Determination of ferric heme-human serum albumin by 1H NMR relaxometry

A new method for the accurate determination of ferric heme-human serum albumin (heme-HSA) at concentrations down to the physiological level, i.e., in the micromolar concentration range, is proposed. This method is based on the H-1 NMR relaxometric properties of heme-HSA. Actually, the binding of the paramagnetic ferric heme to the primary binding site of HSA determines a strong paramagnetic enhancement of the water H-1 NMR relaxation rate. Although a linear relationship may be seen by operating at 20 MHz on conventional electromagnets, the method here reported is improved by working at 0.02 MHz on a field-cycling instrument. This H-1 NMR relaxometric method does not suffer from the presence in serum of heme catabolites (e.g., bilirubin) that affect significantly the optical determination of ferric heme-HSA in the micromolar concentration range. Paramagnetic ferric hemoglobin contribution may be selectively quenched by cyanide binding. (C) 2003 Elsevier Science Inc. All rights reserved.