Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design

Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the β-subunit (F41). We therefore replaced this residue with a tyrosine (βF41Y, Hb Mequon). The βF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, βF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the β-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in βF41Y. NO bioavailability was enhanced in βF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the β-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.</jats:p

The photoreceptor UVR8 mediates the perception of both UV-B and UV-A wavelengths up to 350 nm of sunlight with responsivity moderated by cryptochromes

ABSTRACT The photoreceptors UV RESISTANCE LOCUS 8 (UVR8) and CRYPTOCHROMES 1 and 2 (CRYs) play major roles in the perception of UV-B (280?315?nm) and UV-A/blue radiation (315?500?nm), respectively. However, it is poorly understood how they function in sunlight. The roles of UVR8 and CRYs were assessed in a factorial experiment with Arabidopsis thaliana wild-type and photoreceptor mutants exposed to sunlight for 6?h or 12?h under five types of filters with cut-offs in UV and blue-light regions. Transcriptome-wide responses triggered by UV-B and UV-A wavelengths shorter than 350?nm (UV-Asw) required UVR8 whereas those induced by blue and UV-A wavelengths longer than 350?nm (UV-Alw) required CRYs. UVR8 modulated gene expression in response to blue light while lack of CRYs drastically enhanced gene expression in response to UV-B and UV-Asw. These results agree with our estimates of photons absorbed by these photoreceptors in sunlight and with in vitro monomerization of UVR8 by wavelengths up to 335?nm. Motif enrichment analysis predicted complex signaling downstream of UVR8 and CRYs. Our results highlight that it is important to use UV waveband definitions specific to plants' photomorphogenesis as is routinely done in the visible region. This article is protected by copyright. All rights reserved.Peer reviewe

Human Fetal Hemoglobin : Biochemical Characterization and Recombinant Production

Blood transfusion is a common medical procedure when oxygen supply in the body is impaired. However, blood transfusion is not always without risks. The uncontrolled spreading of blood borne pathogens such as HIV or hepatitis B and C virus has urged researchers to look for “blood substitutes”. Many research groups have put their focuses on hemoglobin-based products (HBOCs). The major source of haemoglobin previous developed products is outdated human blood or bovine blood. However, these haemoglobin (Hb) sources have theirs limit. Therefore, alternative hemoglobin sources for HBOC development have been considered. In this thesis, human fetal hemoglobin (HbF) is introduced as an alternative hemoglobin source for HBOC development. The study of the reaction between ferricHb and H2O2 has indicated that HbF exhibits a better pseudoperoxidase activity compared to that of human adult hemoglobin (HbA). The reaction between oxyHb and nitrite also supports the notion that HbF is a more reactive molecule than HbA. Moreover, the study of the reaction between Hb and Hp using isothermal titration calorimetry (ITC) revealed a high affinity between ferric HbF and Hp 1-1. The results from differential scanning calorimetry (DSC) also revealed that ferricHb is partly inactive at a near body temperature (35˚C) and that may contribute to a lower in Hp binding affinity compared to the binding affinity at 25 ˚C. The enhanced pseudo-peroxidase activity and the higher structural stability of HbF are considered as advantages when considering HBOC development. Furthermore, the observation on a production of recombinant Hb in E. coli supports the original thought that HbF is a suitable platform for recombinant Hb for HBOC development regarding its stability and reasonable production yields in E. coli under well-optimized conditions. Moreover, modified HbF by fusion of α- and γ-subunit (fHbF) can improve a production yield to about 3-fold. The O2 and CO kinetic binding studies have revealed that fHbF can bind small ligands reversibly. Together with alkaline stability and pseudo peroxidative activity tests, we conclude that this fusion globin is a functional protein that may be used as a starting material for further HBOC development

Calorimetric Characterisation of the Binding Reaction Between Human Ferric Haemoglobins and Haptoglobin to Develop a Drug for Removal of Cell-Free Haemoglobin

High levels of cell-free haemoglobin (Hb) may occur in plasma as a consequence of e.g., pathological haemolysis or blood transfusion. These Hb molecules can be removed from blood circulation by forming a complex with the acute-phase protein haptoglobin (Hp) and thereby can also the intrinsic toxicity of free Hb be limited. In this study it is shown that ferric HbA, HbF, HbE and HbS, respectively, all bind firmly to Hp at 25 °C. By using isothermal titration calorimetry (ITC), it is demonstrated that ferric HbF has higher affinity to Hp (Ka = 2.79 ± 0.29 ×109 M−1) compared with HbA and HbS (1.91 ± 0.24 ×109 M−1) and 1.41 ± 0.34 ×109 M−1 for HbA and HbS, respectively. In addition, the affinity constant for HbE is slightly lower than the other haemoglobins (0.47 ± 0.40 ×109 M−1). Since Hp shows a general and high affinity to all Hb variants tested, it can be concluded that Hp may be useful as a therapeutic agent for several different haemolytic conditions by intravenous injection

Possibilities of Using Fetal Hemoglobin as a Platform for Producing Hemoglobin-Based Oxygen Carriers (HBOCs).

The expression levels of fetal hemoglobin (HbF) in bacterial recombinant systems are higher compared with normal adult hemoglobin (HbA). However, heme disorientation in globins are often observed in recombinant production processes, both for HbA and HbF, although the degree of heme oriental disorder is much lower for HbF. In addition, the heme disorientation can be converted to a normal conformation by an oxidation-reduction process. A chromatographic cleaning process involving a strong anion exchanger can be utilized to remove such unstable and nondesirable forms of Hb

Dissection of the radical reactions linked to fetal hemoglobin reveals enhanced pseudoperoxidase activity.

In the presence of excess hydrogen peroxide (H2O2), ferrous (Fe(+2)) human hemoglobin (Hb) (α2β2) undergoes a rapid conversion to a higher oxidation ferryl state (Fe(+4)) which rapidly autoreduces back to the ferric form (Fe(+3)) as H2O2 is consumed in the reaction. In the presence of additional H2O2 the ferric state can form both ferryl Hb and an associated protein radical in a pseudoperoxidative cycle that results in the loss of radicals and heme degradation. We examined whether adult HbA (β2α2) exhibits a different pseudoenzymatic activity than fetal Hb (γ2α2) due to the switch of γ to β subunits. Rapid mixing of the ferric forms of both proteins with excess H2O2 resulted in biphasic kinetic time courses that can be assigned to γ/β and α, respectively. Although there was a 1.5 fold increase in the fast reacting γ /β subunits the slower reacting phases (attributed to α subunits of both proteins) were essentially the same. However, the rate constant for the auto-reduction of ferryl back to ferric for both proteins was found to be 76% higher for HbF than HbA and in the presence of the mild reducing agent, ascorbate there was a 3-fold higher reduction rate in ferryl HbF as opposed to ferryl HbA. Using quantitative mass spectrometry in the presence of H2O2 we found oxidized γ/β Cys93, to be more abundantly present in HbA than HbF, whereas higher levels of nitrated β Tyr35 containing peptides were found in HbA samples treated with nitrite. The extraordinary stability of HbF reported here may explain the evolutionary advantage this protein may confer onto co-inherited hemoglobinopathies and can also be utilized in the engineering of oxidatively stable Hb-based oxygen carriers

Trapping of human hemoglobin by haptoglobin: Molecular mechanisms and clinical applications.

Significance: Haptoglobin (Hp) is an abundant plasma protein controlling the fate of hemoglobin (Hb) released from red blood cells after intravascular hemolysis. The complex formed between Hp and Hb is extraordinary strong, and once formed, this protein-protein association can be considered irreversible. Recent advances: A model of the Hp-Hb complex has been generated and the first steps towards understanding the mechanism behind the shielding effects of haptoglobin have been taken. The clinical potential of the complex for modulating inflammatory reactions and for functioning as an HBOC (hemoglobin-based oxygen carrier) have been described. Critical Issues: The three-dimensional structure of the Hp-Hb complex is unknown. Moreover, Hp is not a homogeneous protein. There are two common alleles at the Hp genetic locus denoted Hp1 and Hp2, which when analyzed on the protein levels result in differences between their physiological behavior, particularly in their shielding against hemoglobin-driven oxidative stress. Additional cysteine residues on the alpha-subunit allow Hp2 to form a variety of native multimers, which influence the biophysical and biological properties of Hp. The multimeric conformations in turn also modulate the glycosylation patterns of Hp by steric hindrance. Future Directions: A detailed analysis of the influence of Hp glycosylation will be instrumental to generate a deeper understanding of its biological function. Several pathological conditions also modify the glycan compositions allowing Hp to be potentially used as a marker protein for these disorders