Metal Metabolism: Transport, Development and Neurodegeneration 1317

Albumin as a zinc carrier: properties

of its high-afnity zinc-binding site
Jin Lu*, Alan J. Stewart, Peter J. Sadler*, Teresa J.T. Pinheiro and Claudia A. Blindauer*1
*Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K., MRC Human Reproductive Sciences Unit, Queens Medical Research Institute,
Edinburgh EH16 4TJ, U.K., and Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.

Abstract
Although details of the molecular mechanisms for the uptake of the essential nutrient zinc into the
bloodstream and its subsequent delivery to zinc-requiring organs and cells are poorly understood, it is clear
that in vertebrates the majority of plasma zinc (914 M; approx. 7585%) is bound to serum albumin,
constituting part of the so-called exchangeable pool. The binding of metal ions to serum albumins has been
the subject of decades of studies, employing a multitude of techniques, but only recently has the identity
and putative structure of the major zinc site on albumin been reported. Intriguingly, this site is located at
the interface between two domains, and involves two residues from each of domains I and II. Comparisons
of X-ray crystal structures of free and fatty-acid bound human serum albumin suggest that zinc binding to
this site and fatty acid binding to one of the ve major sites may be interdependent. Interactive binding
of zinc and long-chain fatty acids to albumin may therefore have physiological implications.

Introduction studies have addressed questions of affinity, stoichiometry

Serum albumin is, with an average concentration of 600 M,
the most abundant protein in the blood plasma of vertebrates
and functions as a carrier for a variety of nutrients, meta-
bolites and xenobiotics, as reviewed previously [1,2]. The
present paper briefly reviews the metal-binding properties
of albumin in general, before highlighting recent advances in
understanding zinc binding to albumin.
Albumin is thought to be the major zinc transporter in
plasma [3], and typically binds approx. 80% of all plasma
zinc [4]. Numerous studies have also demonstrated that
albumin modulates zinc uptake into cells [5,6], and some
suggest that, at least in some cell types, such as endothelial
cells, there is a receptor-mediated endocytosis pathway for
albumin-bound zinc uptake [7], and that zinc transport across
endothelia involves co-transport with albumin [8]. Further-
more, certain cases of familial hyperzincaemia appear to be
due to increased zinc binding to albumin [9].
In the light of the global importance of an adequate zinc
supply for many physiological processes, including cell
division and differentiation, combined with considerable
research activity on albumin, it is therefore surprising that
the major zinc site on HSA (human serum albumin) has been
identified only relatively recently [10].
Metal binding to albumins
The metal-binding capacity of albumins has been ack-
nowledged for a long time [11], and albumin has been shown
to bind a variety of essential and toxic metal ions, including
Ca(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II). Many
Key words: albumin, fatty acid, metal specicity, serum, zinc trafcking.
Abbreviations used: ATCUN, N-terminal copper and nickel-binding; HSA, human serum albumin.
1
To whom correspondence should be addressed (email c.blindauer@warwick.ac.uk).
and specificity [1220].
Although there is still a considerable lack of detailed
structural information, four different metal sites have been
described: (i) the so-called ATCUN (N-terminal copper
and nickel-binding) motif at the N-terminus, the primary
site for Cu(II) and Ni(II) [21]; (ii) the so-called site A, the
primary Zn(II)-binding site that can also bind other 2+ metal
ions [22]; (iii) site B, which displays a high affinity towards
Cd(II), but the location of which is unknown [12,14,18];
and (iv) the reduced thiol of Cys34 , which binds gold [23] and
platinum [24] compounds. Although this site has repeatedly
been suggested to also bind Cd(II), we have demonstrated
that this is not the case by studying both Cys34 -blocked BSA
[18] and the C34A mutant of HSA [10] by 113 Cd or 111 Cd
NMR spectroscopy. Finally, although Ca(II) is thought to be
transported by albumin in the plasma [25], the identity of its
binding site(s) is unknown, although the so far unidentified
site B may play a role [18].
The ATCUN motif is probably the most studied metal-
binding site on albumin (for a comprehensive review, see
[26]). Such sites are not restricted to albumin, and require the
presence of a histidine residue in position 3 of an amino acid
chain. The ATCUN motif is particularly suitable for metal
ions that have a prevalence for tetragonal or square-planar
co-ordination, and provides in total four nitrogen ligands that
include an imidazole nitrogen of His3 , the N-terminal amino
group and two deprotonated backbone amide nitrogens. In
addition, the carboxylate side chain of Asp1 has also been
implicated in metal binding, giving an overall penta-co-
ordinate site [26], although this residue appears to be not
crucial for high-affinity Cu(II) and Ni(II) binding. The X-ray
structures of copper complexes of corresponding tripeptides
confirm that Cu(II) is co-ordinated in a mildly distorted

Biochem. Soc. Trans. (2008) 36, 13171321; doi:10.1042/BST0361317 

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1318 Biochemical Society Transactions (2008) Volume 36, part 6

square-planar fashion, with very weak interaction to a fifth
apical ligand [27]. In principle, as shown in studies involving
tripeptides [28], other metal ions, such as Zn(II) and Co(II),
can also bind to the ATCUN motif, and have been shown to
effect backbone amide deprotonation at physiological pH,
but to a lesser extent than does Cu(II). Interestingly, dog, pig
and chicken albumins are devoid of His3 , and this has been
correlated with the higher susceptibility of dogs to copper
toxicity [17]. Typical affinities for Cu(II) for the ATCUN
site are of the order of log K 11 [17] for the intrinsic
stoichiometric stability constant.
A detailed comparative study of HSA and dog serum
albumin [14] has demonstrated further that neither Zn(II) nor
Cd(II) binds preferentially to the N-terminus, and a recent
study suggests that neither does Co(II) [29], although this is
controversial [30,31]. The equilibrium dialysis experiments
reported by Goumakos et al. [14] also revealed that, in total,
3 mol equiv. of Zn(II) and Cd(II) can be bound to albumin
with non-negligible affinity, but that there is only one
high-affinity site for Zn(II) and two for Cd(II) on both HSA
and dog serum albumin. The apparent stability constants for
the Zn(II) site on HSA was determined as log K = 6.4 0.8,
and that for the two Cd(II) sites as log K = 5.3 0.6. Intrinsic
stoichiometric constants for Zn(II)HSA have been reported
as log K = 7.53 0.45 [16] and log K = 7.1 0.2 [19]. It
should be noted that these constants are independent of the
protonation state of albumin, but still depend on factors
such as temperature and ionic strength, which may account
for discrepancies between the results of experiments from
different laboratories.
Clues to the nature of the preferred binding site for Zn(II)
came mainly from 113 Cd-NMR studies [12,18]. BSA [12] as
well as HSA and albumins from sheep, pigs and horses [18] all
display two strong binding sites for Cd(II). The correspond-
ing 113 Cd spectra contain two peaks, one at 2530 p.p.m. (la-
belled B), and another at 110150 p.p.m. (labelled A), and both
peaks are rather broad (linewidths 200900 Hz). The 113 Cd
chemical shifts observed for the two sites are characteristic of
mixed nitrogen/oxygen ligand sets [32], and site A was sug-
gested to contain no fewer than two nitrogen ligands [12,14],
whereas site B is thought to contain one or no nitrogen ligand.
One of the most crucial conclusions arising from competi-
tion experiments was the pronounced preference for Zn(II)
of site A, as 1 mol equiv. is sufficient to completely suppress
the corresponding 113 Cd or 111 Cd resonance in either BSA
[12] or HSA [10,18] (Figure 1), suggesting replacement
of Cd(II) by Zn(II). In contrast, higher ratios of either
Cu(II) and Ni(II) are required to affect peak A, with
Ni(II) displaying the smallest effect. This is consistent with
preferential binding of Cu(II) and Ni(II) to the N-terminus
as discussed above, and also indicates that site A might not
be particularly suitable for accommodating the d8 ion Ni(II).
CD and EPR spectroscopy and metal competition exper-
iments [22] also demonstrated that the second mol equiv. of
Cu(II) added to BSA or HSA binds to the high-affinity Zn(II)
site. The site displays fairly rigid co-ordination geometry and
binds Zn(II) more strongly than either Ni(II) or Cd(II).
Figure 1 111 Cd NMR spectroscopy of recombinant HSAs in the
presence of 2 mol equiv. of 111 Cd(II)
Albumin defatted either through dialysis or charcoal treatment shows
binding of 111 Cd(II) to two different sites. Addition of 1 mol equiv.
(eq) of Zn(II) leads to complete suppression of peak A, presumably
by displacement of 111 Cd(II). Mutation of the putative zinc ligand His67
to alanine also impairs Cd(II) biding to this site. Saturation of wild-type
albumin with fatty acid (+5 eq octanoate) also interferes with Cd(II)
binding to site A. The preferred Cd(II)-binding site B is not affected by
any of these manipulations.
In summary, these and other competition experiments
allowed the following conclusions: (i) there are, in total,
three sites with non-negligible affinity for Zn(II), Cd(II)
and Cu(II) on both BSA and HSA; (ii) HSA and BSA have
exactly one high-affinity site for Zn(II) and Cu(II), and
two for Cd(II), one of which is identical with the Zn(II)
site; (iii) the high-affinity Zn(II) site also binds other 2+
metal ions, including Cu(II), if the N-terminus is saturated
or unavailable as in dog albumin; and (iv) both Zn(II) and
Cu(II) have a higher affinity than Cd(II) for this site.
The high-afnity zinc site on albumin
In the light of the importance of zinc transport and delivery,
we set out to identify and characterize the high-affinity zinc
site on HSA, using a combination of site-directed muta-
genesis, molecular modelling and NMR spectroscopy [10].
An inspection of published X-ray crystal structures of HSA
revealed that the only region which contains two histidine lig-
ands in close proximity to potential oxygen donors involves
His67 and His247 (Figure 2). Mutation of His67 to alanine had
a major effect on cadmium binding to site A, as judged by
111
Cd NMR of the mutant (Figure 1), suggesting that this
residue may indeed be involved in metal binding to this site.
One of the peculiar characteristics of the NMR resonances
of site A, as reported from various studies, is the relatively


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Figure 2 Location and modelled structure of the high-afnity
zinc-binding site on albumin [10]
(a) The zinc site is at the interface of domains I (orange) and II (blue).
The model is based on PDB code 1AO6 [33]. (b) The zinc (purple) site
is composed of two amino acids from domain I (His67 and Asn99 ), two
amino acids from domain II (His247 and Asp249 ) and a fth exogenous
ligand.
large chemical shift range observed for this site. A close
scrutiny of available data revealed that one of the major
differences between the various preparations was the
composition of the buffers, and our experiments involving
titrations with chloride indeed confirmed that the chemical
shift of 111 Cd(II) in site A is highly dependent on the
availability of exogenous ligands [10], suggesting that Cd(II)
in this site might bind an additional ligand.
The molecular model of Zn(II)-bound albumin shown in
Figure 2 is compatible with all available experimental data. In-
triguingly, the site is located at the domain interface between
domains I and II, and each domain provides two ligands: His67
and Asn99 from domain I, and His247 and Asp249 from domain
II. This site is essentially preformed in all published X-ray
crystal structures of fatty-acid-free albumin. Starting from an
X-ray crystal structure of fatty-acid-free albumin, only minor
adjustments to atom positions are required to model a phys-
ically reasonable zinc site: the root mean square deviation be-
tween the X-ray crystal structure used as starting model (PDB
code 1AO6) [33] and the modelled structure is only 0.73 A
(1 A = 0.1 nm) for all heavy atoms of the zinc ligands [10]. The
inclusion of a fifth ligand, modelled as a water molecule, is
also supported by our modelling studies, as attempts to model
a four-co-ordinate site led to a highly distorted co-ordination
geometry with an empty space towards the exterior.
Compared with the major metal-binding side chains of
cysteine, histidine, aspartate and glutamate, asparagine (as
well as glutamine) is a rare ligand for zinc (or other metals) in
proteins, but a few X-ray crystal structures of Zn(II)-bound
asparagine are available. The active sites of purple acid phos-
phatase [34], calcineurin [35], periplasmic 5 -endonucleo-
tidase [36] and the inactive form of the glycyl-glycine
endopeptidase LytM [37] have the same amino acid set as the
Zn(II) site on albumin. Whereas in the former three proteins,
the Zn(II) ion is in a bimetallic cluster, the active site of
LytM contains a single Zn(II) ion. LytM is an autolysin,
and, notably, the latent form in which Zn(II) is tetrahedrally
co-ordinated by two histidine, one aspartate and one
Metal Metabolism: Transport, Development and Neurodegeneration 1319
asparagine residue is devoid of a water ligand, and displays
very low enzymatic activity. It is thought that activation
involves cleavage of the N-terminus of the protein which
contains the Asn117 ligand, yielding a free co-ordination site
for the substrate. Thus, by analogy to the cysteine-switch
in matrix metalloproteases, the activation mechanism for
LytM has been termed the asparagine-switch. In summary,
considering the apparent requirement for either a second
metal ion or removal of one of the four ligands from a
Zn(His)2 (Asp)(Asn) site, the Zn(II)-binding site on albumin
is not expected to show notable hydrolytic activity.
Finally, sequence comparisons of all available serum
albumins show that the four proposed ligands are conserved
in almost all available mammalian sequences, except for that
of the guinea-pig, which lacks both domain I residues. The
zinc site is also present in the marsupial opossum albumin,
but other vertebrate albumin sequences from fish, snakes,
amphibians and birds also lack one or more of the zinc-
binding residues. These observations suggest that it would
be interesting to compare mechanisms of zinc transport and
metabolism in these different species.
Interactive binding of metal ions
and fatty acids
It had been noted that the appearance of peak A in 113 Cd
NMR spectra is dependent on the fatty acid content of the
albumin preparation used [18], pointing towards the possib-
ility for reduced Cd(II) affinity for albumin in the presence
of fatty acids, although previous studies had not reported a
correlation between the presence of fatty acids and Zn(II)
affinity [14,15]. We tested this hypothesis using recombinant
HSA [10], and compared the 111 Cd NMR spectra of a sample
containing approx. 8 mol equiv. of octanoate with that of an
extensively dialysed sample (Figure 1). Clearly, the presence
of an excess of octanoate had an effect on Cd(II) binding to
site A, but had none on site B. A large number of X-ray crystal
structures with various bound fatty acids is available (e.g.
[38,39]), and a comprehensive analysis of all these structures
reveals that, in each one, the Zn(II) site A is disrupted. This
disruption is caused by a domaindomain movement that can
be described as a rotation of domain I with respect to domain
II [38]. As a consequence, the two zinc-binding braces in
the two domains move apart by 46 A [10] (Figure 3).
Intriguingly, the fatty acid molecule that is largely respons-
ible for this particular conformational change is, like the zinc
ion, bound to a site (FA2) which is formed by two half-sites,
one in each of the two domains (Figure 3). The full fatty-acid-
binding site is only formed when the conformational switch
engages the relevant side chains in both domains (see legend
of Figure 3). Thus binding of long-chain fatty acids to FA2
and zinc binding to site A might be mutually exclusive.
The observations described above raise the question of the
significance of the allosteric relationship between zinc and
fatty acid binding to albumins. HSA contains both high-
and low-affinity fatty-acid-binding sites [40], and there is
agreement about the existence of two high-affinity sites for
medium- and long-chain fatty acids, followed by three to

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1320 Biochemical Society Transactions (2008) Volume 36, part 6

Figure 3 Schematic illustration of the relative domain Idomain II movement induced by fatty acid binding in HSA and its
impact on the zinc site
(a) Residues involved in fatty-acid-binding site FA2 [38] in fatty-acid-free albumin (with modelled zinc site [10]; the zinc
ion is coloured purple), with the van der Waals surfaces of domain I residues in orange and those of domain II residues
in blue. (b) The same residues in albumin containing ve molecules of myristate (PDB code 1BJ5). The straight lines are
drawn to illustrate the rotation of domain I with respect to domain II, and represent arbitrary axes through the middles of
the pocket-forming residues in subdomains IA (the larger portion) and IB (the small portion on the lower right). The small
arrows indicate the resulting movement of the domain I zinc-binding residues in relation to the domain II residues.

four sites with only slightly lower affinities [38,41]. It is also
clear that chain length plays a major role [41,42]. The most
common fatty acids bound to HSA in vivo are the long-
chain fatty acids oleate and palmitate [2], and, under normal
healthy conditions, each albumin molecule in the human
plasma carries one or two fatty acid molecules in their
deprotonated (carboxylate) forms.
The average occupancy of the zinc site on albumin is
much lower, with only approx. 2% of circulating albumin
molecules thought to carry a zinc ion [2]. Also, the apparent
binding constants for the first two equivalents of long-chain
fatty acids are larger than that of zinc [41]. Taking these
considerations together, it seems unlikely that zinc binding
would influence the amount of fatty acid transported under
normal conditions. However, we hypothesize that the
zinc-binding affinity of albumin might be reduced during
conditions of increased free fatty acid mobilization, e.g. after
strenuous exercise, when about four molecules of fatty acid
may be bound to albumin [2]. Changes in plasma levels of
zinc during exercise have been reported (e.g. [43]), although
there does not appear to be clear agreement as to whether
these levels are increased or decreased.
Conclusions
Albumin contains several metal-binding sites with clear
specificities for different metal ions. Specificity is achieved
by exploiting differences in the co-ordination chemistries
of the preferred metal ions. Under physiological conditions,
only Zn(II) will be bound to the high-affinity site, as Cu(II)
binds much more avidly to the ATCUN motif, and other
metal ions, including the toxic Cd(II), have a lower affinity
for the zinc site.
Albumin-bound zinc has been categorized as exchange-
able [11], and this is thought to be important with respect
to transport and delivery of zinc. A site that works in the
physiological transport of zinc has to have thermodynamic
and kinetic properties that are suited to this task. Accordingly,
metal ions bound to site A seem to have rapid exchange
kinetics, as judged from competition experiments involving
UVvisible spectroscopy. The conditional dissociation con-
stant of the zinc albumin complex (approx. 1 M) is relatively
high compared with other zincprotein complexes, but it is
yet about one order of magnitude lower than typical plasma
zinc concentrations (approx. 10 M). Taking into account the
approx. 100-fold excess of albumin and the presence of other
zinc-binding ligands in plasma, this is exactly sufficient to
preclude the existence of significant amounts of free Zn(II)
which could be harmful. Furthermore, there are indications
that the physiological state of the organism influences the
affinity of albumin for zinc via the binding of excess fatty
acids. This might have functional significance, but further
studies are required to demonstrate this link.
We thank the Biotechnology and Biological Sciences Research
Council and Delta Biotechnology Ltd (now Novozymes) for
an Advancement and Support of Education Award (to A.J.S.),
the European Community for a Marie Curie Fellowship (to C.A.B.), the
Engineering and Physical Sciences Research Council and BP (British
Petroleum) for a Dorothy Hodgkin Postgraduate Award (to J.L.), the
Wellcome Trust (Edinburgh Protein Interaction Centre), the Wolfson
Foundation, and the Royal Society (Olga Kennard Fellowship to
C.A.B.) for their support of this work.


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Received 8 July 2008
doi:10.1042/BST0361317

C The Authors Journal compilation 
C 2008 Biochemical Society