PubMed · 14312955
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P M DE TRAVERSE, J F BURI. 1965-03-07. [HEMOGLOBIN].. https://pubmed.ncbi.nlm.nih.gov/14312955/
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The hypertensive effect observed with most cell-free haemoglobins has been proposed to result from NO scavenging. However, a newly developed PEG [poly(ethylene glycol)]-conjugated haemoglobin, MalPEG-Hb [maleimide-activated PEG-conjugated haemoglobin], is non-hypertensive with unique physicochemical properties: high O2 affinity, low co-operativity and large molecular radius. It is therefore of interest to compare the ligand-binding properties of MalPEG-Hb with unmodified cell-free HbA (stroma-free human haemoglobin). NO association rates for deoxy and oxyMalPEG-Hb and HbA were found to be identical. These results confirm the lack of correlation between hypertension and NO for a similar modified haemoglobin with high molecular radius and low p50 (pO2 at which haemoglobin is half-saturated with O2) [Rohlfs, Bruner, Chiu, Gonzales, Gonzales, Magde, Magde, Vandegriff and Winslow (1998) J. Biol. Chem. 273, 12128-12134]. The R-state O2 association kinetic constants were also the same for the two haemoglobins. However, even though the p50 of MalPEG-Hb is approx. half of that of HbA, the biphasic O2 dissociation rates measured at relatively high pO2 (150 Torr) were 2-fold higher, giving rise to a 2-fold lower R-state equilibrium association constant for MalPEG-Hb compared with HbA. Thus the O2 affinity of MalPEG-Hb is higher only at pO2 values lower than the intersection point of the O2 equilibrium curves for MalPEG-Hb and HbA. In summary, the present studies found similar rates of NO binding to HbA and MalPEG-Hb, eliminating the possibility that the lack of vasoactivity of MalPEG-Hb is simply the result of reduced molecular reactivity with NO. Alternatively, the unique O2-binding characteristics with low p50 and co-operativity suggest that the 'R-state' conformation of MalPEG-Hb is in a more T-state configuration and restricted from conformational change.
Elucidating the reaction of nitric oxide (NO) with oxyhemoglobin [HbFe(II)O2] is critical to understanding the metabolic fate of NO in the vasculature. At low concentrations of NO, methemoglobin [HbFe(III)] is the only detectable product from this reaction; however, locally high concentrations of NO have been demonstrated to result in some iron-nitrosylhemoglobin [HbFe(II)NO] and S-nitrosohemoglobin (SNO-Hb) formation. Reductive nitrosylation through a HbFe(III) intermediate was proposed as a viable pathway under such conditions. Here, we explore another potential mechanism based on mixed valenced Hb tetramers. The oxidation of one or two heme Fe(II) in the R-state HbFe(II)O2 has been observed to lower the oxygen affinity of the remaining heme groups, thus creating the possibility of oxygen release and NO binding at the heme Fe(II) sites. This mixed valenced hypothesis requires an allosteric transition of the Hb tetramer. Hence, this hypothesis can account for HbFe(II)NO formation, but not SNO-Hb formation. Here, we demonstrate that cyanide attenuated the formation of SNO-Hb by 30-40% when a saturated NO bolus was added to 0.1-1.0 mM HbFe(II)O2 solutions. In addition, HbFe(II)NO formation under such inhomogeneous conditions does not require allostericity. Therefore, we concluded that the mixed valenced theory does not play a major role under these conditions, and reductive nitrosylation accounts for a significant fraction of the HbFe(II)NO formed and approximately 30-40% of SNO-Hb. The remaining SNO-Hb is likely formed from NO oxidation products.
Two main hypotheses describe the role of hemoglobin in the regulation of nitric oxide (NO) bioavailability. It has been suggested that hemoglobin interacts with circulating NO, forming Fe-nitrosyl hemoglobin and then S-nitrosothiols, which deliver NO extracellularly by an allosterically regulated mechanism. Alternatively, the existence of diffusional barriers that protect NO from hemoglobin-mediated degradation has been proposed. The reliability of each model in vivo is supported by the detection of physiological hematic levels of S-nitrosohemoglobin. However, the measured concentrations of S-nitrosohemoglobin are largely divergent between the two models. Moreover, recent reports suggest that circulating levels of S-nitrosohemoglobin in human blood could be significantly lower than assessed previously. We suggest that solving the methodological controversies that make the field of NO research a 'minefield', even for skilled analysts, is fundamental to understanding the role of S-nitrosothiols in the vasculature.