title: The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins.
abstract: 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(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) 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.
authors.affiliation: University of Connecticut, Department of Chemistry, 55 North Eagleville Road, U-3060, Storrs, Connecticut 06269, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Duff
authors.forename: Challa V
authors.initials: CV
authors.lastname: Kumar
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D023302:Protein Footprinting; D040901:Proteomics; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008670:Metals; D012709:Serum Albumin; D003300:Copper; D007501:Iron