Spin concentration measurements of high-spin (g' = 4.3) rhombic iron(III) ions in biological samples: theory and application.

Electron paramagnetic resonance (EPR) signals at g' = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe3+ ions in sites of low symmetry. The present study was undertaken to develop the experimental method and a suitable g' = 4.3 intensity standard and for accurately quantifying the amount of Fe3+ responsible for such signals. By following the work of Aasa and Vänngård (J. Magn. Reson. 19:308-315, 1975), we present equations relating the EPR intensity of S = 5/2 ions to the intensities of S = 1/2 standards more commonly employed in EPR spectrometry. Of the chelates tested, Fe3+-EDTA (1:3 ratio) in 1:3 glycerol/water (v/v), pH 2, was found to be an excellent standard for frozen-solution S = 5/2 samples at 77 K. The spin concentrations of Cu2+-EDTA and aqua VO2+, both S = 1/2 ions, and of Fe3+-transferrin, an S = 5/2 ion, were measured against this standard and found to agree within 2.2% of their known metal ion concentrations. Relative standard deviations of +/-3.6, +/-5.3 and +/-2.9% in spin concentration were obtained for the three samples, respectively. The spin concentration determined for Fe3+-desferrioxamine of known Fe3+ concentration was anomalously low suggesting the presence of EPR-silent multimeric iron species in solution.