Structure Of Hb Research Articles

The Overriding Roles of Concentration and Hydrophobic Effect on Structure and Stability of Heme Protein Induced by Imidazolium-Based Ionic Liquids.

Spectroscopic and molecular docking investigations were carried out to characterize the effect of imidazolium-based ionic liquids (ILs) with varying chain length of the cation on the thermal stability as well as spectroscopic behavior of heme protein hemoglobin (Hb). The goal of this work is to investigate the role of concentration of ILs, the effect of alkyl chain length of the cation, and the related Hofmeister series on the structure of Hb. To achieve this goal, a series of ILs possessing same Cl(-) anion and a set of cation [Cnmim](+) with increasing chain length such as 1-ethyl-3-methylimidazolium chloride ([Emim][Cl]), 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-hexyl-3-methylimidazolium chloride ([Hmim][Cl]), and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]) were used in this study. It was observed that the stability of the protein was concentration dependent as well as the hydrophobic interactions between [Cnmim](+) of ILs, and the amino acid residues in the protein played a major role in protein unfolding. As a consequence, the destabilization tendency of the ILs toward the Hb increases with increasing chain length of the cation of ILs. Additionally, the cations of the ILs obeyed the Hofmeister series when arranged in the order of providing stability to Hb structure.

Cigarette smokers develop structurally modified hemoglobin: a possible way of increasing oxidative stress

Context: Increased levels of free radicals and various reactive species along with reduced antioxidant defence system are the major threat to erythrocyte in tobacco smokers. Thus, the hemoglobin (Hb) within the erythrocyte is very prone to oxidative damage. Earlier reports suggest that cigarette smoking is related with the glutathionylation and formation of adducts of Hb.Objective: We have highlighted the possible changes in secondary and tertiary structures of Hb in cigarette smokers and its physiological consequences.Material and method: Twenty smokers and 18 non-smokers, aged 25–35 years were volunteered in this study. We used flow cytometry for measuring intracellular reactive oxygen species (IcROS). The purified Hb was subjected to different spectrophotometric, fluoremetric and circular dichroic (CD) analysis. The hydrophobicity, thermal stability, heme release and oxidation of purified Hb were also studied.Result: We observed that the IcROS was also higher in cigarette smokers than non-smokers. The data of intrinsic fluorescence, synchronous fluorescence and tryptophan quenching studies showed that the microenvironments of β37 tryptophan and tyrosine residues of Hb were moved toward more hydrophobic region in cigarette smokers. Increased hydrophobicity and thermal stability furthermore indicated more compactness of smokers' hemoglobin. From CD spectra, we confirmed an overall modification of the secondary and tertiary structures of hemoglobin in smokers. Both auto- and co-oxidation rates of purified Hb were found to be higher in cigarette smokers.Discussion and conclusion: We conclude that the modified Hb in cigarette smokers may further enhance the oxidative insult within the cell.

New look at hemoglobin allostery.

New look at hemoglobin allostery.

Hb Feilding [β12(A9)Thr → Pro; HBB: c.37A>C]: A Novel Unstable β-Globin Chain Variant

We report here a patient heterozygous for a previously unreported β chain variant. A 72-year-old Caucasian female was found to have an abnormal hemoglobin (Hb) as an incidental finding following Hb A1C analysis. There was no family history of anemia or hemoglobinopathy. Her full blood count revealed a mild normochromic anemia with Hb 11.1 g/dL (range 11.5–15.0), mean corpuscular volume (MCV) 93.0 fL (range 80.0–100.0) and mean corpuscular Hb (MCH) 30.0 pg (range 27.0–32.0). Isopropanol stability tests and a variant Hb on high performance liquid chromatography (HPLC) comprizing 37.0% of the total Hb suggested an unstable Hb variant. Sanger sequencing of the β-globin gene revealed a single base substitution, HBB: c.37A>C, causing the missense mutation β12(A9)Thr → Pro in exon 1 of the HBB gene. This mutation changes the threonine residue at position 12(A9) to a proline in the β-globin chain. We propose that this variant be called Hb Feilding after the town where the proband lived. Three dimensional modeling suggested that the disruption of the Hb structure was due to the introduction of a proline at helix A9 which caused distortion of the helical structure and resulted in reduced solubility.

Changes in the Structural Ordering of Hemoglobin under Extreme Conditions of the Arctic Region

The structure of hemoglobin (Hb) was examined by UV and IR Fourier spectroscopy. The electronic characteristics of the -conjugated (aromatic) prosthetic group of heme and the content of the secondary structure elements of globin in Hb were studied. Heme and globin are connected to each other by Van der Waals forces. A 8-10 nm short-wave shift of the heme absorption band at 417±0.5 nm was revealed for the residents of the Arctic Region in different latitudes (the Yamal Peninsula, 64 and 70° North) as compared to the residents of middle latitudes (Novosibirsk, 55° North). This indicates that the ordering of heme structure increases with the latitude of human residence. The IR Fourier spectroscopy study of the content of the secondary structure elements showed that the ordering of hemoglobin in the Arctic residents increases with latitude. The study has demonstrated that affinity of Hb for oxygen in the residents of the Arctic Region is higher than in the residents of middle latitudes. Changes in the structure of Hb and its affinity for oxygen in the Arctic residents can be attributed to heliophysical factors.

Effect of kiwi fruit powder in tenderizing M. Triceps brachii from mature cows

Due to increasing use of lanthanides/actinides in nuclear and civil applications, understanding the impact of these metal ions on human health and environment is a growing concern. Hemoglobin (Hb), which occurs in all the kingdom of living organism, is the most abundant protein in human blood. In present study, effect of lanthanides and actinides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] on the structure and function of Hb has been investigated. Results showed that these metal ions, except Ce(IV) interacted with carbonyl and amide groups of Hb, which resulted in the loss of its alpha-helix conformation. However, beyond 75 μM, these ions affected heme moiety. Metal–heme interaction was found to affect oxygen-binding of Hb, which seems to be governed by their closeness with the charge-to-ionic-radius ratio of iron(III). Consistently, Ce(IV) being closest to iron(III), exhibited a greater effect on heme. Binding constant and binding stoichiometry of Th(IV) were higher than that of U(VI). Experiments using aquatic midge Chironomus (possessing human homologous Hb) and human blood, further validated metal–Hb interaction and associated toxicity. Thus, present study provides a biochemical basis to understand the actinide/lanthanide-induced interference in heme, which may have significant implications for the medical and environmental management of lanthanides/actinides toxicity.

Combined effect of arsenic trioxide and radiation on physical properties of hemoglobin biopolymer

Arsenic trioxide (As2O3) has been recently established as one of the most effective drugs for the treatment of patients with acute promyelocytic leukemia. However, it was widely used in therapeutic of many kinds of cancer by combining it with ionizing radiation. Thus, the purpose of the present study was to explain the combined effect of As2O3 and gamma irradiation on hemoglobin (Hb) structure. Measurements using fourier transform infrared (FTIR) and UV-visible spectra were done. This study included five groups: control, irradiation with single dose of gamma irradiation of 5 Gy, intraperitonial injection with single dose of 10 mg/kg body weight of As2O3, As2O3+5 Gy and 5 Gy+As2O3. The results reported that the absorbance of secondary amide, amide I and amide II of all groups were lowerd than control, whereas the absorbance of amide III and amide IV for As2O3 and 5 Gy followed by As2O3 injection has been increased. For UV-visible spectra, As2O3 injection decreased the absorbance of globin-heme and soret bands and increased β, α and 630 bands compared with control. On injection with As2O3 followed by 5 Gy showed a decrease in globin-heme, soret, β and α bands and increase in 630 band. Moreover, 5 Gy followed by As2O3 demonestrated a decrease in globin-heme, β, α and 630 bands and an increase in soret band, also the ratio of α/β showed an increase in absorbance compared with control. The results concluded that 5 Gy followed by As2O3 showed some sort of repair in the structure of rats hemoglobin rather than injection with As2O3 and 5 Gy both individually.

Structural characterization and electrocatalytic application of hemoglobin immobilized in layered double hydroxides modified with hydroxyl functionalized ionic liquid

Hemoglobin (Hb) was immobilized in Zn2Al-Layered Double Hydroxides (LDH) modified with Hydroxyl Functionalized Ionic Liquid (HFIL) through adsorption and coprecipitation method, respectively. Adsorption experiments showed that the presence of HFIL could enhance the maximum protein loading. However, the Hb loading through coprecipitation technique was far higher than that for adsorption. The role of HFIL on the interaction between Hb and LDH was investigated by XRD, FTIR, UV–vis and fluorescence spectroscopies. Although the quaternary structure of Hb entrapped in HFIL–LDH through coprecipitation technique (denoted as HFIL–LDH–Hbcop) might be altered slightly more than that in LDH (LDH–Hbcop), its secondary structure and redox-active heme groups kept intact. Morphologies of LDH–Hbcop and HFIL–LDH–Hbcop biohybrids were analyzed through SEM and TEM images. The direct electrochemistry of the immobilized Hb indicated that the coprecipitation bioelectrode performed better than that of the corresponding adsorption one. Regardless of adsorption and coprecipitation, the introduction of HFIL could distinctly promote the electron transport. Among all bioelectrodes, HFIL–LDH–Hbcop/GCE displayed the best electrocatalytic activity for H2O2 determination with a larger electroactive Hb percentage (6.76%), higher sensitivity (40.63A/Mcm2) and lower detection limit (0.0054μmol/L). So HFIL–LDH could effectively immobilize enzymes via coprecipitation technique, which had potential applications in the fabrication of electrochemical biosensors.

Calculating The Acidity of Silica Supported Alkyl Sulfonic Acids Considering the Matrix Effect: A Dft Study

Density functional theory (DFT) was used to investigate the acidity of the various silica alkyl sulfonic acids. In this regard, cluster models with various alkyl spacer lengths were selected to mimic the surface of silica gel. The effects of distance from the surface and the role of hydrogen bond (H–bond) on the ΔHacidity values of these catalysts were investigated. DFT calculations revealed that a notable gap of ΔHacidity values exists between the structures considering lateral hydrogen bonding with the surface of the silica HB structure and the structures with omitted surface interactions (non-HB structures). Natural bonding orbital (NBO) and quantum theory of atoms in molecules (QTAIM) analyses were carried out to obtain detailed information about the nature of the H–bonds.

Multiple Gaussian network modes alignment reveals dynamically variable regions: The hemoglobin case

Gaussian network model (GNM) modes of motion are calculated to a dataset of hemoglobin (Hb) structures and modes with dynamics similarity to the T state are multiply aligned. The sole criterion for the alignment is the mode shape itself and not sequence or structural similarity. Standard deviation (SD) of the GNM value score along the alignment is calculated, regions with high SD are defined as dynamically variable. The analysis shows that the α1β1/α2β2 interface is a dynamically variable region but not the α1β2/α2β1 and the α1α2/β1β2 interfaces. The results are in accordance with the T→R2 transition of Hb. We suggest that dynamically variable regions are regions that are likely to undergo structural change in the protein upon binding, conformational transition, or any other relevant chemical event. The represented technique of multiple dynamics-based alignment of modes is novel and may offer a new insight in proteins' dynamics to function relation.

Two New Hemoglobin Variants: Hb Tallahassee [α3(A1)Ser→Tyr; HBA2: c.11C>A] and Hb Madison-NC [β119(GH2)Gly→Ser; HBB: c.358G>A

Of the 1570 reported hemoglobin (Hb) variants detected to date, 390 are α2-globin chain (some variants have yet to be identified by DNA analyses and are therefore presumed) and 827 are the result of mutations of the β-globin chain. Due to their location on the Hb structure, only a minority of these variants result in a clinical phenotype; most are silent and are detected during routine surveillance, are found incidentally during other disease-related investigations or following newborn screening programs. In this report we discuss phenotype/genotype and molecular characteristics of two new Hb variants, both of which were clinically silent. One is an α2-globin chain variant located at codon 3 [α3(A1)Ser→Tyr; HBA2: c.11C > A] named Hb Tallahassee and the other is a β-globin chain variant located at codon 119 [β119(GH2)Gly→Ser; HBB: c.358G > A] called Hb Madison-NC.

Deferiprone: structural and functional modulating agent of hemoglobin fructation

Diabetic complication arises from the presence of advanced glycation end products in different sites of the body. Great attention should be paid to recognizing anti-glycation compounds. Here, deferiprone as an oral iron chelator drug administrated in treatment of β-thalassemic patients was selected to find its effect on the fructation of hemoglobin (Hb). Our results indicated that deferiprone could prevent the AGE and carbonyl formation via inhibition of structural changes in the structure of Hb during the fructation process. Moreover, deferiprone can preserve peroxidase and esterase activities of fructated Hb similar to native Hb. Therefore, deferiprone can be introduced as an anti-glycation drug to prevent the AGE formation.

Oxygen-Affinity of Hemoglobin is Regulated by Effector-Linked Dynamic Modulations of High-Frequency Thermal Fluctuations

Heterotropic effectors reduce the O2-affinity of hemoglobin (Hb) as much as >10\3-folds: P50 = 0.4 → 200 mmHg, Klow = 0.3 → 0.004 mmHg-1, and Khigh = 10 → 0.004 mmHg-1. However, there are no detectable changes in either static T/R-quaternary and associated tertiary structures or the stereochemical and electronic structures of the heme coordination that are attributable to such changes in the O2-affinity of Hb, upon binding of the heterotropic effectors to either T(deoxy)- or R(oxy)-Hb1,2, indicating that the O2-affinity of Hb is not regulated by static T/R-quaternary and associated tertiary structures of Hb and that the reactivity (or the O2-affinity) of the heme Fe itself is not altered1, in sharp contrast to the mainstream idea of the heme-centric regulation of the O2-affinity in Hb3. Our 6ns-molecular dynamics simulations4,5 indicated that the amplitudes of very high-frequency (>GHz) thermal fluctuations of the globin matrix are entropically enhanced by the dissociation of O2 from oxy-Hb and/or binding of heterotropic effectors to deoxy- and oxy-Hb, in parallel to the reduction of the O2-affinity. The rate of dissociation of O2 from oxy-Hb is enhanced by the effector-linked enhanced thermal fluctuations, which simultaneously reduce the rate of geminate-recombination of O2. This results in the reduction of the apparent O2-affinity of Hb by heterotropic effectors.

The Species and Heme Pocket Properties of Sturgeon Hemoglobins Upon Interaction with N-dodecyl Trimethylammonium Bromide

The variations in fish hemoglobin (Hb) structures play a vital role in their respiratory performance under various environmental conditions and are impacted by their physiological properties. The major hemoglobins from two species of sturgeon were studied upon interaction with n-dodecyl trimethyl ammonium bromide (DTAB) using the UV-vis absorption, circular dichroism (CD), fluorescence spectroscopy, and oxygen affinity measurement methods as well as chemometric analysis. The UV-Vis absorption spectra between 500 and 650 nm was used to identify each species of hemoglobin, and to show that the concentration of oxyHb and metHb decreases, while that of deoxyHb increases upon interaction with DTAB. Both reduced oxyHb and oxidized hemichrome of the two Hbs were studied to obtain information about the DTAB efects on their structural features. The circular dichroism (CD) was utilized to obtain secondary structure and compactness for Hb upon interaction with DTAB. Binding of DTAB molecules induced the unfolding of Hb, and was accompanied with exposure of the heme pocket facilitating its oxidation. The differences between unfolding processes for the two Acipenser species were indicated by fluorescence spectroscopy. The chemometric analysis of Hbs was investigated upon interaction with DTAB under titration, using fluorescence spectra allowing determination of the number of components and mole fractions of the oxidized Hb. Our data showed that Acipenser persicus Hb had a more hyperchromic character, more surface area, more loosely folded structure, and therefore, exposed region of heme group compared with Acipenser stellatus oxyHb. In addition, with increasing DTAB the transition of Acipenser stellatus oxyHb to the state of hemichrome occurred at a slower speed than Acipenser persicus oxyHb, and finally more oxygen affinity and compactness. Our results suggest that these differences aroused from the inherent differences between the heme groups which fulfil a potentially important physiological role in these fish.

Small-Angle Neutron Scattering Studies of Hemoglobin Confined Inside Silica Tubes of Varying Sizes

In addition to the chemical nature of the surface, the dimensions of the confining host exert a significant influence on confined protein structures; this results in immense biological implications, especially those concerning the enzymatic activities of the protein. This study probes the structure of hemoglobin (Hb), a model protein, confined inside silica tubes with pore diameters that vary by one order of magnitude (≈20-200 nm). The effect of confinement on the protein structure is probed by comparison with the structure of the protein in solution. Small-angle neutron scattering (SANS), which provides information on protein tertiary and quaternary structures, is employed to study the influence of the tube pore diameter on the structure and configuration of the confined protein in detail. Confinement significantly influences the structural stability of Hb and the structure depends on the Si-tube pore diameter. The high radius of gyration (Rg) and polydispersity of Hb in the 20 nm diameter Si-tube indicates that Hb undergoes a significant amount of aggregation. However, for Si-tube diameters greater or equal to 100 nm, the Rg of Hb is found to be in very close proximity to that obtained from the protein data bank (PDB) reported structure (Rg of native Hb=23.8 Å). This strongly indicates that the protein has a preference for the more native-like non-aggregated state if confined inside tubes of diameter greater or equal to 100 nm. Further insight into the Hb structure is obtained from the distance distribution function, p(r), and ab initio models calculated from the SANS patterns. These also suggest that the Si-tube size is a key parameter for protein stability and structure.

Heme Releasing from Human Hemoglobin upon Interaction with a New Synthesized Complex of 1,10-Phenanthroline-n-butyl Dithiocarbamato Pd(II) Nitrate

In the present study, we investigated the effect of a new anticancer Pd(II) complex, 1,10-phenanthroline-n-butyl dithiocarbamato Pd(II) nitrate, on the heme releasing from human hemoglobin (Hb) as well as alterations in the structure and function of Hb using different spectroscopic methods of UV-Vis, fluorescence and circular dichroism (CD)at two temperatures of 25 and 37 °C. Fluorescence data revealed that the Pd (II) complex is able to quench the intrinsic fluorescence of Hb. The binding constant, number of binding sites and thermodynamic parameters at two temperatures were calculated. The values of ΔH°, ΔS°, and ΔG° indicated that the van der Waals force or hydrogen bond interactions might play a major role in the interaction of complex with Hb. The far-UV-CD studies displayed that the regular secondary structure of Hb had no significant changes. To evaluate the functional changes of Hb via destruction of the heme structure, fluorescence studies were performed at excitation wavelengths of 321 nm and 460 nm with emission wavelengths of 465 nm and 525 nm, respectively. The results demonstrated that two fluorescent heme degradation products are found during the interaction of Pd(II) complex with Hb. The results of thermal behavior of Hb studied at 415 nm confirmed the heme degradation, which referred to decrease in the hemoglobin stability in the presence of Pd(II) complex. Also, the cytotoxic effects and anti-tumor activity of the complex against human breast cell line, T47D were carried out using MTT assay. The Cc50value obtained after different incubation times of 24 and 48 h. The finding related to structural and functional changes of Hb induced by Pd(II) complex may be important to improve understanding of side effects of new designed metal anti-cancer drugs undergoing.

Pathway and Mechanism of pH Dependent Human Hemoglobin Tetramer-Dimer-Monomer Dissociations

Hemoglobin dissociation is of great interest in protein process and clinical medicine as well as in artificial blood research. However, the pathway and mechanisms of pH-dependent human Hb dissociation are not clear, whether Hb would really dissociate into monomers is still a question. Therefore, we have conducted a multi-technique investigation on the structure and function of human Hb versus pH. Here we demonstrate that tetramer hemoglobin can easily dissociate into dimer in abnormal pH and the tetramer → dimer dissociation is reversible if pH returns to normal physiological value. When the environmental pH becomes more acidic (<6.5) or alkaline (>8.0), Hb can further dissociate from dimer to monomer. The proportion of monomers increases while the fraction of dimers decreases as pH declines from 6.2 to 5.4. The dimer → monomer dissociation is accompanied with series changes of protein structure thus it is an irreversible process. The structural changes in the dissociated Hbs result in some loss of their functions. Both the Hb dimer and monomer cannot adequately carry and release oxygen to the tissues in circulation. These findings provide a comprehensive understanding on the pH-dependent protein transitions of human Hb, give guideline to explain complex protein processes and the means to control protein dissociation or re-association reaction. They are also of practical value in clinical medicine, blood preservation and blood substitute development.

Structural studies of haemoglobin from pisces species shortfin mako shark (Isurus oxyrinchus) at 1.9 Å resolution.

Haemoglobin (Hb) is a tetrameric iron-containing protein that carries oxygen from the lungs to tissues and carbon dioxide from tissues back to the lungs. Pisces are the advanced aquatic vertebrates capable of surviving at wide depth ranges. The shortfin mako shark (SMS) is the pelagic, largest, fastest and most sophisticated species of the shark kingdom with well developed eyes. Mostly the pisces species are cold blooded in nature. Distinctly, the SMSs are warm-blooded animals with an advanced circulatory system. SMSs are capable of maintaining elevated muscle temperatures up to 33 K above the ambient water temperatures at a depth of 150-500 m. SMSs have a diverged air-breathing mechanism compared with other vertebrates. The haemoglobin molecule consists of four polypeptide chains, namely two α chains, each with 140 amino acids and two β chains each having 136 amino acids. The SMS Hb was found to crystallize in monoclinic space group P21 using the hanging-drop vapour-diffusion method at room temperature. The crystal packing parameters for the SMS Hb structure contain one whole biological molecule in the asymmetric unit with a solvent content of 47%. The SMS Hb quaternary structural features interface-interface interactions and heme binding sites are discussed with different state Hbs and the results reveal that SMS Hb adopts an unliganded deoxy T state conformation.

Serum haptoglobin in clinical biochemistry: Change of a paradigm

Serum haptoglobin (Hp) was discovered by Max Fernand Jayle as a young assistant professor in the Department of Biochemistry of the Paris Medical Faculty, headed by Professor Michel Polonovski. Jayle showed that Hp was an acute phase glycoprotein and worked out its routine determination in the blood-serum, based on its complex formation with hemoglobin, using the increased peroxidase activity of the hemoglobin–haptoglobin (Hb–Hp) complex, for routine determination in clinical biochemistry for the characterisation of inflammatory processes, together with other acute phase glycoproteins as orosomucoide. Later Smithies described the genetic control of human Hp-isoforms and quite recently Andersen et al. reported the elucidation of the crystal structure of the porcine Hb–Hp complex. In that article there was no mention of the discovery of Hp, neither of its determination in clinical biochemistry as an inflammatory marker. The only biologically significant role assigned to Hp by Andersen et al. is its (hypothetical) role to prevent or minimize the harmful effects of Hb during intravascular hemolysis, by the generation of reactive oxygen species (ROS) and complexing it. This shift of paradigm, not at all exceptional in medical biochemistry, will be described and discussed with its pitfalls and consequences.

Are the Intramolecular O–H···F and O–H···Cl Hydrogen Bonds Maintained in Solution? A Theoretical Study

The present case study aims at calculating the equilibrium conformer compositions for 2X-ethanol and 2X-phenol (X = F, Cl) in solution, and exploring the effect of the applied theoretical method and basis set on the obtained results, as well as considering the usefulness of the continuum solvent approach in comparison with the explicit solvent Monte Carlo model utilizing the free energy perturbation method. Gas-phase optimizations at the DFT/B97D/aug-cc-pvtz and ab initio MP2/aug-cc-pvtz levels predicted structures in good agreement with the available experimental data for three test molecules. Because in-solution geometries change only slightly according to the IEF-PCM continuum solvent calculations in carbon tetrachloride and water, the two theoretical levels were applied further on, and complete basis set (CBS) relative internal free energies were estimated for the conformers under study. The predicted OCCF gauche/trans ratio for 2F-ethanol was well reproduced in comparison with available experimental compositions. The predominant gauche structure maintains an intramolecular hydrogen bond in carbon tetrachloride (HB structure), whereas HB and NoHB gauche conformers appear in nearly the same fraction in aqueous solution. The internally hydrogen-bonded conformer is predominant also for 2X-phenol species, as calculated on the basis of relative CBS internal free energies and IEF-PCM and FEP/MC solvation free energies. Use of a trihydrate supermolecule model for 2F-ethanol conformers leads to the prediction of the aqueous-solution composition in contrast to the experiment. Solution structure modeling predicts weak hydrogen-bond formation capacity for both the covalently bound F and Cl atoms, even in conformations where they are fully exposed to hydration.