Tetrakisphosphates and bispyrophosphates of myo-inositol derivatives as allosteric effectors of human hemoglobin: Synthesis, molecular recognition, and oxygen release

Various 2,5- and 1,4-substituted and unsubstituted myo-inositol tetrakisphosphates and bispyrophosphates were prepared following a general synthetic pathway. All final compounds were tested for their capability to induce oxygen release from human hemoglobin. Most of these proved to be efficient allosteric effectors, with similar affinities for hemoglobin to that of myo-inositol hexakisphosphate, which is one of the best known allosteric effectors of hemoglobin. The efficacy was found to be higher for free phosphates than pyrophosphates. As allosteric Hb effectors, these compounds enable enhanced oxygen release. These effects increase with the strength of Hb binding and correspond primarily to electrostatic interactions. Stereochemical and steric factors also play a significant but secondary role in molecular recognition. In view of the central role played by hypoxia in numerous types of diseases, the exploration of myo-inositol phosphate derivatives represents an important avenue in the search for substances which act on the oxygenation status of tissues and may have significant potential in the discovery and development of novel drug candidates.     

Polyphosphates and pyrophosphates of hexopyranoses as allosteric effectors of human hemoglobin: synthesis, molecular recognition, and effect on oxygen release

Polyphosphorylated and perphosphorylated hexopyranose monosaccharides and disaccharides were synthesized from parent or partially protected carbohydrates as potential allosteric effectors of hemoglobin. A study toward the construction of seven- and eight-membered cyclic pyrophosphates was also performed on the sugars which had the proper orientation, protection, and number of phosphates. All final compounds were tested for their efficiency on oxygen release from human hemoglobin. Several compounds presented higher potency than myo-inositol hexakisphosphate, which is the most efficient of the known allosteric effectors of hemoglobin. Structure-activity relationships were analyzed. The affinity and efficiency depend on the number of phosphates attached to the carbohydrate skeleton and are related primarily to the number of negative charges present. Other effects operate, but play a lesser role.     

myo-Inositol trispyrophosphate: a novel allosteric effector of hemoglobin with high permeation selectivity across the red blood cell plasma membrane

myo‐Inositol trispyrophosphate (ITPP), a novel membrane‐permeant allosteric effector of hemoglobin (Hb), enhances the regulated oxygen release capacity of red blood cells, thus counteracting the effects of hypoxia in diseases such as cancer and cardiovascular ailments. ITPP‐induced shifting of the oxygen–hemoglobin equilibrium curve in red blood cells (RBCs) was inhibited by DIDS and NAP‐taurine, indicating that band 3 protein, an anion transporter mainly localized on the RBC membrane, allows ITPP entry into RBCs. The maximum intracellular concentration of ITPP, determined by ion chromatography, was 5.5×10^?3 M , whereas a drop in concentration to the limit of detection was observed in NAP‐taurine‐treated RBCs. The dissociation constant of ITPP binding to RBC ghosts was found to be 1.72×10^?5 M . All data obtained indicate that ITPP uptake is mediated by band 3 protein and is thus highly tissue‐selective towards RBCs, a feature of major importance for its potential therapeutic use.     

Site-directed mutagenesis in hemoglobin: attempts to control the oxygen affinity with cooperativity preserved

We synthesized three artificial human hemoglobin mutants in which Lys- 66(E10)beta was replaced by Ser, Arg or Thr by site-directed mutagenic protein engineering. These engineered hemoglobins were designated as eHb K66betaS, eHb K66betaR and eHb K66betaT, respectively. By synthesizing these mutants we attempted to control the oxygen affinity of hemoglobin with cooperativity preserved and to clarify the functional role of Lys-66(E10)beta, as well. Such attempts may be useful for creating an oxygen carrier suitable as a blood substitute. The oxygen affinities of eHbs K66betaS, K66betaR and K66betaT were 1.3- , 1.5- and 2.3-fold, respectively, lower than that of reconstituted Hb A. Their allosteric properties such as the Bohr effect and the effect of inositol hexaphosphate were well preserved. Since the oxygen affinity of eHb K66betaT is comparable with that of red cells, it may be a potential candidate for a blood substitute. X-Ray crystallographic data for Hb Chico [Lys-66beta-->Thr], which is identical with eHb K66betaT, together with our computer simulation indicate that an interaction between the introduced Thr and the distal His via a water molecule lowers the oxygen affinity for the T state eHb K66betaT.      

Engineering Allosteric Regulation into Biological Catalysts

Enzymes and ribozymes constitute two classes of biological catalysts. The activity of many natural enzymes is regulated by the binding of ligands that have different structures than their substrates; these ligands are consequently called allosteric effectors. In most allosteric enzymes, the allosteric binding site lies far away from the active site. This implies that communication pathways must exist between these sites. While mechanisms of allosteric regulation were developed more than forty years ago, they continue to be revisited regularly. The improved understanding of these mechanisms has led in the past two decades to projects to transform several unregulated enzymes into allosterically regulated ones either by rational design or directed evolution techniques. More recently, ribozymes have also been the object of similar successful engineering efforts. In this review, after briefly summarising recent progress in the theories of allosteric regulation, several strategies to engineer allosteric regulations in enzymes and ribozymes are described and compared. These redesigned biological catalysts find applications in a variety of areas.     

Allosteric Regulation of Proteases

Allostery is a basic principle of control of enzymatic activities based on the interaction of a protein or small molecule at a site distinct from an enzyme's active center. Allosteric modulators represent an alternative approach to the design and synthesis of small‐molecule activators or inhibitors of proteases and are therefore of wide interest for medicinal chemistry. The structural bases of some proteinaceous and small‐molecule allosteric protease regulators have already been elucidated, indicating a general mechanism that might be exploitable for future rational design of small‐molecule effectors.     

Effect of surface phosphorus functionalities of activated carbons containing oxygen and nitrogen on electrochemical capacitance

Micro/mesoporous activated carbons containing oxygen and phosphorus heteroatoms were modified by incorporation of nitrogen using melamine and urea precursors. The surface chemistry was analyzed by the means of elemental analysis, XPS, and ³¹P MAS NMR. The results indicate that upon the incorporation of nitrogen at high temperatures not only new species involving carbon/nitrogen/oxygen are formed but also the phosphorous environment is significantly altered. Both urea and melamine precursors have similar effects on formation of P-N and P-C bonds. These compounds, although present in small but measurable quantities seem to affect the performance of carbons in electrochemical capacitors. With an increase in the heterogeneity of phosphorus containing species and with a decrease in the content pyrophosphates the capacitance increases and the retention ratio of the capacitor is improved.     

Rates of heat and smoke release of wood in an ohio state university calorimeter

Particleboard, Douglas fir plywood and red oak were tested for heat and smoke release rates under different heat flux levels, piloted and unpiloted conditions, and vertical and horizontal orientations in an Ohio State University calorimeter. The chamber was instrumented to obtain heat release data by both the standard thermal and oxygen consumption methods simultaneously. Heat and smoke release data obtained by both methods are reported. The heat release rates by the thermal method are consistently lower than those by the oxygen consumption method. Variability in results is consistently lower using the oxygen consumption method. The heats of combustion calculated by oxygen consumption are close to those calculated from measurements with the oxygen bomb. The heat release rates measured on particleboard using the standard thermal method are in agreement with measurements on the same material in other Ohio State University calorimeters.     

氮氧自由基定点修饰牛血红蛋白的制备和抗自氧化研究

摘要:以4-（N-马来酰亚胺基）-2,2,6,6-四甲基哌啶-1-氮氧自由基( Mal-Tempol) 与牛血红蛋白（Hb）为原料,制备了氮氧自由基定点修饰在巯基上的血红蛋白( Mal-Tempol-Hb)。利用分光光度法测定了氮氧自由基的修饰度,通过紫外光谱研究了有无叠氮化钠（NaN3）介导下,Hb和Mal-Tempol-Hb中高铁血红蛋白(MetHb)含量随时间变化的过程。结果表明,无介导下,3h时Mal-Tempol-Hb中MetHb的摩尔百分含量由修饰前的44%下降为修饰后的28%;在NaN3介导下,3h时Mal-Tempol-Hb中MetHb的摩尔百分含量从修饰前的50%下降为修饰后的32%。氮氧自由基的修饰有效地降低了血红蛋白的自氧化速率。     

Production and examination of oxygen‐carrier materials based on manganese ores and Ca(OH)2 in chemical looping with oxygen uncoupling

This study concerns production of oxygen‐carrier particles using six different manganese ores. The ores were made to react with Ca(OH)₂ at elevated temperature, forming calcium manganite. The method utilized to manufacture particles was extrusion. Methane and syngas conversion and oxygen release of the samples in inert atmosphere were investigated. The oxygen carrier based on South African (B) manganese ore, showed good methane conversion and was able to transfer oxygen corresponding to 1.5% of its mass during reduction with gaseous fuel. All examined oxygen carriers were capable of converting syngas completely. The ability to release gaseous oxygen was examined by adding wood char in a stream of nitrogen for four selected samples sintered at 1300°C/6 h. These samples released an amount of oxygen corresponding to 0.37–0.68% of their mass. The reactivity of all the ores was improved after the proposed treatments. Reactivity results of the oxygen carrier made from South African (B) ore and Ca(OH)₂, sintered at 1300°C for 6 h were the most promising. Attrition measurements with a jet cup of the oxygen carriers sintered at 1300°C/6 h showed that all the samples made from ores were at least three times more resistant to mechanical attrition compared to particles made from synthetic Mn₂O₃. Producing feasible oxygen carriers directly from ores could potentially cut the cost of chemical looping with oxygen uncoupling and have a significant impact on its competitiveness among other carbon capture technologies. © 2013 American Institute of Chemical Engineers AIChE J 60: 645–656, 2014     

Modulation of Ultrafast Conformational Dynamics in Allosteric Interaction of Gal Repressor Protein with Different Operator DNA Sequences

Although all forms of dynamical behaviour of a protein under allosteric interaction with effectors are predicted, little evidence of ultrafast dynamics in the interaction has been reported. Here, we demonstrate the efficacy of a combined approach involving picosecond‐resolved FRET and polarisation‐gated fluorescence for the exploration of ultrafast dynamics in the allosteric interaction of the Gal repressor (GalR) protein dimer with DNA operator sequences O_E and O_I. FRET from the single tryptophan residue to a covalently attached probe IAEDANS at a cysteine residue in the C‐terminal domain of GalR shows structural perturbation and conformational dynamics during allosteric interaction. Polarisation‐gated fluorescence spectroscopy of IAEDANS and another probe (FITC) covalently attached to the operator directly revealed the essential dynamics for cooperativity in the protein–protein interaction. The ultrafast resonance energy transfer from IAEDANS in the protein to FITC also revealed different dynamic flexibility in the allosteric interaction. An attempt was made to correlate the dynamic changes in the protein dimers with O_E and O_I with the consequent protein–protein interaction (tetramerisation) to form a DNA loop encompassing the promoter segment.     

Nitrogen release during thermochemical conversion of single coal pellets in highly preheated mixtures of oxygen and nitrogen

In this investigation, single coal particles (pellets) were combusted in highly preheated oxidants (873-1273 K) with oxygen concentrations ranging from 0% to 100%, using a small scale‘ batch reactor. In base of the experimental results, the influence of oxygen concentration and oxidizer temperature on total mass conversion, the release of fuel nitrogen and the fraction of fuel nitrogen that is oxidized to NOx, is discussed. For oxygen concentration 5-21%, the rate of the thermochemical conversion was shown to be almost independent oxygen concentration when oxidant temperatures of 1073-1273 K were used. The opposite was true for an oxidant temperature of 873 K. Thus there appears to be an oxidant temperature above which devolatilisation is controlled by convective heat transfer rather than reaction. Further it was shown that the release of fuel nitrogen was promoted by an increased oxygen concentration (from 5% to 21% at 1273 K) and an increase of oxidant temperature (from 1073 K to 1273 K at 21% oxygen). An estimate of the devolatilisation of nitrogen from the measured pellet temperature indicated that the devolatilisation of nitrogen is significantly delayed with respect to other components. In fact, during the very initial part of the thermochemical conversion, most released nitrogen appeared to follow the route via char rather than via devolatilisation. Favorable conditions for NO reduction thanks to a prompt devolatilisation contemporarily to a release of fuel nitrogen via the char route was believed to be one of the explanation for the evidenced low ratios between NOx emissions and fuel nitrogen released, particularly in the beginning of the experiment. The fact that the amount of released fuel nitrogen that is oxidized to NOx was shown to decrease with increasing oxidant temperatures from 1073 K to 1273 K supports this interpretation, though a higher temperature of the oxidant creates higher devolatilisation rates.     

阿魏酸修饰的血红蛋白自氧化的研究(加急)

为研究FA-Hb的自氧化,以阿魏酸酯（FA-NBA）与血红蛋白（Hb）为原料,制备了阿魏酸血红蛋白（FA-Hb）。通过紫外光谱扫描、血氧分析仪等检测手段,分析了修饰前后血红蛋白的特征吸收峰的变化情况和氧饱和曲线的差别;研究了有无叠氮钠、黄嘌呤-黄嘌呤氧化酶介导下,血红蛋白和阿魏酸修饰的血红蛋白的自氧化速率的改变。实验结果表明,阿魏酸修饰血红蛋白,保持了血红蛋白的功能结构,仍具备血红蛋白的载氧活性,有效降低了自氧化速率。     

Development of a biosensor for glycated hemoglobin

The development of an electrochemical piezoelectric sensor for the detection of glycated hemoglobin is presented. The total hemoglobin (Hb) content is monitored with a mass-sensitive quartz crystal modified with surfactants, and the glycated fraction of the immobilized Hb is determined by subsequent voltammetric measurement of the coupled ferroceneboronic acid.Different modifications of the sensor were tested for their hemoglobin binding ability. Deoxycholate (DOCA) was found to be the most suitable among the examined modifiers. Piezoelectric quartz crystals with gold electrodes were modified with DOCA by covalent binding to a pre-formatted 4-aminothiophenol monolayer. The properties of the Hb binding to DOCA and the pH effect on this interaction were studied.In the proposed assay for glycated hemoglobin at first an Hb sample is incubated with ferroceneboronic acid (FcBA), which binds to the fructosyl residue of the glycated Hb. Then this preincubated Hb sample is allowed to interact with the DOCA-modified piezoelectric quartz crystal. The binding is monitored by quartz crystal nanobalance (QCN). The amount of FcBA present on the sensor surface is determined by square wave voltammetry. The binding of FcBA results in well-defined peaks with an E⁰′ of +200 mV versus Ag/AgCl (1 M KCl). The peak height depends on the degree of glycated Hb in the sample ranging from 0% to 20% of total Hb. The regeneration of the sensing surface is achieved by pepsin digestion of the deposited Hb. Thus the sensor can be re-used more than 30 times.     

Direct electrochemistry of hemoglobin in biomembrane-like DHP-PDDA polyion-surfactant composite films

Hb-DHP-PDDA films were assembled by incorporating hemoglobin (Hb) into polyion-surfactant DHP-PDDA composite films on pyrolytic graphite (PG) electrodes, where DHP is an anionic surfactant dihexa decylphosphate, and PDDA is a polycation poly(diallyldimethylammonium). Cyclic voltammetry of Hb-DHP-PDDA films showed a pair of stable and reversible peaks for Hb Fe(III)/Fe(II) redox couple at about −0.34 V versus saturated calomel electrode (SCE) in pH 7.0 buffers. The electron transfer rate between Hb and PG electrodes was greatly facilitated in microenvironment of the films. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) suggest that in both DHP-PDDA and Hb-DHP-PDDA films, DHP is self-assembled into an ordered bilayer structure which is sandwiched by PDDA backbone layers. Positions of Soret absorption band indicate that Hb keeps its secondary structure similar to its native state in the films in the medium pH range. Hb could act as an enzymatic catalyst in DHP-PDDA films to catalyze reduction of oxygen, trichloroacetic acid, and nitrite with significant decrease of overpotential.     

Immobilization, direct electrochemistry and electrocatalysis of hemoglobin on colloidal silver nanoparticles-chitosan film

This paper reports on the fabrication and characterization of hemoglobin (Hb)-colloidal silver nanoparticles (CSNs)-chitosan film on the glassy carbon electrode and its application on electrochemical biosensing. CSNs could greatly enhance the electron transfer reactivity of Hb as a bridge. In the phosphate buffer solution with pH value of 7.0, Hb showed a pair of well-defined redox peaks with the formal potential (E^0′) of −0.325 V (vs. SCE). The immobilized Hb in the film maintained its biological activity, showing a surface-controlled process with the heterogeneous electron transfer rate constant (k_s) of 1.83 s⁻¹ and displayed the same features of a peroxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide (H₂O₂). The linear range for the determination of H₂O₂ was from 0.75 μM to 0.216 mM with a detection limit of 0.5 μM (S/N = 3). Such a simple assemble method could offer a promising platform for further study on the direct electrochemistry of other redox proteins and the development of the third-generation electrochemical biosensors.     

Poly(L‐histidine)‐chitosan/alginate complex microcapsule as a novel drug delivery agent

Novel poly(L ‐histidine)‐chitosan/alginate complex microcapsules were prepared from biodegradable polymers poly(L ‐histidine) (PLHis) in the presence of chitosan at acetate buffer solution pH 4.6. Microcapsules obtained are spherical and well‐dispersed with a smooth surface and a narrow size distribution. The microcapsules can encapsulate the protein model drug hemoglobin (Hb) efficiently. The results show that the complex microcapsules with low, medium, or high molecular weight of chitosan (0.05%, w/v), the highest encapsulation efficiencies obtained are 91.3%, 85.9%, and 94.2% with loading efficiencies of 47.8%, 44.3%, and 39.7%, respectively. The release profiles indicate that Hb‐loaded microcapsules conform to first‐order release kinetic in whole procedure, and 84.8%, 71.4%, and 87.3% of Hb were released during 72‐h incubation in PBS pH6.8 for microcapsules with low, medium, and high molecular weight chitosan (0.05%, w/v), respectively. The results also indicate that particle size and drug loading efficiency have a significant influence on the release profile and encapsulation efficiency. Our results reveal that the PLHis‐chitosan/alginate complex microcapsules are able to encapsulate and release Hb and are potential carriers for protein drugs. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Oxygen binding and other physical properties of human hemoglobin made in yeast

Wagenbach et al. (1991, BioTechnology, 9, 57–61) haverecently developed a system for producing soluble recombinanttetrameric hemoglobin in yeast: hemoglobin begins to appear4– 5 h after induction with galactose,- and ß-globinchains fold in vivo and endogeneously produced heme is incorporatedinto hemoglobin tetramers. We have further characterized theoxygen-binding properties, as well as the tetramer stability,of recombinant human Hb A made in yeast. After purificationby ion-exchange chromatography, a single band at the same positionas normal human Hb A was obtained using cellulose acetate electrophoresis.Although the oxy and deoxy forms of purified recombinant HbA made in yeast were spectrophotometrically identical to nativehuman Hb A, the oxygen-binding curve was shifted slightly leftof that for native human Hb A. Further purification of recombinanthemoglobin by FPLC revealed two fractions: one (fraction B)with low cooperativity and high oxygen affinity, and the other(fraction A) with almost identical cooperativity and oxygenaffinity compared with native human Hb A. The Bohr effect offraction A was also identical to native human Hb A. Hemoglobinin fraction B with lowered cooperativity precipitated -1.5 timesfaster than normal human Hb A during mechanical agitation, whilehemoglobin in fraction A with normal cooperativity precipitatedwith kinetics identical to native human Hb A. These resultssuggest that some of the recombinant molecules made in yeastfold improperly, and that these molecules may exhibit decreasedcooperativity for oxygen binding and decreased stability. Thissystem should now allow the evaluation of the folding differenceswhich promote hemoglobin function, and should also provide anefficient system which will facilitate the production of varioushemoglobin mutants for studying biochemical and biophysicalproperties of hemoglobin.     

Gel-type shell contributing to the high proton conductivity of pyrophosphates

Metal pyrophosphates have attracted considerable interests due to their high proton conductivity and potentially wide applications in the temperature range of 100–400 °C. However, great difference in conductivity was reported by different groups on the same pyrophosphates. The reason for the huge difference is still in debate up to now, and there is no coherent standpoint in literatures on the proton conduction mechanism. In this study, we chose Fe_0.4Nb_0.5P₂O₇, which was reported showing high proton conductivity recently, as an example to disclose the reason inducing the divergence in proton conductivity and conduction mechanism. We found that the as-prepared pyrophosphate grains have three layers, i.e. crystalline pyrophosphate core, amorphous phosphate shell in the middle and gel-type shell composed of amorphous phosphorus species as the outermost layer. The content of amorphous phosphorus species decreases with the increase of the calcination temperature of pyrophosphates, and the calcination temperature-dependent residual soluble phosphorus curve extremely coincides with the conductivity curve. Thus, the proton conduction of pyrophosphates is realized via a gel-type shell formed by residual amorphous phosphorus species on surfaces of pyrophosphate grains. We suggested that the phosphorus content is the key factor to explain the great difference in conductivity of pyrophosphates prepared by different groups.     

A novel mechanism of allosteric regulation of archaeal phosphoenolpyruvate carboxylase: a combined approach to structure-based alignment and model assessment

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) and plays a crucial role in fixing atmospheric CO(2) in C(4) and CAM plants. The enzyme is widespread in plants and bacteria and mostly regulated allosterically by both positive and negative effectors. Archaeal PEPCs (A-PEPCs) have unique characteristics in allosteric regulation and molecular mass, distinct from their bacterial and eukaryote homologues, and their amino acid sequences have become available only recently. In this paper, we generated a structure-based alignment of archaeal, bacterial and eukaryote PEPCs and built comparative models using a combination of fold recognition, sequence and structural analysis tools. Our comparative modeling analysis identified A-PEPC-specific strong interactions between the two loops involved in both allostery and catalysis, which explained why A-PEPC is not influenced by any allosteric activators. We also found that the side-chain located three residues before the C-terminus appears to play a key role in determining the sensitivity to allosteric inhibitors. In addition to these unique features, we revealed how archaeal, bacterial and eukaryote PEPCs would share a common catalytic mechanism and adopt a similar mode of tetramer formation, despite their divergent sequences. Our novel observations will help design more efficient molecules for ecological and industrial use.