Native Hb Research Articles

Cooperative oxygen binding in beta-semihemoglobins caused by a chemical modification in the alpha1beta1 interface

A beta-semihemoglobin is an alpha-beta dimer of hemoglobin (Hb) in which the beta-subunit carries heme, while the alpha-subunit is heme-less, in apo form. It is characterised by displaying a high affinity for oxygen, and absence of cooperative binding of oxygen. We have modified chemically the residue beta112Cys (G14), located adjacent to the alpha1beta1 interface, and studied the impact of such a modification on the oligomeric state and oxygenation properties of the derivatives. We also studied the impact of modifying beta93Cys (F9) since its modification was unavoidable. For this, we used N-Ethyl maleimide and iodoacetamide. For the alkylation of beta112Cys (G14) in isolated subunits, we used N-Ethyl maleimide, iodoacetamide, or additionally, 4,4’-Dithiopyridine. Seven native and chemically modified beta-subunit derivatives were prepared and analysed. Only those derivatives treated with iodoacetamide showed oxygenation properties that were indistinguishable from those of native beta-subunits. These derivatives were then converted into their respective semihemoglobin forms, and four additional derivatives were prepared and analysed .in terms of ligation-linked oligomeric state, and oxygenation function, and contrasted against native Hb and unmodified beta-subunits. Strikingly, beta-semiHbs with modifications in beta112Cys showed indications of cooperative oxygen binding in various degrees, which suggested the possibility of assembly of two beta-semiHbs. The derivative modified with 4-Thiopyridine in beta112Cys showed a highly cooperative binding of oxygen (nmax = 1.67). A plausible allosteric scheme that could explain allostery in beta-semiHb system is suggested.

Hypoxia-alleviating hemoglobin nanoclusters for sensitizing chemo-photodynamic therapy of cervical cancer

Hypoxia-alleviating hemoglobin (Hb) nanoclusters (NCs) have been developed for sensitizing combined chemo-photodynamic therapy (chemo-PDT) of cervical cancer. Hb was conjugated with chlorin e6 and biotinylated polyethylene glycol and adsorbed with doxorubicin (DOX), resulting in the self-assembly of 220 nm diameter protein NCs. These oxygen-carrying NCs, namely DOX@HPBC, exhibited improved colloidal stability in the serum compared to that of native Hb and pH-responsive drug release favorable for cancer treatment. DOX@HPBC alleviated hypoxia by 64.8% in HeLa cells cultured under hypoxia and normalized the levels of related biomarkers, HIF-1α and MDR1, with enhanced cellular uptake via biotin–receptor interactions. Moreover, antitumor efficacy tests performed in HeLa monolayer and spheroid cultures revealed that DOX@HPBC possessed 3.8-fold improved therapeutic efficacy compared to DOX and chlorin e6 physical mixture, showing synergism between chemotherapy and PDT. DOX@HPBC-mediated chemo-PDT significantly suppressed tumor growth in heterotopic (96.6% reduction in tumor volume compared to no-intervention) and orthotopic (comparable uterus weight to the normal group) HeLa-xenografted mice with no significant toxicity. These results showed the hypoxia amelioration effect, which lowered HIF-1α and MDR1 levels in tumor tissues, and efficient DOX distribution to hypoxic regions of the tumor, suggesting that DOX@HPBC could be a promising chemo-PDT strategy for cervical cancer treatment.

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX-MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.

Discrete cis-acting element regulates developmentally timed gene-lamina relocation and neural progenitor competence invivo.

SUMMARYThe nuclear lamina is typically associated with transcriptional silencing, and peripheral relocation of genes highly correlates with repression. However, the DNA sequences and proteins regulating gene-lamina interactions are largely unknown. Exploiting the developmentally timed hunchback gene movement to the lamina in Drosophila neuroblasts, we identified a 250 bp intronic element (IE) both necessary and sufficient for relocation. The IE can target a reporter transgene to the lamina and silence it. Endogenously, however, hunchback is already repressed prior to relocation. Instead, IE-mediated relocation confers a heritably silenced gene state refractory to activation in descendent neurons, which terminates neuroblast competence to specify early-born identity. Surprisingly, we found that the Polycomb group chromatin factors bind the IE and are required for lamina relocation, revealing a nuclear architectural role distinct from their well-known function in transcriptional repression. Together, our results uncover in vivo mechanisms underlying neuroblast competence and lamina association in heritable gene silencing.

Binding Interaction of Glycated, Fructated and Native Human Hemoglobin with Bulk Propolis

Recently, the great interests in application of natural product have led to focus on the potential impact on bio macromolecules. Propolis is one of the natural products with various biological activities such as anti-diabetic effect. Under hyperglycemia, hemoglobin (Hb) frequently undergoes non-enzymatic glycation which affect its structure and function. In this study, the binding interaction between bulk propolis and three forms of Hb, native Hb, glycated Hb (GHb) and fructated Hb (FHb), in two temperatures of 25 and 37 °C have been examined and compared with our previous study (interaction of propolis nanoparticles with Hb, GHb and FHb). The binding studied with applying fluorescence and far UV circular dichroism spectroscopic techniques. Bulk Propolis possess the ability to quench the intrinsic fluorescence of the different types Hb like propolis nanoparticles. The mechanism of interaction bulk propolis with different types Hb was similar to the propolis nanoparticles, dynamic quenching in native Hb and static quenching in glycated and fructated Hb. According to the modified Stern–Volmer equation, the binding constant and number of binding sites were obtained. The result suggested that propolis nanoparticles have more quenching rate constant (kq) in compared to the bulk propolis in interaction different types Hb. In addition, the values of binding constant (Kb) revealed that nanopropolis has more binding affinity in compared to the bulk propolis. Data obtained from thermodynamic parameters implied more portion of electrostatic forces in GHb-propolis than native Hb-propolis and more hydrophobic portion in FHb-propolis. The CD spectra of Hb, GHb and FHb displayed no significant changes in secondary structure after incubated with propolis. These data are agreement with propolis nanoparticles. Results showed that propolis nanoparticles are a stronger quencher and bound to Hb, GHb and FHb with higher affinity than bulk propolis.

Synthesis of Recombinant Human Hemoglobin With NH2 -Terminal Acetylation in Escherichia coli.

The development of new technologies for the efficient expression of recombinant hemoglobin (rHb) is of interest for experimental studies of protein biochemistry and the development of cell-free blood substitutes in transfusion medicine. Expression of rHb in Escherichia coli host cells has numerous advantages, but one disadvantage of using prokaryotic systems to express eukaryotic proteins is that they are incapable of performing post-translational modifications such as NH2 -terminal acetylation. One possible solution is to coexpress additional enzymes that can perform the necessary modifications in the host cells. Here, we report a new method for synthesizing human rHb with proper NH2 -terminal acetylation. Mass spectrometry experiments involving native and recombinant human Hb confirmed the efficacy of the new technique in producing correctly acetylated globin chains. Finally, functional experiments provided insights into the effects of NH2 -terminal acetylation on O2 binding properties. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Gene synthesis and cloning the cassette to the expression plasmid Basic Protocol 2: Selection of E. coli expression strains for coexpression Basic Protocol 3: Large-scale recombinant hemoglobin expression and purification Support Protocol 1: Measuring O2 equilibration curves Support Protocol 2: Mass spectrometry to confirm NH2 -terminal acetylation.

Takashi Yonetani: A stellar biochemist, a man with dignity. The Roman connection

Takashi Yonetani: A stellar biochemist, a man with dignity. The Roman connection

Consequence of macromolecular crowding on aggregation propensity and structural stability of haemoglobin under glycating conditions

Cell interiors are extremely congested with biological macromolecules exerting crowding effect, influencing various physiognomies of protein life. Present work deals with effect of crowding on folding behaviour of haemoglobin (Hb) under glycating conditions. Macromolecular crowding was mimicked by concentrated solutions of dextran 70. Hb with 0.2 M fructose and ribose was incubated separately for 96 h in dilute and crowded solution to analyse conformational changes. Reduced intrinsic and ANS fluorescence, decreased Soret absorbance, enhanced turbidity, browning of protein, red shift in ThT and Congo red spectra significantly unveiled protein aggregation. FTIR and CD results revealed transition from α-helix to β-sheets confirming aggregation. Transmission electron microscopy exhibited incidence of aggregates. Macromolecular crowding was witnessed to defend conformational stability of native Hb under stress condition at 100 mg/ml dextran, noticeably indicating deceleration of aggregation. Stabilising effect of crowding was marginally better in fructosylated Hb than with ribose due to difference in their glycation potential. Contrarily, in over-crowded solution where dextran concentration was 500 mg/ml, heightened aggregation was perceived implying concentration dependant, dual nature of macromolecular crowding. The novelty of this study lies in idea of considering macromolecular crowding as a key player in regulation of protein stability which was safely ignored previously.

Sphingomyelin-induced structural modification of native human hemoglobin and its chemically and thermally disrupted secondary structure: A photophysical exploration

Sphingomyelin-induced structural modification of Human Hemoglobin (Hb) has been investigated in its native and unfolded conformers that are partially denatured in presence of ∼ 4 M urea, completely denatured in ∼ 8 M urea and thermally disrupted (at ∼ 65 °C) state. The absorption studies unveil ground state complexation between Hb and SM. From steady-state fluorescence and quenching studies alteration of the micro-environments around Trp residues of Hb in above mentioned different cases has been determined. Moreover, lesser exposure of Trp residues to SM in thermally disrupted Hb can be accounted for the exceptionally interesting outcomes in other experiments. The alterations in the time-resolved decay profiles of native Hb, partially and totally chemically denatured as well as thermally disrupted Hb with gradual addition of SM also affirm the amendment of the proteinous micro-environment surrounding Trp residues in a view of FRET between Trp residues and heme group. Wavelength-sensitive emission spectral studies reveal that the protein shows red edge effect in its different conformations in presence and absence of SM. Interestingly, the wavelength-responsive time-resolved study at a constant excitation wavelength demonstrates that with addition of lipid the increment of the average fluorescence lifetime signifies a considerable modulation of solvation dynamics of the fluorescent Trp residues in their excited state being greatest in case of thermally disrupted Hb. Nevertheless, the loss of α-helicity of Hb at its various conformers with addition of SM has been portrayed thoroughly by means of far-UV CD spectral studies in a view of disruption of secondary structure of the protein.

Haemoglobin(βK120C)-albumin trimer as an artificial O2 carrier with sufficient haemoglobin allostery.

The allosteric O2 release of haemoglobin (Hb) allows for efficient O2 delivery from the lungs to the tissues. However, allostery is weakened in Hb-based O2 carriers because the chemical modifications of the Lys- and Cys-β93 residues prevent the quaternary transition of Hb. In this paper, we describe the synthesis and O2 binding properties of a recombinant Hb [rHb(βK120C)]–albumin heterotrimer that maintains sufficient Hb allostery. The rHb(βK120C) core, with two additional cysteine residues at the symmetrical positions on its protein surface, was expressed using yeast cells. The mutations did not influence either the O2 binding characteristics or the quaternary transition of Hb. Maleimide-activated human serum albumins (HSAs) were coupled with rHb(βK120C) at the two Cys-β120 positions, yielding the rHb(βK120C)–HSA2 trimer, in which the Cys-β93 residues were unreacted. Molecular dynamics simulation demonstrated that the HSA moiety does not interact with the amino acid residues around the haem pockets and the α1β2 surfaces of the rHb(βK120C) core, the alteration of which retards Hb allostery. Circular dichroism spectroscopy demonstrated that the quaternary transition between the relaxed (R) state and the tense (T) state of the Hb core occurred upon both the association and dissociation of O2. In phosphate-buffered saline solution (pH 7.4) at 37 °C, the rHb(βK120C)–HSA2 trimer exhibited a sigmoidal O2 equilibrium curve with the O2 affinity and cooperativity identical to those of native Hb (p50 = 12 Torr, n = 2.4). Moreover, we observed an equal Bohr effect and 2,3-diphosphoglycerate response in the rHb(βK120C)–HSA2 trimer compared with naked Hb.

Novel manufacturing method for producing apohemoglobin and its biophysical properties

Apohemoglobin (apoHb) is a dimeric globular protein with two vacant heme-binding pockets that can bind heme or other hydrophobic ligands. Purification of apoHb is based on partial hemoglobin (Hb) unfolding to facilitate heme extraction into an organic solvent. However, current production methods are time consuming, difficult to scale up, and use highly flammable and toxic solvents. In this study, a novel and scalable apoHb production method was developed using an acidified ethanol solution to extract the hydrophobic heme ligand into solution and tangential flow filtration to separate heme from the resultant apoprotein. Total protein and active protein yields were >95% and ~75%, respectively, with <1% residual heme in apoHb preparations and >99% purity from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Virtually no loss of apoHb activity was detected at 4°C, -80°C, and in lyophilized form during long term storage. Structurally, size exclusion chromatography (SEC) and circular dichroism indicated that apoHb was dimeric with a ~25% reduction of helical content compared to Hb. Furthermore, mass spectroscopy and reverse-phase chromatography indicated that the mass of the α and β subunits were virtually identical to the theoretical mass of these subunits in Hb and had no detectable oxidative modifications upon heme removal from Hb. SEC confirmed that apoHb bound to haptoglobin at a similar ratio to that of native Hb. Finally, reconstituted Hb (rHb) was processed via a hemichrome removal method to isolate functional rHb for biophysical characterization in which the O2 equilibrium curve, O2 dissociation, and CO association kinetics of rHb were virtually identical to native Hb. Overall, this study describes a novel and improved method to produce apoHb, as well as presents a comprehensive biochemical analysis of apoHb and rHb.

Hemoglobin as substantial object for biomedical studies and diagnostics

Hemoglobin (Hb) is a hemoprotein consisting of the hem prosthetic group and a protein part. Its main function in human organism is oxygen binding and transportation. Different factors can violate native Hb structure. Depending on such factors, hereditary and acquired hemoglobinopathies are distinguished. Acquired ones are associated with the effect of various chemical agents, the modified Hb forms can appear in this case. It is caused by conjunction of different compounds to the molecule (hem and protein ligands). At several conditions Hb binds with the erythrocyte membrane. Changes in Hb structure can disturb its oxygen-carrying function and stability of erythrocytes; furthermore, they hamper the erythrocyte flow through microvessels. These processes take place at many pathological states as well. Because of increasing amount of information, it becomes urgent to use computer medical systems to increase the efficiency of diagnostics, including hemoglobinopathies. In the expert system elaborating by us, the results of the general haematological analysis are used, with addition of the data on modified Hb forms, among them on membrane-bound one.

Probing hemoglobin glyco-products by fluorescence spectroscopy.

Maillard reaction products (MRPs) participate in reactions of carbohydrate intermediates with proteins, resulting in the formation of advanced glycation end-products (AGEs). Dietary Maillard reaction products are recognized as potential chemical modifiers of human proteins. We have investigated the reaction of isolated MRPs from an asparagine–glucose model system with hemoglobin (Hb) to elucidate the binding effect of the MRPs in hemoglobin by fluorescence spectrophotometry. The tryptophan-specific fluorescence obtained for glycated hemoglobin exhibited a Stokes effect since the wavelength of the emission peak was shifted to a higher wavelength than that of native Hb. The formation of new fluorescence emission features indicates the formation of modified hemoglobin species. Fluorescence spectroscopic studies provide evidence that the conformational changes in the β-Trp 37 moiety induce motion of the distal His 64 (E7) in the heme binding pocket. This results in the formation of inactive hemichrome forms of hemoglobin which are related to blood disorders.

Functional Properties of Hb S and Hb C in Stored Cpda-1 Red Blood Cells Concentrate — a Prospective Study

Most of the 1,300 human hemoglobin (Hb) variants are characterized by normal O2 binding, including here the highly prevalent Hbs S [HBB:c.20A>T; p.Glu6Val] and C [HBB:c.19G>A; p.Glu6Lys]. Hb S and Hb C carriers are generally asymptomatic and might be admitted as blood donors. However, the functional efficiency of these variants in standard blood bags - red blood cells (RBC) concentrates - under the interference of anticoagulant and storage is not well established. In this prospective study, three samples of RBC concentrates (phenotypes AA, AS and AC), with citrate phosphate dextrose adenine (CPDA-1) as anticoagulant, were analyzed for equilibrium tests and compared to fresh Hb A without anticoagulant. The Hb samples were extracted after 48 hours and 35 days of storage and purified by exclusion and ion exchange chromatography (Sephadex G-25 and Amberlite MB-3 columns in Hepes buffer without NaCl). O2 affinity was evaluated by spectrophotometry-tonometry method, by determining the p50 in stripped lysates ([Hb]=70 μM/Heme), in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) in all studied pHs. The activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. The p50 of Hb A after 35 days in stored blood bag was decreased in all pHs, in both presence and absence of IHP, when compared to fresh Hb A, suggesting that the anticoagulant and the storage might affect HbA-O2 binding, increasing its affinity for the ligand. No other differences were observed: heme-heme cooperativity and Bohr effect were similar to fresh Hb A without anticoagulant. The same was found when stored Hb AS was compared to Hb A from blood bag (35 days storage, in the stripped state and under the interference of IHP). However, the stripped lysate containing Hb AC revealed an important reduction of the O2 affinity after both 48 hours and 35 days of storage, mainly at pHs ³ 7, thus interfering in the Bohr effect. The heme-heme cooperativity resulted in normal n coefficients, indicating that the Hb-O2 ligation remained cooperative, in spite of the significant reduction of O2 affinity. Similar results were observed in the presence of IHP (48 hours and 35 days), although Bohr effect was reestablished, indicating that the supplementation with a 2,3-BPG-like allosteric effector might restore some Hb-ion interactions, and therefore, Hb C function. As the 2,3-BPG is consumed with storage of RBC concentrates, Hb AC showed an even lower affinity for O2 after 35 days of storage. Although still preliminary, our results indicate a significant functional reduction of Hb C in RBC concentrates in standard blood bag (CPDA-1 anticoagulant), proportional to the time of storage. Considering that the screening for hemoglobinopathies in the blood banks routine is mainly focused in the detection of Hb S, a greater understanding about the properties of native Hb and its most frequent variants in stored RBC concentrates could also contribute with the determination of safe parameters in hemotherapy based on the functional efficiency of each hemoglobin. Financial Support: Fapesp, CNPq, Capes, Faepex-Unicamp. DisclosuresNo relevant conflicts of interest to declare.

Cross-linked hemoglobin bis-tetramers from bioorthogonal coupling do not induce vasoconstriction in the circulation.

Hemoglobin-based oxygen carriers (HBOCs) are potential alternatives to red blood cells in transfusions. Clinical trials using early versions of HBOCs noted adverse effects that appeared to result from removal of the vasodilator nitric oxide (NO). Previous reports suggest that size-enlarged HBOCs may avoid NO-rich regions along the vasculature and therefore not cause vasoconstriction and hypertension. Hemoglobin (Hb) bis-tetramers (bis-tetramers of hemoglobin that are prepared using CuAAC chemistry [BT-Hb] and bis-tetramers of hemoglobin that are specifically acetylated and prepared using CuAAC chemistry [BT-acHb]) can be reliably produced by a bio-orthogonal cyclo-addition approach. We considered that an HBOC derived from chemical coupling of two Hbs would be sufficiently large to avoid NO scavenging and related side effects. The ability of intravenously infused BT-Hb and BT-acHb to remain in the circulation without causing hypertension were determined in wild-type (WT) and diabetic (db/db) mouse models. In WT mice, the coupled oxygen-carrying proteins retained their function over several hours after administration. No significant changes in systolic blood pressure from baseline were observed after intravenous infusion of BT-Hb or BT-acHb in awake WT and db/db mice. In contrast, infusion of native Hb or cross-linked Hb tetramers in both animal models induced systemic hypertension. The results of this study indicate that bis-tetrameric HBOCs derived from the bio-orthogonal cyclo-addition process are likely to overcome clinical issues that arise from NO scavenging by Hb derivatives.

Binding interaction of phenazinium-based cationic photosensitizers with human hemoglobin: Exploring the effects of pH and chemical structure

The present study demonstrates a spectroscopic study on the interaction of two phenazinium-based cationic photosensitizers, namely, phenosafranin (PSF) and safranin-O (SO) with human hemoglobin (Hb) with particular emphasis on exploring the effects of pH and chemical structures of the dye molecules on the binding phenomenon. The protein (Hb) undergoes complex conformational transitions depending on the medium pH. The dye molecules exhibit a prominent fluorescence quenching following interaction with Hb under various experimental conditions (pH 3.5, 7.4, and 9.0). Our combined steady-state and time-resolved spectroscopic results provide persuasive evidence for static quenching mechanism showing that the dye:Hb interaction proceeds through ground-state complex formation. The meticulous investigations on the pH-dependence of the interaction of the dye molecules with the protein reveal a relatively strong binding of PSF as well as SO with Hb at physiological pH and alkaline pH, while the binding is weaker at acidic pH at which Hb predominantly exists as monomeric units. The binding constant for PSF:Hb interaction is K(PSF:Hb) = (1.09 ± 0.06) × 106 M−1 and that of SO:Hb interaction is K(SO:Hb) = (1.34 ± 0.07) × 105 M−1 at pH 7.4. However, at pH 3.5, the binding constant values are K(PSF:Hb) = (3.58 ± 0.18) × 104 M−1 and K(SO:Hb) = (4.29 ± 0.22) × 104 M−1 and at pH 9.0, the values are K(PSF:Hb) = (8.08 ± 0.40) × 104 M−1 and K(SO:Hb) = (5.07 ± 0.25) × 104 M−1. This depicts a much stronger binding interaction of the dyes with the native Hb at pH 7.4 compared to those at pH 3.5 and 9.0. Our results also unveil the effect of chemical structures of the dyes on the interaction phenomenon in the sense that the binding constant of PSF with Hb is found to be higher than that of SO at pH 7.4 and pH 9.0. The present study also focuses on exploring such important aspects of the interaction phenomena as the effect of binding of the dyes on the protein conformation by circular dichroism spectroscopy and probable binding location of the dyes within the protein scaffolds via micropolarity measurements and molecular docking simulation.

D-Ribose induced glycoxidative insult to hemoglobin protein: An approach to spot its structural perturbations

Glycation of biological macromolecule leads to the establishment of advanced glycation end products (AGEs) having implications in metabolic disorders. d‑ribose appears to be the most reactive among the naturally occurring sugars and contribute significantly to the glycation reactions in vivo, however, no report have been published yet to discuss d‑ribose induced glycation of hemoglobin (Hb). Therefore, the present study was designed to investigate d‑ribose induced glycoxidative damage to Hb protein. Briefly, the commercially available Hb was glycated with d‑ribose for varying time intervals. The structural perturbation induced in glycated Hb (GHb) was confirmed by biophysical techniques viz., UV–visible, fluorescence spectroscopy, circular dichroism, Fourier transform infra-red spectroscopy, dynamic light scattering, MALDIthermal denaturation by UV–visible spectrophotometer and DSC. Biophysical techniques confirm the secondary and tertiary structural perturbation in GHb as compared to native Hb. The values of carbonyl content, hydroxy methyl furfural, thiobarbituric acid reactive substance and nitro blue tetrazolium were found to be increased and free lysine and free arginine content were decreased in the GHb due to structural change. Thus, results of this study have established that glycation with d‑ribose lead to the structural changes in the native Hb which might play an important role in pathophysiology metabolic diseases.

The Reaction of Oxy Hemoglobin with Nitrite: Mechanism, Antioxidant-Modulated Effect, and Implications for Blood Substitute Evaluation.

The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb.

Development, characterization and determination of biological properties of crocodile hemoglobin (Crocodylus siamensis ) tablets

Recently, several biological properties of Siamese crocodile (Crocodylus siamensis) hemoglobin (Hb) and its hydrolysate have been discovered. In light of these findings, this study exploring the physical properties and biological activity of C. siamensis Hb tablets is disclosed. Notably, all derived tablet formulations of crocodile Hb were proven to possess antibacterial and antioxidant properties. Investigation of long-term stability indicated mild storage temperature of only 4°C is adequate to maintain full biological activity for more than 3 months. Scanning electron microscopy further revealed induction of abnormalities on bacterial cell membranes upon treatment with tableted crocodile Hb. Digestion of crocodile Hb tablets with artificial gastric juice was found non-significant differences on the antibacterial activity between the digested Hb tablets and intact Hb. Using C18 RP-HPLC purification followed by N-terminal sequence analysis, four active peptides originating from the alpha and six from the beta chain of native crocodile Hb were identified. Practical applications Several studies demonstrated that crocodile hemoglobin (Hb) possesses various biological activities such as antimicrobial, antioxidant, anti-inflammation, and wound healing activity. Hence, this article aims to develop crocodile hemoglobin as food supplementary tablets. Various crocodile Hb tablet formulations have been prepared. After that, the possibility for using tableted crocodile Hb as a food supplement has been investigated in detail. The outcome obtained from this study justified that the development of crocodile Hb tablets provide a protein rich food supplementary product with promising biological activity.

Aggregation of globular protein as a consequences of macromolecular crowding: A time and concentration dependent study

The living cells show profoundly crowded condition, called as macromolecular crowding. Crowding essentially impacts on protein structure and lead to its aggregation. Protein aggregates have been involved in a wide range of diseases including Parkinson, Alzheimer’s, and Huntington’s. Increased in normal physiological macromolecular crowding because of increasing age can be implicated as one of the leading cause of proteopathies. In the present study, we have demonstrated the effect of macromolecular crowding on native structure of hemoglobin using bovine serum albumin as a crowding agent. Conformational changes of Hb at different concentrations of BSA were monitored using intrinsic fluorescence and ATR-FTIR spectroscopy. These results showed the transition of native Hb to a non-native form. Thermodynamic parameters were analyzed by isothermal titration calorimetry. The measurements of turbidity, thioflavin T, congo red and intrinsic fluorescence revealed the formation of significant protein aggregates with time. The kinetics of protein aggregation using relative ThT and 8-anilino-1-naphthalenesulphonic acid spectra clearly showed acceleration of the process with time and in concentration dependent manner. The spectra at 80g/l of BSA incubated for 64h showed formation of maximum Hb aggregates. Transmission electron microscopy results clearly showed the formation of amyloid aggregates structures, thus supporting the spectroscopic data.