Characterization of the Rat Hemoglobin Thiyl Free Radical Formed upon Reaction with Phenylhydrazine

Abstract

The characterization of the radical formed from rat hemoglobin (Hb) by methemoglobin-generating agents and trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has shown it to be a thiyl radical. The two-electron oxidation of hemoglobin forms a ferryl species with one or more free radicals located on the globin moiety. While the radical species has not been observed by use of uv-visible spectrophotometry, the species can be detected by use of electron paramagnetic resonance spectroscopy (EPR). In previous studies, in vitro experiments have shown that the EPR signal from the rat Hb radical adduct formed by t-butyl hydroperoxide decreased following pretreatment of the oxyllb with thiol-blocking agents except for iodoacetamide. In this study the power saturation profile of the DMPO radical adduct obtained from the reaction of rat oxyHb with phenylhydrazine exhibited a pattern similar to that obtained from human oxyHb, in which the β-93 cystiene was labeled with 2,2,6,6-tetramethyl-1-piperidinyloxy-4-maleimide (4-maleimide-TEMPO). EPR spectra were taken at 77 K and computer simulations were performed. The calculated value for aNiso obtained by simulation indicates that the radical adduct is in a hydrophobic region. The value for aHiso has little structural significance, as the steric effect of the protein makes comparison with radical adducts in solution problematical. The value of gx from the rat Hb radical adduct was significantly higher than that obtained from bovine Hb, whose radical is not thiol-derived, as demonstrated by negative thiol-blocking agent experiments. A higher gxvalue is consistent with the radical adduct containing a heavy atom such as sulfur. Rat Hb was analyzed for thiol content by use of iodoacetamide, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and uv-visible spectrophotometry. It was found that 1.15 ± 0.34 thiols/tetramer of Hb were reactive with DTNB, but not iodoacetamide.