Biochemical investigation of the upstream anti-sickling mechanisms of soursop (Annona muricata): 15-acetyl guanacone as an inhibitor of deoxyhaemoglobin polymerisation.

Current sickle cell disease (SCD) therapies are limited and inefficient. The ethnomedicinal values of Annona muricata in the treatment of SCD, leading to this present research. Leaves and fruits of Annona muricata were processed using solvent extraction and partitioning; aqueous, chloroform and ethyl acetate fractions. In vitro (anti-oxidant and anti-sickling), in silico, quantitative (amino acids) and kinetic simulation experiments were done. 15-acetyl guanacone, was used, in silico against 2,3-bisphosphoglycerate (2, 3-BPG) mutase and deoxyhaemoglobin. The ethyl acetate and chloroform fractions better NO● scavengers, iron-chelators and ferric reducing. In vitro unsickling (UT50) had ethyl acetate = 5 h and methanol = 7 h. Chloroform fraction had EC50 1.00 mg/mL (EC50 = 546 mg/mL) to 10.00 mg/mL (EC50 = 99 mg/mL). EC50 and IC50 of ethyl acetate fraction had steady-decrease. At higher concentration, chloroform fraction had higher Bmax (1.48 × 1021 U/mL) and higher Kd (3.66 × 1019 mg/mL), whereas, at a lower concentration, the ethyl acetate fraction demonstrated higher Bmax (7.23 × 1012 U/mL) and lower Kd (2.12 × 1011 mg/mL); The relative affinity (BP) of chloroform fraction increased progressively with concentration. The amino acid profile revealed rich concentrations glycine, valine, leucine, lysine, phenylalanine, histidine, arginine, and tryptophan. From the in silico experiments, 15-acetyl guanacone specifically targeted the A and B chains, with greater affinity for the beta subunit. This suggested that 15-acetyl guanacone might be able to prevent the polymerisation of deoxyHbSS, induce an allosteric conformational change that increases the oxygen affinity, and decrease the cellular 2, 3-BPG concentration.Communicated by Ramaswamy H. Sarma.

Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules.

Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.

Effect of ytterbium amount on LaNbO4:Tm3+,Yb3+ nanoparticles for bio-labelling applications.

PURPOSE: This work investigates how Yb3+ concentration affects the luminescent properties of LaNbO4 nanoparticles for medical imaging applications. Due to the highly transparent optical window for organic tissues in the near infrared region (650-1000 nm), upconversion fluorescence allows near infrared wavelengths to penetrate deeply into tissues, which is useful in biomedical areas such as biodetection, activated phototherapy, and screening. MATERIALS/METHOD: Upconversion nanoparticles based on LaNbO4 doped with Tm3+ and Yb3+ were prepared by the one-step industrial process called Spray Pyrolysis. Samples with different Tm3+:Yb3+ molar ratios (1:4, 1:8 and 1:16) were obtained. RESULTS: The X-ray powder diffractograms of all the samples displayed the typical peaks of a crystalline material (tetragonal phase). Emission bands emerged in the blue, red, and near infrared regions, and they corresponded to the Tm3+1G4 → 3H6 (475 nm), 1G4 → 3F4 (650 nm), 3F2,3 â†’ 3H6 (690 nm), and 3H4 → 3H6 (803 nm) transitions, which indicated a two-photon absorption process. As for bio-labelling application, the results indicated that Yb3+ concentration was directly related to signal intensity. CONCLUSIONS: The intensity of positive conversion emissions depends directly on Yb3+ concentration. The bio-labelling tests pointed to the potential application of these materials. The sample containing the highest amount of Yb3+ provided better results and was easier to detect than the standard sample.

Redox reactions of heme proteins with flavonoids.

Proteins containing heme groups perform a variety of important functions in living organisms. The heme groups are involved in catalyzing oxidation/reduction reactions, in electron transfer, and in binding small molecules, like oxygen or nitric oxide. Flavonoids, low molecular weight plant polyphenols, are ubiquitous components of human diet. They are also components of many plant extracts used in herbal medicine as well as of food supplements. Due to their relatively low reduction potential, flavonoids are prone to oxidation. This paper provides a review of redox reactions of various heme proteins, including catalase, some peroxidases, cytochrome P450, cytochrome c, myoglobin, and hemoglobin with flavonoids. Potential biological significance of these reactions is discussed, in particular when flavonoids are delivered to the body at pharmacological doses.

Photophysical behavior of heme group: Unfolding of hemoglobin and myoglobin in the presence of Gemini surfactants of different molecular architectures.

Fluorescence studies were performed to determine the photophysical behavior of heme group in the presence of cationic Gemini surfactants of different architectures. Both hemoglobin and myoglobin were used to understand the heme group interactions with Gemini surfactants under the influence of temperature variation and were compared with homologous monomeric surfactants. The results were also supplemented from the size and zeta potential measurements of both proteins. Gemini surfactants showed marked effect on the unfolding behavior of hemoglobin that mainly contributed by the stronger hydrophobic interactions of double hydrocarbon chains as well as methylene spacer in the head group region with the hydrophobic domains of hemoglobin. Myoglobin with single polypeptide chain did not show similar unfolding behavior in the presence of Gemini surfactants rather it was readily solubilized in the surfactant solution and that too in the presence of monomeric surfactants rather than Gemini surfactants. The results highlighted the mechanistic aspects by which water soluble globular proteins interact with amphiphilic molecules of different functionalities and thus, helped to predict the interactions of both hemoglobin and myoglobin with the complex biological molecules possessing similar functionalities.

Enhanced Ferroptosis by Oxygen-Boosted Phototherapy Based on a 2-in-1 Nanoplatform of Ferrous Hemoglobin for Tumor Synergistic Therapy.

Photodynamic therapy (PDT) combined with oxygenating strategies is widely employed in cancer treatment; however, oxygen-boosted PDT has failed to achieve an ideal effect due to the complexity, heterogeneity, and irreversible hypoxic environment generated by tumor tissues. With the emergence of Fe-dependent ferroptosis boasting reactive oxygen species (ROS) cytotoxicity as well, such a chemodynamic approach to cancer therapy has drawn extensive attention. In this study, hemoglobin (Hb) is connected with the photosensitizer chlorin e6 (Ce6) to construct a 2-in-1 nanoplatform (SRF@Hb-Ce6) with Sorafenib (SRF, ferroptosis promotor) loaded, combining oxygen-boosted PDT and potent ferroptosis. Benefiting from the intrinsic presence of Fe capable of binding oxygen, hemoglobin concurrently furnishes oxygen for oxygen-dependent PDT and Fe for Fe-dependent ferroptosis. Furthermore, amphiphilic MMP2-responsive peptide is incorporated into the skeleton of the nanoplatform to ensure drug-release specificity for safety improvement. Correlative measurements demonstrate the potentiation of PDT and ferroptosis with SRF@Hb-Ce6. More importantly, PDT strengthens ferroptosis by recruiting immune cells to secrete IFN-γ, which can sensitize the tumor to ferroptosis in our findings. The therapeutic effect of synergistic treatment with SRF@Hb-Ce6 in vitro and in vivo was proven significant, revealing the promising prospects of combined PDT and ferroptosis therapy with the 2-in-1 nanoplatform.

Insights into the binding mechanism of two-dimensional black phosphorus nanosheets-protein associations.

Exploring the protein-nanomaterials interactions is the topic of high relevance for the future applications of new nanomaterials in biological system. Herein, the binding mechanism of bovine serum albumin(BSA) and bovine hemoglobin(BHB) with two-dimensional black phosphorus nanosheets (BP NSs) was reported. Muti-spectral results showed that the combination of BP NPs with protein resulted in the fluorescence quenching of BSA and BHB and induced the extension of the protein peptide chain by van der Waals forces, hydrophobic forces, and electron-transfer forces. Both BSA and BHB retain their structure in α-helix form. The induced circular dichroism (ICD) spectral results showed that the presence of BP NPs partly destroyed the binding domain of BHB with bilirubin and altered the tertiary structure of BHB by BP NPs binding.

Hemoglobin self-assembly and antibacterial activities of bio-modified Ag-MgO nanocomposites by different concentrations of Artemisia haussknechtii and Protoparmeliopsis muralis extracts.

Interaction of metal nanoparticles and metal nanocomposites with blood proteins is important from the perspectives of cytotoxicity and production of novel drug delivery systems. In this study, Ag-MgO nanocomposites were bio-modified by different concentrations of Artemisia haussknechtii medicinal plant and Protoparmeliopsis muralis medicinal lichen extracts. Determination of physicochemical properties of NCs were carried out by UV-Visible spectroscopy, scanning electron microscope (SEM), X-ray powder diffraction (XRD), and Fourier transform infrared (FT-IR) analyses. Antibacterial effects of NCs against methicillin-resistant Staphylococcus aureus (MRSA), E. coli ATCC 25922, and P. aeruginosa ATCC 27853 bacteria were measured by disc diffusion and minimum inhibition/bactericidal concentrations (MIC and MBC). Also, in final section, interaction of bio-modified NCs (BNCs) with hemoglobin (Hb) protein was surveyed by SEM, atomic force microscope (AFM), and FT-IR techniques. BNCs by plant extract (10%w/v) showed highest antibacterial effects on E. coli ATCC 25922 as inhibition zone diameter of (IZD) value of 20 ± 0.89 mm. Aggregation of Hb around BNCs was approved by mentioned analyses. This investigation illustrated contribution of various functional groups including thiol and imidazole groups in self-assembly of Hb surrounding BNCs.

7-Keto-Cholesterol and Cholestan-3beta, 5alpha, 6beta-Triol Induce Eryptosis through Distinct Pathways Leading to NADPH Oxidase and Nitric Oxide Synthase Activation.

BACKGROUND/AIMS: We showed that patho-physiological concentrations of either 7-keto-cholesterol (7-KC), or cholestane-3beta, 5alpha, 6beta-triol (TRIOL) caused the eryptotic death of human red blood cells (RBC), strictly dependent on the early production of reactive oxygen species (ROS). The goal of the current study was to assess the contribution of the erythrocyte ROS-generating enzymes, NADPH oxidase (RBC-NOX), nitric oxide synthase (RBC-NOS) and xanthine oxido-reductase (XOR) to the oxysterol-dependent eryptosis and pertinent activation pathways. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, reactive oxygen/nitrogen species (RONS) and nitric oxide formation from 2',7'-dichloro-dihydrofluorescein (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) -dependent fluorescence, respectively; Akt1, phospho-NOS3 Ser1177, and PKCζ from Western blot analysis. The activity of individual 7-KC (7 µM) and TRIOL (2, µM) on ROS-generating enzymes and relevant activation pathways was assayed in the presence of Diphenylene iodonium chloride (DPI), N-nitro-L-arginine methyl ester (L-NAME), allopurinol, NSC23766 and LY294002, inhibitors in this order of RBC-NOX, RBC-NOS, XOR and upstream regulatory proteins Rac GTPase and phosphoinositide3 Kinase (PI3K); hemoglobin oxidation from spectrophotometric analysis. RESULTS: RBC-NOX was the target of 7-KC, through a signaling including Rac GTPase and PKCζ, whereas TRIOL caused activation of RBC-NOS according to the pathway PI3K/Akt, with the concurrent activity of a Rac-GTPase. In concomitance with the TRIOL-induced .NO production, formation of methemoglobin with global loss of heme were observed, ascribable to nitrosative stress. XOR, activated after modification of the redox environment by either RBC-NOX or RBC-NOS activity, concurred to the overall oxidative/nitrosative stress by either oxysterols. When 7-KC and TRIOL were combined, they acted independently and their effect on ROS/RONS production and PS exposure appeared the result of the effects of the oxysterols on RBC-NOX and RBC-NOS. CONCLUSION: Eryptosis of human RBCs may be caused by either 7-KC or TRIOL by oxidative/nitrosative stress through distinct signaling cascades activating RBC-NOX and RBC-NOS, respectively, with the complementary activity of XOR; when combined, the oxysterols act independently and both concur to the final eryptotic effect.

Effect of Tea Polyphenols on the Oxidation and Color Stability of Porcine Hemoglobin.

The oxidation and color stability of porcine hemoglobin (Hb) in the presence of tea polyphenols (TP), as well as the mechanism, were investigated using the methods of color and oxidation analyses, ultraviolet-visible and fluorescence spectroscopy. Results indicated that TP interacted with the tryptophan and tyrosine residues of Hb through inserting into its hydrophobic pocket. This interaction showed a concentration-dependent effect on Hb, which might lead to completely opposite results. The presence of TP (16 mg/L) disrupted Hb (16 mg/L) structure, and the exposure of heme iron facilitated the oxidation and discoloration of Hb. However, a lower level of TP should not break Hb structure but could work as an antioxidant and restrain the formation of methemoglobin. Consequently, TP (1.6 mg/L) considerably maintained the redness of Hb (16 mg/L, P < 0.05) when stored at pH 7.4 and 25 °C for 72 hr. Results may provide scientific information for the proper use of TP in blood and meat products. PRACTICAL APPLICATION: Proper utilization of tea polyphenols (TP) in food products is beneficial to improve antioxidant capacity and nutrition quality of food. We proved that it was potential to corporate TP into blood and meat products to prevent discoloration and oxidative deterioration.

Sugar beet hemoglobins: reactions with nitric oxide and nitrite reveal differential roles for nitrogen metabolism.

In contrast with human hemoglobin (Hb) in red blood cells, plant Hbs do not transport oxygen, instead research points towards nitrogen metabolism. Using comprehensive and integrated biophysical methods we characterized three sugar beet Hbs: BvHb1.1, BvHb1.2 and BvHb2. Their affinities for oxygen, CO, and hexacoordination were determined. Their role in nitrogen metabolism was studied by assessing their ability to bind NO, to reduce nitrite (NiR, nitrite reductase), and to form nitrate (NOD, NO dioxygenase). Results show that BvHb1.2 has high NOD-like activity, in agreement with the high nitrate levels found in seeds where this protein is expressed. BvHb1.1, on the other side, is equally capable to bind NO as to form nitrate, its main role would be to protect chloroplasts from the deleterious effects of NO. Finally, the ubiquitous, reactive, and versatile BvHb2, able to adopt 'open and closed forms', would be part of metabolic pathways where the balance between oxygen and NO is essential. For all proteins, the NiR activity is relevant only when nitrite is present at high concentrations and both NO and oxygen are absent. The three proteins have distinct intrinsic capabilities to react with NO, oxygen and nitrite; however, it is their concentration which will determine the BvHbs' activity.

The effect of aluminum oxide on red blood cell integrity and hemoglobin structure at nanoscale.

Herein, we explored the interaction of Al2O3 NPs with RBCs and Hb to determine the effect of Al2O3 NPs on hemolytic activity and Hb denaturation. The percentage of hemolysis of extracts and direct contact assays triggered by Al2O3 NPs was calculated by determining supernatant Hb concentration at 540 nm. Far-UV CD and Trp/ANS/acrylamide fluorescence spectroscopic methods were used to determine the structural changes of Hb upon interaction with Al2O3 NPs. Theoretical studies were carried out to display the residues involved in the binding site of Hb with Al2O3 nanocluster as well as the structural changes of Hb after interaction. The results showed that the percentage of hemolysis of extract and direct contact assays induced by Al2O3 NPs were 1.16 and 0.46, respectively. Fluorescence spectroscopy revealed that Al2O3 NPs alter the quaternary structure of the protein; however, CD spectroscopy indicated that the secondary structure of Hb remains almost unchanged. Theoretical study displayed that Al2O3 nanocluster interacts with different residues of protein, and Hb tends to be destabilized at the binding site with nanocluster. This study may be significant in exploring the toxicity profile of Al2O3 NPs for their in vivo implementations.

Sequential biowashing-biopile processes for remediation of crude oil contaminated soil in Kuwait.

The application of biological processes for remediation of the aged crude oil-contaminated soil of Kuwait can be an inefficient way, thus, this study developed 20 d-sequential biowashing and biopile processes where the biowashing step uses an enrichment culture of the indigenous soil bacterial community and the biopile step includes hemoglobin-catalyzed oxidation (HCO). The residual total petroleum hydrocarbons (TPH) concentrations and CO2 generation were measured to determine the removal efficiency, and the bacterial community changes were studied to investigate the effect of the sequential processes on the soil indigenous bacterial community. The enrichment culture grown on hemoglobin showed an increased surface activity, and this promoted desorption and emulsification of crude oil from the soil sample in the biowashing step resulting in 75% TPH removal. Potential surfactant-producing bacterial species were observed in the soil sample after biowashing. The HCO in the beginning of the biopile step removed 21% of the residual TPH, and further TPH removal was observed with a longer biopile period. Overall, the sequential biowashing and biopile processes removed 86% TPH. The results show that the developed sequential biowashing and biopile processes can be used to efficiently remediate the aged crude oil-contaminated soil of Kuwait.

Luminescent, Oxygen-Supplying, Hemoglobin-Linked Conjugated Polymer Nanoparticles for Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising method for cancer treatment. Two parameters that influence the efficacy of PDT are the light source and oxygen supply. Herein, we prepared a system for PDT using hemoglobin (Hb)-linked conjugated polymer nanoparticles (CPNs), which can luminesce and supply oxygen. Hb catalyzes the activation of luminol, the conjugated polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) nanoparticles can absorb the chemiluminescence of luminol through chemiluminescence resonance energy transfer (CRET) and then sensitize the oxygen supplied by Hb to produce reactive oxygen species that kill cancer cells. This system could be used for the controlled release of an anticancer prodrug. The system does not need an external light source and circumvents the insufficient level molecular oxygen under hypoxia. This work provides a proof-of-concept to explore smart and multifunctional nanoplatforms for phototherapy.

Near infrared light-responsive heat-emitting hemoglobin hydrogels for photothermal cancer therapy.

Photothermal therapy (PTT) is an effective means of treating tumors because tumor cells are sensitive to heat. Gold and carbon nanoparticles are used as efficient PTT materials. However, development of a non-toxic biodegradable PTT agent remains a challenge. Here, we developed a hemoglobin (Hb) hydrogel that exhibited excellent PTT effects in vitro and in vivo. Unlike conventional PTT agents, which are toxic and do not decompose completely in the body, the Hb hydrogel was manufactured using only two components: (i) Hb, a natural substance derived from the human body, and (ii) PEG, an FDA-approved polymer. The gelation time of the Hb hydrogels could be controlled by changing the Hb concentration. Because Hb is present at a high concentration (150 mg/ml) in the body, the Hb hydrogel decomposed and was eliminated in vivo without toxicity. The Hb hydrogel showed an excellent PTT effect in response to 808 nm near-infrared (NIR) laser irradiation and had excellent anticancer effects against A549 lung cancer cells both in vitro and in vivo. Blood hematology and blood biochemical assay results from an animal model treated with Hb hydrogel were similar to those of the control group. Importantly, toxicity was not observed based on H&E staining of major organs (heart, liver, spleen, kidneys and lung). Tumors of A549 cell-xenografted mice treated with Hb hydrogel and 808 nm NIR laser irradiation were significantly smaller than those of the control group (23.1 mm3versus 746.5 mm3, respectively). This is a first report of a biocompatible photothermal hydrogel based on hemoglobin, and our overall results suggest that Hb hydrogels are commercially-promising PTT systems that have excellent anti-cancer effects.

Protective properties of Myrtus communis extract against oxidative effects of extremely low-frequency magnetic fields on rat plasma and hemoglobin.

PURPOSE: This study investigates the protective properties of Myrtus communis extract against the oxidative effects of extremely low-frequency magnetic fields (ELFMF). Also, this study is aimed to analyze the conformational changes of hemoglobin, oxidative damages to plasma proteins and antioxidant power of plasma following exposure to ELFMF. MATERIALS AND METHODS: Adult male rats were divided into 3 groups: (1) control, (2) ELFMF exposure, and (3) ELFMF exposure after M. communis extract administration. The magnetic field (0.7 mT, 50 Hz) was produced by a Helmholtz coil for one month, 2 hours a day. The M. communis extract was injected intraperitoneally at a dose of 0.5 mg/kg before exposure to ELFMF. The oxidative effects of ELFMF were studied by evaluating the hemoglobin, methemoglobin (metHb) and hemichrome levels, absorption spectrum of hemoglobin (200-700 nm), oxidative damage to plasma proteins by measuring protein carbonyl (PCO) levels and plasma antioxidant power according to the ferric reducing ability of plasma (FRAP). The mean and standard errors of the mean were determined for each group. One-way ANOVA analysis was used to compare the means of groups. The significance level was considered to be p < .05. Moreover, artificial neural network (ANN) analysis was used to identify the predictive parameters for estimating the oxyhemoglobin (oxyHb) concentration. RESULTS: Exposure to ELFMF decreased the FRAP which was in concomitant with a significant increase in plasma PCO, metHb and hemichrome concentrations (p < .001). Oxidative modifications of Hb were shown by reduction in optical density at 340 nm (globin-heme interaction) and 420 nm (heme-heme interaction). Administration of M. communis extract increased FRAP values and decreased plasma POC, metHb, and hemichrome concentrations. Also, a significant increase in Hb absorbance at 340, 420, 542, and 577 nm showed the protective properties of M. communis extract against ELFMF-induced oxidative stress in erythrocytes. ANN analysis showed that optical absorption of hemoglobin at 520, 577, 542, and 630 nm and concentration of metHb and hemichrome were the most important parameters in predicting the oxyHb concentration. CONCLUSIONS: Myrtus communis extract enhances the ability of erythrocytes and plasma to deal with oxidative conditions during exposure to ELFMF. Also, ANN analysis can predict the most important parameters in relation to Hb structure during oxidative stress.

The hemoglobin adduct N-(2,3-dihydroxypropyl)-valine as biomarker of dietary exposure to glycidyl esters: a controlled exposure study in humans.

Fatty acid esters of glycidol (glycidyl esters) are heat-induced food contaminants predominantly formed during industrial deodorization of vegetable oils and fats. After consumption, the esters are digested in the gastrointestinal tract, leading to a systemic exposure to the reactive epoxide glycidol. The compound is carcinogenic, genotoxic and teratogenic in rodents, and rated as probably carcinogenic to humans (IARC group 2A). Assessment of exposure from occurrence and consumption data is difficult, as lots of different foods containing refined oils and fats may contribute to human exposure. Therefore, assessment of the internal exposure using the hemoglobin adduct of glycidol, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), may be promising, but a proof-of-principle study is needed to interpret adduct levels with respect to the underlying external exposure. A controlled exposure study was conducted with 11 healthy participants consuming a daily portion of about 36 g commercially available palm fat with a relatively high content of ester-bound glycidol (8.7 mg glycidol/kg) over 4 weeks (total amount 1 kg fat, individual doses between 2.7 and 5.2 µg/kg body weight per day). Frequent blood sampling was performed to monitor the 2,3-diHOPr-Val adduct levels during formation and the following removal over 15 weeks, using a modified Edman degradation and ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results demonstrated for the first time that the relatively high exposure during the intervention period was reflected in corresponding distinct increases of 2,3-diHOPr-Val levels in all participants, following the expected slope for hemoglobin adduct formation and removal over time. The mean adduct level increased from 4.0 to 12.2 pmol 2,3-diHOPr-Val/g hemoglobin. By using a nonlinear mixed model, values for the adduct level/dose ratio (k, mean 0.082 pmol 2,3-diHOPr-Val/g hemoglobin per µg glycidol/kg body weight) and the adduct lifetime (τ, mean 104 days, likely the lifetime of the erythrocytes) were determined. Interindividual variability was generally low. 2,3-DiHOPr-Val was therefore proven to be a biomarker of the external dietary exposure to fatty acid esters of glycidol. From the background adduct levels observed in our study, a mean external glycidol exposure of 0.94 µg/kg body weight was estimated. This value is considerably higher than current estimates for adults using occurrence and consumption data of food. Possible reasons for this discrepancy are discussed (other oral or inhalational glycidol sources, endogenous formation, exposure to other chemicals also forming the adduct 2,3-diHOPr-Val). Further research is necessary to clarify the issue.

Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin.

Heme ligation in hemoglobin is typically assumed by the "proximal" histidine. Hydrophobic contacts, ionic interactions, and the ligation bond secure the heme between two α-helices denoted E and F. Across the hemoglobin superfamily, several proteins also use a "distal" histidine, making the native state a bis-histidine complex. The group 1 truncated hemoglobin from Synechocystis sp. PCC 6803, GlbN, is one such bis-histidine protein. Ferric GlbN, in which the distal histidine (His46 or E10) has been replaced with a leucine, though expected to bind a water molecule and yield a high-spin iron complex at neutral pH, has low-spin spectral properties. Here, we applied nuclear magnetic resonance and electronic absorption spectroscopic methods to GlbN modified with heme and amino acid replacements to identify the distal ligand in H46L GlbN. We found that His117, a residue located in the C-terminal portion of the protein and on the proximal side of the heme, is responsible for the formation of an alternative bis-histidine complex. Simultaneous coordination by His70 and His117 situates the heme in a binding site different from the canonical site. This new holoprotein form is achieved with only local conformational changes. Heme affinity in the alternative site is weaker than in the normal site, likely because of strained coordination and a reduced number of specific heme-protein interactions. The observation of an unconventional heme binding site has important implications for the interpretation of mutagenesis results and globin homology modeling.

Synthesis, characterization and nociceptive screening of new VV-hemorphin-5 analogues.

In the present study, some new analogues of VV-hemorphin-5, modified at position 1 and 7 by the non-proteinogenic and/or natural amino acids followed the structures Xxx-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH2 and Val-Val-Tyr-Pro-Trp-Thr-Yyy-NH2, where Xxx is Ile or Aib and Yyy is Lys/Orn/Dap/Dab were synthesized to investigate their potential antinociceptive activities. We report also the redox potentials and the acid/base properties as pKa values of these peptide analogues which were compared toward electrochemical behaviour of tryptophan containing peptides. All analogues showed a short lasting initial antinociceptive effect, however H2 hemorphin analogue is characterized with prolong and strong antinociceptive effect, while the other peptide analogues exerted more variable effects on the visceral nociception depending on the dose or time after the intracerebral injection.

Molecular insights into the effect of ozone on human hemoglobin in autohemotherapy: Highlighting the importance of the presence of blood antioxidants during ozonation.

Ozone has been known for several decades, with its antiseptic and therapeutic effects determined by the hormesis theory. It is shown that the therapeutic efficacy of ozone therapy may be partly due to the controlled and moderate oxidative stress produced by the reaction of ozone with several biological components. In this study, the effect of ozone on healthy human hemoglobin (Hb) in the whole blood environment (in the presence of antioxidants) and in the purified form (in the absence of antioxidants) is investigated using a number of different techniques including intrinsic fluorescence, circular dichroism and UV-VIS absorption spectroscopy as well as SDS- and Native-PAGE and dynamic light scattering. The results show that the presence of antioxidants prevents damage to Hb while its absence means that as the exposure to ozone is increased, Hb is increasingly damaged. These results highlight the importance for the use of appropriate doses of ozone, for patients with different diseases and hence antioxidant levels, in autohemotherapy.