Joseph M. Rifkind

Three hypotheses explain a role for red blood cells (RBCs) in delivering NO to the vasculature: (a) "the SNOHb hypothesis" involves the uptake of NO by RBCs with NO transferred from the heme to the beta-93 thiol in the R quaternary conformation, followed by the release to membrane thiols in the T quaternary conformation; and (b and c) "the nitrite hypotheses" bypass the intrinsic difficulties of transporting the highly reactive NO, by reutilizing the nitrite formed when NO reacts with oxygen. Deoxyhemoglobin reduces this nitrite back to NO. The distinction between the two nitrite mechanisms depends on the importance of intermediate species formed during nitrite reduction. Without bioactive intermediates, the NO must be immediately released to avoid binding to deoxyhemoglobin. The "nitrite intermediate hypothesis" enables the RBCs to store a pool of potentially bioactive NO until it is released from the cell. In this review, we critically compare these different proposals for the transport/delivery of NO by RBCs. We also compare the redox properties in the RBCs associated with NO with the redox properties associated with oxygen.

The interaction of metal‐substituted hemoglobin (MHb), where M  Ni and Cu (T‐state with no O₂ and CO binding capability) and Fe (R‐state when CO is bound), with cationic cityl trimethyl ammonium bromide (CTAB) and anionic (sodium dodecyl sulfate—SDS) surfactants has been studied using spectroscopic techniques—UV‐visible, electron paramagnetic resonance (EPR), and Fourier transform–Raman—with additional supportive evidence coming from conductivity measurements. We observed the loss of 5‐coordination in all three hemoglobins below the critical micelle concentration (CMC) of surfactant, with noticeable differences, suggesting differing mechanisms involved in this process. In addition, above the CMC, Ni‐ and Cu‐hemes were found to leave their proteins more easily than Fe‐heme, presumably due to weaker or no bond with the proximal histidine in the former. The released heme is stabilized by micellar media through a hydrophobic interaction process. Of the two surfactants, CTAB seems to be capable of releasing the heme better than SDS and it is attributed to the greater hydrophobicity of CTAB though the charge of the surfactant plays an important role. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005

Redox reactions of hemoglobin have gained importance because of the general interest of the role of oxidative stress in diseases and the possible role of red blood cells in oxidative stress. Although electron paramagnetic resonance (EPR) is extremely valuable in studying hemoglobin redox reactions it has not been adequately used. We have focused in this review on the important contributions of EPR to our understanding of hemoglobin redox reactions. We have limited our discussion to the redox reactions thought to occur under physiological conditions. This includes autoxidation as well as the reactions of hydrogen peroxide generated by superoxide dismutation. We have also discussed redox reactions associated with nitric oxide produced in the circulation. We have pinpointed the value of using EPR to detect and study the paramagnetic species and free radicals formed during these reactions. We have shown how EPR not only identifies the paramagnetic species formed but can also be used to provide insights into the mechanism involved in the redox reactions.

The role of hemoglobin in transporting oxygen is dependent on the reversible binding of oxygen to Fe(II) hemoglobin with molecular oxygen released at reduced oxygen pressures. The partially oxygenated hemoglobin formed with the release of oxygen from hemoglobin is susceptible to redox reactions where the functional Fe(II) heme is oxidized to Fe(III) and the substrate is reduced. In this article, we review two important redox reactions of hemoglobin and discuss the ramifications of these reactions. The reduction of oxygen to superoxide starts a cascade of oxidative reactions, which are a source for red cell-induced oxidative stress. The reduction of nitrite to nitric oxide produces a labile form of nitric oxide that can be a source for oxidative stress, but can also have important physiological functions.

We have studied the binding of hemoglobin to the red cell membrane by centrifugation and fluorescence methods. The intact red cell was labeled with eosin-5-maleimide (EM), which specifically reacts with lysine 430 of band 3. Even though this residue is not part of the cytoplasmic domain of band 3 (cdb3) associated with hemoglobin binding, fluorescence quenching was observed when hemoglobin bound to inside-out vesicles (IOVs). The use of fluorescence quenching to measure band 3 binding was quantitatively compared with the binding determined by centrifugation, which measures binding to band 3 and non-band 3 sites. For the centrifugation it was necessary to include the non-band 3 association constants determined from chymotrypsin-treated IOVs. The binding of hemoglobin to band 3 was interpreted in terms of the binding of two hemoglobin tetramers to each band 3 dimer. An anticooperative interaction associated with the conformational change produced when hemoglobin binds results in a 2.8-fold decrease in the intrinsic constant of (1.54 ± 0.25) × 10⁷ M^-1 for the binding of the second hemoglobin molecule. From the changes in lifetime produced by binding the first and second hemoglobin molecules, it was possible to show that the conformational change associated with binding the second hemoglobin molecule results in a decrease of the heme−eosin distance from 47.90 to 44.78 Å. Reaction of cyanate with the α-amino group of hemoglobin (HbOCN) is shown to produce a very dramatic decrease in the binding of hemoglobin to both the band 3 and non-band 3 sites. The intrinsic constant for binding the first hemoglobin molecule to band 3 decreases by a factor of 29 to (5.34 ± 0.15) × 10⁵ M^-1. The anticooperative interaction is greater with the intrinsic constant decreasing by a factor of 3.8 for the binding of the second hemoglobin tetramer to band 3. In addition, the nature of the conformational change produced by binding hemoglobin is very different with the second HbOCN increasing the heme−eosin distance to 55.99 Å. The utilization of eosin-5-maleimide-reacted red cell membrane to study hemoglobin binding makes it possible to directly study the binding to band 3. At the same time a sensitive probe of the conformational changes, which occur when hemoglobin binds to band 3, is provided.