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 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...