976.

Beauford, W., Barber, J., and Barringer, A. R. 1975. Heavy metal
release from plants into the atmosphere. Nature 256 (5512),
pp 35-37.

Bertilsson, L., and Neujahr, H. Y. 1971. Methylation of mercury compounds
by methylcobalamin. Biochemistry 10 (14), pp 2805-2808.

Bisogni, J. J., and Lawrence, A. W. 1973. Kinetics of microbially
mediated methylation of mercury in aerobic and anaerobic aquatic
conditions. U.S. Nat. Tech. Inform. Serv., Pb. Rep., No. 222025/9.

Cantoni, G. L. 1955. Methionine-activating enzyme, liver. Colowick,

S. P., and Kaplan, N. 0. eds. In Methods in Enzymology,
pp 254-256. New York: Academic Press.

Cantoni, G. L. 1957. Preparation of S-adenosyli-methionine. Colowick,
; S. P., and Kaplan, N. 0. eds. In Methods in Enzymology. pp 600-602.
New York: Academic Press.

Cantoni, G. L. 1975. Biological methylation: selected aspects. Snell,
E. E. ed. In Ann, Rev. Biochem. 44, pp 435-451. California:
Annual Reviews, Inc.

Gay, D. D. 1975, Methylmercury: Formation in plant tissues. In
International Conferenc: in ce}
New Jersey: IEEE.

Gerdes, R. A., Hardcastle, J. E., and Stabenow, K. T. 1974. Mercury
content of fresh fruits and vegetables. Chemosphere 3, pp 13-18.

Imura, N., Pan, S. K., and Ukita, T. 1972, Methylation of inorganic
mercury with liver homogenates of tuna. Chemosphere 1 (5),
pp 197-201.


Ss “uy
° e
w= 12 TM..
me
as
2 —
=
25 8 e
a
oz 6 e
oF
ew
<=
Ss ay
ot
i)
=e 24
oz
2 t
a 04 } + + 1 + + + 1
= 0 1 2 3 4 5 6 7 8

TIME IN HOURS

Figure 3. Time course for the in vitro methylation of mercuric nitrate.

A time course over an 8-hour period was done to determine when the
maximum amount of methylmercury was formed with S-adenosyl-methionine as
the methyl-donating substrate. Sixty grams of stems and apices from peas
14 days old were harvested. The tissues were ground in 0.01M phosphate
buffer at pH 7.5 using 1.5 milliliter buffer per gram fresh weight of tissue.
The 70% to 92% acetone-precipitated proteins were dissolved in 20 milliliters
of phosphate buffer. S-adenosyl~methionine at a final concentration of
5 x 10 "M and mercuric nitrate at a final concentration of 1.1 x 10 ‘M were
added to the protein solution. Aliquots were removed at 0, 1, 2, 4, 6, and
8 hours. Each aliquot was immediately analyzed for methylmercury. Maximum
formation of methylmercury occurred after 1 hour of incubation with a steady
decline in amount of methylmercury thereafter to 8 hours (Figure 3).

The results correspond to the time course obtained from the infiltra-
tion and incubation experiment as shown in Figure 1. In both in vivo and
in vitro experiments methylmercury concentrations reached a peak and then
declined thereafter, What happens to the methylmercury is unknown at this

time, but this study suggests that methylmercury is an intermediate product
in the mercury pathway in peas.


After harvesting the selected tissues, all procedures up to the incuba-
tions of enzyme fractions with the substrates were done at -4 to 2° C.

The supernatant solution obtained from the centrifugation of the
ground tissues was treated successively with 25%, 50%, 70%, and 92%
acetone. The acetone for each fraction was added dropwise to the super-
natant solution while the solution was being agitated or stirred. The
stirring continued 15 minutes after the addition of acetone. The mixture
was then centrifuged at 15,000 rpm for 30 minutes at -4° C. The super-
natant solution was treated with the next amount of acetone, consecutively,
and the pellet obtained was placed under a stream of nitrogen to drive
off the acetone, The pellet was dissolved in a small volume of buffer
and used as an enzyme preparation.

All reaction mixtures, consisting of the substrates and the enzyme
system, were incubated at 30° C, in a constant temperature bath for the
duration of the incubation period.

RESULTS AND DISCUSSION

A time course of methylmercury formation in pea stems via the
infiltration and incubation procedure was carried out to determine the
incubation time required for the formation of the maximum concentration
of methylmercury. Ten grams of stems from the peas grown for 14 days in
the greenhouse were used for each time determination. The infiltration
and incubation solution contained 10 parts per million mercuric nitrate.
The results obtained for the time course (Figure 1) show that the maximum
formation of methylmercury occurred after 10 hours of incubation and
declined to approximately 25% of the maximum after 20 hours of incubation.
These findings suggest that methylmercury is either lost by the tissues or
it is transformed to another form of mercury which cannot be detected with
the equipment and methodologies used.

In our previous study (Gay, in press) an experiment was set up to
determine whether methylmercury or ethylmercury was being liberated from
the incubation mixture. A nitrogen stream was directed into the incuba-
tion solution and subsequently into a carbonate-phosphate trap as described
by Kimura and Miller (1960) and into a cysteine trap, After 20 hours of
incubation, the traps were analyzed. No methylmercury or ethylmercury was
detected. The total mercury balance in the infiltrated and incubated
tissues has not been determined at this time, so the question of the
transformation of methylmercury to a volatile mercury species which is lost
or the transformation of methylmercury to a species which is not detected
cannot be answered at this time.

The supernatant solution obtained after the centrifugation of pea
tissues ground in 0.01M phosphate buffer with a mortar and pestle was
used initially as a crude enzyme preparation, Methionine was selected
as the first compound to be used as a substrate for a methyl-donating
compound with which to methylate mercuric nitrate. After various periods
of incubation, no methylmercury was found. Several cofactors were added
in various combinations and molarities in an attempt to complete the
enzyme systen,


DISCLAIMER

This report has been reviewed by the Environmental Monitoring and
Support Laboratory-Las Vegas, U.S. Environmental Protection Agency, and
approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.


TECHNICAL REPORT DATA

(Please read Instructions on the reverse before completing)

- REPORT NO. 2.

EPA~600/3-76-082

3. RECIPIENT'S ACCESSIONNO.

Ed

TITLE AND SUBTITLE

BIOTRANSFORMATION AND CHEMICAL FORM OF MERCURY IN
PLANTS

56. REPORT DATE
July 1976

6. PERFORMING ORGANIZATION CODE

x

- AUTHOR(S)
Don D. Gay

lg. PERFORMING ORGANIZATION REPORT NO.

}. “ERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Monitoring & Support Laboratory
Office of Research and Development

U.S. Environmental Protection Agency

Las Vegas, NV 89114

10. PROGRAM ELEMENT NO.

1AA602

WT. CONTRACT/GRANT NO.

12, SPONSORING AGENCY NAME AND ADDRESS
Same as above

13. TYPE OF REPORT AND PERIOD COVERED
Final

14, SPONSORING AGENCY CODE
EPA-ORD, Office of Health

and Ecological Effects

16. SUPPLEMENTARY NOTES

76. ABSTRACT

methionine as the methyl-donating compound.

of the substrates with the enzyme system.

in the mercury pathway in peas (Pisum sativum).

The in vitro methylation of inorganic mercury has been demonstrated using
an acetone precipitated protein fraction from Pisum sativum and S-adenosyl-

A time course of the enzymatic formation of methylmercury has shown that
the maximum methylmercury concentration occurred after 1 hour of incubation
At all subsequent incubation times
reduced concentrations of methylmercury in the reaction mixture were observed.

When a time course for the in vivo methylation of inorganic mercury was
done using an infiltration and incubation procedure, the maximum concentration
of methylmercury was observed after 10 hours of incubation. All subsequent
incubation periods produced lesser amounts of methylmercury.

The results suggest that the methylmercury is an intermediate compound

Leguminous plants
Absorption

17. KEY WORDS AND DOCUMENT ANALYSIS

a. DESCRIPTORS jb. IDENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Plant physiology Methylmercury 064A
Biochemistry Mercurial transformation] 06C
Plant chemistry Mercury uptake O6F
Mercury Biotransformation 073
Trace elements Phenylmercury o7¢

Pisum sativum

8. DISTRIBUTION STATEMENT

RELEASE TO PUBLIC

21, NO. OF PAGES

. ITY CLASS
SSUNCLASSTFIED 16

20. SECURITY CLASS (This page)
UNCLASSIFIED

22. PRICE

EPA Form 2220-1 (9-73)



EPA-600/3-76-082
July 1976

BIOTRANSFORMATION AND CHEMICAL FORM OF MERCURY IN PLANTS

by

Don D. Gav
Monitoring Systems Research and Development Division
Environmental Monitoring and Support Laboratorv
Las Vegas, Nevada 89114

U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
LAS VEGAS, NEVADA 89114


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously

planned to foster technology transfer and a maximum interface in related fields.
The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.

This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.


Imura, N., Sukegawa, E., and Pan, S. 1971. Chemical and methylation
of inorganic mercury with methylcobalamin, a vitamin B)) analog.
Science 172, pp 1248-1249.

Ingraham, L. L. 1964. Byj9 coenzymes: biological Grignard reagents.
Ann. N. Y. Acad. Science 112, p 713.

Jensen, S., and Jernelov, A. 1969. Biological methylation of mercury
in aquatic organisms. Nature 223, p 753.

John, M. K. 1972. Mercury uptake from soil by various plant species.
Bull. Environ. Contam. Toxicol. 8 (2), pp 77-80.

Kimura, Y., and Miller, V. L. 1960. Vapor phase separation of methyl
or ethyl mercury compounds and metallic mercury. Anal. Chem. 32,
p 420,

Langley, D. G. 1973. Mercury methylation in an aquatic environment.
J. Water Pollut. Contr. Fed. 45 (1), pp 44-51.

Lee, D. F., Thomas, B., Roughan, J. A., and Watters, E. D. 1972.
Mercury content of some foodstuffs of vegetable origin, Pestic.
Science 3, pp 13-17.

Mudd, S. H. 1973. The adenosyltransferases. Boyer, P. D. ed. In The
Enzymes, Vol. VIII, Part A. pp 121-154. New York: Academic Press.

Siegel, S. M., Puerner, N. J., and Speitel, T. W. 1974. Release of

volatile mercury from vascular plants. Physiol. Plantarum 32 (2),
p 174,

Smart, N. A. 1964. Mercury residues in potatoes following application
of foliar spray containing phenylmercuric chloride. J. Sci. Food

Agr. 15, pp 102-108.

Smith, W. H. 1972. Lead and mercury burden of woody plants. Science
176, p 1237.

Tkachuk, R,, and Kuzina, F. D. 1972. Mercury levels in wheat and other

cereals, oilseed and biological samples. J. Sci. Food Agr. 23,
pp 1183-1185.

Van Loon, J. C. 1974a. Agricultural use of sewage treatment plant
sludge, a potential source of mercury contamination. Environ.
Lett. 6 (3), pp 259-265. .

Van Loon, J. C. 1974b. Mercury contamination of vegetation due to
application of sewage sludge as fertilizer. Environ. Lett. 6 (3)
pp 211-218,

Wood, J. M., Kennedy, F. S., and Rosen, C. G. 1968. Synthesis of
methylmercury compounds by extracts of a methanogenic bacterium.
Nature 220, pp 173-174.