Formation of Free Radicals and Nitric Oxide Derivative of Hemoglobin in Rats During Shock Syndrome

Free radicals have been postulated to play an important role as mediators in the pathogenesis of shock syndrome and multiple-organ failure. We attempted to directly detect the increased formation of radicals by Electron Spin Resonance (ESR) in animal models of shock, namely the endotoxin (ETX) shock or the hemorrhagic shock of the rat. In freeze-clamped lung tissue, a small but significant increase of a free radical signal was detected after ETX application. In the blood of rats under ETX shock, a significant ESR signal with a triplet hyperfine structure was observed. The latter ESR signal evolved within several hours after the application of ETX and was localized in the red blood cells. This signal was assigned to a nitric oxide (NO) adduct of hemoglobin with the tentative structur ((a2+ NO)/23+)2. The amount of hemoglobin-NO formed, up to 0.8% of total hemoglobin, indicated that under ETX shock a considerable amount of NO was produced in the vascular system. This NO production was strongly inhibited by the arginine analog N^G-monomethyl-arginine (NMMA). The ESR signal of Hb-NO was also observed after severe hemorrhagic shock. There are three questions, namely (i) the type of vascular cells and the regulation of the process forming such a large amount of NO during ETX shock, (ii) the pathophysiological implications of the formed NO, effects which have been described as cytotoxic mediator, endothelium-derived relaxing factor (EDRF) or inhibitor of platelet aggregation, and (iii) the possible use of Hb-NO for monitoring phases of shock syndrome.     

Oxygen-derived free radicals and hemolysis during open heart surgery

Reperfusion injury occurs during open-heart surgery after prolonged cardioplegic arrest. Cardiopulmonary bypass also is known to cause hemolysis. Since reperfusion of ischemic myocardium is associated with the generation of oxygen free radicals, and since free radicals can attack a protein molecule, it seems reasonable to assume that hemolysis might be the consequence of free radical attack on hemoglobin protein. The results of this study demonstrated that reperfusion following ischemic arrest caused an increase in free hemoglobin and free heme concentrations, simultaneously releasing free iron and generating hydroxyl radicals. In vitro studies using pure hemoglobin indicated that superoxide anion generated by the action of xanthine oxidase on xanthine could release iron from the heme ring and cause deoxygenation of oxyhemoglobin into ferrihemoglobin. This study further demonstrated that before the release of iron from the heme nucleus, oxyhemoglobin underwent deoxygenation to ferrihemoglobin. The released iron can catalyze the Fenton reaction, leading to the formation of cytotoxic hydroxyl radical (OH·). In fact, the formation of OH. in conjunction with hemolysis occurs during cardiac surgery, and when viewed in the light of the in vitro results, it seems likely that oxygen-derived free radicals may cause hemolysis during cardiopulmonary bypass and simultaneously release iron from the heme ring, which can catalyze the formation of OH·.     

The quaternary structure of tetrameric hemoglobin regulates the oxygen affinity of polymerized hemoglobin

This study focuses on the effect of the initial quaternary structure of bovine hemoglobin (Hb) on the physical properties of glutaraldehyde polymerized Hb (PolyHb) solutions. Tense (T) state PolyHb was synthesized by maintaining the pO₂ of Hb before and after polymerization at 0 mm Hg. In contrast, relaxed (R) state PolyHb was generated by maintaining the pO₂ of Hb before and after polymerization to >749 mm Hg. PolyHb solutions were characterized by measuring the pO₂, methemoglobin (metHb) level, molecular weight distribution, O₂ affinity and cooperativity coefficient. The metHb level of all PolyHb solutions was low (<2%). Analysis of the molecular weight distribution of PolyHb solutions indicates that in general, the molecular weight of PolyHb solutions increased with increasing cross‐link density. T‐state PolyHb solutions exhibited lower O₂ affinity compared to unmodified Hb, whereas R‐state PolyHb solutions exhibited higher O₂ affinity compared to unmodified Hb. In addition, the polymerization reaction resulted in a significant decrease in cooperativity that was more pronounced at higher cross‐link densities. All of these results were explained in terms of the quaternary structure of Hb. Taken together, our results yield more insight into the importance Hb's quaternary structure plays in defining the physical properties of glutaraldehyde PolyHb solutions. This information will be useful in designing optimized glutaraldehyde PolyHb oxygen carriers for various applications in transfusion medicine. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: time-resolved UV resonance Raman evidence for intra-dimer coupling

The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy. The Trp alpha 14...Thr alpha 67 and Trp beta 15...Ser beta 72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (Rdeoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues. Mutation of the acceptor residues Thr alpha 67 and Ser beta 72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions. However, the mutations have profound and unexpected effects on the character of the Rdeoxy intermediate, and on the dynamics of the subsequent steps leading to the T state. Formation of the initial quaternary contact (RT intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2. These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the alpha beta dimer behaves as a mechanically coupled dynamical unit. Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected. A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1) (and alpha(2)beta(2)) interface. The present observations establish that quaternary motions can occur on the approximately 100 ns time-scale. They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts.     

The pro-oxidant role of protein SH groups of hemoglobin in rat erythrocytes exposed to menadione.

Menadione is selectively toxic to erythrocytes. Although GSH is considered a primary target of menadione, intraerythrocyte thiolic alterations consequent to menadione exposure are only partially known. In this study alterations of GSH and protein thiols (PSH) and their relationship with methemoglobin formation were investigated in human and rat red blood cells (RBC) exposed to menadione. In both erythrocyte types, menadione caused a marked increase in methemoglobin associated with GSH depletion and increased oxygen consumption. However, in human RBC, GSH formed a conjugate with menadione, whereas, in rat RBC it was converted to GSSG, concomitantly with a loss of protein thiols (corresponding to menadione arylation), and an increase in glutathione-protein mixed disulfides (GS-SP). Such differences were related to the presence of highly reactive cysteines, which characterize rat hemoglobin (cys beta125). In spite of the greater thiol oxidation in rat than in human RBC, methemoglobin formation and the rate of oxygen consumption elicited by menadione in both species were rather similar. Moreover, in repeated experiments under N2 or CO-blocked heme, it was found that menadione conjugation (arylation) in both species was not dependent on the presence of oxygen or the status of heme. Therefore, we assumed that GSH (human RBC) and protein (rat RBC) arylation was equally responsible for increased oxygen consumption and Hb oxidation. Moreover, thiol oxidation of rat RBC was strictly related to methemoglobin formation.     

Zinc content of normal human serum and its correlation with some hematic parameters

Numerous studies have demonstrated that several diseases and stress conditions are associated with changes in the levels of zinc in the blood plasma and cellular elements. In this research the association between serum zinc concentrations and other hematic parameters of diagnostic interest has been evaluated. Quantitative determinations of zinc, total plasmatic proteins, albumin, hemoglobin and calculation of mean corpuscular volume were performed on blood samples from 58 males aged 20–61 years. Concentrations measured in our sample are comparable with reference values. Statistically significant correlation coefficients were found between age and albumin (r = - 0.562, P < 0.001), serum zinc and albumin (r = 0.328, P < 0.05), serum zinc and hemoglobin (r = 0.291, P < 0.05), and total plasmatic proteins and albumin (r = 0.463, P < 0.001). These correlation coefficients were significant even after adjustment for age effect. The determination of serum zinc concentration may be useful in the assessment of clinical scenarios. Particularly, it may provide additional information for the diagnosis of specific pathologies, such as hepatic malfunctions. It could also be useful in the identification of different stages of anemia.     

New insights into deleterious impacts of in vivo glycation on albumin antioxidant activities

Background

Albumin constitutes the most abundant circulating antioxidant and prevents oxidative damages. However, in diabetes, this plasmatic protein is exposed to several oxidative modifications, which impact on albumin antioxidant properties.

Methods

Most studies dealing on albumin antioxidant activities were conducted on in vitro modified protein. Here we tried to decipher whether reduced antioxidant properties of albumin could be evidenced in vivo. For this, we compared the antioxidant properties of albumin purified from diabetic patients to in vitro models of glycated albumin.

Results

Both in vivo and in vitro glycated albumins displayed impaired antioxidant activities in the free radical-induced hemolysis test. Surprisingly, the ORAC method (Oxygen Radical Antioxidant Capacity) showed an enhanced antioxidant activity for glycated albumin. Faced with this paradox, we investigated antioxidant and anti-inflammatory activities of our albumin preparations on cultured cells (macrophages and adipocytes). Reduced cellular metabolism and enhanced intracellular oxidative stress were measured in cells treated with albumin from diabetics. NF-kB –mediated gene induction was higher in macrophages treated with both type of glycated albumin compared with cells treated with native albumin. Anti inflammatory activity of native albumin is significantly impaired after in vitro glycation and albumin purified from diabetics significantly enhanced IL6 secretion by adipocytes. Expression of receptor for advanced glycation products is significantly enhanced in glycated albumin-treated cells.

Conclusions and general significance

Our results bring new evidences on the deleterious impairments of albumin important functions after glycation and emphasize the importance of in vivo model of glycation in studies relied to diabetes pathology.     

Primary structure of hemoglobin β-chain fromColumba livia (gray wild pigeon)

Primary structure of -chain of pigeon is presented. It was determined by amino acid sequence analysis of intact -chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows 14 Ile, 61 Lys, and 113 Ile as residues specific to pigeon. One important replacement at ₁₁ contact is 55 MetSer.     

The 2/2 hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 with covalently attached heme: Comparison of X‐ray and NMR structures

The X‐ray structures of the hemoglobin from Synechococcus sp. PCC 7002 (GlbN) were solved in the ferric bis‐histidine (1.44 Å resolution) and cyanide‐bound (2.25 Å resolution) states with covalently attached heme. The two structures illustrate the conformational changes and cavity opening caused by exogenous ligand binding. They also reveal an unusually distorted heme, ruffled as in c cytochromes. Comparison to the solution structure demonstrates the influence of crystal packing on several structural elements, whereas comparison to GlbN from Synechocystis sp. PCC 6803 shows subtle differences in heme geometries and environment. The new structures will be instrumental in elucidating GlbN reactivity. Proteins 2014; 82:528–534. © 2013 Wiley Periodicals, Inc.     

Coarse‐grained and all‐atom modeling of structural states and transitions in hemoglobin

Hemoglobin (Hb), an oxygen‐binding protein composed of four subunits (α1, α2, β1, and β2), is a well‐known example of allosteric proteins that are capable of cooperative ligand binding. Despite decades of studies, the structural basis of its cooperativity remains controversial. In this study, we have integrated coarse‐grained (CG) modeling, all‐atom simulation, and structural data from X‐ray crystallography and wide‐angle X‐ray scattering (WAXS), aiming to probe dynamic properties of the two structural states of Hb (T and R state) and the transitions between them. First, by analyzing the WAXS data of unliganded and liganded Hb, we have found that the structural ensemble of T or R state is dominated by one crystal structure of Hb with small contributions from other crystal structures of Hb. Second, we have used normal mode analysis to identify two distinct quaternary rotations between the α1β1 and α2β2 dimer, which drive the transitions between T and R state. We have also identified the hot‐spot residues whose mutations are predicted to greatly change these quaternary motions. Third, we have generated a CG transition pathway between T and R state, which predicts a clear order of quaternary and tertiary changes involving α and β subunits in Hb. Fourth, we have used the accelerated molecular dynamics to perform an all‐atom simulation starting from the T state of Hb, and we have observed a transition toward the R state of Hb. Further analysis of crystal structural data and the all‐atom simulation trajectory has corroborated the order of quaternary and tertiary changes predicted by CG modeling. Proteins 2013. © 2012 Wiley Periodicals, Inc.