BIOCHEMICAL

Vol. 74, No. 4, 1977

CADMIUM S. R. Watkins, Department Received

R. M.

-BINDING Hodge,

of Chemistry,

October

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SERUM D.

PROTEIN

C . Cowman

Coe College,

Cedar

and

P. P. Wickham

Rapids,

Iowa

52402

25,1976

Human serum alpha-2-macroglobulin has been found to be a major SUMMARY: cadmium-binding protein in vitro. Serum and alpha-2-macroglobulin equilibrated -__ with cadmium at the 0.20 ppm level were chromatographed over Sephadex and agarose gels to separate and estimate the molecular weights of the proteins. Alpha-2-macroglobulin was found to fragment into reproducible fragments when chromatographed on agarose gels showing different metal-binding fractions for cadmium and endogenous zinc. The distribution of cadmium on serum protein chromatograms was correlated with alpha-2-macroglobulin chromatograms. Cadmium was bound to fractions with molecular weights as high as 800,000 daltons with an affinity greater than that observed for serum albumin. INTRODUCTION: extensively

studied,

systems. human

The metal-binding with

the greatest

Cadmium-binding serum

albumin

studies

(1,2,3)

from --~ in vitro

studies

that

to albumin.

--In vitro

studies

ing to alpha-globulins the finding affinity

loosely

(5).

than

of serum

emphasis

bound

(4,5).

show

It is the purpose protein

proteins

cadmium

on rat plasma

that

proteins

on copper

on serum

and rat plasma

of a cadmium-binding

for cadmium

properties

Giroux

been

limited

to

(2) has suggested is bound

a predominant

of this

been

and zinc-binding

have

in serum

have

communication

has a substantially

primarily

cadmium

bind-

to report greater

albumin.

MATERIALS AND METHODS: Tris buffer, 0. 1M (Sigma) adjusted to pH 7.4 with concentrated HCl, 0.02% sodium azide (NaN3), was used for preparation of standards, solutions and gel elutant. Metal contamination was minimized by passing all buffer through a Chelex-100 resin (Bio-Rad) column before use. Cadmium extraction was carried out with Eastman technical grade methyl isobutyl ketone (MIBK) and ammonium pyrrolidine dithiocarbamate (APDC). Three separate gel media were employed. Sephadex G-150, 100-200 mesh (Pharmacia), prepared as per manufacturer’s instructions, was used in a 2.5 x 110 cm Kontes Kromaflex glass column. The column had a bed height of 104 cm, void-volume of 140 ml and a flow of 70 ml/hr. A similar column was packed with Bio-Gel A-Sm, 200-400 mesh (Bio-Rad). The column had a bed height of Copyright All rights

0 I977 by Academic Press, Inc. of reproduction in any form reserved.

1403

ISSN

0006-291X

Vol. 74, No. 4, 1977

BIOCHEMICAL

Fraction

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Number

Fig. 1. Elution pattern of human serum (-) on Sephadex G-150 the distribution of Cd (-----) and 2 n (* . . * .) . Cd concentration ppm; Zn concentration, 1.6 ppm.

showing is 0.2 1

95 cm, void-volume of 156 ml and a flow of 18 ml/hr. Bio-Gel A-1.5 m (BioRad) was packed in a 2.5 x 55 cm Kontes Kromaflex glass column. The column had a bed height of 50 cm, void-volume of 100 ml and a flow of 36 ml/hr. Each column was calibrated by the method of Andrews (6) using fribrinogen (Schwartz/Mann), albumin (Pentex), alpha-2-macroglobulin (American National Red Cross) and blue dextran (Sigma). Prior to running a sample of the metal-incubated serum or protein, the column was eluted with a 3 .O ml sample of 225 mM EDTA (ethylenediaminetetraacetic acid) followed by Tris buffer until two EDTA elution volumes of buffer ran through the column. Blood collected from healthy donors was pooled, allowed to clot and the serum extracted. 37.0 ml of the whole serum was incubated with 60~1 of 100 ppm cadmium stock standard on a RSCO shaker bath at 37OC for 72 hr. Solutions of alpha-2-macroglobulin were prepared by dissolving 0.050 g of 74% pure protein (American National Red Cross) in 1.5 ml Tris buffer and were incubated Zinc with 40~1 of 10 ppm cadmium stock standard for 24 to 76 hr at 37OC. present in the samples was endogenous. Serum chromatograms used 3.0 ml of the incubated sample, alpha-2-macroglobulin chromatograms used 1.5 ml. 80 drop fractions (4.0 ml) were collected by a model 1200 ISCO Pup Golden Retriever fraction collector. Fractions were collected in acid-washed, demineralized water-rinsed polypropylene tubes to minimize latent metal contamination. Protein determination was carried out using 2.0 ml of alternate fractions in quartz cells on a Beckman DB spectrophotometer 280 me.

1404

BIOCHEMICAL

Vol. 74, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.5. IV 1.2-

s 5 06z 2 -a

I: ,:

I :I :I .I :I :’

I: ;; I: 1: 1:

0.3-

:I :I.,

I: 90 Froctlon

v

120

140

Number

() on Sephadex G-150 Elution pattern of human serum Fig. 2. between the proteins the distribution of Cd (-----) and Zn ( **=..) ligand EDTA (30mM concentration). Cd concentration is 0.20 ppm; centration, 1.6 ppm.

showing and the Zn con-

A Perkin Elmer Model 103 atomic absorption spectrophotometer with a Model 56 recorder were used for cadmium and zinc analysis. Fractions analyzed for protein were also analyzed for zinc by a direct aspiration method. Cadmium determinations were performed by extraction of the cadmium-APDC complex into MIBK and analyzing the extract using a Delves cup microsampling assembly (7). Protein absorbance and metal concentrations were then plotted vs. fraction number. RESULTS: ___-

Under

volume

fraction

graphed

sample and

being

of 300,000

Bio-Gel than

A-5m

described,

in addition (peaks with

daltons was

300,000

bound

cadmium

to the albumin I and III of Fig.

30 mM EDTA

as the EDTA complex

cadmium

weights

G-150

equilibrated

zinc

remaining

greater

of serum

on Sephadex

serum mium

the conditions

(peak

shows

IV,

Fig.

to one or more

or greater

(peak

I, Fig. proteins

to resolve

serum

(Fig.

3),

cadmium-incubated

1405

to bind

fractions

when

to a voidchromato-

1 respectively). the partial 2), with

proteins

used

When

is seen

A similar

removal virtually

having

of cadall

the

molecular

2). with

molecular serum

was

weights fraction-

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I.?.-

03-

Fraction

Number

Fig. 3. Elution pattern of human serum (-) the distribution of Cd (-----) and Zn (* ’ . * s). ppm; Zn concentration, 1.3 ppm .

on Bio-Gel A-5m showing Cd concentration is 0.26

1.5-

l.2-

:c 0.6x b u) 9

Ill :. II

.

I

:

. ,

0.3-

130 Fraction

Number

) on Bio-Gel A-5m (Fig. 4. Elution pattern of alpha-2-macroglobulin showing the distribution of Cd (-----) and Zn ( * * * * *) . Cd concentration is 0.22 ppm; Zn concentration, 0.4 1 ppm; protein concentration, 3.2%.

1406

BIOCHEMICAL

Vol. 74, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 15

. . . . . . . . . . . . . . . . . . . .f(

1.2. 2 Cl : 0.9.

: 5

0.6

1’

0.3

:, i II.

:. . . . . . . . . . . . . . . . . . ; 1: 1. I: I’ I: r

8 v) 4”

I

I’0

.

I

3’0

5’0 Number

Fraction

() on Sephadex (* * * * .) . Cd concentration concentration, 3.5%.

Fig. 5. Elution pattern of alpha-2-macroglobulin showing the distribution of Cd (-----) and Zn 0.19 ppm; Zn concentration, 0.3 6 ppm; protein

ated on this

column,

the most

cadmium

molecular

weight

being

with

observed:

instead,

Solutions bated G-150 when with

with (Fig.

the

cadmium-binding

associated

800,000 zinc

weight

was

with

(peak

dalton

region

found

of 75,000

cadmium

an approximate

peak II

3.

3).

observed,

an approximate The expected

zinc

(alpha-2-macroglobulin) with

was

peakIII,

havtig

A-5m

weight

When

This

endogenous

on Bio-Gel

m (Fig.

cadmium.

of Fig.

Fig.

containing

Bio-Gel

molecular

having

were

is

not

an approxi-

daltons.

5) and Bio-Gel A-1.5 over

II,

exclusively

and chromatographed

chromatographed

regions

a region

daltons

of alpha-2-macroglobulin

90% of the recovered binding

distinct

of 400,000

association

mate molecular

three

G-150

6). (peak

This II,

of 400,000 corresponds

alpha-Z-macroglobulin

1407

A-5m protein Fig.

zinc (Fig.

were

4),

Sephadex

appears 4) forming

daltons

which

to the

400,000

incu-

to fragment a fragment

binds

almost

dalton

is chromatographed

cadmium on

Vol. 74, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-10

I .5-

-BN0 x r; E -6 0

1.2E’ s cd 0.9-

a

r;

OS

() on Bio-Gel A-l (-. a a a). Cd concentration concentration, 4.8%.

Fig. 6. Elution pattern of alpha-Z-macroglobulin showing the distribution of Cd (-----) and Zn 0.23 ppm; Zn concentration, 0.39 ppm; protein

Sephadex tions

G-150

having

fragments graphed

(Fig.

S), both

molecular

of lower

weights

molecular

on Bio-Gel

A-l

bound

to a fraction

with

(peak

I, Fig.

Although bands

6),

observed

with

two-thirds

appeared

daltons

do not bind

an approximate that

either

molecular with

the Bio-Gel

on Bio-Gel

A-l.

5 m is less

of 400,000

and

of the recovered

metal.

weight

70,000

cadmium

frac-

Impurities

shows

observed

weights

in void-volume

or greater.

.5 m, alpha-2-macroglobulin

unlike

molecular

and cadmium

of 300,000 weight

the fragmentation with

zinc

When

.5m is

or

chromato-

cadmium

and zinc

of 800,000

daltons

A-5 m chromatogram. extensive, daltons

bound

two

protein

respectively

to the 70,000

are dalton

band. DISCUSSION: -__-._---more

proteins

as much

It is concluded of higher

molecular

as 50% of the serum-bound

that

cadmium

weight

than

cadmium.

1408

associates albumin:

strongly these

The affinity

with

proteins

of these

one or can bind

proteins

for

Vol. 74, No. 4, 1977

cadmium (Fig. (2),

BIOCHEMICAL

is demonstrated

2).

Giroux

reports

and Sugiura

EDTA,

removed

the metal

tein

modification Because

cadmium

was

on the agarose

to strongly

G-150, gels

show

for zinc, tion

bound

binding

have

li-

effectively indicating

bound

zinc

(B),

found

to have

by some pro-

considerations

for this

with

study

the chelating

avoided.

from 35 to 113%. with

metals,

were

ligand, balance

Cadmium

it appears requires

metal

the metal

the associated

with

suggestdifferent

in the metal

of cadmium

ranged studies

the incorporation equilibration.

cadmium

1409

to behave

profiles,

fragments

the level

to other

extended

appear

and alpha-2-macroglo-

of the metals

that

with

binding

the time of incubation

recoveries

In comparison

the alpha-2-macroglobulin is incorporated,

different

constituent

for cadmium.

and cadmium

due to a difference

and the material

was

affinity

of serum

into

weight

its association

a high

zinc

is breaking possibly

saturation

of proteins

metal

molecular

bind

significantly

of the metals,

equilibration

contamination

stronger

high

chromatograms

ing that the alpha-2-macroglobulin

to avoid

the con-

the cadmium

and not irreversibly

is a major

and has been

on Sephadex

mium,

at 30 times

acid)

the alpha-2-macroglobulin-bound

Important

histidine

experiment,

from alpha-2-macroglobulin,

exchangeable

shown

studied

distributions

with

In this

used

with

30 mM EDTA

process.

and has been

similarly

(3).

in removing

studies

and cadmium

is in fact

of serum

bulin

active

with

cadmium

1 mM EDTA

ineff

Similar

alpha-2-macroglobulin

Although

bound

(cyclohexanediaminetetraacetic

all of the zinc

that

of cadmium

for cadmium,

was

fractions.

such as, CyDTA

with

histidine

by Sugiura,

to the void-volume gands

than

removal of albumin

96% removal

ligand

reported

partial

50% removal

reports

a stronger

centration

by only

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

cad-

necessary

to ensure from

sites.

that

loss

60 to 104% and

involving

incuba-

of cadmium However,

is as strongly

bound

into

once as the

or

BIOCHEMICAL

Vol. 74, No. 4, 1977

endogenous zinc.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This behavior for cadmium is different

than that reported for

zinc in that --in vitro incubation of serum with zinc is ineffective ting the zinc into the alpha-Z-macroglobulin These preliminary

complex.

in incorpora-

(8)

studies support the conclusion that when serum is incu-

bated --in vitro with cadmium for at least 24 hr at 37”C,

a major fraction of the

cadmium is associated with a high molecular weight macroligand other albumin.

In addition,

the similarity

of the cadmium distribution

grams of serum and alpha-2-macroglobulin the zinc-binding

protein,

ACKNOWLEDGEMENT:

than

in chromato-

indicates that this macroligand is

alpha-2 -macroglobulin. Alpha-2-macroglobulin

used in these studies was

provided by the American Red Cross National Fractionation

Center.

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8.

Perkins, D. J. (1961) Biochem. J., 80, 668-672. Giroux, E. L. and Henkin, R. I. (1972) Bioinorg. Chem., L, 125-133. Sugiura, Y. and Tanaka, H. (1970) Radioisotopes, 19, 7-12. Shaikh, Z. A. and Lucus, 0. J . (l972) Arch. Environ. Health, 24, 410-418. Gasiewicz, T. A. and Smith, J. C. (1976) Biochim. et Biophys. Acta, 428, 113-122. Andrews, P. (1965) Biochem. J. 96, 595-606. Ediger, R. D. and Coleman, R. L. (1973) At. Absorption Newslett., 12, 3-6. Parisi, A. R. and Vallee, B. L. (1970) Biochemistry, 2, 2421-2426.

1410

Cadmium-binding serum protein.

BIOCHEMICAL Vol. 74, No. 4, 1977 CADMIUM S. R. Watkins, Department Received R. M. -BINDING Hodge, of Chemistry, October AND BIOPHYSICAL RESEARC...
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