Production, Purification, and Characterization of Recombinant Human Hemoglobin Rainier Expressed inSaccharomyces cerevisiae

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the β145 tyrosine is substituted with cysteine. The α and β^Rainierglobin cDNAs were cloned in a high copy number vector and expressed inSaccharomyces cerevisiaeunder the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual α and β^Rainierglobin chains. Coexpression of both α and β^RainiercDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the α and β chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A₀. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.     

Study of chemically modified hemoglobin as an artificial oxygen carrier in the model of hemorrhagic shock in dogs]

Hemodynamic and gas-transporting properties of the chemically modified hemoglobin solution have been studied on the model of hemorrhagic shock in dog. It has been shown that the polymerized hemoglobin solution exerts the hemodynamic action just as the plasma substitute "polyglucin" does. However, in contrast to the latter, polyhemoglobin circulating in the vascular bed for a prolonged period of time increases the blood oxygen capacity and oxygen delivery to tissues with the resultant increase in body total oxygen taking-up.     

Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock

Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has yielded promising results in Phase II clinical trials, and is entering a pivotal Phase III clinical trial for the treatment of shock associated with systemic inflammatory response syndrome (SIRS). Shock associated with SIRS is a NO-induced shock. PHP, a new mechanism-based therapy, has been demonstrated in clinical trials to have the expected hemodynamic activity of raising blood pressure and reducing catecholamine use, consistent with its mechanism of action as a NO scavenger. PHP is conjugated with polyoxyethylene, which results in a surface-decorated molecule with enhanced circulation time and stability as well as in attachment of soluble red blood cell enzymes, including catalase and superoxide dismutase. PHP thus contains an antioxidant profile similar to the intact red blood cell and is therefore resistant to both initial oxidative modification by oxidants such as hydrogen peroxide and subsequent ferrylhemoglobin formation. These studies suggest both that the redox activity of modified hemoglobins can be attenuated and that modified hemoglobins containing endogenous antioxidants, such as PHP, may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including ischemia-reperfusion injury and hemorrhagic shock.     

Effect of NaBH4 concentration and reaction time on physical properties of glutaraldehyde-polymerized hemoglobin

The US is about 1.5 days away from exhausting its entire blood supply. Hence, there is an urgent need for the development of universal blood substitutes. One such blood substitute is glutaraldehyde-polymerized bovine hemoglobin. Despite the commercial development of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes, there has been little published work on the effect of reaction conditions on the physical properties of the polymerized hemoglobin dispersion. In this study, the effect of varying the concentration of the quenching agent NaBH(4), glutaraldehyde concentration, and reaction time on the physical properties of PolyHb dispersions was studied by measuring the absolute molecular weight distribution, as well as oxygen-binding properties such as P(50), Hill coefficient, and methemoglobin level (MetHb) of these dispersions. Bovine hemoglobin was polymerized with glutaraldehyde using a parallel synthetic approach. Asymmetric flow field-flow fractionation (AFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight distribution of the PolyHb dispersions. In general, high glutaraldehyde concentrations (20-30 times the molar concentration of hemoglobin) adversely affected the oxygen-binding properties of PolyHb dispersions, while NaBH(4) concentrations (up to 300 times the molar concentration of hemoglobin) and reaction times (up to 24 h) did not appear to have any effect on the oxygen-binding properties of PolyHb dispersions.     

Effect of glutaraldehyde on hemoglobin: functional aspects and M?ssbauer parameters

Glutaraldehyde is widely used for the cross-linking of hemoglobin for blood substitute research or for technological purposes. The effects of this reagent on the biochemical properties of hemoglobin were correlated with M?ssbauer data. Human hemoglobin was cross-linked by glutaraldehyde as soluble polymers and insoluble particles. Effects of cross-linking on oxygen affinity, oxidation-reduction potential, autoxidation kinetics, and thermal stability were studied. Stability of cross-linked hemoglobin was specifically studied by M?ssbauer spectroscopy. Oxygen affinity is increased, redox potential is decreased, autoxidation rates are increased, and stability towards thermal denaturation is increased. The regeneration of partially denatured hemoglobin by glutaraldehyde cross-linking is shown. Effects of cross-linking on biochemical properties are explained by the hypothesis of the opening of the heme pocket on the distal-histidine side and the concomitant charge transfer from the iron to the oxygen.     

Liposome-Encapsulated Hemoglobin: A Red Blood Cell Substitute

Abstract

A safe and effective red blood cell (RBC) substitute would have broad implications in the practice of emergency medicine, trauma management, surgery, and several other areas of medicine. Several hemoglobin-based RBC substitutes have been developed that can deliver oxygen to peripheral tissues. However, although these RBC substitutes have desirable biophysical properties, their in vivo efficacy is limited by their significant toxicity. In view of the very high doses of blood substitute that are likely to be used clinically, toxicity as well as other safety issues that include hemostasis and thrombosis are critical considerations for the development and ultimate application of RBC surrogates.

Recent work conducted in our laboratories has demonstrated that administration of liposome-encapsulated hemoglobin (LEH) in rats was efficacious. Also our results have demonstrated that the replacement of more conventional lipids with the sterically-stabilizing lipid polyethyleneglycol distearoylphosphatidyl-ethanolamine in the LEH results in a significant decrease in LEH immunotoxicity, as measured by host resistance to infectious insult.     

Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae

Due to limitations associated with whole blood for transfusions (antigen compatibility, transmission of infections, supply and storage), the use of cell-free hemoglobin as an oxygen carrier substitute has been in the center of research interest for decades. Human hemoglobin has previously been synthesized in yeast, however the challenge is to balance the expression of the two different globin subunits, as well as the supply of the prosthetic heme required for obtaining the active hemoglobin (α₂β₂). In this work we evaluated the expression of different combinations of α and β peptides and combined this with metabolic engineering of the heme biosynthetic pathway. Through evaluation of several different strategies we showed that engineering the biosynthesis pathway can substantially increase the heme level in yeast cells, and this resulted in a significant enhancement of human hemoglobin production. Besides demonstration of improved hemoglobin production our work demonstrates a novel strategy for improving the production of complex proteins, especially multimers with a prosthetic group.     

An (R)-specific N-methyltransferase involved in human morphine biosynthesis

Chemically or genetically modified hemoglobins are a therapeutic class indicated for the treatment of a variety of hypo-oxygenation pathologies, severe trauma-related hemorrhages or elective surgery when blood transfusions are refused or not available. Recombinant heterologous hemoglobins offer the possibility of a potentially unlimited production and genetically optimized properties in terms of oxygen affinity, NO reactivity and resistance to autoxidation. Hemoglobin Polytaur is an autopolymerizing human-bovine hybrid mutant, previously obtained as a 500 kDa polymer, shown to reduce the infarct volume from focal cerebral ischemia in in vivo animal models. In this work, hemoglobin Polytaur polymerization, carried out under conditions to minimize heme oxidation and modification, resulted in a 180 kDa cyclic homogeneous trimer of hemoglobin tetramers. This novel oligomer was characterized by electrophoresis, MALDI-TOF mass spectrometry and gel filtration. The size and the oxygen binding properties were shown to be ideally suited for its use as a blood substitute. Co-expression with the human α hemoglobin-stabilizing protein (AHSP), a chaperone that assists hemoglobin folding in vivo, resulted in an unexpected decrease in yield and in unusual spectroscopic and functional properties, suggesting the formation of strong protein–protein interactions that reduce the expression, hinder the tetramer assembly and prevent purification.     

Ferric species of the giant extracellular hemoglobin of Glossoscolex paulistus as function of pH: an EPR study on the irreversibility of the heme transitions

The present article is focused on the transitions of ferric heme species of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) induced by successive alterations in pH, involving alkaline and acid mediums. Electron paramagnetic resonance (EPR) is the spectroscopy used to evaluate the transitions that occur in the first coordination sphere of ferric ion as a consequence of ligand changes in a wide range of pH, since this tool is very sensitive to slight changes that occur in the heme pocket of paramagnetic species. This approach is adequate to obtain information regarding the reversibility/irreversibility that involves the heme transitions induced by pH, since the degree of reversibility is associated to the intensity of the changes that occur in the spatial configuration of the polypeptide chains, which is clearly associated to the first coordination sphere. The results demonstrate a significant degree of irreversibility of heme transitions, since the final species, which do not present any change after 6 h of its respective formations, are quite different of the initial species. The results denote that the more stable species are the bis-histidine (hemichrome) and pentacoordinate species, due to the properties of their ligands and to the mechanical influence of the respective subunits. EPR spectra allow to distinguish the types of hemichrome species, depending on the reciprocal orientation between the histidine axial ligands, in agreement with Walker's Classification [Walker, F.A., 1999. Magnetic spectroscopic (EPR, ESEEM, M?ssbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord. Chem. Rev. 185-186, 471-534]. However, these transitions are not completed, i.e., the appearance of a determined species does not mean the total consumption of its precursor species, implying the coexistence of several types of species, depending on pH. Furthermore, it is possible to conclude that a "pure" EPR spectrum of aquomet ferric species is an important indicator of a high level of conservation referent to the "native" configuration of whole hemoglobin, which is only encountered at pH 7.0. The results allow to infer important physico-chemical properties as well as to evaluate aspects of the structure-activity relationship of this hemoprotein, furnishing information with respect to the denaturation mechanism induced by drastic changes in pH. These data are very useful since HbGp has been proposed as prototype of substitute of blood, thus requiring wide knowledge about its structural and chemical properties.     

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.     

Novel method for early signs of clinical shock detection by monitoring blood capillary/vessel spatial pattern

The ability to monitor capillary/vessel spatial patterns and local blood volume fractions is critical in clinical shock detection and its prevention in Intensive Care Units (ICU). Although the causes of shock might be different, the basic abnormalities in pathophysiological changes are the same. To detect these changes, we have developed a novel method based on both spectrally and spatially resolved diffuse reflectance spectra. The preliminary study has shown that this method can monitor the spatial distribution of capillary/vessel spatial patterns through local blood volume fractions of reduced hemoglobin and oxyhemoglobin. This method can be used as a real‐time and non‐invasive tool for the monitoring of shock development and feedback on the therapeutic intervention. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Protective effect of extractive and biotechnological chondroitin in insulin amyloid and advanced glycation end product-induced toxicity

Glycosaminoglycans are extracellular matrix components related to several biological functions and diseases. Chondroitin sulfate is a sulphated glycosaminoglycan synthesized as part of proteoglycan molecules. They are frequently associated with amyloid deposits and possess an active role in amyloid fibril formation. Recently, a neuroprotective effect of extracellular matrix components against amyloid toxicity and oxidative stress has been reported. Advanced glycation end products (AGEs), the end products of the glycation reaction, have been linked to amyloid-based neurodegenerative disease as associated with oxidative stress and inflammation. In this study we have analyzed the effect of chondroitin sulfate isolated from different species, in comparison with a new biotechnological unsulfated chondroitin, in the amyloid aggregation process of insulin, as well as the ability to prevent the formation of AGEs and related toxicity. The results have showed a determining role of chondroitin sulfate groups in modulating insulin amyloid aggregation. In addition, both sulfated and unsulfated chondroitins have shown protective properties against amyloid and AGEs-induced toxicity. These data are very relevant as a protective effect of these glycosaminoglycans in the AGE-induced toxicity was never observed before. Moreover, considering the issues related to the purity and safety of chondroitin from natural sources, this study suggests a new potential application for the biotechnological chondroitin.     

Using polymeric materials to control stem cell behavior for tissue regeneration

Patients with organ failure often suffer from increased morbidity and decreased quality of life. Current strategies of treating organ failure have limitations, including shortage of donor organs, low efficiency of grafts, and immunological problems. Tissue engineering emerged about two decades ago as a strategy to restore organ function with a living, functional engineered substitute. However, the ability to engineer a functional organ is limited by a limited understanding of the interactions between materials and cells that are required to yield functional tissue equivalents. Polymeric materials are one of the most promising classes of materials for use in tissue engineering, due to their biodegradability, flexibility in processing and property design, and the potential to use polymer properties to control cell function. Stem cells offer potential in tissue engineering because of their unique capacity to self‐renew and differentiate into neurogenic, osteogenic, chondrogenic, and myogenic lineages under appropriate stimuli from extracellular components. This review examines recent advances in stem cell–polymer interactions for tissue regeneration, specifically highlighting control of polymer properties to direct adhesion, proliferation, and differentiation of stem cells, and how biomaterials can be designed to provide some of the stimuli to cells that the natural extracellular matrix does. (Part C) 96:63–81, 2012. © 2012 Wiley Periodicals, Inc.     

Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) was developed in the 1980s as an oxygen carrier and is now under development for treatment of nitric oxide-dependent, volume refractory shock. PHP is made by derivatizing human stroma-free hemoglobin with pyridoxal-5-phosphate and polyoxyethylene (POE). A unique aspect of using POE for modification is that unlike its mono-methoxy polyethylene glycol (PEG) relatives, POE is bifunctional. The result of derivatization of stroma-free hemoglobin is a complex mixture of modified hemoglobin and other red cell proteins. The molecular weight profile, based on size exclusion chromatography, is bimodal and has a number average molecular weight of approximately 105? omitted?000 and a weight average molecular weight of approximately 187? omitted?000. The mixture of hemoglobin molecules has on average 3.3 pyridoxal and 5.0 polyoxyethylene units per tetramer. A portion of the tetramers are linked by POE crosslinks. The hemoglobin tetramers retain their ability to dissociate into dimer pairs and only a small percentage of the dimer pairs are not modified with POE. The SDS-PAGE profile exhibits the ladder-like appearance commonly associated with polyethylene glycol-modified proteins. The isoelectric focusing profile is broad, demonstrating a pI range of 5.0-6.5. The hydrodynamic size of PHP was determined to be approximately 7.2 nm by dynamic light scattering. Soluble red blood cell proteins, such as catalase, superoxide dismutase, and carbonic anhydrase, are present in PHP and are also modified by POE.     

Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus

A novel truncated hemoglobin has been identified in the thermophilic bacterium Geobacillus stearothermophilus (Gs-trHb). The protein has been expressed in Escherichia coli, the 3D crystal structure (at 1.5 Angstroms resolution) and the ligand binding properties have been determined. The distal heme pocket displays an array of hydrogen bonding donors to the iron-bound ligands, including Tyr-B10 on one side of the heme pocket and Trp-G8 indole nitrogen on the opposite side. At variance with the highly similar Bacillus subtilis hemoglobin, Gs-trHb is dimeric both in the crystal and in solution and displays several unique structural properties. In the crystal cell, the iron-bound ligand is not homogeneously distributed within each distal site such that oxygen and an acetate anion can be resolved with relative occupancies of 50% each. Accordingly, equilibrium titrations of the oxygenated derivative in solution with acetate anion yield a partially saturated ferric acetate adduct. Moreover, the asymmetric unit contains two subunits and sedimentation velocity ultracentrifugation data confirm that the protein is dimeric.     

Evaluation of target-specific natural compounds for drug discovery against Leishmaniasis

Leishmaniasis is a parasitic disease with no effective vaccine still now. Globally, it has affected millions of people, precisely in the undeveloped and developing countries. The control strategy for leishmaniasis depends only on chemotherapeutic methods that are associated with several side effects. Therefore, to overcome these negative impacts natural products are the best alternative for developing effective herbal-based drugs, which can act as one of the safest and effective alternative options to treat this particular disease. Leishmania, the causative agent of this disease possesses unique enzymes and metabolic pathways that are different from its mammalian host. Moreover, these unique enzymes, along with the signaling molecules and metabolic pathways that are crucial for its survival, serve as a suitable drug target for the evaluation of specific natural inhibitors to overcome leishmaniasis. Hence, in this review, we have discussed various specific targets of Leishmania, along with their natural inhibitors which can play a significant role in anti-leishmanial drug discovery.     

Correlations of blood selenium with hematological parameters in West German Adults

The serum selenium and the whole blood selenium of 72 healthy persons (47 women, 25 men) was determined. There exist sex specific differences of the whole blood selenium between men (98±19 μg Se/L) and women (89±17 μg Se/L). The serum selenium did not show sex specific differences, but sex specific differences are found if the total amount of extracellular selenium is calculated by correction of the serum selenium with the hematocrit. Women have more extracellular selenium/L whole blood (40±8 μg Se) than men (36±7 μg Se). Men have more intraerythrocyte selenium (cellular selenium=67±14 μg Se) in one L whole blood than women (52±17 μg Se). There exist also sex specific differences if the cellular selenium is calculated/g hemoglobin (men .44 μg Se/g Hb, women .37 μg Se/Hb) or per erythrocyte (men 136.1×10^?19 g Se/Ery, women 113.9×10^?19 g Se/Ery). In the cellular compartment of one L whole blood on the average 1.56 times more selenium is present than in the extracellular compartment. Most of the intraerythrocyte selenium is hemoglobin bound (84%) and utmost 16% glutathione peroxidase associated. An erythrocyte contains about 3500 mol glutathione peroxidase, or, for every 80000 mol hemoglobin one mol glutathione peroxidase. A standard man needs about 2.5 μg selenium/d for the synthesis of the hemoglobin and the erythrocyte. The hematological parameters hemoglobin and the erythrocyte number are correlated with the cellular selenium and the ratio cellular selenium/extracellular selenium. Positive significant correlations are found that are best if a parabolic model is used to interpret the shape of the curves. From the shape of the best correlation lines it can be concluded that selenium may be beneficial for hemoglobin synthesis and erythropoesis. The extracellular selenium may have influence on the volume of the erythrocyte by protecting the outer erythrocyte membrane from lipid peroxidation. A method is reported based on the carbon furnace atomic absorption spectroscopy, which is able to determine without wet digestion selenium in whole blood.     

Direct Measurement of Extracellular Lactate in the Human Hippocampus During Spontaneous Seizures

Abstract: The effect of clinical, spontaneous-onset seizures on extracellular fluid lactate was investigated by the method of lactography, the in vivo on-line measurement of lactate levels using microdialysis. Studies of experimental animals have suggested that generation of extracellular lactate as measured by microdialysis is an index of local glucose utilization and is dependent on the activity of neurons under physiological conditions. Patients with medically refractory complex partial epilepsy underwent stereo-tactic implantation of combination depth electrode/micro-dialysis probes into both hippocampi for 7–16 days. During spontaneous complex partial seizures with secondary generalization, extracellular lactate levels rose by 91 β 32%. Moreover, this increase persisted for 60–90 min. During a unilateral hippocampal seizure that did not propagate to the contralateral hippocampus, the increase in lactate content was restricted to the side of seizure activity. Between seizures, extracellular lactate levels correlated with the frequency of interictal spikes. In summary, these data suggest that brief clinical seizures increase nonoxidative glucose metabolism significantly as measured by the generation of extracellular lactate. Furthermore, the increase in extracellular lactate level is limited to the site of seizure activity. Lactate is transported extracellularly via a lactate/proton cotransporter; therefore, the rise in extracellular lactate level may mediate the drop in pH_o associated with seizure activity. As acidification of the extracellular compartment has an inhibitory effect on neuronal excitability, the rise in extracellular lactate content may be a mechanism of seizure arrest and postictal refractoriness. Moreover, extracellular lactate may also mediate the decreased seizure susceptibility associated with frequent interictal spikes.     

In vitro inhibition of human erythrocyte hexokinase by various hyperglycemic drugs

Hemolysis is the red blood cell abnormality most often associated with adverse effect of drug therapy. Drug‐induced or drug‐associated hyperglycemia could decrease the activity of hexokinase. The aim of this study was to investigate the inhibitory effects of some commonly used drugs that have hyperglycemic side effect on the human erythrocyte hexokinase enzyme in vitro. Hexokinase was purified from human erythrocytes using sequential chromatography, with a specific activity of 0.96 ± 0.18 U/g hemoglobin, and assayed in the presence of selected drugs that have hyperglycemic side effect. The IC₅₀ were determined from the regression analysis graph. Correlation analysis showed that there was positive correlation between the hyperglycemic side effect of some of the tested drugs and decrease of hexokinase activity. This suggests that, at least in part, these drugs exert their hyperglycemic effect by inhibiting glucose phosphorylation by the hexokinase, which consequently causes the glucose accumulation.