J Biol Inorg Chem (2009) 14:61–74
DOI 10.1007/s00775-008-0424-1
ORIGINAL PAPER
Simultaneous Cu-, Fe-, and Zn-specific detection of metalloproteins
contained in rabbit plasma by size-exclusion chromatography–
inductively coupled plasma atomic emission spectroscopy
Shawn A. Manley Æ Simon Byrns Æ Andrew W. Lyon Æ
Peter Brown Æ Jürgen Gailer
Received: 16 April 2008 / Accepted: 23 August 2008 / Published online: 10 September 2008
Ó SBIC 2008
Abstract Analytical methods which are capable of deter-
mining the plasma or serum metalloproteome have inherent
diagnostic value for human diseases associated with
increased or decreased concentrations of specific plasma
metalloproteins. We have therefore systematically devel-
oped a method to rapidly determine the major Cu-, Fe-, and
Zn-containing metalloproteins in rabbit plasma (0.5 mL)
based on size-exclusion chromatography (SEC; stationary
phase Superdex 200, mobile phase phosphate-buffered sal-
ine pH 7.4) and the simultaneous online detection of Cu, Fe,
and Zn in the column effluent by an inductively coupled
plasma atomic emission spectrometer (ICP-AES). Whereas
most previous studies reported on the analysis of serum, our
investigations clearly demonstrated that the analysis of
Parts of the work described in this paper were presented at HPLC
2007 in Ghent, Belgium.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-008-0424-1) contains supplementary
material, which is available to authorized users.
S. A. Manley
S. Byrns
J. Gailer (&)
Department of Chemistry,
University of Calgary,
2500 University Drive NW,
Calgary, AB T2N 1N4, Canada
e-mail: jgailer@ucalgary.ca
A. W. Lyon
Department of Pathology and Laboratory Medicine,
University of Calgary and Calgary Laboratory Services,
9, 3535 Research Rd NW,
Calgary, AB T2L 2K8, Canada
P. Brown
Teledyne Leeman Labs,
6 Wentworth Drive,
Hudson, NH 03051, USA
plasma within 30 min of collection results in the detection of
one more Cu peak (blood coagulation factor V) than has
been previously reported (transcuprein, ceruloplasmin,
albumin-bound Cu, and small molecular weight Cu). The
average amount of Cu associated with these five proteins
corresponded to 21, 18, 21, 30 and 10% of total plasma Cu,
respectively. In contrast, only two Fe metalloproteins (fer-
ritin and transferrin, corresponding to an average of 9 and
91% of total plasma Fe) and approximately five Zn metal-
loproteins (a2-macroglobulin and albumin-bound Zn, which
corresponded to an average of plasma Zn) were detected.
Metalloproteins were assigned on the basis of the coelution
of the corresponding metal and protein identified by
immunoassays or activity-based enzyme assays. The SEC-
ICP-AES approach developed allowed the determination of
approximately 12 Cu, Fe, and Zn metalloproteins in rabbit
plasma within approximately 24 min and can be applied to
analyze human plasma, which is potentially useful for
diagnosing Cu-, Fe-, and Zn-related diseases.
Keywords Blood plasma
Size-exclusion
chromatography
Inductively coupled plasma atomic
emission spectrometry
Metalloproteins
Introduction
All organisms must regularly ingest sufficient quantities of
essential trace elements, such as Cu, Fe, and Zn, to main-
tain the continuous in vivo assembly of biologically active
metalloproteins, which are inherently associated with
health [1, 2]. In humans, for instance, about 1% of the total
body Zn content is replenished daily by the diet [3]. Fol-
lowing the absorption of essential trace elements from the
gastrointestinal tract into the systemic blood circulation,
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62 J Biol Inorg Chem (2009) 14:61–74

Table 1 Molecular properties and relative abundances of the major metalloproteins and metallopeptides in human plasma or serum
Metal Metalloprotein or entity which Molecular Number of metal atoms Plasma or serum References
contains bound metal mass (kDa) bound per protein protein concentration

Fe Ferritin 450 B4,500 10–250 lg/L [9]

Transferrin 79.7 2 1.8–3.7 g/L [9]
Cu Blood coagulation factor V 330 1 *10 mg/L [34]
Transcuprein 270 0.5 *180 lg/La [4, 32]
Ceruloplasmin 132 6 0.2–0.6 g/L [9]
Albumin 66 1 36.1–53.6 g/L [4, 9]
EC-SOD 165 4 – [40, 49]
Cu,Zn-SOD 31 – – [40]
Peptides and amino acids \5 – – –
Zn a2 macroglobulin 725 5 1.1–3.7 g/L [4, 9]
Albumin 66 1 36.1–53.6 g/L [9]
EC-SOD 165 4 – [40]
Cu,Zn-SOD 31 – – [40]
SOD superoxide dismutase, EC-SOD extracellular Cu,Zn superoxide dismutase
a
Rat plasma

specific plasma proteins subsequently distribute these ele-
ments to internal organs [4], where absorption occurs by
highly specific uptake mechanisms, such as endocytosis in
the case of Fe [5, 6]. Among the most abundant transition
metals present in human plasma are Cu, Fe, and Zn, which
are present at total concentrations of 0.84–1.45 lg Cu/mL,
0.46–1.67 lg Zn/mL, and 2.47–13.1 lg Fe/mL [7, 8].
Therefore, the analysis of plasma for the contained major
Cu-, Fe-, and Zn-containing metalloproteins (Table 1) will
provide insight into the roles of trace elements in the bio-
chemistry and pathophysiology of both healthy and
diseased states.
It is well known that numerous genetic human diseases
are associated with increased or decreased plasma
concentrations of specific metalloproteins [9]. Wilson’s
disease, for instance, is a Cu-overload disease associated
with low plasma Cu [10] and hereditary hemochromatosis
is an Fe-overload disease associated with elevated plasma
Fe [6]. Conversely, it is chemically feasible that the
exposure of humans to certain environmental pollutants or
that the physiological response to infection could also
result in increased or decreased plasma concentrations of
specific Cu, Fe, and Zn metalloproteins [9]. Therefore, an
instrumental analytical method which can rapidly deter-
mine the major Cu-, Fe-, and Zn-containing plasma
metalloproteins would represent an innovative tool to assist
in screening or diagnosing human diseases associated with
altered essential trace elements, regardless of whether the
latter have a genetic origin or are the result of exposure to
environmental pollutants (chemical or bacterial).
From an analytical point of view, the direct liquid
chromatography (LC) analysis of plasma in conjunction
with an element-specific detector, such as a flame atomic
absorption spectrometer, a graphite furnace atomic
absorption spectrometer, an inductively coupled plasma
atomic emission spectrometer (ICP-AES), or an induc-
tively coupled plasma mass spectrometer (ICP-MS), should
allow the detection of individual plasma metalloproteins
assuming that the metal–protein bond(s) in the latter
remain intact during the LC separation process [11–14].
Despite this attractive proposition, and even though
numerous investigations have been reported on the speci-
ation of metals and metalloid compounds in other
biological fluids and tissues, comparatively few studies
have been carried out to attempt this goal in undiluted
mammalian plasma or serum [15].
All studies which reported on the direct LC analysis of
mammalian plasma or serum for metalloproteins are lis-
ted in Table 2 (only those which detected at least two
of the elements of interest are listed). In particular, size-
exclusion chromatography (SEC), anion-exchange chro-
matography (AEX), and reversed-phase chromatography
have been employed in conjunction with various element-
specific detectors. The pioneering work of Dawson et al.
[16], published in 1981, represents the first study of its
kind to detect metalloproteins in human plasma and
involved SEC analysis (Sephacryl S-300) followed by the
detection of Cu and Zn in the collected fractions by
flame atomic absorption spectrometry. The reconstruction
of a Cu- and Zn-specific chromatogram revealed one Cu
and three Zn peaks [16]. The same approach (Sephadex
G-100) was applied for the analysis of human serum and
graphite furnace atomic absorption spectrometry of the
fractions showed one Cu, one Fe, and three Zn peaks

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J Biol Inorg Chem (2009) 14:61–74 63

Table 2 Applications of liquid chromatography coupled with element-specific detectors for the identification of metalloproteins in mammalian
plasma/serum reported in the literature (in chronological order)
Biological fluid Elements Separation Column Stationary phased Mobile-phase composition Reference
analyzedb detectedc mechanism dimensions
(cm)

Human plasma (0.5) Cu (1) Zn (3) SEC 57 9 0.9 Sephacryl S-300 (25–75) 0.1 M Tris/HCl pH 8.0 ? 0.5 M [16]
NaCl
Human serum (1.0) Cu (1) Fe (1) SEC 100 9 2.6 Sephadex G-100 (40–120) 0.05 M Tris/HCl pH 7.4, 4 °C [17]
Zn (3)
Human serum (5.0) Cu (2) Fe (1) SEC 100 9 2.6 Sephacryl S-300 (25–75) 0.1 M Tris/HCl pH 7.4, 22 °C [18]
Zn (3)
Human serum (0.25) Cu (1) Fe (2) SEC – TSK G 3,000 SW (10) 0.1 M HEPES ? 0.1 M NaCl pH [19]
Zn (4) 7.4
Rat serum (0.1) Cu (4) Zn (3) SEC 30 9 0.7 TSK G 3,000 SW (10) 0.1 M Tris/HCl pH 7.2 [20]
Human serum Cu (4) Fe (1) SEC 25 9 0.2 SynChropak GPC 300 (5) 0.1 M Tris/HCl pH 6.9 [22]
SRM (0.002) Zn (6)
Human plasma? (0.25) Cu (3) Fe (2) SEC 60 9 1.6 Fractogel EMD BioSEC 0.02 M NaH2PO4 ? 0.3 M NaCl [8]
650 (20–40) pH 6.8, 30 °C
Human serum (0.1) Cu (1)a Fe (2) AEX 5 9 0.5 Mono Q HR (10) 15 min linear gradient A (0.05 M [24]
Zn (4)a Tris/HCl pH 7.4) ? B (0.25 M
NH4Ac in A)
Human serum (0.2) Cu (2) Zn (2) SEC – Superose 12 HR (8–12) 0.1 M Tris/HCl ? 2.5 mM CaCl2 [25]
pH 7.4
Human serum (0.02) Cu (1) Fe (1) RP 25 9 0.46 CHAPS-coated ODS 0.2 mM Tris/HCl ? 0.2 mM [21]
Zn (1) CHAPS pH 7.4
Human serum (0.1) Cu (1)a Fe (2) AEX 5 9 0.5 Mono Q HR (10) 15 min linear gradient [23]
Zn (4)a A (0.05 M Tris/HCl pH
7.4) ? B (0.25 M NH4Ac in A)
SEC size-exclusion chromatography, AEX anion-exchange chromatography, RP reversed-phase chromatography, Tris tris(hydroxy-
methyl)aminomethane, HEPES N-(2-hydroxyethyl)piperazine-N0 -ethanesulfonic acid, CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate, ODS octadecyl silica, SRM standard reference material
a
Artifact
b
Volume in mL
c
No. of peaks
d
Bead diameter in lm

[17]. The separation of human serum on another SEC
stationary phase (Sephacryl S-300) and the utilization of
direct current plasma atomic emission spectrometry
resulted in the detection of two Cu, one Fe, and three Zn
peaks [18]. With use of a sequential ICP-AES as the
online multielement-specific detector, the SEC analysis
(TSK G 300 SW) of human serum brought to light one
Cu, two Fe, and four Zn peaks [19]. Applying the same
stationary phase for the analysis of rat serum in con-
junction with an ICP-MS revealed four Cu and three Zn
peaks [20]. Reversed-phase chromatography (octadecyl
silica stationary phase) was also employed to analyze
human serum using an ICP-MS as the Cu-, Fe-, and
Zn-specific detector, but revealed that all metalloproteins
containing these elements were essentially coeluted [21].
Yet another SEC material (SynChropak GPC 300) was
used for the analysis of a reconstituted human serum
standard reference material by an ICP-MS and uncovered
four Cu (one major and three minor), one Fe, and six
non-baseline-separated Zn peaks [22]. Human plasma
analysis by SEC (Fractogel EMD BioSEC 650) with
offline analysis of the fractions by an ICP-AES identified
three Cu and two Fe peaks (all baseline-separated) [8]. A
double focusing ICP-MS was also employed as an online
Cu-, Fe-, and Zn-specific detector for human serum
analysis using AEX (MonoQ HR) [23, 24] and employ-
ing a mobile-phase gradient. AEX, however, altered the
speciation of Cu and Zn and must therefore be avoided
when plasma/serum metalloproteins are to be determined
for potential diagnostic applications. More recently,
human serum analysis by SEC (Superose 12HR) followed
by the online detection of Cu and Zn by an ICP-MS
revealed two Zn and two poorly separated Cu peaks [25].
In view of the reported variability in the number of Cu,
Fe, and Zn metalloprotein peaks that were detected by LC
analysis of mammalian plasma or serum (Table 2) and to

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develop this analytical approach into a clinically useful
diagnostic tool, two key questions must be investigated.
First, it should be examined if plasma and serum generate
consistent analytical results (whichever contains more
individual metal peaks in the corresponding Cu-, Fe-, and
Zn-specific chromatogram inherently contains more infor-
mation). Second, the stability of metalloproteins in plasma/
serum over time must be investigated to establish the
maximum time that plasma samples can be kept (e.g., at
room temperature) before ex vivo degradation of certain
metalloproteins will occur.
We have systematically developed a novel LC method
for the determination of Cu, Fe, and Zn metalloproteins
in rabbit plasma which can be potentially applied to any
mammalian plasma. In view of the fact that the binding
of transition metals in plasma metalloproteins can be
weak [4, 26] and to maintain the integrity of the Cu, Fe,
and Zn metalloproteins throughout the entire LC separa-
tion process [19], we chose SEC as the separation
mechanism. This is because SEC minimizes direct
interactions between the analyte molecules and the sta-
tionary phase and therefore represents a comparatively
gentle separation mechanism for the separation of bio-
molecules. In addition, the utilization of physiologically
relevant buffers will provide an environment where
conformational changes of the plasma metalloproteins—
which could potentially lead to the loss of the metal—are
least likely to occur. Therefore, dissociation of a metal
from its parent plasma protein is minimized. Furthermore,
SEC can be employed in an isocratic separation mode
(which inherently increases sample throughput), whereas
AEX often requires salt gradients to elute proteins which
can—in turn—sever weak transition metal–protein link-
ages. Finally, the availability of SEC stationary phases
with much smaller particle sizes (approximately 13 lm)
and particle size distributions today compared with those
that were used in some previous studies (Table 2) will
inherently allow better separations owing to the increased
chromatographic resolution. Following these consider-
ations to least disrupt weak metal–protein binding
equilibria during the SEC separation process, we
employed phosphate-buffered saline (PBS, pH 7.4) as the
isocratic mobile phase. With regard to the detection of
the separated Cu-, Fe-, and Zn-containing metalloproteins
in the SEC column effluent, we utilized a state-of-the-art
charge injection device based ICP-AES because this
multielement-specific detector could be directly hyphen-
ated to the separation column for the simultaneous online
detection of Cu, Fe, and Zn. In addition, this detector is
compatible with LC separations involving mobile phases
containing more than 0.5% salt [27], which is required to
preclude irreversible adsorption of plasma proteins to the
stationary phase.
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J Biol Inorg Chem (2009) 14:61–74
Materials and methods
Chemicals and solutions
Blue dextran, PBS (10 mM phosphate, 2.7 mM KCl, 137
mM NaCl) tablets, sodium chloride (more than 99.5%
pure), sodium acetate trihydrate (more than 99% pure),
N,N-dimethyl-p-phenylenediamine monohydrochloride
(highly toxic), lysozyme (from chicken egg white), heparin
(sodium salt), and the bicinchoninic acid protein determi-
nation kit were purchased from Sigma-Aldrich (St Louis,
MO, USA), bovine serum albumin (BSA) was from
Amersham Pharmacia Biosciences (Little Chalfont, UK),
glacial acetic acid (more than 97.7% pure, to make 1 M
acetic acid) was from Fisher Scientific (Nepean, ON,
Canada), and Plasma PURE HCl (34–37%) was from SCP
Science (Baie D’Urfé, QC, Canada). A mixture of protein
standards for SEC column calibration was obtained from
Bio-Rad Laboratories (Hercules, CA, USA) which con-
tained thyroglobulin (670 kDa), c-globulin (158 kDa),
ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12
(1.35 kDa). All solutions were prepared with water from a
Simplicity water purification system (Millipore, Billerica,
MA, USA). PBS of pH 7.4 (10 mM phosphate, 2.7 mM
KCl, and 137 mM NaCl) was prepared by dissolving PBS
tablets in the appropriate volume of water (followed by pH
adjustment if necessary) and filtration through 0.45-lm
nylon filter membranes (Mandel Scientific, Guelph, ON,
Canada). Ceruloplasmin oxidase activity was measured in
collected fractions according to a published procedure [28].
Development of an isocratic SEC separation method
of the major plasma proteins using UV detection
Screening of prospective stationary phases
All separations were carried out at room temperature
(22 °C) and a mobile phase flow rate of 1.0 mL/min
(peristaltic pump). Since irreversible binding of plasma
proteins to the stationary phase represents a major obstacle
that must be overcome when plasma is to be directly
analyzed by SEC, we systematically screened three com-
mercially available SEC stationary phases (Sephacryl
S-500, Superose 6 prep grade, and Superdex 200 prep
grade, 25 cm 9 1.0 cm column) for their ability to analyze
rabbit plasma (0.5 mL) using various PBS concentrations
(39, 29 and 19) and a UV detector (280 nm). Each
column was equilibrated with at least 50 mL of the
mobile phase before plasma was injected. To detect irre-
versible binding of plasma proteins to the stationary
phases, a standard protein mixture (BSA and lysozyme)
was chromatographed before and after the analysis of six
consecutive plasma samples. A shift of the retention times
J Biol Inorg Chem (2009) 14:61–74
of the standard proteins after the six plasma injections
(compared with those obtained before the six plasma
injections) would indicate irreversible binding of plasma
proteins to the stationary phase. In addition, the column
effluent of each plasma injection was analyzed for total
protein (bicinchoninic acid assay) and compared with the
total protein contained in 0.5 mL of the plasma to establish
the percentage protein recovery. After six consecutive
plasma injections, no irreversible protein binding was
detected with any stationary phase–mobile phase combi-
nation for the seventh injection (protein recovery 99 ± 1%,
see the supplementary material). On the basis of the
chromatograms obtained and our objective to separate
plasma proteins into as many chromatographic protein
bands as possible (the term ‘‘band’’ is used rather than the
term ‘‘peak’’ since hundreds of proteins constitute a single
chromatographic ‘‘band’’), Superdex 200 prep grade and
PBS (19) were identified as the ideal stationary phase–
mobile phase combination. In particular, Superdex 200
prep grade resulted in four protein bands, corresponding
to 15% (band 1), 14% (band 2), 69% (band 3), and 2%
(band 4) of total protein. In contrast, Superose 6 prep
grade produced only three protein bands, with 10, 89,
and 1% of total protein, and Sephacryl S-500 resulted in
only two protein bands, with 7 and 93% of total protein,
respectively.
Increase of column efficiency by using a higher-resolution
column
The commercially available stationary phase Superdex 200
prep grade is composed of 34-lm particles. In view of the
fact that stationary phases with a smaller particle size
generally result in a better chromatographic resolution, we
improved the separation of plasma bands by using a pre-
packed Superdex 200 column (30 cm 9 1.0-cm inner
diameter) which contained 13-lm particles. Again a mix-
ture of BSA and lysozyme (1.2 and 0.62 mg in 5.0 mL of
water) was used to check the column integrity before and
after the injection of six plasma samples. In addition, the
number of theoretical plates was calculated (using the
lysozyme peak) and increased from approximately 1,000
for Superdex 200 prep grade (25.4 cm 9 1.0-cm inner
diameter, 34-lm particles) to approximately 23,000 for the
prepacked Superdex 200 column (30 cm 9 1.0-cm inner
diameter, 13-lm particles). At least 30 consecutive plasma
analyses could be carried out per column without loss of
chromatographic resolution of the metal peaks.
Animal experiments
The Animal Care Committee of the University of Calgary
approved the procedure to collect blood from New Zealand
65
white rabbits (Protocol Approval #BI 2005-27). Male New
Zealand white rabbits were purchased from Casey Van-
dermeer (Edmonton, AB, Canada) and fed ad libitum on a
‘‘high-fiber’’ diet (Lab Diet 5321, Canadian Lab Diets,
Leduc, AB, Canada). Blood (5.0 mL) was collected from
the marginal ear vein with 20-gauge stainless steel blood
collection needles (211 monoject, Sherwood Medical, St
Louis, MO, USA) into BD Vacutainer blood collection
tubes (no additive, BD Vacutainer, Franklin Lakes, NJ,
USA) to which 0.5 mg heparin had been added for the
preparation of plasma. For the preparation of serum, the
blood clot was removed by centrifugation (described
below). The injection of a heparin blank onto the SEC-ICP-
AES system (control experiment) revealed no detectable
Fe, Cu, or Zn (data not shown). Blood was collected from
4.5-h-fasted rabbits at approximately 13:30 and was cen-
trifuged at 1,100g (22 °C) for 10 min and the plasma (or
serum) obtained was analyzed using the SEC-ICP-AES
system within 30 min after blood collection, or at the time
points indicated later. Only straw-yellow plasma (free of
the characteristic red color of hemoglobin from ruptured
erythrocytes) was used throughout the study. To establish
the interanimal variation, 18 rabbit plasma samples were
consecutively analyzed using the SEC-ICP-AES system.
To qualitatively identify the detected Cu, Fe and Zn
metalloproteins in collected fractions after the analysis of
rabbit plasma, one human plasma sample was chromato-
graphed; this was obtained from a healthy male volunteer.
The collection of human blood was approved by the Uni-
versity of Calgary Conjoint Health Research Ethics Board
(approval no. E-21198).
Experimental setup of the optimized SEC-ICP-AES
system
The SEC-ICP-AES system (Fig. 1) consisted of a Waters
(Milford, MA, USA) model 510 high-performance LC
pump, a Rheodyne 9010 PEEK injection valve (Rheodyne,
Rhonert Park, CA, USA) equipped with a 0.5-mL PEEK
injection loop, and a prepacked SuperdexTM 200 10/300 GL
TricornTM high-performance column (30.0 cm 9 1.0-cm
inner diameter, separates globular proteins between
approximately 600 and 10 kDa; GE Healthcare, Piscataway,
NJ, USA). The exit of the SEC column was connected to the
Meinhard concentric glass tube nebulizer of the ICP-AES
with fluorinated ethylene–propylene Teflon tubing (30 cm,
inner diameter 0.5 mm). Simultaneous multielement-
specific detection of C (193.091 nm), S (180.731 nm), Zn
(213.856 nm), Fe (259.940 nm), Cu (324.754 nm), and P
(213.618 nm) in the column effluent was achieved with a
Prodigy, high-dispersion, radial-view ICP-AES (Teledyne
Leeman Labs, Hudson, NH, USA) at an Ar gas-flow rate of
19 L/min, an RF power of 1.3 kW, and a nebulizer gas
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Fig. 1 The instrumental
analytical size-exclusion
chromatography (SEC)–
inductively coupled plasma
atomic emission spectrometer
(ICP-AES) setup. HPLC high-
performance liquid
chromatography
pressure of 35 psi. The detector technology utilized in the
Prodigy allows the simultaneous measurement of the peak
and the background emissions to generate the net emission
intensity. This capability is critical in experiments where
the background emission intensity changes (e.g., when a
major protein peak reaches the ICP-AES) so the operator is
not mislead into believing that an analytically significant
event has occurred when in fact it has not. This advantage,
together with the ability of the ICP-AES to handle salt-
containing solutions, makes the Prodigy ideally suited for
the LC analysis of solutions containing metalloproteins.
Time scans were performed using the time-resolved-anal-
ysis mode (Salsa version 3.0) and a data acquisition rate of
one data point per 2 s. The raw data were imported into
SigmaPlot 10 and smoothed using the bisquare algorithm.
According to the void volume of the Superdex 200 column
(blue dextran), a 7.0-min delay was implemented between
injection and the beginning of data acquisition (1,080-s
acquisition window). Figure 2 depicts the C-specific SEC-
ICP-AES chromatograms of rabbit plasma obtained on a
Superdex 200 prep grade (34 lm) and a prepacked (13 lm)
column, which clearly displays the increased resolution of
80000
Albumin (~66 kDa)
60000
Intensity (counts/s)
40000
20000 V0
0
600 800 1000 1200 1400
Time (s)
Fig. 2 C-specific chromatograms of rabbit plasma on Superdex 200
prep grade (1.0 cm 9 25 cm; 34-lm particle size) (dashed line), and
Superdex 200 10/300 GL SEC column (1.0 cm 9 30 cm; 13-lm
particle size) (solid line). Phosphate-buffered saline mobile phase (pH
7.4, 22 °C), flow rate 1.0 mL/min, injection volume 500 lL, ICP-
AES detector (C emission at 193.091 nm). Void volume 600 kDa,
inclusion volume 10 kDa
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J Biol Inorg Chem (2009) 14:61–74
the latter stationary phase. The marginal increase in the
retention time for the small molecular weight C peak in the
13-lm column compared with the 34-lm column is caused
by the difference in column length of approximately 5 cm.
We size-calibrated the analytical Superdex 200 column
with known molecular weight protein standards. In addi-
tion, the analysis of rabbit blood plasma provided internal
molecular weight standards as several proteolytically stable
plasma metalloproteins, such as ferritin and transferrin (Fe
metalloproteins), ceruloplasmin (Cu metalloprotein), and
the most abundant plasma protein albumin (which can be
easily identified on the basis of the most intense C and S
peaks in the chromatogram), are naturally present in
plasma. These can therefore serve as a rough proxy to
estimate the size of a detected metalloprotein. Furthermore,
it is likely that owing to the sheer complexity of plasma
(more than 3,700 proteins), a detected unknown plasma
metalloprotein is unlikely to have the same retention time
as is suggested from a calibration curve because of its
unavoidable interactions with other plasma proteins.
Therefore, deductions of the molecular mass of an unknown
metalloprotein from its retention time alone should be
interpreted with caution.
Identification and quantification of metalloproteins
in SEC column effluent
In terms of qualitatively identifying the separated plasma
metalloproteins in the column effluent, we did not utilize
electrospray ionization mass spectrometry because of the
salt content (approximately 1% or approximately 164 mM)
of the mobile phase. In general, the salt concentration of
aqueous samples that can be analyzed by electrospray
ionization mass spectrometry must be below 10 mM. We
therefore qualitatively identified the Cu metalloprotein
ceruloplasmin in collected fractions using an established
ceruloplasmin oxidase activity assay (based on the oxida-
tion of N,N-dimethyl-p-phenylenediamine) [28]. Since an
antibody-based approach (e.g., enzyme immunoassay) for
the identification of rabbit ferritin, transferrin, a2 macro-
globulin, and factor V was not readily available, an
alternative way of identifying these metalloproteins had to
be pursued and we therefore analyzed human plasma using
J Biol Inorg Chem (2009) 14:61–74
the SEC-ICP-AES system and collected fractions for pro-
tein identification purposes.
The two major Fe-containing proteins and all Zn-con-
taining species had essentially the same retention times as
those obtained for rabbit plasma. Fractions were collected
for each Fe peak at time points corresponding to the max-
imum, shoulders on either side of the maximum, and at the
baseline before and after each peak. Similarly, altogether
eight fractions were collected of all Zn-containing entities.
Human ferritin was quantified in the collected fractions by
microparticle enzyme immunoassay technology with an
Axsym analyzer (Abbott Diagnostics, Mississauga, ON,
Canada) using the manufacturer’s method and calibrators.
Human transferrin was measured by immunoturbidometric
assay with a Cobas Integra 700 analyzer (Roche Diagnos-
tics Canada, Laval, QC, Canada) using the manufacturer’s
method and calibrators, and human a2-macroglobulin was
measured with a Dade Behring BN2 Prospect rate nephe-
lometric immunoassay using the manufacturer’s reagents
(Dade Behring Canada, Mississauga, OC, Canada).
With regard to the identification of factor V, fractions
were collected corresponding to the baseline before and
after Cu peak 1 as well as the peak itself. Factor Va
coagulation activity was determined by performing a
modified prothrombin time assay. In this assay, correction
of the clotting time of factor V-deficient plasma is pro-
portional to the concentration (activity percentage) of
factor Va in test plasma, interpolated from a calibration
curve [29]. Factor Va coagulation activity was determined
using an ACL TOP analyzer (Beckman Coulter, Palo Alto,
CA, USA) using factor V-deficient plasma, reagents, and a
HemosIL factor V assay protocol supplied by Instrumen-
tation Laboratory USA (Lexington, MA, USA).
To quantify the metal that corresponded to a detected
chromatographic metal peak, we injected increasing doses
of each metal onto the chromatographic system without a
column and measured the area under each ‘‘peak’’ using
SigmaPlot. This allowed us to establish a calibration curve,
which was used to calculate the total amount of metal (in
micrograms per 0.5 mL plasma) that was associated with a
detected metal peak (based on its peak area) in the sub-
sequent analysis. Finally, these data were used to calculate
the number of micrograms of metal that was present in
form of a certain metalloprotein per 1.0 mL of plasma for
comparison with literature data [7, 8].
Results and discussion
Although mammalian blood plasma can be easily obtained
and contains critical information about the essential trace
element status of the organism from which it was obtained,
the sheer complexity of the plasma proteome makes it
67
extremely difficult to extract relevant information about the
organism’s health status. In fact, the human serum prote-
ome comprises at least 3,700 proteins [30], which poses an
almost insurmountable problem from an analytical sepa-
ration viewpoint. The very complexity of analyzing plasma
for the proteins contained within it, however, can be
reduced dramatically if one is able to selectively analyze
for a subproteome, such as the ‘‘metalloproteome’’ (in the
context of this paper this term refers to all major plasma
proteins with bound Cu, Fe, and Zn). This would require
the separation of these metalloproteins from each other,
which—since the molecular masses of all major metallo-
proteins in plasma are known—can be achieved by
choosing a SEC stationary phase with the appropriate
fractionation range. Following this basic approach, we
have developed a rapid SEC-ICP-AES method to directly
analyze plasma for the major Cu-, Fe-, and Zn-containing
metalloproteins (by essentially determining the retention
time of the metals corresponding to these metalloproteins).
Conceptually, the detection of metal peaks within the
chromatographic window (between the exclusion volume
and the inclusion volume) together with the established
stability of the major plasma metalloproteins of Cu, Fe, and
Zn [1, 31–33] would imply that each detected metal peak
corresponds to a plasma metalloprotein. Furthermore, the
absence of tailing in the detected Cu, Fe, and Zn peaks
would further substantiate that each metalloprotein
remained intact during the entire LC separation process.
With regard to the analysis of rabbit plasma, interanimal
variation was expected and experimentally quantified
(Table 3). Excluding the standard deviation of the diag-
nostically inadequate Cu peak 1 (factor V, which disappears
after 0.5 h), the average relative standard deviation for all
Table 3 Average concentration of Cu, Fe, and Zn associated with
metalloproteins derived from size-exclusion chromatography–induc-
tively coupled plasma atomic emission spectrometry analysis of
rabbit plasma samples (N = 18)
Metal Protein(s) Average metal
concentration
(lg/mL plasma) ± SD
Cu Factor V and transcuprein 0.85 ± 0.73
Ceruloplasmin 0.46 ± 0.14
Albumin 0.65 ± 0.29
Small molecular weight 0.21 ± 0.12
Fe Ferritin 0.27 ± 0.16
Transferrin 2.66 ± 0.99
Zn a2-Macroglobulin and 0.84 ± 0.19
unidentified peaks 2–4
Albumin 1.10 ± 0.24
The peak areas of metalloproteins that were not distinct were com-
bined for integration
SD standard deviation
123
68 J Biol Inorg Chem (2009) 14:61–74

detected Cu, Fe, and Zn metalloproteins was 39%. In con-
trast to this, the method reproducibility is excellent (see
‘‘Stability of plasma metalloproteins’’).
The task of qualitatively identifying the detected plasma
metalloproteins is simplified as only approximately ten
major Cu, Fe, and Zn metalloproteins have so far been
reported in mammalian plasma (Table 1) [1]. In principle,
two strategies can be employed to qualitatively identify an
individual metalloprotein. First, it can be definitively
identified on the basis of either its enzymatic activity or a
specific antibody target site on its surface (e.g., using an
enzyme immunoassay). However, if neither of these tech-
niques is applicable because the metalloprotein has no
inherent enzymatic activity (e.g., if it functions exclusively
as a transport protein) or because no enzyme immunoassay
is readily available (for the organism of interest; in our case
rabbits), the second strategy to tentatively identify a
metalloprotein must be used. The latter involves the utili-
zation of information that is derived from the Cu-, Fe-, and
Zn-specific chromatogram in conjunction with literature
data. For instance, the retention time of an unknown
metalloprotein relative to a known and abundant protein,
such as albumin (66 kDa), can be indicative of its hydro-
dynamic radius and thus its approximate molecular mass
(assuming minimal protein–protein interactions). In addi-
tion, the intensity of a metal peak (corresponding to a
metalloprotein) relative to another metal peak (of a dif-
ferent metalloprotein containing the same metal) contains
information about the relative abundance of metal atoms
that are associated with these two proteins in plasma. In
this instance, the experimentally determined relative
retention time and abundance of both metalloproteins can
be compared with their known molecular mass and abun-
dance from handbooks on human metalloproteins [9] to
tentatively identify both metalloproteins.
Plasma versus serum metalloprotein analysis
To establish whether plasma or serum contains a larger
number of individual Cu-, Fe-, and Zn-containing entities
and therefore more information with regard to the health
status of an organism, we applied the SEC-ICP-AES

Fig. 3 Simultaneous
multielement-specific
chromatograms of rabbit plasma
on a Superdex 200 10/300 GL
(13 lm particle size) SEC
column with a phosphate-
buffered saline mobile phase
(pH 7.4, 22 °C); flow rate
1.0 mL/min, injection volume
500 lL, ICP-AES detector.
Emission lines for a C at
193.091 nm (black), S at
180.731 nm (orange), and P at
213.618 nm (pink) and for b Cu
at 324.754 nm (green), Fe at
259.940 nm (blue), and Zn at
213.856 nm (red). Both a and b
were obtained from the same
rabbit plasma sample. The SEC
column was size-calibrated with
a mixture of standards
(thyroglobulin 670 kDa,
c-globulin 158 kDa, ovalbumin
44 kDa, myoglobin 17 kDa, and
vitamin B12 1.35 kDa). The
qualitative identification of the
metalloproteins factor V,
a2-macroglobulin,
ceruloplasmin, ferritin, and
transferrin in collected fractions
by various enzyme-based assays
(see ‘‘Materials and methods’’)
is indicated by horizontal bars

123
J Biol Inorg Chem (2009) 14:61–74 69

method developed to analyze plasma (n = 3) and serum 250

(n = 3) of 4.5-h-fasted rabbits. Cu peak 1 in the plasma Cu
chromatogram at the bottom of Fig. 3 was absent in the 200
serum chromatogram (data not shown). Because the num-
ber and intensity of all other detected peaks remained
150

Counts/s
unchanged (data not shown), the Cu metalloprotein corre-
sponding to Cu peak 1 must be either directly or indirectly
(e.g., by specific adsorption) involved in the blood clotting 100

process. This Cu peak could possibly represent blood Vo

coagulation factor V, which is a single-chain glycoprotein 50
that contains one Cu per molecule [34] and is known to be
sensitive to proteolysis [35]. Although factor V has a 0
molecular mass of 330 kDa, it is known to self-associate to 0.5 h

form higher multimers [36, 37], which could explain its Fe 1.0 h
1.5 h
elution in essentially the void volume. On the basis of these 300 2.0 h

results, plasma contains more information than serum for

the desired application of the instrumental analytical

Counts/s
method developed for diagnostic purposes. 200

Stability of plasma metalloproteins Vo

100

To address a possible degradation of metalloproteins at

room temperature (22 °C) over time, plasma was analyzed
0
using the SEC-ICP-AES system at 0.5, 1, 1.5, and 2 h after
140
blood collection. This experiment was carried out twice
Zn
and the results essentially showed the same overall trend. 120
Typical Cu-, Fe-, and Zn-specific time-course chromato-
100
grams are shown in Fig. 4.
Counts/s

As depicted in this chromatogram, the Cu that was eluted 80

prior to 800 s disappeared from plasma after the 0.5-h time
point. In addition, the peak corresponding to Cu that was 60
Vo
eluted at approximately 900 s (it likely corresponds to 40
albumin-bound Cu; see the discussion below) and the one
corresponding to the Cu that was eluted at approximately 20

1,200 s (small molecular weight Cu; see the discussion 0

below) decreased, whereas the most intense Cu peak
(ceruloplasmin; see the discussion below) increased to some
extent. The discrepancy between the reduction in intensity
of some Cu peaks over time versus the increase of the most
intense Cu peak must be attributed to the loss of Cu (net Cu
loss of approximately 30%) either to the container wall (that
the plasma was kept in prior to analysis) or to the stationary
phase of the SEC column. These, however, are only the two
most likely explanations and at present the exact cause is
unknown. Therefore, if one aims to detect all Cu metallo-
proteins (including the labile ones) in plasma, the latter
must be analyzed within 0.5 h after blood collection.
The intensity of the Fe and Zn peaks remained virtually
unchanged over the 2-h time period (Fig. 4). These results
strongly suggest that the corresponding metalloproteins are
stable and—more importantly—that the analytical method
itself produces results that are sufficiently reproducible for
diagnostic applications.
600 800 1000 1200 1400
Retention Time (s)
Fig. 4 Simultaneous Cu-, Fe-, and Zn-specific chromatograms of
rabbit plasma over a 2-h time period (after collection) on a Superdex
200 10/300 GL (13-lm particle size) SEC column with a phosphate-
buffered saline mobile phase (pH 7.4, 22 °C); flow rate 1.0 mL/min,
injection volume 500 lL, ICP-AES detector. Cu-, Fe-, and Zn-
specific chromatograms were obtained in 0.5-h intervals at room
temperature and the emission lines of each element (Cu at
324.754 nm, Fe at 259.940 nm, and Zn at 213.856 nm) were plotted
on top of each other
C-, S-, and P-specific chromatogram of plasma
The analysis of plasma with the method developed and the
simultaneous online detection of C, S, and P using the ICP-
AES resulted in the three-element-specific chromatogram
shown at the top of Fig. 3 and revealed four major
C-containing protein bands. The S-specific chromatogram

123
70
closely resembled that of the first three C-containing pro-
tein bands, which is expected since most mammalian
proteins contain the S-containing amino acids L-cysteine
and/or L-methionine. Interestingly, the fourth S-containing
entity was eluted before the fourth C-containing entity
(both of these correspond to small molecular weight pep-
tides and amino acids). Since albumin is by far the most
abundant mammalian plasma protein (approximately 50 g/L)
and comprises more than half of the total protein in plasma
[9], the most prominent C band—band 3—must be pre-
dominantly composed of albumin (a BSA standard had the
same retention time). Owing to the utilization of a P-con-
taining mobile phase (PBS), the P-specific chromatogram
displayed an elevated P baseline throughout the entire
chromatographic window (Fig. 3, top). The P-emission
intensity also provided an effective measure of the mass
transfer of droplets from the nebulizer chamber to the
plasma. The detection of the albumin peak did not affect
the intensity of the P-emission line, which demonstrates
that the injected total protein did not adversely affect the
mass transfer from the nebulizer to the plasma (e.g., by a
surfactant effect), which is an important prerequisite to
accurately measure the total metal that is associated with
an eluting plasma metalloprotein. In addition, the P-spe-
cific chromatogram also revealed a characteristic dip at a
retention time of about 1,210 s, which coincides with the
elution of the small molecular weight C band 4, and
therefore corresponds to the injected plasma ‘‘plug’’ (which
contains less P than the mobile phase) reaching the
detector. A similar phenomenon has previously been
observed [38].
Cu, Fe, and Zn metalloproteins in plasma
The analysis of plasma (eight different animals) by SEC
and the simultaneous online detection of Cu, Fe, and Zn
using the ICP-AES resulted in a three-element-specific
chromatogram, a representative of which is shown at the
bottom of Fig. 3. At first glance, five Cu-containing, two
Fe-containing, and approximately five poorly separated Zn-
containing entities were detected. The majority of the
detected metal peaks displayed an ideal peak shape, which
suggests that the metals did not dissociate from their parent
protein during the chromatographic separation process.
However, the second Fe peak, the last Zn peak, and Cu
peaks 2 and 4 displayed a hump on the long retention end,
which can be rationalized by the elution of a slightly
smaller metalloprotein containing the same metal. Given
the inherent limitations of SEC with regard to the chro-
matographic resolution of proteins of almost similar size
from each other, this is not unexpected. Nevertheless, the
detection of approximately 12 metalloproteins demon-
strates that the optimized mobile phase–stationary phase
123
J Biol Inorg Chem (2009) 14:61–74
combination is well suited to separate the major Cu-, Fe-,
and Zn-containing entities that are contained in rabbit
plasma. Importantly, and in contrast to the peaks in the
chromatograms at the top Fig. 3, the individual Cu, Fe, and
Zn peaks in the chromatograms at the bottom of Fig. 3
correspond to individual metalloproteins and—since Cu
peak 5 was in the small molecular mass region—
metallopeptides.
The Cu-specific chromatogram revealed five peaks,
which is one more than has been reported in other studies
(Table 2) (peak 1: approximately 515 s, approximately
28% of total Cu, approximately 0.9 lg Cu/mL; peak 2:
approximately 605 s, approximately 13% of total Cu,
approximately 0.3 lg Cu/mL; peak 3: approximately 775 s,
approximately 27% of total Cu, approximately 0.9 lg
Cu/mL; peak 4: approximately 890 s, approximately
19% of total Cu, approximately 0.6 lg Cu/mL; peak 5:
approximately 1,230 s, approximately 13% of total Cu,
approximately 0.3 lg Cu/mL; Fig. 3, bottom). The sum of
all Cu peaks in this particular plasma sample amounted to
3.0 lg Cu/mL plasma, which is higher than the average total
Cu concentration (2.14 lg/mL) in the 18 plasma samples
that were analyzed (Table 3). Thus, the total rabbit plasma
Cu concentration was higher than what has been reported for
other mammalian species (range 0.2–2.0 lg/mL) [7]. Cu
peak 1 appeared close to the void volume and was not
observed in previous studies as evidenced by comparing the
relative intensities of the observed Cu peaks with the Cu-
specific chromatograms of previous studies [20, 22, 32].
This discrepancy, however, can be easily explained by the
fact that previous studies analyzed either aged plasma or
serum, which according to our investigations both lack Cu
peak 1 (Fig. 4). On the basis of the observation that Cu peak
1 was absent from serum and disappeared from plasma after
30 min (Fig. 4), it was tentatively identified as factor V,
which is known to be labile, contains Cu [34], and is present
in human plasma at approximately 10 mg/L [35]. This was
corroborated by analyzing collected fractions for factor V
coagulation activity (bar in Fig. 3, bottom), which was
highest at the maximum intensity of Cu peak 1. The activity,
however, extended beyond this peak, which can be ratio-
nalized by the fact that under the circumstances of blood
sampling factor V is expected to be activated into factor Va,
which has a smaller molecular mass (approximately
221 kDa) and could explain the observed tailing of the
activity. Factor V is known to be very labile and prone to
proteolysis, which may explain the rapid disappearance of
Cu peak 1 in the time-course experiments (Fig. 4) [39].
According to our analytical data and assuming an identical
stoichiometry in rabbit and human factor V (Table 1), we
calculated a plasma concentration of 4.7 g/L, which is sev-
eral-fold higher than its concentration in human plasma and
is therefore in apparent disagreement. We note, however,
J Biol Inorg Chem (2009) 14:61–74
that evidence in favor of significant interanimal species
differences of certain plasma metalloprotein concentrations
have been reported [40]. Furthermore, a different metal-
to-protein stoichiometry between human and rabbit factor V
could significantly affect the calculated plasma con-
centration. With regard to the retention time of this
metalloprotein (Fig. 3, bottom) it is noteworthy that
dimerization of this protein has been observed by others [36,
37]. This would result in a 660-kDa entity and could explain
the elution of factor V close to the void volume. On the basis
of the previously reported order of elution of Cu plasma
metalloproteins from a SEC column [32], Cu peak 2 was
tentatively identified as the 270-kDa protein transcuprein,
which is also known to dimerize (540 kDa) [4]. This Cu
peak had a small shoulder on the long retention end, which is
in accord with the Cu-specific chromatogram in Fig. 4. Cu
peak 3 had ceruloplasmin oxidase activity (bar in Fig. 3,
bottom) and was therefore identified as the glycoprotein
ceruloplasmin. On the basis of the experimentally deter-
mined total Cu associated with ceruloplasmin in 1.0 mL of
plasma and the known stoichiometry of Cu in this protein
(Table 1), the plasma ceruloplasmin concentration was
calculated at 0.31 g/L, which is within the concentration
range reported for human serum. Cu peak 4 was compara-
tively broad and appeared 11 s after albumin (dotted line in
Fig. 3, bottom). The misalignment of this Cu peak with the
albumin peak can be rationalized either by a rather weak
binding of Cu to albumin (which has been reported by others
[41]) or by the presence of a smaller Cu-containing entity in
addition to the expected albumin-bound Cu [4, 32]. On the
basis of the simultaneous appearance of Cu peak 5 (Fig. 3,
bottom) with the last C band (Fig. 3, top), this Cu peak
represents Cu bound to small non-S-containing peptides and
amino acids, such as L-histidine [42] (the retention time for S
band 4 in Fig. 3, top was different from that for Cu peak 5 in
Fig. 3, bottom) and is in general agreement with other
studies [32]. We note that the observed order of elution for
all major Cu entities is as expected on a SEC column
and follows decreasing molecular masses from 660 kDa
(putative factor V dimer), 540 kDa (transcuprein dimer),
132 kDa (ceruloplasmin), 66 kDa (albumin), and small
molecular weight Cu.
The Fe-specific chromatogram revealed two baseline-
separated peaks, which is identical to the maximum num-
ber of Fe peaks that was previously reported (Table 2)
[8, 19, 23, 24, 43, 44] (peak 1: approximately 670 s, 11%
of total Fe, approximately 0.3 lg Fe/mL; peak 2: approx-
imately 870 s, 89% of total Fe, approximately 2.6 lg
Fe/mL; sum of all Fe peaks: 2.9 lg Fe/mL plasma) (Fig. 3,
bottom). On the basis of the known molecular size and
plasma abundance of the two major Fe-containing metal-
loproteins ferritin and transferrin (Table 1), Fe peak 1 is
identified as ferritin and Fe peak 2 as transferrin. This peak
71
assignment was confirmed by enzyme immunoassay and
immunoturbidometric assay (bars in Fig. 3, bottom). On
the basis of the experimentally derived total Fe associated
with ferritin and transferrin in 1.0 mL of plasma and
assuming that both Fe metalloproteins were fully loaded
with Fe (Table 1), the rabbit plasma concentration of fer-
ritin was calculated as 535 lg/L and that of transferrin as
1.8 g/L. Even though these results are in overall accord
with the established concentrations of these metallopro-
teins in mammalian plasma/serum (Table 1), we point out
that the method developed cannot inherently determine the
metal loading of a metalloprotein in which the metal
loading may vary. It is therefore impossible to distinguish
if the Fe that is associated with, e.g., ferritin is attributable
to (1) 50% apoferritin and 50% fully loaded holoferritin or
(2) the case where all ferritin is 50% loaded with Fe.
Nevertheless, our method allows us to determine the dis-
tribution of a metal among various metalloproteins, which
has inherent diagnostic value that cannot be obtained by
conventional antibody-based enzyme assays. The distinct
shoulder on the long retention end of Fe peak 2 indicates
the presence of another Fe-containing entity. On the basis
of previous studies which demonstrated that Fe is bound to
human serum albumin in serum [23], this additional Fe-
containing entity could be albumin-bound Fe especially
since albumin is approximately 14 kDa smaller than
transferrin, which would therefore explain its elution after
transferrin. In contrast to Cu, however, no detectable Fe
was eluted in the small molecular weight range, which
indicates that Fe is not bound to peptides and amino acids
in rabbit plasma.
With respect to Zn, approximately five non-baseline-
separated peaks were detected (peak 1: approximately
613 s, 10% of total Zn, approximately 0.1 lg Zn/mL;
peaks 2–4: approximately 655 s, approximately 700 s,
approximately 770 s, 34% of total Zn, approximately
0.6 lg Zn/mL; peak 5: approximately 880 s, 56% of total
Zn, approximately 1.1 lg Zn/mL; sum of all Zn peaks:
1.8 lg Zn/mL plasma) (Fig. 3, bottom), which is more than
the average number of Zn peaks that has previously been
reported (Table 2) [31, 44, 45]. This finding can be ratio-
nalized with the higher-resolution SEC column that was
used in the present study (13-lm particles) compared with
earlier studies. Zn peak 1 likely represents the 725-kDa a2-
macroglobulin [46] since between 12 and 31% of human
plasma Zn has previously been reported to be tightly
incorporated in this protein, which is in accord with our
results [25, 31]. This peak assignment was confirmed by
enzyme immunoassay (bars in Fig. 3, bottom) and is in
accord with another study, which also identified the first Zn
compound that was eluted from a SEC column as a2-
macroglobulin [31]. Even though the putative a2-macro-
globulin was eluted before ferritin (450 kDa), it was eluted
123
72
after the void volume, which is in discord with what would
be predicted if its retention were solely based on its
molecular mass. We note, however, that nonideal interac-
tions between a2-macroglobulin and a SEC stationary
phase (TSK-G4000SW) have been observed using PBS
when the native protein was treated with chemicals which
exposed hydrophobic amino acids to the surface [47] and
resulted in the adsorption of this protein to the stationary
phase. Similar behavior could also occur between the
components of plasma and a2-macroglobulin and subse-
quently the stationary phase in our experiments. Using the
experimentally determined total Zn that is contained in
1.0 mL of plasma in form of this metalloprotein (approx-
imately 0.1 lg Zn/mL), we calculated the rabbit plasma a2-
macroglobulin concentration (using the stoichiometry
delineated in Table 1) at 222 mg/L, which is approxi-
mately one tenth of its concentration reported for human
plasma (Table 1). We note, however, that significant in-
teranimal species differences regarding certain plasma
metalloprotein concentrations have been reported [40, 48].
On the basis of the identical retention times for Zn peak 5
and albumin (dotted lines in Fig. 3) and since albumin-
bound Zn represents the major Zn entity in plasma (56% of
total plasma Zn in this study, which is in good accord with
previous studies on humans [26, 31] and pigs [44]), this Zn
entity likely represents albumin-bound Zn (transferrin does
not bind Zn2? [44]). Zn peaks 2–4 and the Zn shoulder on
the long retention end of Zn peak 5 could not be qualita-
tively identified. However, the existence of a 165-kDa
extracellular secretory glycoprotein Cu,Zn superoxide
dismutase (EC-SOD) and that of a 31-kDa Cu,Zn super-
oxide dismutase (Cu,Zn-SOD) have been reported in
guinea pig and human plasma [40, 49]. It is therefore likely
that EC-SOD represents one of the unidentified Zn peaks
2–4 and that the shoulder on the long retention end of Zn
peak 5 could possibly be Cu,Zn-SOD. Similar to the results
obtained for Fe, no Zn was detected bound to small
molecular weight peptides and amino acids in rabbit
plasma.
Practical applications
Owing to the fact that the SEC-ICP-AES method devel-
oped allows one to determine the plasma Cu, Fe, and Zn
metalloproteome within approximately 24 min, two major
practical applications of this method can be envisioned.
The first application is its utilization as a clinical tool to
screen for early- or advanced-stage human diseases by
the direct analysis of human plasma or serum [50–57].
Even though assays exist to quantify individual
plasma metalloproteins, such as ceruloplasmin (e.g., by a
spectrophotometric activity assay), few methods have
been reported that can simultaneously determine all
123
J Biol Inorg Chem (2009) 14:61–74
metalloproteins of one element, let alone those of more
than one element simultaneously. Therefore, this method
has the obvious advantage of extracting more information
(namely, the relative abundance of the metalloproteins of
the three major essential trace metals in plasma as well as
the concentration of those metalloproteins in which the
metal-to-protein ratio is fixed given that no other metal-
loprotein containing the metal of interest is coeluted) from
a single analysis in a given amount of time than is possible
with other methods that are currently in use. This, in turn,
can be helpful to more accurately diagnose the severity of a
disease since Wilson’s disease, for instance, is not only
associated with decreased plasma concentrations of the Cu
metalloprotein ceruloplasmin [58], but can also result in an
increased plasma concentration of the Fe metalloprotein
hemoglobin during episodes of acute hemolysis [52].
The second application is the utilization of the method
developed to probe the nonenzymatic bioinorganic chem-
istry of environmentally abundant pollutants, such as toxic
metals and metalloid compounds, in the mammalian
bloodstream to better understand their chronic toxicity,
individually and—more importantly—cumulatively. This
latter application appears particularly relevant since bio-
inorganic processes in the mammalian bloodstream are
likely to be fundamentally involved in the origin of
numerous human diseases that are associated with chronic
exposure to toxic metals and metalloid compounds [59,
60].
Conclusions
The daunting analytical task of extracting health-relevant
information from plasma can be considerably simplified
by determining a ‘‘subproteome’’, such as the Cu, Fe, and
Zn metalloproteome. To this end, we have developed a
rapid SEC-based separation of the metalloproteins con-
tained in rabbit plasma followed by the online analysis of
the column effluent by an ICP-AES, which served as the
simultaneous Cu-, Fe-, and Zn-specific detector. This
novel SEC-ICP-AES method has allowed us to directly
analyze rabbit plasma in order to generate the Cu, Fe, and
Zn metalloproteome, which is composed of approximately
12 metalloproteins and metallopeptides, within approxi-
mately 24 min. From a clinical perspective, this simple
and rapid technique to establish the Cu, Fe, and Zn me-
talloproteome offers important advantages over individual
metalloprotein assays since much more information can
be extracted with this method from a single plasma
sample. Thus, the detection of the majority of the
expected Cu-, Fe-, and Zn-containing entities in rabbit
plasma by the SEC-ICP-AES system constitutes an
important first step in the development of an instrumental
J Biol Inorg Chem (2009) 14:61–74
analytical technique for the efficient detection of the
plasma metalloproteome for potential diagnostic applica-
tions in humans. The method developed can also be used
to directly probe the bioinorganic chemistry of toxic
metals in whole blood and thus has considerable potential
to provide exciting new insights into the origin of toxic-
metal-related human diseases.
Acknowledgments This research was funded by the Natural Sci-
ences and Engineering Research Council (NSERC) of Canada.
Teledyne Leeman Labs is gratefully acknowledged for funding the
attendance of S.A.M. and J.G. at HPLC 2007 in Ghent, Belgium.
Katie L. Pei is gratefully acknowledged for help regarding the col-
lection of fractions. We would also like to extend thanks to Raymond
J. Turner and especially Arvi Rauk for constructive feedback on the
final draft of the manuscript. The staff of the Animal Health Unit
(LESARC) at the University of Calgary is gratefully acknowledged
for the maintenance of and the drawing of blood from the rabbits. We
would also like to extend sincere thanks to one anonymous reviewer
who provided valuable comments to significantly improve the final
manuscript.
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