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 overlappin g signals. The method requires double integration of a number of spectra co ntaining the signals in different proportions and the subsequent solution o f 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 concen trations of all the paramagnetic species present. There is no requirement t o deconvolute the whole spectrum into its individual components. The method is employed to quantify different heme species in methemoglobin and metmyo globin preparations. A significantly greater intensity of the high-spin sig nal in metmyoglobin, compared to methemoglobin at the same heme concentrati on, 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 concentrat ions obtained by optical spectroscopy. (C) 2000 Academic Press.