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Abstract
Background: Nitric oxide (NO) is a gaseous transmitter playing numerous physiological roles and characterized by a short half-life. Its binding to endogenous thiols increases its stability, facilitating its storage and transport. The purpose of this study was to investigate the nitrosated serum albumin (SA-SNO) and to provide a reference for its easy preparation for further use in in vitro studies.
Methods: Serum albumin (SA) was S-nitrosated by reacting with (i) NaNO2 in acidic medium; (ii) different low-molecular weight S-nitrosothiols (RSNO) (S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), and S,S'-dinitrosobucillamine (Buc(NO)2)); and (iii) diethylamine NONOate (DEA/NO). SA-SNO was purified by size exclusion chromatography and the S-nitrosation site and the rate were studied by mass spectrometry and Griess–Saville assay, respectively. Then, SA-SNO was characterized by spectrofluorimetry, dynamic light scattering, and circular dichroism. Finally, SA-SNO reactivity with citrate stabilized gold nanoparticles (AuNP-citrate) was investigated via determination of NO release.
Results: S-nitrosation rates of SA were 90.1 ± 3.3, 76.8 ± 2.7, 80.3 ± 3.2, 84.8 ± 5.0, and 15.4 ± 1.9% (n = 5), when SA was reacted with acidified NaNO2, CysNO, GSNO, Buc(NO)2, and DEA/NO, respectively. The physicochemical characterization indicated that the resulting product corresponded to a mono-S-nitrosothiol (on cysteine-34), and the conformational construction remained unchanged. Stability studies showed that the NO content was preserved over 1 week. AuNP-citrate reacted with SA-SNO with increase of its hydrodynamic diameter but preservation of SNO bond.
Conclusions: SA-SNO prepared and stored under the reported conditions affords a well-defined reference suitable for in vitro studies.
Acknowledgements
Authors would also like to acknowledge Miss Agnieszka Lyczykowska for her support.
Disclosure statement
The authors report that they have no conflicts of interest.
Funding information
This work was financially supported by Région Lorraine (CPER) and Université de Lorraine. Authors are grateful to the program of China Scholarships Council for its financial support of Ming Luo’s PhD thesis; to Dr Alexandre Kriznik (Service Commun de Biophysicochimie des Interactions Moléculaires (SCBIM) de l'Université de Lorraine) for CD measurements, and to Pr Jolivet Yves and Mrs Gérard Joelle (UMR 1137, Ecologie et Ecophysiologie Forestières de l'Université de Lorraine) for providing Clark electrode.