Evidence that the Principal Co^II-Binding Site in Human Serum Albumin Is Not at the N-Terminus:  Implication on the Albumin Cobalt Binding Test for Detecting Myocardial Ischemia

Abstract

Human serum albumin (HSA) is the most abundant protein in the blood plasma and is involved in the transport of metal ions. Four metal-binding sites with different specificities have been described in HSA: (i) the N-terminal site provided by Asp1, Ala2, and His3, (ii) the site at the reduced Cys34, (iii) site A, including His67 as a ligand, and (iv) the nonlocalized site B. HSA can bind CoII, and HSA was proposed to be involved in CoII transport. Recently, binding of CoII to HSA has attracted much interest due to the so-called albumin cobalt binding (ACB) test approved by the Food and Drug Administration for evaluation of myocardial ischemia. Although the binding of CoII to HSA is important, the binding of CoII to HSA is not well-characterized. Here the binding of CoII to HSA was studied under anaerobic conditions to prevent CoII oxidation. Electronic absorption, EPR, and NMR spectroscopies indicate three specific and well-separated binding sites for CoII in HSA. CoII ions in all three sites are in a high-spin state and coordinated in a distorted octahedral geometry. Competition experiments with CdII (known to bind to sites A and B) and CuII (known to bind to the N-terminal site) were used to identify the sites of binding of CoII to HSA. They revealed that the first two equivalents of CoII bind to sites A and B. Only the third may be bound to the N-terminal site. The repercussions of these results on the understanding of the ACB test and hence the myocardial ischemia are discussed.

The Clinical Assessment of Ischaemia Modified Albumin and Troponin I in the Early Diagnosis of the Acute Coronary Syndrome

“…This reduces the ability of albumin to bind to cobalt and hence, this accounts for the generation of IMA [17].…”

Alterations in the functional capacity of albumin in patients with decompensated cirrhosis is associated with increased mortality†‡§

“…Previous research into the areas of albumin affected to result in increased IMA have focused on the metal binding region at the N terminus of the protein. However, a recent study 28 has suggested that although cobalt may bind at this site transiently, its electronic configuration is better suited to binding at the fatty acid binding sites. We observed a significant relationship between the IMAR and the functionality of these sites, supporting the hypothesis that this is the binding location (Fig.…”

Randomised Assessment of Treatment using Panel Assay of Cardiac markers – Contemporary Biomarker Evaluation (RATPAC CBE)

“…Three binding sites for cobalt were identified, two of which showed greater avidity than the N-terminal binding site. 235 Fatty acid binding to albumin occurs at one of the additional cobalt binding sites with a negative allosteric interaction. It is hypothesised that in myocardial ischaemia, the release of fatty acids results in binding of fatty acids to albumin.…”

“…This would then reduce the ability of albumin to take up cobalt and would account for the presence of IMA. 235 If this also produced a conformational change in the albumin affecting the …”