Evidence that the principal CoII-binding site in human serum albumin is not at the N-terminus:: Implication on the albumin cobalt binding test for detecting myocardial ischemia

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 Co-II, and HSA was proposed to be involved in Co-II transport. Recently, binding of Co-II 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 Co-II to HSA is important, the binding of Co-II to HSA is not well-characterized. Here the binding of Co-II to HSA was studied under anaerobic conditions to prevent Co-II oxidation. Electronic absorption, EPR, and NMR spectroscopies indicate three specific and well-separated binding sites for Co-II in HSA. Co-II ions in all three sites are in a high-spin state and coordinated in a distorted octahedral geometry. Competition experiments with Cd-II (known to bind to sites A and B) and Cu-II (known to bind to the N-terminal site) were used to identify the sites of binding of Co-II to HSA. They revealed that the first two equivalents of Co-II 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.