Circular dichroism and magnetic circular dichroism of bismuth-induced, metallothionein-like proteins

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

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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