Key Points• Hb-conformation-dependent interaction with band 3 protein regulates glycolysis in RBCs.• In hypoxia, HbS disrupts this system, disabling RBC antioxidant defense. Energy metabolism in RBCs is characterized by O 2 -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 O 2 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 (O 2 -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 O IntroductionSickle cell anemia (SCA) arises from a single amino acid substitution (Glu6Val) in the ␤-globin chain. Although the change to hemoglobin (Hb) is simple and uniform, SCA is characterized by broad differences in clinical manifestation. Phenotype variation in SCA is thought to arise from both environmental and genetic factors (eg, ␤-gene cluster haplotype, degree of HbF expression, or effects of other epistatic genes). The environmental factor that most clearly influences SCA phenotype is hypoxia, which drives sickle Hb (HbS) polymerization and the resulting well-characterized alterations in RBC physiology and the microcirculation. However, the influence of hypoxia on the SCA phenotype appears to be insufficiently explained by HbS polymerization alone. 1 Moreover, we lack a clear mechanistic understanding of the significant oxidative stress complicating SCA, a key feature of phenotype variation, both at rest and in association with hypoxia. 2 Nonpolymerized, solution-phase HbS may promote oxidative stress, even in RBCs under normal physiologic O 2 gradients. 3 Specifically, the low redox potential for heme in HbS 4 and avid binding affinity of HbS for the cytoplasmic regulatory domain of the Band 3 membrane protein (cdB3) 5,6 strongly affect RBC energetics and antioxidant systems [7][8][9] and, notably, do so as a function of RBC O 2 content. Therefore, both the genesis and the disposal of reactive oxygen species are abnormal in SCA, creating a baseline state of oxidative stress, which worsens in hypoxia.In particular, consideration of metabolic control in RBCs suggests O 2 -dependent HbS-cdB3 interaction as a relatively ...

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 chemiluminscence, 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), NO species inter-conversion, 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.

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Energy metabolism in red blood cells (RBCs) is characterized by oxygen (O2)‐responsive variation in flux through the Embden Meyerhof Pathway (EMP) or the Hexose Monophosphate Pathway (HMP). As such, generation of ATP, NADH, and 2,3‐DPG (EMP) or NADPH (HMP) shifts with RBC O2 content, due to competition between deoxy‐hemoglobin (Hb) and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxy RBCs favors HMP flux and NADPH generation (maximizing glutathione (GSH)‐based antioxidant systems). We hypothesized that sickle hemoglobin (HbS) disrupts cdB3‐based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia (SCA), we demonstrate constrained NADPH and GSH recycling and reduced resilience to oxidative stress. We further illustrate abnormal association of HbS to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. These findings are confirmed by EMP/HMP 1H NMR glucose flux analysis in addition to RBC immunofluorescent imaging during 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 SCA phenotype.