A new method for quantitation of spin concentration by EPR spectroscopy: application to methemoglobin and metmyoglobin.

A new method of EPR spectral analysis is developed to quantitate overlapping signals. The method requires double integration of a number of spectra containing the signals in different proportions and the subsequent solution of a system of linear equations. The result gives the double integral values of the individual lines, which can then be further used to find the concentrations of all the paramagnetic species present. There is no requirement to deconvolute the whole spectrum into its individual components. The method is employed to quantify different heme species in methemoglobin and metmyoglobin preparations. A significantly greater intensity of the high-spin signal in metmyoglobin, compared to methemoglobin at the same heme concentration, is shown to be due to larger amounts of low-spin forms in methemoglobin. Three low-spin types in methemoglobin and two in metmyoglobin are present in these samples. When their calculated concentrations are added to those of the high-spin forms, the results correspond to the total heme concentrations obtained by optical spectroscopy.