The detection of superoxide anion from the reaction of oxyhemoglobin and phenylhydrazine using EPR spectroscopy

Summary The low temperature EPR spectrum of a quickly reacted mixture of oxyhemoglobin and phenylhydrazine was studied. With the use of a computer, the spectral contribution of methemoglobin in the region of g=2 was subtracted. The remaining spectrum was that of an axial free radical (g=2.00, g=2.06) having the magnetic parameters of superoxide anion. In the presence of superoxide dismutase, this axial radical was not seen, confirming that superoxide anion is indeed generated by the reaction.The portion of this investigation carried out at the Albert Einstein College of Medicine was supported in part by US Public Health Service Research Grant HL-93399 from the Heart and Lung Institute and by National Institute Contract Nol-CP-55606 to J.P. This is communication No. 378 from the Joan and Lester Avnet Institute of Molecular Biology.Predoctoral fellow in the Medical Scientist Training Program (United States Public Health Service Grant 5-TO-5-GM 01669-12) at the New York University School of Medicine.Recipient of a grant-in-aid from the New York Heart Association.