Michael R.
Duff, Jr.
and
Challa V.
Kumar
*
University of Connecticut, Department of Chemistry, 55 North Eagleville Road, U-3060, Storrs, Connecticut 06269, USA. E-mail: Challa.Kumar@uconn.edu; Web: http://jasmin.chem.uconn.edu
First published on 2nd September 2009
Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(II) and Cu(II), for example, mediate protein cleavage at their respective binding sites on serum albumins , in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na+, Mg2+, Ca2+, Al3+, Cr3+, Mn2+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+, Pb2+, and Ce3+ to albumins , and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins . The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins . Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.
To address these challenges, we describe a novel metallomics approach to rapidly identify if a given metal ion binds to a particular site on a target protein, and also quantify the relative affinities of a small set of metal ions to these sites. Our strategy is two-fold. The first part is to use a well-known, metal-based reagent to cleave the peptide backbone, and then identify the binding site by amino acid sequencing of the daughter fragments. For example, metal-ions,1,2 metal-oxo species3 and metal complexes,4,5 are activated to cleave the peptide backbone at the metal binding site, thereby providing a footprint of the metal site on the protein. We chose the well-known cleavage reactions of serum albumins 2,5 by Fe(II) or Cu(II)-mediated Fenton chemistry6 or Haber–Weiss chemistry.7 In these reactions, the proteins are cleaved into two or more peptide fragments near the metal binding site which facilitates the identification of the metal binding site on the protein. In addition to using the cleavage chemistry mediated by the metal ions, one could use metal mediated oxidation of amino acid residues at the metal binding site. For example, the residues surrounding the Cu(II) binding site in Cu/Zn superoxide dismutase were identified through the analysis of the Cu(II)-mediated oxidation.8 Thus, metal-mediated chemical footprinting methods could be exploited to identify their binding sites on proteins. However, one limitation of this approach has been that it is limited to a few metal ions which can either cleave the protein backbone or modify the surrounding residues.
The second part of our strategy, which is novel, is to use specific metal ions to evaluate the extent of inhibition of the above described metal-mediated protein cleavage via competitive inhibition, and assess the relative binding affinities of specific metal ions to particular sites. By combining the cleavage chemistry with competitive inhibition, the binding sites of metal ions which do not induce protein cleavage or modify amino acid residues can be footprinted by this approach. The data show that the binding of a number of transition metal ions as well as alkaline earth metal ions to specific sites on serum albumins can be examined by this approach.
Therefore, metal-footprinting with Fenton chemistry is a versatile tool to search for metal binding sites (known or unknown sites) on proteins by an indirect approach, where the metal ion of interest may not produce a spectroscopic or chemical signature to identify its interaction with the target protein. Even in cases where specific signatures of metal binding can be monitored, identifying the binding site, and its relationship to other known metal binding sites can be still challenging. Using a competitive cleavage-inhibition approach, the relative affinities of metal ions to particular sites on proteins can be quantified, rapidly and accurately. The approach is rapid (<24 hours from start of the reaction to data analysis), does not require the metal to be spectroscopically or chemically active on binding to the protein, and the method is amenable to evaluating the relative affinities of a set of ions to particular sites on proteins.
Serum albumins (SA) are chosen for the current studies because they are the most abundant proteins in the serum, they bind and transport a multitude of metal ions,9 and have a well characterized metal binding site at their N-terminus (N-Site). SA bind Cu(II) and Ni(II) at the N-site and a second binding site (Site II) has not been firmly established.10 Site II binds Cu(II), Ni(II), Cd(II) and Zn(II) and it is suggested to consist of His105, His147, and His246 in bovine serum albumin (BSA)11 or His67, Asn99, His247, Asp249 in human serum albumin (HSA).12 Recently, a third site has been suggested for Ni(II) and Zn(II) but not identified, and relative affinities of multiple metal ions to specific sites on albumins have not been established.13 Because of the multitude of metals known to bind to serum albumins , at multiple sites,14 this protein is a good candidate to quantify the relative affinities of multiple ions. The metallomics approach outlined above can be effective in rapidly assessing the relative affinities of a small set of metal ions to particular sites on serum albumins . Such data could be useful to evaluate the intrinsic metallo-biochemistry of metal ions and in assessing the toxicities of these ions via binding to the serum proteins such as SA.
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Fig. 1 (A) Binding of Fe(II) (1.5 mM) to HSA (25 μM) by ITC (10 mM NaMES pH 6.4). (B) Integrated heat release was analyzed with a number of models and indicated that the single-set of indistinguishable binding sites model gave best fits with a binding constant of 2.0 ± 0.3 × 104 M−1, enthalpy change of −2.9 ± 1.0 kcal/mol and entropy change of 9.9 ± 3.7 cal/mol K. The titration was repeated three times and the data averaged. Note that no precipitate was noted at the end of the titration. |
The enthalpy data were analyzed using different binding models by non-linear least squares method, and the best fit to the data indicated the validity of a single-set of indistinguishable binding sites model. The binding parameters extracted from the best fit are, Kb = 2.0 ± 0.3 × 104 M−1, ΔH = −2.9 ± 1.0 kcal/mol, stoichiometry (n) = 1.1 ± 0.1, and ΔS = 9.9 ± 3.7 cal/mol/K. Note that the binding is weakly exothermic, but it is accompanied by an increase in the entropy of the system which is likely due to protein unfolding and/or solvent release from the binding surfaces. Thus, binding of Fe(II) to HSA is both enthalpy and entropy driven.
In a similar manner, titration of FeSO4 into a solution of BSA (50 μM, 10 mM PIPES pH 6.1) indicated a binding constant of 2.4 ± 0.6 × 105 M−1, ΔH of 0.8 ± 0.3 kcal/mol, stoichiometry of 3.3 ± 0.3, and ΔS of 26.8 ± 1.6 cal/mol/K. In contrast to the exothermic binding of Fe(II) to HSA, its binding to BSA is endothermic and entirely entropy driven. Protein structural changes and desolvation play a major role in controlling Fe(II) binding to BSA. Note that binding of Fe(II) to BSA is nearly an order of magnitude stronger than to HSA, under similar conditions of pH/ionic strength, and this observation is interesting.
If Fe(II) binds primarily to the N-terminal site on serum albumin , then binding to the bovine form with residues Asp-Thr-His-Lys at this site is better than binding to Asp-Ala-His-Lys of the corresponding human protein. Thus, the binding sites for Fe(II) on BSA and HSA are different enough to alter the affinity for Fe(II). However, the binding of Fe(II) to SA is not well documented, thermodynamic data are sketchy, but the binding of Fe(III) to BSA has been reported to be stronger than Fe(II).15,16 In contrast, Zn(II) has essentially the same affinity for BSA and HSA, but it binds to Sites A/B on albumin but not at the N-Site.17 On the other hand, UO22+ binds to an internal site on BSA but does not bind to HSA where this site is somehow altered.13 Thus, multiple metal binding sites exist on albumins , and their binding to metal ions can be quite complex and varied.
Note that ITC studies of Cu(II) binding to BSA (100 mM TrisBorate at pH 9) suggested that BSA has only one detectable Cu(II)-binding site with an estimated affinity of 1013 M−1.18 We used these metal binding data in conjunction with the footprinting data shown below to assess the relative affinities of metal ions to particular sites on SA.
The reaction was quenched after 1.5 hours by the addition of 2-mercaptoethanol (1 mM) and EDTA (1 mM) and the products analyzed by SDS PAGE (Fig. 2). The appearance of two product bands, one at ∼39 kDa (20%) and another at ∼27 kDa (20%), is immediately clear (lane 3). A significant portion of the starting material is left unreacted and low conversions are not unsual for these reactions.1 For example, partial cleavage of pigeon liver malic enzyme by FeSO42 into two fragments and the Fe(II) cleavage of poly(A)-specific ribonuclease into two fragments and a majority of uncleaved starting material has been noted previously.5 As demonstrated in Fig. 2, the cleavage reaction required all three reagents (Fe(II), ascorbate, and H2O2, lane 3) and the reaction did not occur when any one of these were omitted (lanes 4–9).
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Fig. 2 Fe(II)-induced cleavage of HSA with FeSO4–H2O2–ascorbate. Lane 1 contained molecular weight markers listed in kDa, and each lane contained multiple reagents. For example, lanes 2 through 9 contained 15 μM HSA; lanes 3, 4, 7, and 8 contained 15 μM FeSO4, lanes 3, 5, 7, and 9 contained 500 μM H2O2; and lanes 3, 6, 8, and 9 contained 500 μM ascorbate. Thus, lane 3 contained the protein, metal ion, hydrogen peroxide and ascorbate, reacted for 1.5 h. |
In contrast to the low reactivity of Fe(II)-mediated cleavage of HSA, Cu(II)-mediated cleavage of HSA, under similar conditions, produced at least five distinct product bands (Fig. 3), and these bands are similar to those reported earlier, from this laboratory.19 Lanes 4–9 served as controls where one of the reagents is absent. Significant activity was noted in lane 8, which contained HSA, Cu(II) and sodium ascorbate but not hydrogen peroxide. This reactivity in the absence of hydrogen peroxide is mostly likely due to O2 dissolved in the air saturated solutions where it could serve as the oxidant to promote the oxidative cleavage reactions with Cu(II). Note that such reactivity is not noticed with Fe(II). Thus, there are some distinctions between the reactivities of Fe(II) and Cu(II) as well as the bovine versus human forms of serum albumins . Taken together, these four systems served as excellent reagents to footprint metal binding sites, and they provided opportunities to quantify relative affinities of multiple metal ions to particular sites.
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Fig. 3 Cu(II)-induced cleavage of HSA with CuSO4–H2O2–ascorbate. Lane 1 contained molecular weight markers listed in kDa, and each lane contained multiple reagents. For example, lanes 2 through 9 contained 15 μM HSA; lanes 3, 4, 7, and 8 contained 15 μM CuSO4, lanes 3, 5, 7, and 9 contained 500 μM H2O2; and lanes 3, 6, 8, and 9 contained 500 μM ascorbate. Thus, lane 3 contained the protein, metal ion, hydrogen peroxide and ascorbate, reacted for 1.5 h. |
To test the hypothesis that the protein cleavage in Fig. 2–3 is due to the metal ions bound to the protein and not due to ions that are free in the solution, quenching experiments were conducted. Attempts were made to quench the reactive oxygen species in these reactions by the addition of radical scavengers such as ethanol, 2-propanol and D-mannitol.20 There was only minimal quenching (<15%) by any of the quenchers (up to 3 M), and thus any reactive oxygen species generated in the solution or on the exterior of the protein is unlikely to be responsible for the observed protein cleavage. Given the fact that these metal ions bind to SA, and that the quenchers are not able to inhibit the chemistry to a measurable extent, data suggest that protein cleavage occurs essentially at metal binding sites on proteins which are not readily accessible to solution-borne quenchers .
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Fig. 4 Inhibition of Fe(II) cleavage of HSA by specific metal ions. Lane 1 contained molecular weight markers. Lane 2 is 15 μM HSA, lanes 3–9 contained 15 μM HSA, 15 μM (NH4)2Fe(SO4)2, 500 μM H2O2, and 500 μM ascorbate, in 10 mM TrisHNO3 pH 7.2. Lanes 4–14 contained 1 mM Zn(II), Cd(II), Co(II), Ni(II), Mn(II), Mg(II), Cr(III), Al(III), Ce(III), Hg(II) and Pb(II), respectively. Lane 15 contained 10 mM NaCl and Na(I) did not inhibit the reaction. |
The cleavage site on HSA was identified by isolating, purifying and N-terminal sequencing of the two product bands from the Fe(II)–HSA reaction. The analysis showed that the 39 kDa band had the sequence DAHKS (60% of total product) which is the known N-terminal sequence of HSA. In addition, this product band had an additional peptide with the N-terminal sequence, XXKDL (40% of total product), and XXKDL is an interior sequence of HSA which uniquely occurs at residue 10. Thus, two distinct fragments are responsible for the 39 kDa band. N-terminal sequencing of the second product (band 2) indicated one peptide with the sequence DAHKS (100%), the known N-terminal sequence of HSA, and there were no other products found in the sequencing studies. Thus, Fe(II) cleaves HSA near the N-terminal metal binding site (residues 9–10) and two other interior sites. Modeling with Rasmol (v. 2.7.2 by Roger Sayle, freeware) using the known crystal structure of HSA indicates that metal binding to the N-terminal site could account for all the three products identified in the sequencing studies, but the involvement of additional metal binding sites in the protein cleavage cannot be ruled out.
The bands in Fig. 4 were quantified and percentage inhibition estimated for each lane, and these have been collected in Table 1. The data clearly show that the order of inhibition for the Fe(II)–HSA reaction is: Cr(III) ∼ Pb(II) > Ce(II) > Co(II) ∼ Ni(II) > Zn(II) ∼ Hg(II) ∼ Mn(II) > Cd(II) ⋙ Na(I), Mg(II), Ca(II) and Al(III). Specific metal ions inhibited the cleavage reaction to a much greater extent than others, but the formation of both product bands was inhibited to similar extents, thereby supporting the hypothesis that metal binding to one site could be responsible for both the product bands.
Redox system | Buffer | Percent inhibitionabc | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
a Calculated with NIH Image v. 1.62. b Calculated as a percentage of the difference of the redox system with and without metal over just the redox system. c Estimated error is ±10%. | ||||||||||||||
Na+ | Mg2+ | Ca2+ | Al3+ | Cr3+ | Mn2+ | Co2+ | Ni2+ | Zn2+ | Cd2+ | Hg2+ | Pb2+ | Ce3+ | ||
HSA–Fe(II) | TrisNO3 | <10 | <10 | <10 | <10 | 100 | 50 | 60 | 60 | 50 | 40 | 50 | 100 | 70 |
BSA–Fe(II) | TrisNO3 | <10 | <10 | <10 | 90 | 90 | 60 | 100 | 90 | 50 | <10 | 90 | 40 | 40 |
HSA–Cu(II) | TrisNO3 | <10 | <10 | <10 | 20 | 40 | <10 | 100 | <10 | 30 | <10 | 100 | 30 | 50 |
BSA–Cu(II) | TrisNO3 | <10 | <10 | <10 | <10 | 30 | <10 | 40 | 20 | 10 | <10 | <10 | 10 | <10 |
HSA–Fe(II) | None | <10 | <10 | <10 | <10 | 100 | 80 | 100 | <10 | 100 | 40 | 30 | 90 | 50 |
BSA–Fe(II) | None | <10 | <10 | <10 | <10 | 100 | 60 | 80 | 70 | 100 | <10 | 80 | 60 | 100 |
HSA–Cu(II) | None | <10 | <10 | <10 | <10 | 50 | 100 | 20 | 10 | 20 | <10 | <10 | <10 | <10 |
BSA–Cu(II) | None | <10 | <10 | <10 | 30 | 100 | 100 | 100 | 20 | 20 | <10 | 70 | 10 | 100 |
To test if the cleavage inhibition is due to metal ions or due to metal coordinated buffer ions, we also carried out the cleavage reactions in non-buffered, pH adjusted de-ionized water (Table 1). Overall, there has been greater inhibition in the absence of buffer ions, except for Ni(II), presumably due to higher affinities at lower ionic strengths. These data show that the metal coordinated buffer complexes are unlikely to be responsible for the observed inhibition.
Cleavage inhibition is most likely due to the competitive binding of specific metal ions to particular sites, although allosteric effects can not be ruled out. Therefore, one hypothesis is that the extent of inhibition is proportional to the corresponding affinities of the metal ions to particular sites on the protein. Accordingly, Cr(III) and Pb(II) show the highest affinity for the Fe(II) site on HSA, while Co(II), Ni(II), Mn(II), Zn(II), Hg(II), Cd(II) and Ce(III) show only moderate affinities, and Al(III), Na(I), Mg(II) and Ca(II) show poor or no affinity. Lack of inhibition by 10 mM Na(I) is important because it shows that a mere increase in ionic strength is not responsible for the observed inhibition of the reaction.
Having obtained encouraging results with the Fe(II)–HSA reaction, we then examined the selectivity for the metal binding to Fe(II) binding sites on BSA. SDS PAGE analysis of the reaction mixtures, followed by quantitation of the product band intensities indicated the following trend in the cleavage inhibition for the Fe(II)–BSA reaction: Co(II) > Hg(II) ∼ Cr(III) ∼ Al(III) ∼ Ni(II) > Mn(II) > Zn(II) > Pb(II) ∼ Ce(III) >>> Cd(II), Na(I), Mg(II) and Ca(II). This order is quite different from that of the Fe(II)–HSA reaction. Co(II) competed efficiently with the BSA reaction, but it inhibited only 60% of the HSA reaction. The two proteins differ considerably in their affinities to specific metal ions. Again, the alkaline earth metals and Na(I) did not inhibit the reaction to an appreciable extent. One notable feature is that Al(III) inhibited the Fe(II)–BSA reaction very efficiently (90%), to nearly the same extent as that of Cr(III), Ni(II) or Hg(II). This approach of determining the relative affinities of metal ions to particular binding sites on proteins is further tested by using Cu(II)-mediated protein cleavage.
The data in Table 1 further show that the observed trend in the inhibition of the Cu(II)–BSA reaction is: Co(II) > Cr(III) > Ni(II) > Zn(II) ∼ Pb(II), while the other metals ions examined do not inhibit the reaction appreciably. Indeed, none of the metal ions examined indicated complete inhibition of the Cu(II)–BSA reaction and this particular reaction is least sensitive to the presence of other metal ions. Since the affinity of Cu(II) is generally greater than that of Fe(II) to albumins , it is not surprising that the former reaction is less sensitive to the addition of metal ions but there are more interesting subtle trends.
There are additional, important differences between the human and bovine forms of serum albumins . For example, Al(III) inhibited the Fe(II)–BSA reaction (90%) but not the Fe(II)–HSA reaction (<10%), while Pb(II) inhibited the Fe(II)–HSA reaction (100%) but inhibited only weakly the Fe(II)–BSA reaction (40%). Therefore, the percent inhibition depended on the protein, as well as the type of metal binding site under consideration.
The metallomics method shown here allows for quick (<24 hours) analysis of metal binding to proteins in a parallel manner. Competitive protein-cleavage inhibition could be a useful chemical tool for the rapid determination of the relative affinities of multiple metal ions for a given site on a specific protein. The method developed here complements the direct chemical footprinting of metal binding sites on proteins.13 However, caution about allosteric effects should be born in mind. Even so, this is a powerful method to rapidly screen a number of metal ions for their relative affinities to specific sites on a given protein.
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