Structure, Properties, and Engineering of the Major Zinc Binding Site on Human Albumin

Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn2+ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His67 and Asn99 from domain I, His247 and Asp249 from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn2+ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn2+ binding but alter protein stability, whereas mutation of His67 to alanine removes an interdomain H-bond and weakens Zn2+ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures. Most blood plasma zinc is bound to albumin, but the structure of the binding site has not been determined. Zn K-edge extended x-ray absorption fine structure spectroscopy and modeling studies show that the major Zn2+ site on albumin is a 5-coordinate site with average Zn-O/N distances of 1.98 Å and a weak sixth O/N bond of 2.48 Å, consistent with coordination to His67 and Asn99 from domain I, His247 and Asp249 from domain II (residues conserved in all sequenced mammalian albumins), plus a water ligand. The dynamics of the domain I/II interface, thought to be important to biological function, are affected by Zn2+ binding, which induces cooperative allosteric effects related to those of the pH-dependent neutral-to-base transition. N99D and N99H mutations enhance Zn2+ binding but alter protein stability, whereas mutation of His67 to alanine removes an interdomain H-bond and weakens Zn2+ binding. Both wild-type and mutant albumins promote the safe management of high micromolar zinc concentrations for cells in cultures. Zinc is not only required for hundreds of essential extra- and intracellular proteins and enzymes but is also recruited by toxins such as anthrax lethal factor (1Turk B.E. Wong T.Y. Schwarzenbacher R. Jarrell E.T. Leppla S.H. Collier R.J. Liddington R.C. Cantley L.C. Nat. Struct. Mol. Biol. 2004; 11: 60-66Crossref PubMed Scopus (176) Google Scholar) and staphylococcal enterotoxin (2Petersson K. Håkansson M. Nilsson H. Forsberg G. Svensson L.A. Liljas A. Walse B. EMBO J. 2001; 20: 3306-3312Crossref PubMed Scopus (86) Google Scholar). There is a need to understand how zinc transport and distribution is controlled (3Ford D. Proc. Nutr. Soc. 2004; 63: 21-29Crossref PubMed Scopus (48) Google Scholar). Although considerable progress has been made in the identification and study of membrane-bound zinc transporters, the molecular mechanism of extracellular zinc transport is still obscure. The total concentration of zinc in blood is high, ∼15–20 μm (4Caroli S. Alimonti A. Coni E. Petrucci F. Senofonte O. Violante N. Crit. Rev. Anal. Chem. 1994; 24: 363-398Crossref Scopus (259) Google Scholar), and plasma zinc concentrations are maintained at a relatively constant level, except during periods of dietary zinc depletion and acute responses to stress or inflammation, when they are depressed (5Cousins R.J. Clin. Phys. Biochem. 1986; 4: 20-30PubMed Google Scholar). In humans, ∼98% of so-called “exchangeable” zinc in blood plasma (9–14 μm) is bound to serum albumin (6Giroux E.L. Henkin R.I. Bioinorg. Chem. 1973; 2: 125-133Crossref Scopus (42) Google Scholar). Studies on perfused rat intestine have implicated albumin in the transport of newly absorbed zinc in portal blood, from the intestine to the liver (5Cousins R.J. Clin. Phys. Biochem. 1986; 4: 20-30PubMed Google Scholar). Albumin has also been shown to promote zinc uptake by endothelial cells, with receptor-mediated endocytosis as the most likely mechanism (7Rowe D.J. Bobilya D.J. Proc. Soc. Exp. Biol. Med. 2000; 224: 178-186Crossref PubMed Scopus (37) Google Scholar). Albumin, the most abundant protein in blood plasma (∼40 mg ml−1 and 0.6 mm), is synthesized in the liver and secreted into the blood stream as a 585-residue, single-chain protein after loss of a 24-residue propeptide (8Peters Jr., T. All about Albumin: Biochemistry, Genetics and Medical Applications. Academic Press, New York1996: 9-54Google Scholar). The protein is largely α-helical and folds into three structurally homologous domains (I, II, and III), each of which contains two subdomains (A and B) (see Fig. 1A) (9Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1523) Google Scholar). There are 35 Cys residues that form six disulfide bridges in each domain, except for domain I, which contains only five bridges and a free thiol at Cys34. Zinc binding to albumin has also been demonstrated in vitro, and the so-called high-affinity site for zinc on albumin displays a binding constant of K ≈ 107m−1 (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 11Goumakos W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, 12Ohyoshi E. Hamada Y. Nakata K. Kohata S. J. Inorg. Biochem. 1999; 75: 213-218Crossref PubMed Scopus (79) Google Scholar, 13Bal W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar). Although over 50 x-ray structures of albumin have been reported to date (9Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1523) Google Scholar, 14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1190) Google Scholar, 15Carter D.C. He X.M. Munson S.H. Twigg P.D. Gernert K.M. Broom M.B. Miller T.Y. Science. 1989; 244: 1195-1198Crossref PubMed Scopus (536) Google Scholar, 16Ghuman J. Zunszain P.A. Petitpas I. Bhattacharya A.A. Otagiri M. Curry S. J. Mol. Biol. 2005; 353: 38-52Crossref PubMed Scopus (1488) Google Scholar), no experimental structural data are available for the zinc site on albumin. On the basis of 111Cd NMR studies, site-directed mutagenesis, and molecular modeling, we have proposed that the major zinc site on albumin, termed site A, is located at the interface of domains I and II and is formed by the side chains of His67 and Asn99 from domain I and by His247 and Asp249 from domain II (Fig. 1B) (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, 18Lu J. Stewart A.J. Sadler P.J. Pinheiro T.J. Blindauer C.A. Biochem. Soc. Trans. 2008; 36: 1317-1321Crossref PubMed Scopus (194) Google Scholar). Here, we report the first direct structural characterization of a zinc site on albumin, using Zn K-edge x-ray absorption fine structure (EXAFS) 2The abbreviations used are: EXAFSx-ray absorption fine structureN-to-Bneutral-to-base transitionrHArecombinant human albumin. spectroscopy. We also explored the possibility of engineering albumins with increased or decreased zinc-binding affinity by mutating the postulated zinc-binding ligands. NMR methods were used to identify mutated and zinc-binding histidines and to probe the effects of zinc binding on the conformational dynamics of the protein. Finally, we show that albumin at physiological concentrations promotes the culture of hepatocytes at otherwise toxic zinc concentrations. x-ray absorption fine structure neutral-to-base transition recombinant human albumin. All albumin samples were prepared from purified, recombinant proteins produced as described previously (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar). albumin were prepared from a human albumin by site-directed were by of samples with and NMR were at K on a at using a and probe using albumin in at to the for a in using for be to by 50 50 and as to were for the and domain data using a for water data are shown and described in 111Cd NMR were on of mutant albumins in 50 50 and of prepared by in the of NMR were at K using a probe with as an Albumin samples were in 50 which been using to albumin of and were from Zn K-edge data were on and at at K and in data were and The data were and as described previously M. I. W. D.J. Sadler P.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) using We used of the zinc site (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar), on a structure at Å of (9Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1523) Google as a for data The the side chains of and as as the side and of His247 and all of the bond to Asp249 required of the In to data and the molecular modeling using an of the The of as by J. Google Scholar) and G. PubMed Scopus Google Scholar). The of the were by to the wild-type (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar), using a were using R. M. K. J. Mol. PubMed Scopus Google Scholar) and The affinity of albumin for zinc using an C.A. 2004; PubMed Scopus Google Scholar). of μm recombinant human albumin for at K of and The free zinc concentration by using concentrations of The free zinc concentrations were using the of the G. and Academic Scholar), and the for the J. Chem. Soc. Scopus Google Scholar). the samples were to to or the zinc zinc that bound to albumin the The albumin concentration by the at using a of J. NMR Studies of of Scholar). The zinc concentration using the zinc μm Zn2+ in were in to albumin The data are the are on in albumin cells were from the and maintained in culture in with serum and and which the zinc-binding and at a concentration of were at in an and with or as cells they were using and were culture at using of (see Fig. or of zinc Fig. and to The and the with were with of with or μm in the or of wild-type or mutant albumin with as a a the were using to samples to to were and using a The total of after to for All were in on in as the residues in high-affinity zinc binding on albumin, we the of wild-type and the three and N99H by Zn K-edge and 111Cd and NMR spectroscopy. The mutant to a of and to zinc and in a wild-type and the two Asn99 were to the weak with the or the wild-type we Zn K-edge data for three and (see and Fig. is that albumin contains (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 13Bal W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar), important to that the methods zinc binding to high-affinity of on previously data (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 11Goumakos W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, 13Bal W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar, T. Inorg. Scopus Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar), the is to albumin with bound to the Sarkar B. Chem. Scopus Google Scholar), bound to the so-called site P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar), and Zn2+ to site (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar). The for albumins are that the the first of Zn2+ to site A. and for the mutant proteins were on of Zn2+ and albumin. The data for the and are with that of wild-type in Fig. The data show that each mutation the zinc of the data a structural of the zinc site in wild-type albumin. of the first or for wild-type of bound Zn2+ the zinc as a 5-coordinate O/N site with an average Zn-O/N of 1.98 Å, plus O/N at 2.48 molecular on Protein in which zinc is bound by the postulated and a water The data for an of with to the experimental to as the and data (Fig. with of the of the a of the site (Fig. The of His67 the side of Asn99 of His247 and of Asp249 with a water the first whereas the of His247 a bound to the data by by the or of Asp249 or Asn99 as the sixth not and residues are in to all of residues Å of the Zn2+ in to for all to the not be from the data for a of the and a of related with structures were the of the of residues shown in Fig. are of a of which a to the data The of the mutant (Fig. from the of wild-type and is consistent with a The loss of is also in the of the first in the In for the Asn99 form the (Fig. of wild-type All data for the were using on the in mutation of the residues Fig. The affinity of the mutated albumins for zinc by The high zinc affinity of albumins reported in the K (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 11Goumakos W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google E. Hamada Y. Nakata K. Kohata S. J. Inorg. Biochem. 1999; 75: 213-218Crossref PubMed Scopus (79) Google W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar) be to the of the postulated site and the of the side of as of the a binding that is relatively weak and in of Asn99 to or of which have high for to zinc whereas the of the His67 to Fig. a of zinc from the of K be for the binding constant of the first of Zn2+ to wild-type at a in the as those reported previously for human serum albumin (10Masuoka J. Saltman P. J. Biol. Chem. 1994; 269: 25557-25561Abstract Full Text PDF PubMed Google Scholar, 11Goumakos W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, 12Ohyoshi E. Hamada Y. Nakata K. Kohata S. J. Inorg. Biochem. 1999; 75: 213-218Crossref PubMed Scopus (79) Google Scholar, 13Bal W. Christodoulou J. Sadler P.J. Tucker A. J. Inorg. Biochem. 1998; 70: 33-39Crossref PubMed Scopus (265) Google Scholar). all three mutations have a on the affinity of albumin for The the N99H The binding of the Asn99 are that of wild-type whereas that of the mutant is data are in with the on side chains with affinity and on the and affinity on the into the postulated zinc binding site G. and Academic Scholar). 111Cd and NMR spectroscopy were used to probe the binding of and Zn2+ to the mutant albumins in to wild-type 111Cd and NMR have been used previously to probe zinc in proteins albumin W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, T. Inorg. Scopus Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar). with previously reported data W. Laussac J.P. Sarkar B. Biochem. Cell Biol. 1991; 69: 809-820Crossref PubMed Scopus (73) Google Scholar, A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, T. Inorg. Scopus Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar), wild-type albumin with 111Cd to two in 111Cd NMR at and B) (Fig. The 111Cd NMR of as that for wild-type at and (Fig. that site is affected by the Asn99 whereas site The of Zn2+ and are also of Zn2+ were not to from site Fig. in to wild-type for which of after of of Zn2+ (17Stewart A.J. Blindauer C.A. Berezenko S. Sleep D. Sadler P.J. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 3701-3706Crossref PubMed Scopus (166) Google Scholar, 27Sadler P.J. Viles J.H. Inorg. Chem. PubMed Scopus (166) Google Scholar). affinity for with Zn2+ on of an by the in the N99H mutant is in with the J. Chem. Soc. Scopus Google Scholar). The 111Cd NMR of at K only which is by the in of the affinity of mutant for Zn2+ and affinity for of of and the to two at and (Fig. The at be to a by with a Fig. whereas the at is in the for a site with and three to five Biochem. Cell Biol. 1998; PubMed Google Scholar). The of the that in the N99D which are at an on the NMR at K. We used NMR spectroscopy to identify residues of and to probe the of Zn2+ on residues of wild-type and mutant NMR studies of albumin are by and are to but with the of and site-directed mutagenesis, of in the are The of the wild-type and mutant is (Fig. but are to two and and in the and for all three mutant that to the two histidines in the mutated zinc His67 and in Fig. also Fig. for a total the for at residues are by Zn2+ binding to most the of and and In the for of to on Zn2+ binding to effects from in the direct effects of Zn2+ or in is that are also on of of zinc Fig. The effects of Zn2+ on the of the mutant Fig. were to those for wild-type consistent with zinc binding to the as by the studies of Asn99 were by protein at K. that is a structural at the interface domains I and II and the of the protein the of albumin in the of mammalian cells, we the of human cells on culture human liver a to cultures. Zn2+ concentrations μm were toxic to cells in with serum Fig. and as by loss of and effects to those of Zn2+ on E. K. J. J. M. 2003; PubMed Scopus Google Scholar, E. S. J.H. A. B. H. J. Clin. 2000; PubMed Scopus Google Scholar). concentrations of μm) were to the effects of Zn2+ on at Zn2+ concentrations as high as μm (Fig. and that the is in and of Zn2+ distribution in cultures. be that the culture used in contains the and mm), which Zn2+ with micromolar affinity K ≈ or ≈ G. and Academic the in Fig. (A and B) are to the of albumin All albumin were also by cells, in the and of The of site-directed mutagenesis, molecular modeling, and has into the structure of the important high-affinity zinc site on human albumin. The residues site are conserved in all mammalian albumin and Fig. except for that of the which domain I human albumin with mutations of of residues are the of site with of affinity but high for likely to to biological transport The coordination of zinc be described as the of His247 is as the sixth ligand. the zinc coordination be as The distances are the for zinc and protein structures D. PubMed Scopus Google Scholar, K. Y. P. J. 2008; Scopus Google Scholar). the is relatively with those for but has in zinc in as an of distances in the K. Y. P. J. 2008; Scopus Google Scholar). that bound be and have a a of zinc Albumin is to but has previously been to and M. Y. K. N. S. T. Otagiri M. 2005; PubMed Scopus Google Scholar). The of zinc albumin has not been possibility be of of with the of as the zinc site in albumin are in in in the the zinc site of a site with and and displays a to that in Fig. site with the of protein is in the in form I. M. M. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). In to albumin, in is no for coordination of a for The form of the is formed only of the which a for In the side of is only as a protein for or for but coordination is during for in the of and such as Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus (86) Google Scholar). In the interdomain zinc site in albumin, Asn99 has a in the of with the of Asn99 of the and likely interdomain in albumin. The that the from albumin of the N99D mutant three for the and with the of NMR for a that interdomain H-bond is important for of the protein The N99D mutation of a by a the interdomain and a and the of His247 Fig. In the N99H mutation an H-bond to be in Fig. is that albumin a for zinc binding in blood but is to that zinc binding in has a on the of albumin, as by the NMR data (Fig. and Fig. The effects of zinc on the NMR of human serum albumin a to the effects by the (see 1986; PubMed Scopus Google and Zn2+ to the of the has been that residues are implicated in the conformational transition J. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). and allosteric effects are important for the of albumin to and transport and M. Curry S. E. M. G. P. S. P. 2005; PubMed Scopus Google Scholar, K. T. K. Y. R. A. Otagiri M. 1999; PubMed Scopus Google Scholar) and have direct on the of W. F. M. Biochem. PubMed Scopus Google Scholar). the transition has been shown to the affinity of albumin J. Biochem. PubMed Scopus Google Scholar) and J. Otagiri M. J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Curry S. PubMed Scopus Google Scholar), which to site I. site contains a binding located in which is also the binding site for and and M. Curry S. E. M. G. P. S. P. 2005; PubMed Scopus Google Scholar, W. F. M. Biochem. PubMed Scopus Google Scholar). the of an important and binding is also affected by conformational H. Mol. of Scholar). Although x-ray structures of albumin in the form have been in the no structural data are available for the of available experimental a a of the molecular of the transition as as the effects of zinc binding. be that studies have that the structure of albumin is by the x-ray structures R. B. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is thought that the transition is with a of the structure in domain I with a from domain II J. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, R. B. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). loss of α-helical in the form has been to the interdomain and has been that in form domain M. D.C. F. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), which be in the The molecular mechanism of the transition is thought to the of from residues J. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). of residues have high J. NMR Studies of of Scholar) are to be and H-bond at The loss of the of affinity J. PubMed Scopus (42) Google Scholar) as as the increased M. D.C. F. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) of the of x-ray structures of albumin that five residues are at domain and have the to form side and (Fig. and the of residues in the transition has previously been reported as five 1986; PubMed Scopus Google Scholar). All five interdomain histidines are located domains I and II an consistent with NMR studies of and of albumin J. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). experimental not only for the of of five in the transition but also for on binding to site I Curry S. PubMed Scopus Google Scholar). of to a not only the of the transition from the wild-type of to a of but also the affinity is located in domain II and a bond with (Fig. which is located in the domain Albumin contains interdomain that not and domains I and II are also is likely that the domain I/II interface is controlled by the of the interdomain and zinc binding, of interdomain histidines have effects on the is that two of the five interdomain histidines are of the zinc-binding The zinc His247 not form an interdomain H-bond in albumin but is to in albumin (9Sugio S. Kashima A. Mochizuki S. Noda M. Kobayashi K. Protein Eng. 1999; 12: 439-446Crossref PubMed Scopus (1523) Google Scholar, 14Curry S. Mandelkow H. Brick P. Franks N. Nat. Struct. Biol. 1998; 5: 827-835Crossref PubMed Scopus (1190) Google Scholar). In His247 is to which also an interdomain H-bond to of His67 in albumin. The of Asn99 is an H-bond to the of His247 as the of domains I and II, is not that the binding of a zinc at interface have as is from the NMR We that zinc the interdomain and a of the two be to have an on the of residues at interface, the interdomain in the and of site is of for the and of domains I and a conformational thought to be to the transition also when albumin with J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, T. T. S. S. J. Scholar), and has been that in the of to liver that to the binding of zinc and and is in zinc with in blood have important effects on the transport of of studies show that physiological concentrations of albumin cells from (Fig. at a of of serum albumin the toxic effects of zinc has been shown with S. Y. 2005; PubMed Scopus Google Scholar) and cells E. in 2004; PubMed Scopus Google Scholar). the for a zinc-binding to be in physiological such as blood plasma and the affinity of albumin for zinc is only zinc has been to be of the so-called zinc affinity is likely to the of zinc to The molecular by which is are not in and is that albumin, in to has as a zinc in physiological the of the site is and for zinc and to zinc with the affinity to albumin to and zinc when with