EPA-600/1-78-010
January 1978
Environmental Health Effects Research Series
RADIOIMMUNOASSAY OF METALLOTHIONEIN
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
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ances of man for unhealthful substances or conditions. This work is generally
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clude biomedical instrumentation and health research techniques utilizing ani-
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This document is available to the public through the National Technical Informa-
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EPA-600/1-78-010
January 1978
RADIOIMMUNOASSAY OF METALLOTHIONEIN
by
Felix Friedberg
Department of Biochemistry
College of Medicine
Howard University
520 W Street, N.W.
Washington, D.C. 20059
Grant No. R-803201
Project Officer
Larry L. Hall
Toxic Effects Branch
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial
products consitute endorsement or recommendation for use.
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance
the quality of our environment in order to promote the public health and
welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation
of affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
The goal of this project was to develop a radioimmunoassay for metallo-
thionein. As this protein is involved with the transport of cadmium in
biological systems and may in fact protect against cadmium poisoning, the
ability to monitor the levels in the human population is of the utmost
importance to our evaluation of the hazards of environmental cadmium
exposure.
John H. Knelson, M.D,
Director,
Health Effects Research Laboratory
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INTRODUCTION
Metallothionein, a cadmium and zinc containing (6-11%), cysteine-rich
(30-35%) protein of low molecular weight was first described for equine renal
cortex (1). This protein is devoid of histidine, aromatic amino acids, leu-
cine and isoleucine. It is also present in large amounts in human kidneys as
well as human and equine liver. After administration of salts of cadmium,
zinc or certain other heavy metals, the metal lothionein accumulates in the
liver and kidney of a variety of animals in which normally it is not readily
detectable.
The equine renal metal lothionein exists in at least two types whose
sizes and total metal contents are identical but which differ in at least 7
amino acids and in their cadmium/zinc ratio. The one with the higher ratio
exhibits the following amino acid composition Cys2Q Serg Lysy Arg-| Alay
Val3 Asp2 Asn-| Glu-j Gln2 Prog Thr^ Met-] (Cd + Zn)y (2).
The 20 cysteinyl residues are distributed along the entire chain but are
closer to each other in the center portion. Fourteen form part of 7 Cys x Cys
tripeptides. There are 3 Cys X-X-cys and 3 cys-cys sequences. (Cys X-X cys
occur also at the iron-binding sites of many iron-sulfur proteins.) (The
native protein contains no disulfide bonds.) The cysteine is associated par-
ticularly with serine, lysine and arginine.
Binding of Cd or In ion displaces 3 protons and formation of a trimer-
captide has been suggested.
There are also two major variants of metallothionein in livers of Cd
treated rats. But to complicate the picture, it has been reported that ad-
ministration of Cu2+ to rats leads to the accumulation of a similar but dif-
ferent protein: "Cu-chelatin." The chelatin does contain histidine, phenyl-
alanine, leucine, isoleucine and tyrosine (3). Other workers claim that even
if a Cu2+ inducible protein for rat liver exists, administration of this metal
results in appreciable amounts of the copper being held by copperthionein (4).
Among the many hypotheses for the functions of the metallothionein, the
protective role suggested by Nordberg et al. (5) is the most likely one. They
reported that while intravenously administered cadmium salts cause testicular
necrosis in mice, animals excreting protein in the urine (induced by repetitive
ingestion of the cadmium salts) showed no significant damage of testicular
tissue. Another suggested role for metallothionein is its involvement in the
transport of cadmium to the kidneys which was also proposed by Nordberg (6).
Such transport, in excess, may result in the degeneration of the proximal
renal tubular lining cells and cause proteinuria.
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EXPERIMENTAL PROCEDURE
Rats were dally injected intraperitoneally with CdCl2 (2.5 mg/kg body
weight) over a period of one week. After sacrifice of the anesthetized ani-
mals, livers and kidneys were removed and frozen immediately. Approximately
1000 rats were sacrificed and isolation of the metallothionein from the accu-
mulated tissues was performed as described below. Extraction and fractiona-
tion were done at 4°C.
The tissue (1:1 w/v) was homogenized in 0.001 M Tris-buffer (pH 8.6)
(containing 0.25 M Sucrose) followed by centrifugation at 15,000 g for 1 hr.
Next, the supernatant was centrifuged at 100,000 g for 2 hrs. Upon applica-
tion of the supernatant resulting from the second centrifugation to a Sephadex
G-75 column (equilibrated and eluted with 0.001 M Tris-buffer (pH 8.6) the
emerging fractions comprising peak B (see Fig. 1) were pooled and lyophilized.
Further purification of B resulted from rechromatography utilizing
Sephadex 6-50. The same Tris-buffer was employed. The eluant labeled M£
(see Fig. 2) was pooled and lyophilized after desalting by passage through
Sephadex G-25.
Finally, the material was applied to a DEAE Sephadex A-25 column. Elu-
tion by a linear gradient Tris-buffer pH 8.6 (0.05 0.25 M) allowed resolu-
tion of two fractions Q and R (see Fig. 3). This result is in agreement with
that reported by Kimura e£ al_. (7) who claim that both Q and R represent
metallothionein and imply that further purification is not necessary.
Because of difficulty in reproducibility, in later experiments, DEAE
cellulose rather than DEAE Sephadex A-25 was utilized and the linear gradient
obtained with Tris-buffer (pH 8.6) was extended on both sides (0.01 0.4 M).
Two peaks were resolved (DEAE I and II; see'Fig. 4). In turn, each peak was
rechromatographed on DEAE cellulose. Thereafter, the protein labelled DEAE I
showed a single band when examined by 7.5% polyacrylamide disc gel electro-
phoresis while that labelled DEAE II exhibited sometimes one and sometimes two
bands when applied to the gel. The final yield for each protein (DEAE I and
II) was approximately a couple of mgs/100 g of liver.
We evaluated also the modification suggested by Cherian (8), i.e., heating
and ammonium slufate treatment as means of facilitating the preparation of
metallothionein. When we heated the material at 70 C for 20 sec., the DEAE I
peak was not affected but the DEAE II was much smaller than when the heat
treatment was not applied.
The following instruments were employed in this project:
Atomic Absorption Perkin Elmer 360
Gilford Model 220 Spectrophotometer
Baird Atomic Sealer 135, plus Scintillation Detector 8109
1KB Fraction Collector 7000
Serval RC-2 Centrifuge
New Brunswick Freeze Dryer B67
Beckman L2-65 Ultracentrifuge
-2-
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All chemicals were Fisher Scientific products.
Rats (both males and females, 100-200 grams) were primarily of Sprague-
Dawley strain even though some mutants have also been used by us. They were
kindly furnished by N.I.H.
-3-
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CONCLUSION
eliminary amino acid analysis (see Table I) suggests that our
ein" appears similar in composition to Riordan and Gower's (9)
ns isolated from copper loaded liver of rats (where the cysteine
out four times less than that of metallothionein), it must be
that these workers used a mixture of three proteins for their
action of CuSO^ into rats, Bremner and Young (4) isolated "copper
ilar in composition to that of "zinc thioneins" as described by
avies (10) and unlike that of the copper chelatin described by
(3).
ile workers do not agree whether "copper thioneins" and "zinc
rat are identical, we find in preliminary analysis that our
appears to have a composition similar to that of copper chelatins
those who claim that copper thioneins and zinc thioneins are
teins.
-4-
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RECOMMENDATION
Any radioimmunoassay is as specific as the antigen that was used. Today,
many laboratories employ antibodies to antigens which were not properly ana-
lysed and characterized and the radioimmunoassay does not really test what
the investigator claims it does. We are not referring here to impurities
carried by the antigen (they can be adsorbed), but to the identification of
the main components.
We recommend that development of a radioimmunoassay should not be
attempted until the putative metallothionein is clearly characterized. There
are too many kinds of metallothionein induced by the injection of different
metals according to the reports in the literature. They must be properly
identified first.
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REFERENCES
1. Margoshes, M. and Vallee, B. L., J. Amer. Chem. Soc. 79, 4813 (1957).
2. Kojima, Y., Berger, C., Vallee, B. L. and Kagi, J. H. R., Proc. Natl.
Acad. Sci. 73, 3413 (1976).
3. Winge, D. R., Premakumar, R., Wiley, R. D. and Rajagopalan, K. V.,
Arch. Biochem. Biophys. 170, 253 (1975).
4. Bremner, I. and Young, B. V., Biochem. J-. 157, 517 (1976).
5. Nordberg, G. F., Goyer, R. and Nordberg, M., Arch. Pathol. 99_, 192
(1975).
6. Nordberg, G. F., Environ. Physio!. 1, 171 (1971).
7. Kimura, M., Otaki, N., Yoshiki, S. et al., Arch. Biochem. Biophys. 165,
340 (1974).
8. Cherian, G. M., Biochem. Biophys. Res. Comm. 61_, 920 (1974).
9. Riordan, J. R. and Gower, I., Biochem. Biophys. Res. Comm. 66_, 678 (1975),
0. Bremner, J. and Davies, N. T., Biochem. J. 149, 733 (1975).
-6-
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TABLE I
Amino Acid Composition of Putative Rat Metal!othiohein
Amino Acid Composition
(Neutral and Acidic Amino Acid only)
Composition in Relative Moles
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Half Cys
Val
Met
lieu
Leu
Tyr
Phe
9.1
11
9.8
12
11.3
4.4
8.5
3.3
4.1
4.3
3.6
1.8
3.4
-7-
-------
3.0
2.0 -
o
<
CO
DC
O
CO
QQ
1.0 -
0
50
TUBE NUMBER
Fig. 1. Liver supernatant (after centrifugation) applied to a Sephadex
6-75 column (2.5 x 100 cm). Flow rate .15 ml/hr. Elution
volume: 10 ml/tube. Elution buffer: 0.001 M Tris-HCl (pH 8.6).
Radioactivity (resulting from radioactive cadmium chloride) given
by the curve showing A and B. The other curve represents absor-
bance measurements.
-8-
-------
1.2 -
o.
o
cc
o
o
I
cc
0.6 -
50
TUBE NUMBER
Fig. 2. Purification of B (liver) (lyophilized fraction from Sephadex
G-75) on a Sephadex G-50 column (2.5 x 60 cm). Equilibrated
and eluted with 0.001 M Tris-HCl (pH 8.6). Elution volume:
5 ml/tube. Upper curve gives absorbance at 252 nm. Lower
curve indicates radioactivity (cadmium).
-9-
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1.2
CO
o
a.
O
cc
O
>
O
0.8
CO
cc
o
C/5
S 0.4
R
0.25
LU
5
6.15
0.05
40
TUBE NUMBER
80
Fig. 3. Further purification of M2 using DEAE-A25 Sephadex column (1.5 x
45 cm). Equilibrated and sample applied with 0,05 M Tris-HCl
(pH 8.6). The linear salt gradient was established by gradually
mixing 200 ml 0,05 M Tris-HCl. Higher curve: radioactivity.
Lower curve: absorbance.
-10-
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1.0
D
0.5
I
DEAE~N
DEAEI
40
50 60
TUBE NUMBER
- .228
-.124 -
Fig. 4. Further purification of M? using a DEAE cellulose column (2.2 x
5 cm) equilibrated with 0.01 M Tris-HCl buffer (pH 8.6). The
limiting buffer for the linear salt gradient was 0,4 M,
Solid curves:
Highest peaks: Absorbancy 250 nm
Peaks just below those highest peaks: Cadmium determined by
Atomic Absorption
Stipled peaks:
Zinc determined by Atomic Absorption
Absorbancy: 280 nm
-11-
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-78-010
2.
4. TITLE AND SUBTITLE
Radioimmunoassay of metallothionein
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
.lannavw 1Q7Q
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Felix Friedberg
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Biochemistry, College of Medicine
Howard University
520 W Street, N.W.
Washington. D.C. 20059
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
R-803201
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP.NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The goal of this project was to develop a radioimmunoassay for metal!othionein.
Since this protein is involved with the transport of cadmium in biological systems
and may in fact protect against cadmium poisoning, the ability to monitor the levels
in the human population is of the utmost importance to our evaluation of the hazards
of environmental cadmium exposure.
While researchers do not agree whether "copper thioneins" and "zinc thioneins"
of rats are identical, this study found in preliminary analysis that zinc thionein
appears to have a composition similar to that of copper chelatins.
It is recommended that development of a radioimmunoassay should not be
attempted until the putative metallothioneing is clearly characterized. There are
too many kinds of metallothionein induced by the injection of different metals
according to the reports in the literature. They must be properly identified first.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Proteins.
amtno acids
assaying
cadmium
zinc
metallothionein
06 A, T
14 B
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
15
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
12
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