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.
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