The importance of bCys93 in processes of hemoglobin oxidation and clearance

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	FDA/CBER	OXIDSTRESS-8

Authors

• CP Pereira
• PW Buehler

Abstract

Introduction: Oxidative processes at hemoglobin’s (Hb) heme groups result in free radical generation, radical migration and amino acid oxidation that are potentially critical to extracellular Hb clearance. Recent data suggests that extensive changes to Hb occur following H2O2 exposure. Critical to these findings were the identification and quantitation of βCys93 as the most extensively modified (approximately 75%) amino acid following glucose/glucose oxidase (GOX) exposure.. Because βCys93 is the primary location for modification following oxidant exposure, we investigated the effect of a bCys93 (WT) à bAla93 mutation on oxidative modifications, haptoglobin (Hp) binding and CD163 interaction. Methods: The reaction of Hb with GOX-derived H2O2 were studied by spectrophotometry. Free radical migration was evaluated by immuno spin trapping and amino acid oxidations were quantified by mass spectrometry. Hb structural changes were evaluated by circular dichroism. Additionally, binding of oxidized and non-oxidized Hbs to Hp and CD163 were studied by surface plasmon resonance. Results and conclusions: The bAla93 mutant Hb was found to be more resistant to oxidation, radical formation and structural changes than the bCys93 (WT). These observations suggest that modification of bCys93 in Hb is the major end product of peroxidative stress and alters the normal pathway of Hb clearance.

back to top