Chimeric Antibody-Binding Vitreoscilla Hemoglobin (VHb) Mediates Redox-Catalysis Reaction: New Insight into the Functional Role of VHb

Experimentation was initiated to explore insight into the redox-catalysis reaction derived from the heme prosthetic group of chimeric Vitreoscilla hemoglobin (VHb). Two chimeric genes encoding chimeric VHbs harboring one and two consecutive sequences of Fc-binding motif (Z-domain) were successfully constructed and expressed in E. coli strain TG1. The chimeric ZVHb and ZZVHb were purified to a high purity of more than 95% using IgG-Sepharose affinity chromatography. From surface plasmon resonance, binding affinity constants of the chimeric ZVHb and ZZVHb to human IgG were 9.7 x 10(7) and 49.1 x 10(7) per molar, respectively. More importantly, the chimeric VHbs exhibited a peroxidase-like activity determined by activity staining on native PAGE and dot blotting. Effects of pH, salt, buffer system, level of peroxidase substrate and chromogen substrate were determined in order to maximize the catalytic reaction. From our findings, the chimeric VHbs displayed their maximum peroxidase-like activity at the neutral pH (~7.0) in the presence of high concentration (20-40 mM) of hydrogen peroxide. Under such conditions, the detection limit derived from the calibration curve was at 250 ng for the chimeric VHbs, which was approximately 5-fold higher than that of the horseradish peroxidase. These findings reveal the novel functional role of Vitreoscilla hemoglobin indicating a high trend of feasibility for further biotechnological and medical applications

Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical alpha- and two identical gamma-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the alpha- and gamma-subunits of HbF were fused in order to stabilize the alpha-subunits and facilitate balanced expression of alpha- and gamma-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes

One-Step Purification of Recombinant Human Amelogenin and Use of Amelogenin as a Fusion Partner

Amelogenin is an extracellular protein first identified as a matrix component important for formation of dental enamel during tooth development. Lately, amelogenin has also been found to have positive effects on clinical important areas, such as treatment of periodontal defects, wound healing, and bone regeneration. Here we present a simple method for purification of recombinant human amelogenin expressed in Escherichia coli, based on the solubility properties of amelogenin. The method combines cell lysis with recovery/purification of the protein and generates a >95% pure amelogenin in one step using intact harvested cells as starting material. By using amelogenin as a fusion partner we could further demonstrate that the same method also be can explored to purify other target proteins/peptides in an effective manner. For instance, a fusion between the clinically used protein PTH (parathyroid hormone) and amelogenin was successfully expressed and purified, and the amelogenin part could be removed from PTH by using a site-specific protease

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

Redox Chemistry of Hemoglobin-Associated Disorders

This Forum addresses oxidative reactions of hemoglobin (Hb) and explores the underlying mechanisms of some of these reactions that contribute to the pathophysiology associated with hemolytic anemia and Hb-based oxygen therapeutics. A special focus of this Forum is on the understanding of naturally occurring mutations in human Hb and how these mutations were influenced overtime by variety of oxidative stresses. What emerges from these contributions is that some hemoglobinopathies involve mutant Hb that resists oxidative challenges, whereas the majority often result in circulatory disorder. The contributors provide in-depth and comprehensive overviews on selected key mechanisms underlying Hb oxidative reactions in health and in disease states and how this knowledge may help in the design of countermeasures against these oxidative and toxicological pathways. Antioxid. Redox Signal. 26, 745-747

Molecular Modeling of the Human Hemoglobin- Haptoglobin Complex Sheds Light on the Protective Mechanisms of Haptoglobin

Hemoglobin (Hb) plays a critical role in human physiological function by transporting O2. Hb is safe and inert within the confinement of the red blood cell but becomes reactive and toxic upon hemolysis. Haptoglobin (Hp) is an acute-phase serum protein that scavenges Hb and the resulting Hb-Hp complex is subjected to CD163-mediated endocytosis by macrophages. The interaction between Hb and Hp is extraordinarily strong and largely irreversible. As the structural details of the human Hb-Hp complex are not yet available, this study reports for the first time on insights of the binding modalities and molecular details of the human Hb-Hp interaction by means of protein-protein docking. Furthermore, residues that are pertinent for complex formation were identified by computational alanine scanning mutagenesis. Results revealed that the surface of the binding interface of Hb-Hp is not flat and protrudes into each binding partner. It was also observed that the secondary structures at the Hb-Hp interface are oriented as coils and a-helices. When dissecting the interface in more detail, it is obvious that several tyrosine residues of Hb, particularly b145Tyr, a42Tyr and a140Tyr, are buried in the complex and protected from further oxidative reactions. Such finding opens up new avenues for the design of Hp mimics which may be used as alternative clinical Hb scavengers

SDS-PAGE analysis of samples from different stages of purification of recombinant amelogenin from <i>E. coli</i> cells.

<p>Lanes marked with (A) represent samples from cells expressing amelogenin and lanes marked with (N) refers to negative control cells harboring empty expression vector. Lanes marked with (1) represent whole cell samples after cultivation. Lanes marked (2), (3) and (4) represent samples after different treatments of the intact cells. (2): soluble fractions of cells sonication in 3% HAc. (3): soluble fractions after heat treatment of the sonicated samples in lanes 2A and 2N. (4): soluble fractions of cells directly heat treated at 80°C. This generates the most pure amelogenin. Lanes marked (M) contain molecular weight markers.</p

Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein

Hemoglobin (Hb) is well protected inside the red blood cells (RBCs). Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA). Hb induced cleavage of supercoiled pDNA (sc pDNA) which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2) and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA) could easily induce complete pDNA cleavage while fetal hemoglobin (HbF) was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals

Expression, Purification and Initial Characterization of Functional alpha(1)-Microglobulin (A1M) in Nicotiana benthamiana

alpha(1)-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function

Structure superimposition of modeled (cyan) and crystal (green) structure of Hb-Hp complex (a) as well as Hp (b) and Hb (c) structures.

<p>Structure superimposition of modeled (cyan) and crystal (green) structure of Hb-Hp complex (a) as well as Hp (b) and Hb (c) structures.</p