ABSTRACT
Energy metabolism in RBCs is characterized by O2-responsive variations in flux through the Embden Meyerhof pathway (EMP) or the hexose monophosphate pathway (HMP). Therefore, the generation of ATP, NADH, and 2,3-DPG (EMP) or NADPH (HMP) shift with RBC O2 content because of competition between deoxyhemoglobin and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxygenated RBCs favors HMP flux and NADPH generation (maximizing glutathione-based antioxidant systems). We tested the hypothesis that sickle hemoglobin disrupts cdB3-based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia, we demonstrate in the present study constrained HMP flux, NADPH, and glutathione recycling and reduced resilience to oxidative stress manifested by membrane protein oxidation and membrane fragility. Using a novel, inverted membrane-on-bead model, we illustrate abnormal (O2-dependent) association of sickle hemoglobin to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. This finding was confirmed by immunofluorescent imaging during RBC O2 loading/unloading. Moreover, selective inhibition of inappropriately dispersed GAPDH rescues antioxidant capacity. Such disturbance of cdB3-based linkage between O2 gradients and RBC metabolism suggests a novel mechanism by which hypoxia may influence the sickle cell anemia phenotype.
Subject(s)
Antioxidants/metabolism , Erythrocytes/metabolism , Glycolysis , Hemoglobin, Sickle/physiology , Oxygen/metabolism , Adolescent , Adult , Case-Control Studies , Child , Child, Preschool , Erythrocytes/drug effects , Glycolysis/drug effects , Glycolysis/physiology , Hemoglobin, Sickle/adverse effects , Hemoglobin, Sickle/pharmacology , Humans , Models, Biological , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Oxidative Stress/physiology , Young AdultABSTRACT
This chapter summarizes the principles of RSNO measurement in the gas phase, utilizing ozone-based chemiluminescence and the copper cysteine (2C)±carbon monoxide (3C) reagent. Although an indirect method for quantifying RSNOs, this assay represents one of the most robust methodologies available. It exploits the NO detection sensitivity of ozone based chemiluminescence, which is within the range required to detect physiological concentrations of RSNO metabolites. Additionally, the specificity of the copper cysteine (2C and 3C) reagent for RSNOs negates the need for sample pretreatment, thereby minimizing the likelihood of sample contamination (false positive results), or the loss of certain highly labile RSNO species. Herein, we outline the principles of this methodology, summarizing key issues, potential pitfalls and corresponding solutions.