Optimizing the lyophilization of Lumbricus terrestris erythrocruorin.

Haemorrhagic shock is a leading cause of death worldwide. Blood transfusions can be used to treat patients suffering severe blood loss but donated red blood cells (RBCs) have several limitations that limit their availability and use. To solve the problems associated with donated RBCs, several acellular haemoglobin-based oxygen carriers (HBOCs) have been developed to restore the most important function of blood: oxygen transport. One promising HBOC is the naturally extracellular haemoglobin (i.e. erythrocruorin) of Lumbricus terrestris (LtEc). The goal of this study was to maximise the portability of LtEc by lyophilising it and then testing its stability at elevated temperatures. To prevent oxidation, several cryoprotectants were screened to determine the optimum formulation for lyophilisation that could minimise oxidation of the haem iron and maximise recovery. Furthermore, samples were also deoxygenated prior to storage to decrease auto-oxidation, while resuspension in a solution containing ascorbic acid was shown to partially reduce LtEc that had oxidised during storage (e.g. from 42% Fe3+ to 11% Fe3+). Analysis of the oxygen equilibria and size of the resuspended LtEc showed that the lyophilisation, storage, and resuspension processes did not affect the oxygen transport properties or the structure of the LtEc, even after 6 months of storage at 40 °C. Altogether, these efforts have yielded a shelf-stable LtEc powder that can be stored for long periods at high temperatures, but future animal studies will be necessary to prove that the resuspended product is a safe and effective oxygen transporter in vivo.

Probability Density Functional Method of Enzyme Effect on Denatured Protein Soil.

Protein soils must be removed for both appearance and hygienic reasons. They are denatured by heat treatment or bleaching and cleaned using enzymes. Among the various types of protein soils, blood soils are the most noticeable and known to be denatured by heat and bleaching by oxidation. We verified herein that the detergency of heat and oxidatively denatured hemoglobin is greatly improved by the enzyme immersing treatment in the detergency with SDS and can be analyzed using the probability density functional method. The probability density functional method evaluates the cleaning power by assuming that the adhesion and cleaning force of soils are not uniquely determined, but instead have a distribution in intensity, with a usefulness that had recently been demonstrated. This analytical method showed that the cleaning power of the enzyme immersing treatment improved when the soil adhesive force was decreased due to denatured protein degradation, even though the cleaning power of the SDS remained unchanged, and the values were consistent with those in the cleaning test. In conclusion, the probability density functional method can be used to analyze enzymatic degradation of denatured protein soils and the resulting changes in their detergency.

How to Personalize General Anesthesia-A Prospective Theoretical Approach to Conformational Changes of Halogenated Anesthetics in Fire Smoke Poisoning.

Smoke intoxication is a central event in mass burn incidents, and toxic smoke acts at different levels of the body, blocking breathing and oxygenation. The majority of these patients require early induction of anesthesia to preserve vital functions. We studied the influence of hemoglobin (HMG) and myoglobin (MGB) blockade by hydrochloric acid (HCl) in an interaction model with gaseous anesthetics using molecular docking techniques. In the next part of the study, molecular dynamics (MD) simulations were performed on the top-scoring ligand-receptor complexes to investigate the stability of the ligand-receptor complexes and the interactions between ligands and receptors in more detail. Through docking analysis, we observed that hemoglobin creates more stable complexes with anesthetic gases than myoglobin. Intoxication with gaseous hydrochloric acid produces conformational and binding energy changes of anesthetic gases to the substrate (both the pathway and the binding site), the most significant being recorded in the case of desflurane and sevoflurane, while for halothane and isoflurane, they remain unchanged. According to our theoretical model, the selection of anesthetic agents for patients affected by fire smoke containing hydrochloric acid is critical to ensure optimal anesthetic effects. In this regard, our model suggests that halothane and isoflurane are the most suitable choices for predicting the anesthetic effects in such patients when compared to sevoflurane and desflurane.

Physicochemical, structural and functional properties of low methoxyl pectin­iron (III) complex and its effect on rats with iron deficiency anemia.

Iron deficiency anemia (IDA) is the most common nutritional disease worldwide. In this study, a low methoxyl pectin (LMP)­iron(III) complex was prepared. The physicochemical and structural properties were characterized by HPSEC, HPIC, CV, FTIR, 1H NMR, XRD, SEM and CD. The results showed that iron increased the molecular weight of the LMP­iron(III) from 11.50 ± 0.32 to 12.70 ± 0.45 kDa and improved its crystallinity. Moreover, the findings demonstrated that -OH and -COOH groups in LMP coordinate with Fe3+ to form ß-FeOOH. The water-holding capacity, emulsion stability, and antioxidant activities of the LMP­iron(III) were lower than those of LMP. Furthermore, the therapeutic effects of LMP­iron(III) on IDA were investigated in rats. Following LMP­iron(III) supplementation, compared with the model group, the administration of LMP­iron(III) significantly increased the body weight, hemoglobin concentration, and serum iron concentration as well as decreased free erythrocyte protoporphyrin concentration. Therefore, the LMP­iron(III) can potentially treat IDA in rats experiments, providing a theoretical basis for the development of a promising iron supplement.

Comparison of the Backbone Dynamics of Dehaloperoxidase-Hemoglobin Isoenzymes.

Dehaloperoxidase (DHP) is a multifunctional hemeprotein with a functional switch generally regulated by the chemical class of the substrate. Its two isoforms, DHP-A and DHP-B, differ by only five amino acids and have an almost identical protein fold. However, the catalytic efficiency of DHP-B for oxidation by a peroxidase mechanism ranges from 2- to 6-fold greater than that of DHP-A depending on the conditions. X-ray crystallography has shown that many substrates and ligands have nearly identical binding in the two isoenzymes, suggesting that the difference in catalytic efficiency could be due to differences in the conformational dynamics. We compared the backbone dynamics of the DHP isoenzymes at pH 7 through heteronuclear relaxation dynamics at 11.75, 16.45, and 19.97 T in combination with four 300 ns MD simulations. While the overall dynamics of the isoenzymes are similar, there are specific local differences in functional regions of each protein. In DHP-A, Phe35 undergoes a slow chemical exchange between two conformational states likely coupled to a swinging motion of Tyr34. Moreover, Asn37 undergoes fast chemical exchange in DHP-A. Given that Phe35 and Asn37 are adjacent to Tyr34 and Tyr38, it is possible that their dynamics modulate the formation and migration of the active tyrosyl radicals in DHP-A at pH 7. Another significant difference is that both distal and proximal histidines have a 15-18% smaller S2 value in DHP-B, thus their greater flexibility could account for the higher catalytic activity. The distal histidine grants substrate access to the distal pocket. The greater flexibility of the proximal histidine could also accelerate H2O2 activation at the heme Fe by increased coupling of an amino acid charge relay to stabilize the ferryl Fe(IV) oxidation state in a Poulos-Kraut "push-pull"-type peroxidase mechanism.

Structural dynamics of the heme pocket and intersubunit coupling in the dimeric hemoglobin from Scapharca inaequivalvis.

Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe-proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI.

Quantification of Glycated Hemoglobin in Total Hemoglobin by a Simultaneous Dual-Signal Acquisition Approach.

The glycated hemoglobin (HbA1c) level, which is defined as the ratio of HbA1c to total hemoglobin (tHb, including glycated and unglycated hemoglobin), is considered one of the preferred indicators for diabetes monitoring. Generally, assessment of the HbA1c level requires separate determination of tHb and HbA1c concentrations after a complex separation step. This undoubtedly increases the cost of the assay, and the loss or degradation of HbA1c during the separation process results in a decrease in the accuracy of the assay. Therefore, this study explored a dual-signal acquisition method for the one-step simultaneous evaluation of tHb and HbA1c. Quantification of tHb: graphene adsorbed carbon quantum dots and methylene blue were utilized as the substrate material and linked to the antibody. tHb was captured on the substrate by the antibody. The unique heme group on tHb catalyzed the production of •OH from H2O2 to degrade methylene blue on the substrate, and a quantitative relationship between the tHb concentration and the methylene blue oxidation current signal was constructed. Quantification of HbA1c: complex labels with HbA1c recognition were made of ZIF-8-ferrocene-gold nanoparticles-mercaptophenylboronic acid. The specific recognition of the boronic acid bond with the unique cis-diol structure of HbA1c establishes a quantitative relationship between the oxidation current of the label-loaded ferrocene and the concentration of HbA1c. Thus, the HbA1c level can be assessed with only one signal readout. The sensor exhibited extensive detection ranges (0.200-600 ng/mL for tHb and 0.100-300 ng/mL for HbA1c) and low detection limits (4.00 × 10-3 ng/mL for tHb and 1.03 × 10-2 ng/mL for HbA1c).

Spin Trapping of Nitric Oxide by Hemoglobin and Ferrous Diethyldithiocarbamate in Model Tumors Differing in Vascularization.

Animal tumors serve as reasonable models for human cancers. Both human and animal tumors often reveal triplet EPR signals of nitrosylhemoglobin (HbNO) as an effect of nitric oxide formation in tumor tissue, where NO is complexed by Hb. In search of factors determining the appearance of nitrosylhemoglobin (HbNO) in solid tumors, we compared the intensities of electron paramagnetic resonance (EPR) signals of various iron-nitrosyl complexes detectable in tumor tissues, in the presence and absence of excess exogenous iron(II) and diethyldithiocarbamate (DETC). Three types of murine tumors, namely, L5178Y lymphoma, amelanotic Cloudman S91 melanoma, and Ehrlich carcinoma (EC) growing in DBA/2 or Swiss mice, were used. The results were analyzed in the context of vascularization determined histochemically using antibodies to CD31. Strong HbNO EPR signals were found in melanoma, i.e., in the tumor with a vast amount of a hemorrhagic necrosis core. Strong Fe(DETC)2NO signals could be induced in poorly vascularized EC. In L5178Y, there was a correlation between both types of signals, and in addition, Fe(RS)2(NO)2 signals of non-heme iron-nitrosyl complexes could be detected. We postulate that HbNO EPR signals appear during active destruction of well-vascularized tumor tissue due to hemorrhagic necrosis. The presence of iron-nitrosyl complexes in tumor tissue is biologically meaningful and defines the evolution of complicated tumor-host interactions.

Identification of histidine residues that affect the T/R-state conformations of human hemoglobin using constant pH molecular dynamics simulations.

Human hemoglobin (Hb) is a tetrameric protein consisting of two α and two ß subunits that can adopt a low-affinity T- and high-affinity R-state conformations. Under physiological pH conditions, histidine (His) residues are the main sites for proton binding or release, and their protonation states can affect the T/R-state conformation of Hb. However, it remains unclear which His residues can effectively affect the Hb conformation. Herein, the impact of the 38 His residues of Hb on its T/R-state conformations was evaluated using constant-pH molecular dynamics (CpHMD) simulations at physiological pH while focusing on the His protonation states. Overall, the protonation states of some His residues were found to be correlated with the Hb conformation state. These residues were mainly located in the proximity of the heme (α87 and ß92), and at the α1ß2 and α2ß1 interfaces (α89 and ß97). This correlation may be partly explained by how easily hydrogen bonds can be formed, which depends on the protonation states of the His residues. Taken together, these CpHMD-based findings provide new insights into the identification of titratable His residues α87, α89, ß92, and ß97 that can affect Hb conformational switching under physiological pH conditions.

Zinc-Substituted Hemoglobin-Albumin Cluster as a Porphyrin-Carrier for Enhanced Photodynamic Therapy.

Apohemoprotein is focused on the field of theranostics, serving as a porphyrin carrier. Hemoglobin (Hb) consists of α2ß2 tetramer with iron(II)-protoporphyrin IX (heme) bound to each globin. However, heme-removed Hb (apoHb) causes dissociation at αß interfaces and aggregation under physiological conditions. We synthesized a stable apoHb derivative comprising intramolecular-crosslinked apoHb (apoXHb) and human serum albumin (HSA), apoXHb-HSA3. ApoXHb-HSA3 engendered no aggregates in the physiological solutions. Moreover, apoXHb-HSA3 was reconstituted with zinc(II)-protoporphyrin IX (ZnP), generating ZnXHb-HSA3, a potent photosensitizer for photodynamic therapy (PDT). The photophysical properties of ZnXHb-HSA3 were identical to those of zinc-substituted XHb (ZnXHb). Cellular uptake behavior was evaluated using various cancer cell lines. ZnXHb-HSA3 released ZnP around the cells, and the free ZnP penetrated cell membranes. In contrast, protein units were not observed within the cells. ZnXHb-HSA3 showed no cytotoxicity under dark conditions and demonstrated superior PDT activity in comparison to naked ZnXHb. ZnXHb-HSA3 acts as an innovative porphyrin carrier for enhanced PDT.

Redox Reactivity of Nonsymbiotic Phytoglobins towards Nitrite.

Nonsymbiotic phytoglobins (nsHbs) are a diverse superfamily of hemoproteins grouped into three different classes (1, 2, and 3) based on their sequences. Class 1 Hb are expressed under hypoxia, osmotic stress, and/or nitric oxide exposure, while class 2 Hb are induced by cold stress and cytokinins. Both are mainly six-coordinated. The deoxygenated forms of the class 1 and 2 nsHbs from A. thaliana (AtHb1 and AtHb2) are able to reduce nitrite to nitric oxide via a mechanism analogous to other known globins. NsHbs provide a viable pH-dependent pathway for NO generation during severe hypoxia via nitrite reductase-like activity with higher rate constants compared to mammalian globins. These high kinetic parameters, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant hemoglobins could indeed serve as anaerobic nitrite reductases in vivo. The third class of nsHb, also known as truncated hemoglobins, have a compact 2/2 structure and are pentacoordinated, and their exact physiological role remains mostly unknown. To date, no reports are available on the nitrite reductase activity of the truncated AtHb3. In the present work, three representative nsHbs of the plant model Arabidopsis thaliana are presented, and their nitrite reductase-like activity and involvement in nitrosative stress is discussed. The reaction kinetics and mechanism of nitrite reduction by nsHbs (deoxy and oxy form) at different pHs were studied by means of UV-Vis spectrophotometry, along with EPR spectroscopy. The reduction of nitrite requires an electron supply, and it is favored in acidic conditions. This reaction is critically affected by molecular oxygen, since oxyAtHb will catalyze nitric oxide deoxygenation. The process displays unique autocatalytic kinetics with metAtHb and nitrate as end-products for AtHb1 and AtHb2 but not for the truncated one, in contrast with mammalian globins.

Histidine Protonation States Regulate the State Transition from R State Hemoglobin.

The objective of our work is to investigate the impact of pH on the structural changes of hemoglobin that affect its O2 affinity, known as the Bohr effect. We conducted molecular dynamics (MD) simulations to explore the transition between various hemoglobin states based on the protonation states (PSs) of two histidine residues (ßHis143 and ßHis146). We conducted the MD simulations from the R and R2 states with three sets of PSs assuming pH values of 7.0, 6.5, and 5.5, aiming to investigate the influence of pH on hemoglobin behavior. Our results demonstrated that the protonated His residues promote the state transition from the R state to the R2 state and encourage elongation of the distance between the ß1-ß2 subunits by weakening the inter-subunit interactions in the R state. These observations, aligning with the experimental evidence, indicate that the R2 state typically crystallizes under low pH conditions. Our findings suggest that the relationship between the PSs and the structural stability of the R state plays a role in the acid and alkaline Bohr effect.

Rapid measurement of hemoglobin-oxygen dissociation by leveraging Bohr effect and Soret band bathochromic shift.

Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.

Investigating the influence of solvent type and pH on protein adsorption onto silica surface by evanescent-wave cavity ring-down spectroscopy.

Several studies have explored the adsorption of various proteins onto solid-liquid interfaces, revealing the crucial role of buffer solutions in biological processes. However, a comprehensive evaluation of the buffer's influence on protein absorption onto fused silica is still lacking. This study employs evanescent-wave cavity ring-down spectroscopy (EW-CRDS) to assess the influence of buffer solutions and pH on the adsorption kinetics of three globular proteins: hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt-C) onto fused silica. The EW-CRDS tool, with a ring-down time of 1.4 µ s and a minimum detectable absorbance of 1 × 10 - 6 , enabled precise optical measurements at solid-liquid interfaces. The three heme proteins' adsorption behavior was investigated at pH 7 in three different solvents: deionized (DI) water, tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), and phosphate buffered saline (PBS). For each protein, the surface coverage, the adsorption and desorption constants, and the surface equilibrium constant were optically measured by our EW-CRDS tool. Depending on the nature of each solvent, the proteins showed a completely different adsorption trend on the silica surface. The adsorption of Mb on the silica surface was depressed in the presence of both Tris-HCl and PBS buffers compared with unbuffered (DI water) solutions. In contrast, Cyt-C adsorption appears to be relatively unaffected by the choice of buffer, as it involves strong electrostatic interactions with the surface. Notably, Hb exhibits an opposite trend, with enhanced protein adsorption in the presence of Tris-HCl and PBS buffer. The pH investigations demonstrated that the electrostatic interactions between the proteins and the surface had a major influence on protein adsorption on the silica surface, with adsorption being greatest when the pH values were around the protein's isoelectric point. This study demonstrated the ability of the highly sensitive EW-CRDS tool to study the adsorption events of the evanescent-field-confined protein species in real-time at low surface coverages with fast resolution, making it a valuable tool for studying biomolecule kinetics at solid-liquid interfaces.

Layered assembly of PEGylated graphene oxide and Mg-Al layered double hydroxide nanosheets with superior adsorption capacity and selective isolation of hemoglobin.

This study developed a novel organic-inorganic hybrid composite, shortly as GO-PEG-LDHs, by self-assembly of exfoliated Mg-Al layer double hydroxide (LDHs) on the polyethylene glycol (PEG) grafted graphene oxide (GO) to achieve the selective adsorption of hemoglobin (Hb). The prepared GO-PEG-LDHs has a hierarchical structure with a homogeneous loading of exfoliated LDHs nano-sheets on its surface. The adsorption test reveals that GO-PEG-LDHs exhibits an adsorption efficiency of 95.03% for Hb and 3.45% for bovine serum albumin (BSA). The adsorption of Hb follows the Langmuir model, with an ultrahigh adsorption capacity of 55248.6 mg/g, which is higher than any previously reported materials. Meanwhile, the adsorbed Hb can be efficiently recovered through elution with a 50 mM Tris-HCl buffer, with an elution efficiency of 80.77%. Circular dichroism (CD) spectra indicate no conformational change for Hb during the process of adsorption/desorption. Furthermore, the composite demonstrates the ability to selectively isolate Hb in the presence of interfering protein BSA, indicating its potential for practical applications.

The Protection of Enzyme Activity for the Preparation of Humanized Polymerized Hemoglobin-Superoxide Dismutase-Catalase-Carbonic Anhydrase.

This work finds suitable enzyme activity protectants to improve the recovery rate of enzyme activity in the preparation of human polymerized hemoglobin-superoxide dismutase-catalase-carbonic anhydrase (PolyHb-SOD-CAT-CA), including trehalose, sucrose, glucose, hydroxypropyl-ß-cyclodextrin, and mannitol.Different types and concentrations of enzyme activity protective agents were added during polymerization to compare their protective ability to enzyme activity and the effect on the properties of hemoglobin. The study found that compared with trehalose, the protective effect of sucrose on CA enzyme activity is non-significant to that on hemoglobin, the recovery rate of SOD, and CAT enzyme activity has significant increased. Glucose, hydroxypropyl-ß-cyclodextrin, and mannitol are unsuitable for the added enzyme activity protective agent of PolyHb-SOD-CAT-CA.The protective effect of sucrose on CA was non-significant with trehalose. The protective effect of sucrose on SOD and CAT enzyme activity was higher than trehalose, and the protective effect reached the maximum when the concentration reached 1.5%.

Binding of Carbon Monoxide to Hemoglobin in an Oxygen Environment: Force Field Development for Molecular Dynamics.

Carbon monoxide (CO) is a byproduct of the incomplete combustion of carbon-based fuels, such as wood, coal, gasoline, or natural gas. As incomplete combustion in a fire accident or in an engine, massively produced CO leads to a serious life threat because CO competes with oxygen (O2) binding to hemoglobin and makes people suffer from hypoxia. Although there is hyperbaric O2 therapy for patients with CO poisoning, the nanoscale mechanism of CO dissociation in the O2-rich environment is not completely understood. In this study, we construct the classical force field parameters compatible with the CHARMM for simulating the coordination interactions between hemoglobin, CO, and O2, and use the force field to reveal the impact of O2 on the binding strength between hemoglobin and CO. Density functional theory and Car-Parrinello molecular dynamics simulations are used to obtain the bond energy and equilibrium geometry, and we used machine learning enabled via a feedforward neural network model to obtain the classical force field parameters. We used steered molecular dynamics simulations with a force field to characterize the mechanical strength of the hemoglobin-CO bond before rupture under different simulated O2-rich environments. The results show that as O2 approaches the Fe2+ of heme at a distance smaller than ∼2.8 Å, the coordination bond between CO and Fe2+ is reduced to 50% bond strength in terms of the peak force observed in the rupture process. This weakening effect is also shown by the free energy landscape measured by our metadynamics simulation. Our work suggests that the O2-rich environment around the hemoglobin-CO bond effectively weakens the bonding, so that designing of O2 delivery vector to the site is helpful for alleviating CO binding, which may shed light on de novo drug design for CO poisoning.

Nanoscale dynamical investigation of the hemoglobin complex with the bacterial protein IsdB: is their interaction stabilized by catch bonds?

Iron-regulated surface determinant B (IsdB) is a surface protein of Staphylococcus aureus that plays essential roles in host cell invasion by mediating both bacterial adhesion and hemic iron acquisition. Single-molecule experiments have recently revealed that the binding of IsdB to vitronectin and integrins is dramatically strengthened under mechanical stress conditions, promoting staphylococcal adhesion. Here we conducted atomic force spectroscopy (AFS) measurements of the interaction between IsdB and hemoglobin (Hb), in both its oxidized (metHb) and reduced forms (HbCO). While the former represents the natural substrate for IsdB, the latter is resistant to heme extraction. For the unbinding between IsdB and HbCO, we obtained a linear trend in the Bell-Evans plot, indicative of a weakening of the interaction upon mechanical stress. For the unbinding between IsdB and metHb, we found similar behavior at low loading rates. Remarkably, a non-linear trend of the complex interaction force was detected at higher force-pulling rates. Such behavior may provide some cues to the ability of IsdB to form stress-dependent bonds also with Hb, possibly enabling a more efficient heme transfer through stabilization of the transient (in vivo) IsdB-Hb complex.

Decomposition of 2,4-dihalophenols by dehaloperoxidase activity and spontaneous reaction with hydrogen peroxide.

The enzyme dehaloperoxidase (DHP) found in the marine worm Amphitrite ornata is capable of enzymatic peroxidation of 2,4-dichlorophenol (DCP) and 2,4-dibromophenol (DBP). There is also at least one parallel oxidative pathway and the major products 2-chloro-1,4-benzoquinone (2-ClQ) and 2-bromo-1,4-benzoquinone (2-BrQ) undergo aspontaneous secondary hydroxylation reaction. The oxidation and hydroxylation reactions have been monitored by UV-visible spectroscopy, High Performance Liquid Chromatography (HPLC), and mass spectrometry. Evidence from time-resolved UV-visible spectroscopy suggests that the hydroxylations of 2-ClQ and 2-BrQ in the presence of hydrogen peroxide (H2O2) are non-enzymatic spontaneous processes approximately ∼10 and âˆ¼ 5 times slower, respectively, than the enzymatic oxidation of DCP or DBP by DHP in identical solvent conditions. The products 2-ClQ and 2-BrQ have λmaxat 255 nm and 260 nm, respectively. Both substrates, DCP and DBP, react to form a parallel product peaked at 240 nm on the same time scale as the formation of 2-ClQ and 2-BrQ. The 240 nm band is not associated with the hydroxylation process, nor is it attributable to the catechol 3,5-dihalobenzene-1,3-diol observed by mass spectrometry. One possible explanation is that muconic acid is formed as a decomposition product, which could follow decomposition either the catechol or hydroxyquinone. These reactions give a more complete understanding of the biodegradation of xenobiotics by the multi-functional hemoglobin, DHP, in Amphitrite ornata. SYNOPSIS: The decomposition of 2,4-dihalophenols catalyzed by dehaloperoxidase was studied by UV-visible spectroscopy, High Performance Liquid Chromatography and Liquid Chromatography-Mass Spectrometry. Spectroscopic evidence suggests two major products, which we propose are 2-halo-1,4-benzoquinone and 2-halomuconic acid. These complementary techniques give a high-level view of the degradation of xenobiotics in marine ecosystems.

The interaction of perfluorooctane sulfonate with hemoproteins and its relevance to molecular toxicology.

Perfluorooctane sulfonate (PFOS), a representative of perfluorinated compounds in industrial and commercial products, has posed a great threat to animals and humans via environmental exposure and dietary consumption. Herein, we investigated the effects of PFOS binding on the redox state and stability of two hemoproteins (hemoglobin (Hb) and myoglobin (Mb)). Fluorescence spectroscopy, circular dichroism and UV-vis absorption spectroscopy demonstrated that PFOS could induce the conformational changes of proteins along with the exposure of heme cavity and generation of hemichrome, which resulted in the increased release of free hemin. After that, free hemin liberated from hemoproteins led to reactive oxygen species formation, lipid peroxidation, cell membrane damage and loss of cell viability in vascular endothelial cells, while neither Hb nor Mb did show cytotoxicity. Chemical inhibitors of ferroptosis effectively mitigated hemin-caused toxicity, identifying the hemin-dependent ferroptotic cell death mechanisms. These data demonstrated that PFOS posed a potential threat of toxicity through a mechanism which involved its binding to hemoproteins, decreased oxygen transporting capacity, and increased hemin release. Altogether, our findings elucidate the binding mechanisms of PFOS with two hemoproteins, as well as possible risks on vascular endothelial cells, which would have important implications for the human and environmental toxicity of PFOS.