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

Abstract

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2'-Azobis(2-amidinopropane) (AAPH) and 4,4'-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, 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 free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin. Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.

Analysis of 2,2’-Azobis (2-Amidinopropane) Dihydrochloride Degradation and Hydrolysis in Aqueous Solutions

“…14). However, k h1 plateaus above pH 10, likely due to the deprotonation of the amidine moieties in AAPH (p K a 10.4) 25. The loss of the positive charge makes the amidine moieties less electrophilic, which explains the dampened hydrolysis rate.…”

Free Radical‐Induced Protein Degradation of Erythrocyte Membrane is Influenced by the Localization of Radical Generation

“…If, however, the cells were hemolyzed prior to adding the peroxyl radical generator, a signi cant increase in protein oxidation was observed (13). In this context, it is also interesting to note that intact red blood cells do not produce methemoglobin during AAPH treatment, whereas puri ed hemoglobin produced methemoglobin after 60 min of AAPH treatment (14,15). These data suggest a lack of accessibility of AAPH with intact cells.…”

Red Cell Membrane Lipid Changes at 3500 m and on Return to Sea Level

“…After centrifugation, plasma and buffy coat were removed and red blood cells were washed three times with isotonic Krebs-HEPES buffer (KHB), pH 7.4. AAPH treatment was performed as has been previously described (Celedón et al, 2001b). Cells were resuspended in KHB at 16% hematocrit and incubated either with or without AAPH at 37°C for 90 min.…”