Abstract
NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFe(II)) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N',N",N"-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of approximately 100% heme-nitrosylated Hb (HbFe(II)NO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysbeta(93) of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.
Highlights
Possible exchange of NO between thiols and hemoglobin (Hb)1 in red blood cells (RBCs) has been the focus of intense interest recently [1, 2]
We report the results of a detailed examination of HbFeII/GSNO incubates after 5 min in the presence of preferential chelators of Cu(I) and Cu(II) (DTPA)
HbSNO formation does occur in the HbFeIIO2/GSNO incubates in the absence of metal chelators as we reported previously [4]
Summary
Possible exchange of NO between thiols and hemoglobin (Hb) in red blood cells (RBCs) has been the focus of intense interest recently [1, 2]. We have suggested that neocuproine, a Cu(I)-specific chelator, inhibits NO release from GSNO in solutions of HbFeIIO2 [4]. To distinguish between direct reductive cleavage of GSNO by HbFeII (Reaction 2) and Cu(I)-catalyzed release (Reaction 4), it is necessary to remove all trace copper or prevent its turnover via redox cycling using GSH (Reaction 5) or another donor in the Hb-containing solutions. We report the results of a detailed examination of HbFeII/GSNO incubates after 5 min in the presence of preferential chelators of Cu(I) (neocuproine) and Cu(II) (DTPA). DeoxyHb and GSNO lease from GSNO is Ͻ2% in the presence of neocuproine in HbFeII/GSNO solutions containing dialyzed Hb. HbFeIINO formation, and GSNO breakdown, is ϳ100% within 5 min in the absence of chelators. Because trace copper was found in all reagents by ICP-MS, these observations are consistent with Cu(I)-catalyzed release of NO (Reaction 4)
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