THE GLOBIN-BASED FREE-RADICAL OF FERRYL HEMOGLOBIN IS DETECTED IN NORMAL HUMAN BLOOD

Normal human venous blood was studied by electron paramagnetic resonan ce (EPR) spectroscopy at -196 degrees C. The EPR signal of free radica ls in frozen blood is shown to have the same radiospectroscopic parame ters and properties as the signal of the globin based free radical, 'H b(Fe(IV)=O), formed in the reaction of purified methemoglobin (metHb) with H2O2 and therefore has been assigned as such. The globin-based ra dicals and metHb exhibited significant variation (fluctuations) in dif ferent frozen samples taken from the same liquid blood sample. In any given sample a high concentration of free radicals was associated with a low concentration of metHb and vice versa, i.e. the fluctuations we re always of opposite sense. No such fluctuations were observed in the concentration of two other paramagnetic components of blood, transfer rin and ceruloplasmin. The time course of free radical formation and d ecay upon the addition of H2O2 to purified metHb was studied at three different molar ratios H2O2/metHb. This kinetic study together with th e results of an annealing experiment allow us to propose a mechanism f or the formation and decay of the globin-based radical in blood. Withi n this mechanism, the source of H2O2 in blood is considered to be dism utation of O-2(radical anion) radicals produced via autoxidation of Db . We postulate that the dismutation is intensified on the phase separa tion surfaces during cooling and freezing of a blood sample. The fluct uations are explained within this hypothesis.