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Spectroscopy-based characterization of Hb–NO adducts in human red blood cells exposed to NO-donor and endothelium-derived NO

Abstract

Red blood cells (RBCs) play an important role in the processing and delivery of nitric oxide (NO), but insight into the formation of hemoglobin (Hb) adducts with NO (Hb–NO) inside human RBCs is still weakly explored due to methodological constraints. This work presents a complementary approach based on Resonance Raman Spectroscopy (RRS) supported by Blood Gas Analysis, Electron Paramagnetic Resonance Spectroscopy, UV–Vis Absorption Spectroscopy and Mössbauer Spectroscopy to characterize the formation of various Hb species, with the focus on Hb–NO adducts formed inside isolated human RBCs exposed to NO, in experimental conditions of low and high levels of oxygen Hb saturation. In the present work, we induced Hb–NO adducts using the PAPA–NONOate, a NO-donor with known chemistry and kinetics of NO release, and confirmed the formation of Hb–NO adducts in RBCs incubated with Human Aortic Endothelial Cells (HAECs) stimulated to produce NO. Our results provide a new insight into the formation of Hb–NO adducts after exposure of RBCs with high oxyHb content to exogenous NO with the special attention to the formation of LSHbIIINO in addition to LSHbIINO and metHb (HS/LSHbIIIH2O). We also point out that reliable characterization of Hb–NO adducts requires complementary techniques. Among them, RRS as a label-free and non-destructive tool, appears as an important discriminating technique in studies of Hb–NO adducts inside intact RBCs.

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Publication details

The article was received on 15 Feb 2018, accepted on 16 Jul 2018 and first published on 25 Jul 2018


Article type: Paper
DOI: 10.1039/C8AN00302E
Citation: Analyst, 2018, Accepted Manuscript
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    Spectroscopy-based characterization of Hb–NO adducts in human red blood cells exposed to NO-donor and endothelium-derived NO

    J. Dybas, P. Berkowicz, B. Proniewski, K. Dziedzic-Kocurek, J. Jan Stanek, M. Baranska, S. Chlopicki and K. M. Marzec, Analyst, 2018, Accepted Manuscript , DOI: 10.1039/C8AN00302E

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