Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity

We have investigated the influence of the free radical initiator characteri stics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2'-Azobis(2-amidinopropane) (AAPH) and 4,4'-az obis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilic ity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively. AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient fre e radical-mediated process, particularly for ACV-derived radicals, where ne arly one heme moiety was modified per radical introduced into the system, s uggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-com pounds lead to different oxi dation products. Methemoglobin, hemichromes and choleglobin were produced i n AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominan tly form hemichromes, with very low production of choleglobin. Red cell damage was evaluated at the level of hemoglobin and membrane const ituents modification, and was expressed in terms of free radical doses. Bef ore the onset of the lytic process, ACV leads to more lipid peroxidation th an AAPH, and induces a moderate oxidation of intracellular Hb. This intrace llular oxidation is markedly increased if ACV hydrophilicity is decreased b y lowering the pH. On the other hand, AAPH-derived radicals are considerabl e more efficient in promoting protein band 3 modification and cell lysis, w ithout significant intracellular hemoglobin oxidation. These results show t hat the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant f actor leading to red blood cell lysis.