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Review
. 2018 Aug:135:147-157.
doi: 10.1016/j.plefa.2018.07.014. Epub 2018 Jul 20.

Ischemia-modified albumin: Crosstalk between fatty acid and cobalt binding

James P C Coverdale  1 , Kondwani G H Katundu  2 , Amélie I S Sobczak  3 , Swati Arya  3 , Claudia A Blindauer  4 , Alan J Stewart  5
Affiliations
Review

Ischemia-modified albumin: Crosstalk between fatty acid and cobalt binding

James P C Coverdale et al. Prostaglandins Leukot Essent Fatty Acids. 2018 Aug.

Abstract

Myocardial ischemia is difficult to diagnose effectively with still few well-defined biochemical markers for identification in advance, or in the absence of myocardial necrosis. "Ischemia-modified albumin" (IMA), a form of albumin displaying reduced cobalt-binding affinity, is significantly elevated in ischemic patients, and the albumin cobalt-binding (ACB) assay can measure its level indirectly. Elucidating the molecular mechanism underlying the identity of IMA and the ACB assay hinges on understanding metal-binding properties of albumin. Albumin binds most metal ions and harbours four primary metal binding sites: site A, site B, the N-terminal site (NTS), and the free thiol at Cys34. Previous efforts to clarify the identity of IMA and the causes for its reduced cobalt-binding capacity were focused on the NTS site, but the degree of N-terminal modification could not be correlated to the presence of ischemia. More recent work suggested that Co2+ ions as used in the ACB assay bind preferentially to site B, then to site A, and finally to the NTS. This insight paved the way for a new consistent molecular basis of the ACB assay: albumin is also the main plasma carrier for free fatty acids (FFAs), and binding of a fatty acid to the high-affinity site FA2 results in conformational changes in albumin which prevent metal binding at site A and partially at site B. Thus, this review advances the hypothesis that high IMA levels in myocardial ischemia and many other conditions originate from high plasma FFA levels hampering the binding of Co2+ to sites A and/or B. This is supported by biophysical studies and the co-association of a range of pathological conditions with positive ACB assays and high plasma FFA levels.

Keywords: Albumin cobalt binding assay; Free fatty acids; Human serum albumin; Molecular diagnostics; Myocardial ischemia.

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Figures

Fig 1
Fig. 1
Location of the three metal binding sites that have been successfully identified on human serum albumin,PDB: 5IJF. Site A, the multi-metal binding site (MBS) (blue); NTS/ATCUN motif (green); Cys34 (red). The precise location of site B is not yet known. The boxed labels indicate the six sub-domains of albumin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 2
Fig. 2
Contrasting geometries of metal binding sites on albumin. Left: square planar coordination of Cu2+ or Ni2+ at the NTS site; the structure shown is derived from molecular modelling. The N-terminal amino group, two deprotonated backbone amide N atoms and the N(delta) of the imidazole ring of His3 form a square plane around the central metal ion. Right: tetrahedral coordination of Zn2+ at site A in human serum albumin (pdb 5ijf). His67 uses its N(epsilon) N atom, whilst His247 binds via N(delta). Asp249 binds in mono-dentate fashion, with the second carboxylate O at ca. 2.6 Å distance, too long for a metal-ligand bond. Typically for zinc sites in proteins, angles between ligands deviate substantially from the ideal tetrahedral angle (109.5°) and vary between 95° and 125°. Metal ions are rendered in gold, N atoms in blue, O atoms in red, carbon atoms in grey. No H atoms are shown.
Fig 3
Fig. 3
Co2+ competes with both Cd2+ and Zn2+ for albumin binding under physiological conditions (pH 7.4, 50 mM Tris-Cl, 50 mM NaCl) but not with Cu2+. (a) 111 Cd NMR spectra of Cd2BSA (1.5 mM) with increasing addition of Co2+. 111-Cd resonances corresponding to both site A and B (∼140 ppm and 35 ppm, respectively) are affected by Co2+. (b) Isothermal calorimetry experiments performed in the presence of 1 mol. equiv. of Cu2+ (●) or Zn2+ (formula image) demonstrate that addition of Zn2+ decreases albumin's affinity and capacity for Co2+-binding, while addition of Cu2+ has no significant effect.
Fig 4
Fig. 4
(a) Location of fatty acid (FFA) binding sites FA1-7 on human serum albumin (PDB: 1E7H), complexed with hexadecanoic (palmitic) acid . High (magenta) and low (green) affinity sites are shown. (b) Location of site A, the multi-metal binding site (PDB: 5IJF), occupied by Zn2+ (blue) . Site A and FA2 are both located between subdomains IA-IIA. The inter-domain nature and the proximity of FA2 to site A allows for the allosteric switching of metal ion binding .
Fig 5
Fig. 5
Isothermal titration calorimetry experiments demonstrate the mutual modulation of metal and fatty acid binding to bovine albumin. The presence of the C14:0 fatty acid myristate (○, 0 mol. equiv.; ●, 1 mol. equiv.; ▽, 3 mol. equiv.; and ★ 5 mol. equiv.) affects the binding capacity of albumin for Zn2+ (a) and Co2+ (b) under near-physiological conditions (pH 7.4, 50 mM Tris-Cl, 50 mM NaCl). Co2+ binding to albumin is not only weaker than that of Zn2+, but the effect of FFAs on Zn2+ binding is also much more pronounced than that of Co2+. (c) The presence of 1 mol. equiv. of Zn2+ (formula image) or Co2+ (●) affects the energetics of fatty acid binding relative to the metal-free experiment (○), likely due to the need to remove the metal before the FFA can bind. Notably, again the effect for Zn2+ is larger than that for Co2+.
Fig 6
Fig. 6
Increasing FFA (myristate, C14:0) decreases the total Co2+ binding capacity of BSA, (a) reflected in the number of apparent binding sites of albumin for Co2+ (No data for 4 mol. eq. Myr). (b) In turn, this affects the formation of the Co-DTT complex as part of the ACB assay (b), used for the detection of myocardial ischemia.
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