Vol. 108, No. 3, 1982 October 15, 1982
BIOCHEMICAL
Circular Dichroism of Bismuth-Induced,
Department
August
RESEARCH COMMUNICATIONS Pages 919-925
and Magnetic Circular Metallothionein-Like
A. Szymanska $ and Martin
Jadwiga
Received
AND BIOPHYSICAL
Dichroism Proteins *+
Stillman
J.
of Chemistry, University of Western London, Ontario, Canada N6A 5B7
Ontario,
2, 1982
SUMMARY: Optical studies have been carried out on bismuth-containing proteins which were isolated from the livers and kidneys of rats following injections of BiC13. Absorption, circular dichroism and magnetic circular dichroism spectra of hepatic Bi,Zn-metallothionein 1 and 2 indicate that the spectra are dominated by transitions from the zinc thiolate chromophore. The data from the renal Bi,Cu-metallothionein 2 are quite different and it is suggested that these spectra involve a mixture of transitions from the bismuth and copper thiolate binding sites.
INTRODUCTION Metallothioneins hanced
are widely
concentrations
are observed
(Cd,Zn,Cu,Hg,Ag,Au,Bi) metabolism
(l-12).
of bismuth
excretion
of this
attention
has been
isolated
from
proteins
in this
ions
after
the kidneys organ
bismuth as mercury,
kidneys,
and that
weight lated
given
protein
both
(9,19,20).
and characterised
following
exposure
concentrated
mainly
to the properties of rats, increases
because well
bismuth
The renal (11).
These
on the
Recently,
bismuth-binding preliminary
concentrat-
was found
copper
are bound
greater protein
physiological
the
and
of metallothionein-like
It
enhance
ions
distribution
of the bismuth-binding
above normal
and copper
studies
(11-17).
the level
En-
metal
on the
(11,12,18,19).
and cadmium,
organisms.
to various
time,
in humans and in animals
administration gold
in eucaryotic
Up to the present
have been
metal
as well
distributed
content
by the
in
bismuth, rat
same low-molecule
protein studies
that
has been
classified
isothe low-
*
Address correspondence to this author. +Mbem er of the Centre for Interdisciplinary $Visiting Fellow, Centre for Interdisciplinary Home Department: Department of Toxicological LsdG, POLAND.
Studies in Chemical Physics. Studies in Chemical Physics. Chemistry, Medical Academy,
0006-291X/82/190919-07$01.00/0 919
Copyright 0 1982 by Academic Press, Inc. All rrghrs of reproduction in any form reserved.
Vol. 108, No. 3, 1982
BIOCHEMICAL
molecular
weight,
protein.
However,
whether
this
bismuth-binding
Bismuth which
for is
appears
Despite
(ie.
the
available where
The data
of a bismuth-induced from both
a comparison like
proteins
viva
induced
in the
that
optical
There
.&
paper
the metal-binding by the
MATERIALS
This
will
induce
effect (2,21).
metallothionein
full
optical
studies
of naturally
isolated
from
various
of mixed
metal
metallothioneins,
reports
(27,28). the for
of the
properties ion
of rats
however,
data
same metal
liver
also,
represent
and kidneys
(12).
are
viiX0
We report
liver
of mercury
Cd,Zn-MT
properties
exchanged
livers
(12).
MCD spectra)
to hepatic
(11).
in the
to metallothionein,
Zn+T(26,27).
protein.
between
zinc-metallothionein
injections ions
to conclude
same as a classical
of bismuth
more recently,
in this
the
studies
was the
following
limited
have been
described
these
of bismuth
of metal
&
Cu-MT(25),
the metals
isolated
number
the
as a metallothionein-like
cadmium,
amounts
observed
have been
describing
RESEARCH COMMUNICATIONS
of a zinc-metallothionein
and CD, with,
(22-24),
protein
the binding
and can be bound
proteins
animals
large
from
hepatic
small
to that
absorption
loaded
about
protein
possible renal
to contain
similar
synthesis
was not
biosynthesis
found
renal
example,
known
induces is
it
bismuth-induced
metallothionein, Little
AND BIOPHYSICAL
first
optical
studies
bismuth-binding same animals,
proteins this
allows
of the metallothionein-
exposure
in these
two organs.
AND METHODS
Ptrottin phepatrtionh. Low-molecular weight, bismuth-binding proteins were isolated from the kidneys and livers of rats exposed to BiC13 (3mg Bi/kg). Control levels were 0.15 mg/g of kidney and 0.10 mg/g of liver with no metals added and 0.50 mg/g and 0.21 mg/g after injections of BiC13 (19). Bismuth was applied subcutaneously, every second day, over 14 days. The proteins were isolated according to the method of Zelazowski et al. (29). Polyacrylamide gel electrophoresis of the protein preparations was performed according to Jovin et al. (30), the renal protein was separated into three isoforms of R,: 0.35, 0.57 and 0.80. Each of these isoforms contained considerable amounts of bismuth and copper, the hepatic protein was separated into two isoforms of Rm: 0.57 and 0.72. Total protein concentration was estimated using tannic acid (31) and the molecular weight was determined to be 10,000 daltons from gel filtration studies. This value was used to determine the concentrations of solutions that were used for metal analysis. Both of these isoforms contained mainly zinc. The metal ion contents (Bi, Zn,Cu) were estimated using a plasma emission spectrometer (Spectra Span IIIB). We wish to thank the Canada Centre for Inland Waters, Burlington, Ontario for allowing us to use this instrument. Contamination by free Bi is considered most unlikely in view of the highly selective purification procedures used. Analysis for bismuth was carried out from all fractions 920
Vol. 108, No. 3, 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The majority of the collected from a G-75 column during purification. bismuth was detected in tubs containing the low molecular weight protein, no bismuth was detected in the region expected for free bismuth or bismuth bound to free amino acids (10). The renal, bismuth-binding protein [Bi, Cu-MT 21 contained: 34.1 ug Bi/mg, 10.5 ng Cu/mg and 3.75 ng Zn/mg of protein. Isoform 1 of the bismuth-binding hepatic protein contained: 33.5 ng Zn/mg, 2.5 ng Cu/mg and ca. 1 ng Bi/mg of protein, and isoform 2 contained: 45.5 ng Zn/mg, 1.7 ng Cu/mg and 3.6 ug Bi/mg of protein. Both isoforms contain 0.1 ng Cd/mg. The renal protein isoform of Rm = 0.57 [Bi,Cu-MT 21 and both isoforms of the hepatic protein [Bi,Zn-MT 1 and 21 were used for the spectroscopic studies. .?pec,tim5copic meauhemmevks.Absorption spectra were recorded on a Cary 219 spectrophotometer. CD measurements were made with a JASCO CD/ORD-5 spectrometer which had been modified to Sproul SS-20 specifications. MCD spectra were recorded using an Oxford Instruments SM 2 superconducting magnet operating at 5.5T. Each MCD spectrum reported here has had the corresponding zero field CD spectrum subtracted from it. The pH of protein The units for the absorption spectra samples was between 7.0 and zi3. ordinates, E, are litre mole cm-1, for the CD spectra ordinates, AE, the units are litre mole-l cm-l and for the MCD spectra ordinates, AEM, the units are litre mole-l cm-l tesla-l. For CD spectra the following approximate relationship obtains: deg cm2 3300 AEL-R = [e], where [8] has units dmol-1. RESULTS AND DISCUSSION Fig.
1 shows
the absorption,
both
isoforms
of Bi,Zn-MT
spectra
in this
coworkers
closely for
sample
addition
mainly was incuded
contained
mole
ratio
Fig.
1 were
for
Bi,Zn-MT
samples,
Bi,Zn-MT
features
from
the zinc
component.
24),
the MCD spectrum
region presume
arising that
from this
also
in
the samples
used
a small
in the
will
lack
of detail
is
mainly
the
921
to
in and
our bismuth
is
the
found;
spectral
Cd,Zn-MT
220-250
is
at all
resolved,
the overlap
effects. arise
MCD data
in the
both
The
Zn(5.2),
undoubtedly
not
in
reported
proteins
clusters due
but
In both
of cadmium
unlike
and copper,
zinc
the spectra
of the Bi,Zn-MT
by zinc,
of copper.
be no significant
spectra
thiolate
of cadmium
Zn(7.0).
of the bismuth-binding zinc
was induced
Cu(O.4):
amount
Unfortunately,
the
for
and
the zinc-
concentration
Cu(O.3):
there
ratio
Bi(0,05):
Bi(0.18):
that
which
and CD
by Weser
unlike
contained
and a significant
Zn-MT,
so low
The major
it
reported
et al.,
(MCD) for
The absorption
However,
low mole
1 Cd(cO.01):
2 Cd(cO.01):
is
and a very
bismuth
and in Weser's
concentration
Zn-MT.
by bismuth,
of the metals for
liver
dichroism
livers.
the data
used by Weser zinc
circular
from rat
resemble
rat
metallothionein
and contained our
isolated
figure
(22,25,27)
containing
CD and magnetic
(23, nm
of the weak
we
Vol. 108, No. 3, 1982
BIOCHEMICAL
AND BIOPHYSICAL
7”““““”
(BI,ZN)
5.0
MT
0.20
RESEARCH COMMUNlCATlONS /
5.0.
1
/
I
( BLZN
mgtml
I
I
I
1 MT
0.20
,
2
mglml
A 250= 0.250 * ,o .” ”
*o . w
2.5
0.0 -2,0y T
MCD
MCD
-2.0I
W” w” a
W’ a
-4.0
-4.0
lO.O-
0.0 W a
-lO.O-
W a
CD
CD
-10.0
-20.0 I
200
Figure
zinc
1.
absorption
increase
measurement for
in absorbance
(24)
indicated
Cd charge
exist
for
Zn-MT we would
under
the
S -->
below
bands.
There
spectra
of both
isoforms
is
2, a second
300
transfer
chain
effectively Model
3
and the
precludes
should
no comparable
to observe
bands,
compound
envelope
while
model
the
MCD spectra be seen
in
compound
a similar
derivative
envelope
there
are a series
of over-
data
bands. spectrum
no associated
CD intensity.
show a negative negative
peptide
235 nm.
a derivative
region,
expect
intense
245 nm which
the absorption
lapping
250
dichroism (MCD) and circular rat hepatic, bismuth-induced
the
below
that
transfer
Zn charge
Above 250 nm in
Bi,Zn-MT
with
of CD or MCD spectra
Cd,Zn-MT
:f 200
WAVELENGTH/NM
bands
the S -->
for
I I 350
Absorption, magnetic circular dichroism (CD) spectra of the metallothionein-like protein.
thiolate
rapid
I 1 I 300 WAVELENGTH/NM
250
, I
band 922
band is
However,
at 270 nm and in
observed
at 310 nm.
the MCD the spectrum These
two
Vol. 108, No. 3, 1982
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
( BLCU 1 MT 2 5.0.
0.20
mg:ml
A,,;0.760 -0 < 0
2.5
15.010.0. 5.0. w a
o.o-
CD
-5.0.
-
-1o.o200
Figure
bands
lie
late
2.
in the
region
expected
chromophores
(22,25).
Fig.
the data
2 shows
protein
pared
of protein)
to 1 mole
little
significant
and renal
ent,
a positive
300 nm.
Both
Because
in this
addition
the bismuth spectrum
like
for
has a mole
ratio
of Zn(0.6): in the
the disulfide
Bi,Cu-MT for
the metals
absorption of the
are
protein
there
it
seems likely
and copper
sites.
the data
shown
is
that Copper
in Fig.
kidney.
bound
renal
to it
between
protein
is
resolved with
amount
is
2 (22,27). 923
there
different also
negative
differband at
the new CD bands.
of bismuth
and copper,
the CD and MCD intensity metallothionein
(com-
the hepatic
quite
The MCD spectrum
thio-
This
Although
spectrum
associated
a large
rat
Bi(1.7).
at 330 nm and a poorly
features
and the Cu(1)
2 from
Cu(1.5):
at 290 nm and 330 nm. band
of these
to zinc,
both
the CD spectrum
new bands
with
for
measured
change
proteins,
exhibiting
500
Absorption, magnetic circular dichroism (MCD) and circular dichroism (CD) spectra of the rat renal, bismuth-induced metallothionein-like protein.
bismuth-induced
is
400 WAVELENGTH/NM
300
arises
does not To account
in
for
from
exhibit the
a CD CD
Vol. 108, No. 3, 1982
BIOCHEMICAL
spectrum
shown
in Fig.
by bands
arising
from
modify
the protein
of the copper(I) of copper
2 we suggest the bismuth
structure CD are
and bismuth
AND BIOPHYSICAL
or that
this
spectrum
the
effect
to such an extent
that
changed,
related
that
RESEARCH COMMUNICATIONS
the observed
is
either
dominated
of the bismuth the distinctive
CD spectrum
being
is
to
features a mixture
transitions.
ACKNOWLEDGEMENTS The authors Chemical Program.
gratefully Physics We thank
acknowledge
financial
support
at the UWO and NSERC of Canada Mr.
Chris
Fahrner
for
technical
from
through
the Centre the Stratetic
for Grant
assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Metallothionein (Kagi J.H.R. and Nordberg M., eds.) 1979, Experientia supp. 34. Winge D.R., Premakumar R. and Rajagopalan K.V. (1975) Arch. Biochem. Bioph. 170, 242-252. Winge D.R., Premakumar R. and Rajagopalan K.V. (1978) Arch. Biochem. Bioph. 188, 466-475. B.L. and Garvey J. (1981) Arch. Biochem. Bioph. Winge D.R., Geller 208, 160-166. (1980) Biochim. Biophys. Acta 625, Zelazowski A.J. and Piotrowski J.K. 89-99. (1980) Biochim. Pharmacol. 29, 2017-2021. Sharma R.P. and McQueen E.G. (1977) Biochem. Pharmacol. 26, Mogilnicka E.M. and Piotrowski J.K. 1819-1820. Mogilnicka E.M. and Piotrowski J.K. (1979) Biochem. Pharmacol. 28, 2625-2631. Mogilnicka E.M. and Webb M. (1981) J. App. Toxicol. 1, 42-49. Szymanska J.A., Mogilnicka E.M. and Kaszper B.W. (1977) Biochem. Pharmacol. 26, 257-258. Szymanska J.A. and Piotrowski J.K. (1980) Biochem. Pharmacol. 29, 2913-2918. Szymanska J.A., Zelazowski A.J. and Kawiorski S. (1981) Clin. Toxicol. 18, 1291-1298. Sollman T., Cole H.N. and Henderson K.J. (1933) Archs. Derm. Syph. 28, 615-630. (1981) Clin. Martin-Bouyer G., Foulon G., Guerbois H. and Barin C. Toxicol. 18, 1277-1283. Russ G., Bigler R.E., Tilbury R.S. Wooders H.Q. and Laughlin J.S. (1975) Radiat. Res. 63, 443-453. Pieri F. and Wegmann R. (1981) Cell. Molec. Biol. 27, 57-60. Chaleil D., Lefevre F., Allain P. and Martin G.J. (1981) J. Inorg. Biochem. 15, 213-221. Piotrowski J.K. and Szymanska J.A. (1976) J. Toxicol. Environm. Hlth. 1, 991-1002. Szymanska J.A. and Zelazowski A.J. (1979) Environm. Res. 19, 121-127. Stonard M.D. and Webb M. (1976) Chem.-Biol. Interact. 15, 349-363. Martin M. and Brady F.O. (1977) Proc. S.D. Acad. Sci. 56, 72-76. Rupp H. and Weser U. (1978) Biochim. Biophys. Acta 533, 205-226. 924
Vol. 108, No. 3, 1982 23. 24. 25. 26. 27. 28. 29. 30. 31.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Law A.Y.C. and Stillman M.J. (1980) Biochem. Biophys. Res. Comm. 94, 133-143. Law A.Y.C. and Stillman M.J. (1981) Biochem. Biophys. Res. Comm. 102, 397-402. Hartmann H.J. and Weser U. (1977) Biochim. Biophys. Acta 491, 211-222. Buhler R.H.O. and Kagi J.H.R. in Metallothionein (Kagi, J.H.R. and Nordberg, M., eds.) 1979, Experientia Supp. 34, pp. 211-220. Sokolowski G. and Weser LJ. (1975) Hoppe-Seyler's 2. Physiol. Chem. 356, 1715-1726. Holmquist B. and Vallee B.L. (1981) BiochemVasak M., Kagi J.H.R., istry 20, 6659-6664. Prep. Biochem. Zelazowski A.J., Szymanska J.A. and Witas H. (1980). 10, 495-505. (1964) Anal. Biochem. 9, Jovin T., Chrombach A. and Naughton M.A. 351-369. Pol. 2, 279-290. Mejbaum-Katzemellenloogen W. (1955) Acta Biochim.
925