Search

Antioxidant assays are typically based on non-physiologically relevant reagents. We describe here a quantitative assay based on the inhibition of the liposome autooxidation in the presence of myoglobin (ILA-Mb), an oxidative process with direct biomedical relevance. Additional advantages of the assay include the use of standard and readily available reagents (lecithin and myoglobin) and the applicability to lipophilic antioxidants. The ILA-Mb assay is based on previously reported qualitative or semi-quantitative ones that employed cytochrome c instead of myoglobin. A number of antioxidants are tested, and their IC 50 parameters are discussed and interpreted to involve direct interaction with both myoglobin and the liposomes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Radu Silaghi-Dumitrescu reports financial support was provided by Romanian Government Ministry of Research Innovation and Digitization. Alina Matei reports financial support was provided by European Social Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The NO dioxygenation reaction catalyzed by heme-containing globin proteins is a crucial aerobic detoxification pathway. Accordingly, the second order reaction of NO with oxymyoglobin and oxyhemoglobin has been the focus of a large number of kinetic and spectroscopic studies. Stopped-flow and rapid-freeze-quench (RFQ) measurements have provided evidence for the formation of a Fe(III)-nitrato complex with millisecond lifetime prior to release of the nitrate product, but the temporal resolution of these techniques is insufficient for the characterization of precursor species. Most mechanistic models assume the formation of an initial Fe(III)-peroxynitrite species prior to homolytic cleavage of the OO bond and recombination of the resulting NO 2 and Fe(IV)=O species. Here we report vibrational spectroscopy measurements for the reaction of oxymyoglobin with a photolabile caged NO donor at cryogenic temperatures. We show that this approach offers efficient formation and trapping of the Fe(III)-nitrato, enzyme-product, complex at 180 K. Resonance Raman spectra of the Fe(III)-nitrato complex trapped via RFQ in the liquid phase and photolabile NO release at cryogenic temperatures are indistinguishable, demonstrating the complementarity of these approaches. Caged NO is released by irradiation <180 K but diffusion into the heme pocket is fully inhibited. Our data provide no evidence for Fe(III)-peroxynitrite of Fe(IV)=O species, supporting low activation energies for the NO to nitrate conversion at the oxymyoglobin reaction site. Photorelease of NO at cryogenic temperatures allows monitoring of the reaction by transmittance FTIR which provides valuable quantitative information and promising prospects for the detection of protein sidechain reorganization events in NO-reacting metalloenzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Carbene transfer reactions have emerged as pivotal methodologies for the synthesis of complex molecular architectures. Heme protein-catalyzed carbene transfer reactions have shown promising results on model compounds. However, their limited substrate scope has hindered their application in natural product functionalization. Building upon the foundation of previously published work on a carbene transferase-myoglobin variant, this study employs computer-aided protein engineering to design myoglobin variants, using either docking or the deep learning-based LigandMPNN method. These variants were utilized as catalysts in carbene transfer reactions with a selection of monoterpene substrates featuring C-C double bonds, leading to seven target products. This cost-effective methodology broadens the substrate scope for heme protein-catalyzed reactions, thereby opening novel pathways for research in heme protein functionalities and offering fresh perspectives in the synthesis of bioactive molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Carbon monoxide (CO) is an innate signaling molecule that can regulate immune responses and interact with crucial elements of the circadian clock. Moreover, pharmacologically, CO has been substantiated for its therapeutic advantages in animal models of diverse pathological conditions. Given that an excessive level of CO can be toxic, it is imperative to quantify the necessary amount for therapeutic use accurately. However, estimating gaseous CO is notably challenging. Therefore, novel techniques are essential to quantify CO in therapeutic applications and overcome this obstacle precisely. The classical Myoglobin (Mb) assay technique has been extensively used to determine the amount of CO-release from CO-releasing molecules (CORMs) within therapeutic contexts. Nevertheless, specific challenges arise when applying the Mb assay to evaluate CORMs featuring innovative molecular architectures. Here, we report a fluorinated photo-CORM (CORM-FBS) for the photo-induced CO-release. We employed the 19 F NMR spectroscopy approach to monitor the release of CO as well as quantitative evaluation of CO release. This new 19 F NMR approach opens immense opportunities for researchers to develop reliable techniques for identifying molecular structures, quantitative studies of drug metabolism, and monitoring the reaction process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Reaction between the polymeric [RuCl 2 (CO) 2 ] n and the N,N-bidentate ligand, 8-amino-quinoline (Quin), in methanol, afforded the photoactivated CO releasing molecule with the formula of trans-(Cl,Cl)-[RuCl 2 (CO) 2 Quin]. In the presence of biomolecules or in solvents with varying polarity and coordinating abilities, the solvatochromic characteristics and dark stability were investigated. A new board band emerged in the visible spectrum during the illumination, and its position varies according to the type of solvent used, indicating the role of the solvent in controlling the nature of the CO-depleted species. Spectral methods were used in combination with density functional theory simulations to get insight into the local minimum structure and the electronic properties of the Ru(II) complex. The results of the myoglobin assay showed that within the first two hours of illumination, one of the two CO molecules was released. The cytotoxic properties of the Ru(II)-based complex were investigated against normal mice bone marrow stromal cells and malignant human acute monocytic leukaemia cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

New-to-Nature biocatalysis has emerged as a promising tool in organic synthesis thanks to progress in protein engineering. Notably, hemeproteins have been evolved into robust catalysts for carbene and nitrene transfers and related sigmatropic rearrangements. In this work, we report the first example of a [2,3]-sigmatropic Sommelet-Hauser rearrangement initiated by a carbene transfer of the sperm whale myoglobin mutant L29S,H64V,V68F that was previously reported to catalyze the mechanistically similar [2,3]-sigmatropic Doyle-Kirmse rearrangement. This repurposed heme enzyme catalyzes the Sommelet-Hauser rearrangement between ethyl diazoacetate and benzyl thioethers bearing strong electron-withdrawing substituents with good yields and enantiomeric excess. Optimized catalytic conditions in the absence of any reductant led to an increased asymmetric induction with up to 59% enantiomeric excess. This myoglobin mutant is therefore one of the few catalysts for the asymmetric Sommelet-Hauser rearrangement. This work broadens the scope of abiological reactions catalyzed by iron-carbene transferases with a new example of asymmetric sigmatropic rearrangement., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this paper, the effects of chitosan film containing star anise essential oil nanofiltration (CFSAO) and superchilled (SC) temperature on the changes of physicochemical and microbiological indexes of rabbit meat patties within 15 days of storage were studied. The total aerobic bacteria counts, malondialdehyde content, protein carbonyl content, total sulfhydryl content, and metmyoglobin content continued to grow throughout the entire experimental period, and the maximum absorption peak at the soret region of myoglobin gradually decreased. Along with the storage time extended, the brightness and redness of rabbit meat significantly decreased (P < 0.05), while the yellowness significantly increased (P < 0.05). The results of storage experiments showed that chitosan composite films and SC temperature had good inhibition on lipid oxidation, myoglobin oxidation and degradation, sulfhydryl content reduction, and microbial growth of rabbit meat after 15 days of storage, and could slow down the change of rabbit meat color., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Carbon nanomaterials (CNMs), including carbon quantum dots (CQDs), have found widespread use in biomedical research due to their low toxicity, chemical tunability, and tailored applications. Yet, there exists a gap in our understanding of the molecular interactions between biomacromolecules and these novel carbon-centered platforms. Using gelatin-derived CQDs as a model CNM, we have examined the impact of this exemplar nanomaterial on apo-myoglobin (apo-Mb), an oxygen-storage protein. Intrinsic fluorescence measurements revealed that the CQDs induced conformational changes in the tertiary structure of native, partially unfolded, and unfolded states of apo-Mb. Titration with CQDs also resulted in significant changes in the secondary structural elements in both native (holo) and apo-Mb, as evidenced by the circular dichroism (CD) analyses. These changes suggested a transition from isolated helices to coiled-coils during the loss of the helical structure of the apo-protein. Infra-red spectroscopic data further underscored the interactions between the CQDs and the amide backbone of apo-myoglobin. Importantly, the CQDs-driven structural perturbations resulted in compromised heme binding to apo-myoglobin and, therefore, potentially can attenuate oxygen storage and diffusion. However, a cytotoxicity assay demonstrated the continued viability of neuroblastoma cells exposed to these carbon nanomaterials. These results, for the first time, provide a molecular roadmap of the interplay between carbon-based nanomaterial frameworks and biomacromolecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sulfonamide antibiotics, a family of broad-spectrum antibiotic drugs, are increasingly used in aquaculture and are frequently detected in aquatic environments. This poses a potential threat to organisms and may cause the evolution of antimicrobial resistance. Therefore, it is important to develop an environmentally friendly and efficient biocatalyst to degrade sulfonamides (SAs) such as sulfadiazine (SD) and sulfathiazole (ST). Here, we realized the direct and efficient degradation of SD and ST using a hydrogen peroxide-dependent artificial catalytic system based on myoglobin (Mb). The arrangements of amino acids at positions 29, 43, 64, and 68 were found to influence catalytic activity. An L29H/H64D/V68I myoglobin mutant showed the best catalytic efficiency (i.e., k cat /K m = 720.42 M -1 s -1 ) against SD. Next, mutant H64D/V68I showed the best degradation rate against SD (i.e., 91.45 ± 0.16%). Moreover, L29H/H64D/V68I Mb was found to efficiently catalyze ST oxidation (k cat /K m = 670.08 M -1 s -1 ), while H64D/V68I had the best degradation rate against ST (i.e., 99.45 ± 0.23%). Our results demonstrate that SAs can be efficiently degraded by artificial peroxygenases constructed using a myoglobin scaffold. This therefore provides a simple and economical method for the biodegradation of SD and ST., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

C-H bond amination is an effective way to obtain nitrogen-containing products. In this work, we demonstrate that myoglobin reconstituted with iron porphycene (rMb(FePc)) catalyzes intramolecular C(sp 3 )-H bond amination of arylsulfonyl azides to yield corresponding sultam analogs. The total turnover number of rMb(FePc) is up to 5.7 × 10 4 for the C-H bond amination of 2,4,6-triisopropylbenzenesulfonyl azide. Moreover, rMb(FePc) exhibits higher selectivity for the desired C-H bond amination than the competing azide reduction compared to native myoglobin. Kinetic studies reveal that the k cat value of rMb(FePc) is 4-fold higher than that of native myoglobin. Furthermore, H64A, H64V and H64I mutants of rMb(FePc) enhance the turnover number (TON) and enantioselectivity for the C-H bond amination of 2,4,6-triethylbenzenesulfonyl azide. The present findings indicate that iron porphycene is an attractive artificial cofactor for myoglobin toward the C-H bond amination reaction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Most hemoproteins display an all-α-helical fold, showing the classical three on three (3/3) globin structural arrangement characterized by seven or eight α-helical segments that form a sandwich around the heme. Over the last decade, a completely distinct class of heme-proteins called nitrobindins (Nbs), which display an all-β-barrel fold, has been identified and characterized from both structural and functional perspectives. Nbs are ten-stranded anti-parallel all-β-barrel heme-proteins found across the evolutionary ladder, from bacteria to Homo sapiens. Myoglobin (Mb), commonly regarded as the prototype of monomeric all-α-helical globins, is involved along with the oligomeric hemoglobin (Hb) in diatomic gas transport, storage, and sensing, as well as in the detoxification of reactive nitrogen and oxygen species. On the other hand, the function(s) of Nbs is still obscure, even though it has been postulated that they might participate to O 2 /NO signaling and metabolism. This function might be of the utmost importance in poorly oxygenated tissues, such as the eye's retina, where a delicate balance between oxygenation and blood flow (regulated by NO) is crucial. Dysfunction in this balance is associated with several pathological conditions, such as glaucoma and diabetic retinopathy. Here a detailed comparison of the structural, spectroscopic, and functional properties of Mb and Nbs is reported to shed light on the similarities and differences between all-α-helical and all-β-barrel heme-proteins., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nitrobindins (Nbs) represent an evolutionary conserved all-β-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom, coordinated by a proximal His residue. Interestingly, even if the distal side is exposed to the solvent, the value of the second order rate constants for ligand binding to the ferrous derivative is almost one order of magnitude lower than those reported for myoglobins (Mbs). Noteworthy, nitric oxide binding to the sixth coordination position of the heme-Fe(II)-atom causes the cleavage or the severe weakening of the proximal His-Fe(II) bond. Here, we provide a computer simulation investigation to shed light on the molecular basis of ligand binding kinetics, by dissecting the ligand binding process into the ligand migration and the bond formation steps. Classical molecular dynamics simulations were performed employing a steered molecular dynamics approach and the Jarzinski equality to obtain ligand migration free energy profiles. The formation of the heme-Fe(II)-NO bond took into consideration the iron atom displacement from the heme plane. The ligand migration is almost unhindered, and the low rate constant for NO binding is due to the large displacement of the Fe(II) atom with respect to the heme plane responsible for the barrier for the Fe(II)-NO bond formation. In addition, we investigated the weakening and breaking of the proximal His-Fe(II) bond, observed experimentally upon NO binding, by means of a combination of classical molecular dynamics simulations and quantum-classical (QM-MM) optimizations. In both human and M. tuberculosis Nbs, a stable alternative conformation of the proximal His residue interacting with a network of water molecules was observed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Kinetic and structural investigations of the flavohemoglobin-type NO dioxygenase have suggested critical roles for transient Fe(III)O 2 complex formation and O 2 -forced movements affecting hydride transfer to the FAD cofactor and electron-transfer to the Fe(III)O 2 complex. Stark-effect theory together with structural models and dipole and internal electrostatic field determinations provided a semi-quantitative spectroscopic method for investigating the proposed Fe(III)O 2 complex and O 2 -forced movements. Deoxygenation of the enzyme causes Stark effects on the ferric heme Soret and charge-transfer bands revealing the Fe(III)O 2 complex. Deoxygenation also elicits Stark effects on the FAD that expose forces and motions that create a more restricted NADH access to FAD for hydride transfer and switch electron-transfer off. Glucose also forces the enzyme toward an off state. Amino acid substitutions at the B10, E7, E11, G8, D5, and F7 positions influence the Stark effects of O 2 on resting heme spin states and FAD consistent with the proposed roles of the side chains in the enzyme mechanism. Deoxygenation of ferric myoglobin and hemoglobin A also induces Stark effects on the hemes suggesting a common 'oxy-met' state. The ferric myoglobin and hemoglobin heme spectra are also glucose-responsive. A conserved glucose or glucose-6-phosphate binding site is found bridging the BC-corner and G-helix in flavohemoglobin and myoglobin suggesting novel allosteric effector roles for glucose or glucose-6-phosphate in the NO dioxygenase and O 2 storage functions. The results support the proposed roles of a ferric O 2 intermediate and protein motions in regulating electron-transfer during NO dioxygenase turnover., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Apomyoglobin (apoMb), a model protein in biochemistry, exhibits a strong propensity to bind various ligands, which makes it a good candidate as a carrier of bioactive hydrophobic drugs. The stability of its hydrophobic pocket determines its potential as a carrier of bioactive compounds. High pressure (HP) is a potent tool for studying protein stability, revealing the specific role of hydrophobic cavities in unfolding. We probed the effects of biliverdin (BV) binding and its complex with Zn 2+ ions on the structure and HP stability of apoMb. CD spectroscopy and SAXS measurements revealed that BV and BV-Zn 2+ complexes make the apoMb structure more compact with higher α-helical content. We performed in situ HP measurements of apoMb intrinsic fluorescence to demonstrate the ability of BV to stabilise apoMb structure at HP conditions. Furthermore, the presence of Zn 2+ within the apoMb-BV complex significantly enhances the BV stabilisation effect. In situ visible absorption study of BV chromophore confirmed the ability of Zn 2+ to increase the stability of apoMb-BV complex under HP: the onset of complex dissociation is shifted by ∼100 MPa in presence of Zn 2+ . By combining HP-fluorescence and HP-visible absorption spectroscopy, our strategy highlights the crucial role of tetrapyrrole-metal complexes in stabilising apoMb hydrophobic pocket., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

X-ray fluorescence holography (XFH) is a relatively new technique capable of providing unique three-dimensional structural information around specific atoms that act as a light source in crystalline samples. So far, XFH has typically been applied to inorganic materials such as dopants in metals and semiconductors. Here, we investigate the possibility of using XFH to visualize the metal active site in sperm whale myoglobin (Mb), a monomeric oxygen storage heme protein. We demonstrate that the atomic images reconstructed from the hologram data of crystals of carbonmonoxy myoglobin (MbCO) are moderately consistent with the crystal structure, which is also determined in this study by X-ray crystallography in the near-atomic resolution, as well as simulation results. These results open up a new avenue for the application of XFH to local atomic and electronic structure imaging of metal-sites in biomolecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We examine Hofmeister specific ion effects of electrolytes added to protein solution under conditions minimizing electrostatic attraction between cations and positively charged protein. Hemoglobin (Hb) in aqueous solution at the denaturing pH = 2.7 is investigated in the presence of several metal chlorides, along with sodium and potassium bromides, iodides and thiocyanates, using electrospray ionization mass spectrometry (ESI-MS). Salt concentration was varied to maximize peak intensity and bell-shaped profile in the ESI-MS spectrum. The α-chain of myoglobin is identified as the main pattern of the ESI-MS spectra in all Hb-salt systems. Both peak intensity and quality of the bell-shaped profile of the protein spectrum decrease in the cation order: K + > > Mg 2+  > Li + > > Na +  > Ca 2+  ≈ Cs +  > Rb + for Hb-Metal Chloride systems, and decrease in the anion order: Cl -  > Br -  > I -  > SCN - for systems of both Hb-NaX and Hb-KX salts. To quantify salt addition effects two Hofmeister specific electrolyte parameters H S , and P S are proposed. H S is the mean (Hb-salt)/Hb peak intensity ratio, measured for the nine peaks used for ESI-MS spectra deconvolution, taken at the same m/z values of the Hb profile. P S is the ratio between H S standard deviation and H S , and provides a specific perturbation parameter measuring the loss of protein structure. These two Hofmeister parameters give clear evidence of the effects induced either by KCl, MgCl 2 and LiCl that enhance protein peak intensity, or by NaBr, NaI, NaSCN and KSCN that induce the protein fragmentation, due to electrolyte-mediated dissociation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dehaloperoxidase (DHP) is a multi-functional catalytic globin from the marine worm A. ornata, whose physiological functions include oxygen transport and oxidation of toxic substrates present in its habitat. In the Fe(III) state, DHPA has an isomer shift of 0.42 mm/s, characteristic for high-spin heme proteins. Changes in pH have subtle effects on the electronic structure of DHP in the Fe(III) state detectable in the high-field spectra, which show a pH-dependent mixture of species with different zero-field splittings between 5 and 18 cm -1 . The short-lived intermediate obtained by direct reaction of the Fe(III) enzyme with H 2 O 2 has an isomer shift of 0.10 mm/s, indicative of an Fe(IV)-oxo state and of an S = 1 electronic ground state confirmed by variable field studies. The O 2 -bound state of DHP has an isomer shift of 0.28 mm/s and a high-field spectrum characteristic for diamagnetic heme complexes, similarly to other haemoglobins. Overall, the isomer shift and quadrupole splitting of DHP in the four states studied are expectedly similar to both peroxidases and to myoglobin. The differences in electronic structure between DHP and other heme proteins and enzyme are observed in the high-field Mössbauer spectra of the ferric state, which show pH-dependent zero-field splittings suggesting a heme site in which the ligand field strength at the iron ion is tuned by pH. This tunability is correlated with variable electron-donating properties of the iron, which can perform multiple functions., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Globins play a key role in regulating nitric oxide (NO) levels in all forms of life. Five key reactions of NO with mammalian muscle myoglobin (Mb) and red blood cell hemoglobin (Hb) have been examined: (1) reversible NO binding to Fe(II) forms; (2) reversible NO binding to Fe(III) forms; (3) NO dioxygenation by Fe(II)O 2 complexes; (4) autoxidation of Fe(II)NO complexes in the presence of O 2 ; and (5) autoreduction of Fe(III)NO complexes. NO reacts rapidly and almost irreversibly with deoxyMb(FeII) in the absence of O 2 , whereas it reacts much more slowly and weakly with metMb(FeIII). The reaction of NO with Mb(FeII)O 2 is very rapid and results in oxidation of the iron atom and dioxygenation of NO to nitrate. Autoxidation of Mb(FeII)NO in air is determined by the slow rate of NO dissociation from the Fe(II)NO complex, which is followed by rapid O 2 binding to the newly formed deoxyMb(FeII) and dioxygenation of the displaced NO to generate NO 3 - and metMb(FeIII). MetMb(FeIII)NO autoreduces slowly by addition of a hydroxide ion to bound NO to generate nitrous acid and reduced deoxyMb(FeII), which immediately binds another NO to generate Mb(FeII)NO as the final product. The reverse of this process involves nitrite reduction to NO by deoxyMb(FeII), which can occur on physiological time scales when the globin concentration is in the millimolar range. The relevance of these processes to the regulation of NO metabolism by hemoglobins and myoglobins in humans and other organisms is discussed., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The heme propionates in myoglobin (Mb) form a H-bonding network among several residues within its second-sphere coordination, providing a key structural role towards Mb's functional properties. Our work aims to understand the role of the heme propionates on the nitrite reductase (NiR) activity (e.g. reduction of NO 2 - to NO) of this globin by studying an artificial dimethylester heme-substituted horse heart Mb (DME-Mb). The minor structural change brought about by esterification of the heme propionates causes the NiR rate to increase by more than over two-fold (5.6 ± 0.1 M -1  s -1 ) relative to wildtype (wt) Mb (2.3 ± 0.1 M -1  s -1 ). The lower pK a observed in DME-Mb may enhance the tendency of His64 towards protonation, therefore increasing the NiR rate. In addition, the nitrite binding constant (K nitrite ) for DME-Mb III is greater than wt Mb III (350 M -1 versus 120 M -1 ). The disparity in the NiR activity correlates with the differences in electrostatic behavior, which influences the system's reactivity towards the approaching NO 2 - ion, and thus the formation of the Fe II -NO 2 - intermediate., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Methylglyoxal (MG) is a highly reactive α-dicarbonyl compound which reacts with proteins to form advanced glycation end products (AGEs). MG-induced AGE (MAGE) formation is particularly significant in diabetic condition. In the current study, we have undertaken a time-dependant characterization of MG-modified myoglobin following incubation of the heme protein with the α-dicarbonyl compound for different time periods. Interestingly, mass spectrometric studies indicated modifications at two specific lysine residues, Lys-87 and Lys-133. The AGE adducts identified at Lys-87 were carboxymethyllysine and carboxyethyllysine, while those detected at Lys-133 included pyrraline-carboxymethyllysine and carboxyethyllysine, respectively. Far-UV CD studies revealed a decrease in the native α-helical content of the heme protein gradually with increasing time of MG incubation. In addition, MG modification was found to induce changes in tertiary structure as well as surface hydrophobicity of the heme protein. MG-derived AGE adducts thus appear to alter the structure of Mb considerably. Considering the increased level of MG in diabetic condition, the current study appears physiologically relevant in terms of understanding AGE-mediated protein modification and subsequent structural changes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Myoglobin is an essential transport protein of heart and muscle tissues that acts as a local oxygen reservoir and a marker in different diseased conditions. On the other hand, Vitamin B12 is a vital nutrient that helps synthesize red blood cells, DNA, and proteins. To understand the ability of vitamin B12 to bind to the excess of myoglobin produced in the body under certain conditions (muscle injuries, severe trauma, etc.), it is essential to dig into the interaction between them. Therefore, the present study reports the binding interaction of vitamin B12 and myoglobin employing different spectroscopic and computational methods. The myoglobin's intrinsic fluorescence is quenched by vitamin B12 via static nature as observed from steady-state as well as time-resolved fluorescence measurements. The microenvironment of myoglobin's tryptophan residue gets affected, but there is no change observed in its α-helical content by vitamin B12 as seen from synchronous fluorescence and circular dichroism measurements. The probable binding of vitamin B12 on myoglobin was elucidated through molecular docking, and the interaction stability was studied by molecular dynamics simulation. The determination of vitamin B12's affinity to myoglobin and its effect on the conformational transitions of myoglobin might afford valuable insight for clinical pharmacology., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Macromolecular crowding can have significant consequences on the structure and dynamics of a protein. The size and shape of a co-solute molecule and the nature of protein contribute significantly in macromolecular crowding, which results in different outcomes in similar conditions. The structure of apo-myoglobin (apo-Mb) both in the absence and presence of denaturants (GdmCl and urea) was investigated in crowded conditions at pH 7.0, with a comparable size of crowders (~70 kDa) but of different shapes (ficoll and dextran) at various concentrations using spectroscopic techniques like absorption and circular dichroism to monitor changes in secondary and tertiary structure, respectively. The crowders in the absence of denaturants showed structural stabilization of the tertiary structure while no significant change in the secondary structure was observed. The effect of crowders on the stability of the protein was also investigated using probes such as Δε 291 and θ 222 using chemical denaturants. The analysis of chemical-induced denaturation curves showed that both the crowders stabilize apo-Mb by increasing the values of the midpoint of transition (C m ) and change in free energy in the absence of denaturant (∆G D °), and it was observed that dextran 70 shows more stabilization than ficoll 70 under similar conditions. In this study apo-Mb showed stabilization under crowded conditions, which is a deviation from earlier work from our group where holo form of the same protein was destabilized. This study emphasizes that volume exclusion is a dominant force in a simple protein while soft interactions may play important role in the proteins that are possessing prosthetic group. Hence, the effect of crowders is protein-dependent, and excluded volume plays a great role in the stabilization of apo-Mb, which does not interact with the crowders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The metal active site is precisely designed in metalloproteins. Here we applied 3D domain swapping, a phenomenon in which a partial protein structure is exchanged between molecules, to introduce metal sites in proteins. We designed multiple metal-binding sites specific to domain-swapped myoglobin (Mb) with His mutation. Stable dimeric Mbs with metal-binding sites were obtained by shifting the His position and introducing two Ala residues in the hinge region (K78H/G80A/H82A and K79H/G80A/H81A Mbs). The absorption and circular dichroism spectra of the monomer and dimer of K78H/G80A/H82A and K79H/G80A/H81A Mbs were similar to the corresponding spectra, respectively, of wild-type Mb. No negative peak due to dimer-to-monomer dissociation was observed below the denaturation temperature in the differential scanning calorimetry thermograms of K78H/G80A/H82A and K79H/G80A/H81A Mbs, whereas the dimer dissociates into monomers at 68 °C for wild-type Mb. These results show that the two mutants were stable in the dimer state. Metal ions bound to the metal-binding sites containing the introduced His in the domain-swapped Mb dimers. Co 2+ -bound and Ni 2+ -bound K78H/G80A/H82A Mb exhibited octahedral metal-coordination structures, where His78, His81, Glu85, and three H 2 O/OH - molecules coordinated to the metal ion. On the other hand, Co 2+ -bound and Zn 2+ -bound K79H/G80A/H81A Mb exhibited tetrahedral metal-coordination structures, where His79, His82, Asp141, and a H 2 O/OH - molecule coordinated to the metal ion. The Co 2+ -bound site exists deep inside the protein in the K79H/G80A/H81A Mb dimer, which may allow the unique tetrahedral coordination for the Co 2+ ion. These results show that we can utilize domain swapping to construct artificial metalloproteins., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Developing safe and efficient iron supplements is significant for the alleviation of iron-deficient anemia (IDA). Myoglobin (Mb) is a heme-protein rich in bioavailable iron. Pneumatophorus japonicus (P. japonicus), one important economic fish in China, contain a high Mb level in its dark meat normally discarded during processing. The present study aimed to determine the structure, physicochemical properties, and iron bioavailability of Mb extracted from P. japonicus. Meanwhile, the effects of glycosylation, a commonly applied chemical modification of proteins, on these parameters were evaluated. Using Box-Behnken design, the optimal conditions for Mb-chitosan glycosylation were obtained: 45.07 °C, pH 6.10 and Mb/chitosan mass ratio of 6.29. The structure and functional properties of the glycosylated Mb (Mb-gly) were investigated. Compared with the original Mb, Mb-gly obtained a more ordered secondary structure. The surface hydrophobicity of Mb-gly was found to be decreased together with the observations of elevated water solubility. Moreover, glycosylation enhanced the Mb antioxidant capacity, and improved its stability in enzymatic digestion system. Regarding to the iron bioavailability, the cellular uptake of Mb‑iron was significantly higher than FeSO 4 , and further elevated by glycosylation. These results provided a basis for the development of Mb-based iron supplements, promoting the utilization of fish-processing industries wastes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Human Islet Amyloid Polypeptide (hIAPP) or amylin, can bind heme and the resultant complexes are prone to generate partially reduced oxygen species (PROS). The formation of PROS and the related oxidative stress highlight the importance of Heme-hIAPP in the onset and development of Type 2 Diabetes mellitus (T2Dm) in humans. In this study, the interaction of Heme-hIAPP with apomyoglobin (ApoMb) has been investigated using a combination of spectroscopic and electrophoresis techniques. Absorption, resonance Raman data and gel electrophoresis results confirm that ApoMb can uptake heme from Heme-hIAPP and constitute a six-coordinate high-spin ferric heme active site identical to that of myoglobin (Mb). The heme transfer reaction has two distinct kinetic steps. A possible mechanism of this reaction involves heme transfer to the apoprotein in the first step followed by a reorganisation of the protein chain to form the active site of native Mb. Increase in the pH of the reaction medium enhances the rate of the second step of heme transfer. This possibly corresponds to the deprotonation of a propionate side chain of the heme moiety at high pH which facilitates secondary interactions with the conserved distal Lys45 residue of horse heart Mb. Additionally, ApoMb sequesters ligand bound heme from Heme-hIAPP. After the heme transfer reaction, the amount of PROS formed by Heme-hIAPP complex diminishes significantly. This not only potentially diminishes heme-induced toxicity in the pancreatic β-cells but also produces Mb which has well-documented functions throughout the respiratory system and can thereby likely reduce the risks associated with T2Dm., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The effects of the retail display temperature (8 °C, 3 °C and - 1 °C) on the discoloration of the Longissimus thoracis et lumborum of rabbits and the associations among such effects with microbial spoilage, myoglobin autoxidation, lipid oxidation, and protein oxidation were investigated. The total aerobic count, total volatile basic nitrogen content, metmyoglobin content, protein carbonyl content, and contents of thiobarbituric acid-reactive substances steadily increased during retail display. Moreover, the lightness and redness of the rabbit meat significantly (P < 0.05) declined over time, whereas the yellowness increased considerably (P < 0.05) with prolonged retail time. Canonical correlation analysis suggested that microbial spoilage, myoglobin autoxidation, lipid oxidation, and protein oxidation jointly affected rabbit meat color. Linear mixed models further revealed that microbial spoilage, myoglobin autoxidation, lipid oxidation and protein oxidation positively affected yellowness, and they inversely impacted lightness and redness., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nitrite (0.25-2%) incorporated thermoplastic starch (TPS) and linear low-density polyethylene (LLDPE) blend films were produced by the conventional blown-film extrusion process. Films were characterized and determined for efficacy as active packaging for pork steak. Scanning electron micrographs showed enhanced starch granule disruption due to melting. Increased nitrite contents enhanced the starch network and improved mechanical properties. Water vapor and barrier properties of blend films were improved by nitrite incorporation, with increased compatibility between TPS and LLDPE networks. Films containing 0.5% nitrite effectively improved redness and corresponded to 0.06 ppm residual nitrite and 75 ppm nitrosomyoglobin in packaged pork. Nitrite addition modified protein secondary conformation involving CO stretching bonds via H-bonding, while films with 1% and 2% nitrite significantly inhibited growth of lactic acid bacteria, yeast and mold, and retained softer texture during storage. Nitrite incorporated films were demonstrated as efficient active packaging to improve the quality of red meat products., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Screening and monitoring endogenous peptides from complicated biosamples is still a major challenge in mass spectrometry-based proteomics research, mainly due to their low concentration and the interference of high-abundance proteins and other contaminants in biological samples. Herein, a facile and novel approach was described for rapid fabrication of C 18 -functionalized magnetic nanocomposites (C 18 -MNCs) based on one-step aqueous coating of C 18 -Val-Lys-Val-Lys-Val-Lys (C 18 -VK-VI) for the highly selective enrichment of low-abundance endogenous peptides from biological samples. C 18 -VK-VI can readily self-assemble into complete monolayers mainly composed of β-sheets with C 18 hydrophobic chains erecting on the surface of GO@Fe 3 O 4 MNCs under the physiological conditions. The resulting C 18 VK-VI-GO@Fe 3 O 4 MNCs exhibited good performance for peptides enrichment from digests of standard protein (myoglobin, MYO) and human serum, such as high sensitivity (0.05 fmol μL -1 ) and selectivity (mass ratio of MYO digests and MYO = 1:500), rapid separation, and good reproducibility. Such a simple mild and rapid one-step aqueous coating method on the basis of oligopeptides self-assembly showed great potential in surface functionalization of various nanoadsorbents for proteome/peptidome researches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Consumers consider beef color to be an indicator of freshness and therefore it is a major factor when purchasing beef. The ideal conditions for maintaining color throughout retail display following frozen storage have yet to be well established. Therefore, the objective of this study was to determine the effects of myoglobin oxygenation levels (especially high oxygenation) during freezing on color stability of thawed steaks during retail display (RD) and to determine the impact that frozen storage duration, aging, and packaging films have on meat color after thawing under RD conditions. USDA Choice strip loins (n = 36) were aged for 4 or 20 d. Steaks were randomly assigned to a myoglobin oxygenation level [deoxymyoglobin (DeOxy; packaged within 3 min), oxygenation (Oxy; oxygenated in air for 30 min), or high oxygenation (HiOxy; packaged for 24 h in 80% O 2 )]. Steaks were then vacuum packaged in oxygen permeable or impermeable film and immediately frozen (-10 °C). Following either 0, 2, 4, or 6 months of frozen storage at -5 °C, steaks were removed from the packaging and immediately placed under simulated RD conditions for 7 d. During RD, instrumental color and subjective color were measured every day after the initial 24 h thaw period. Steaks were analyzed for instrumental color (L*, a*, b*), a*:b* ratio, percentage oxymyoglobin, metmyoglobin, and deoxymyoglobin, delta E, redness ratio, subjective discoloration, and lipid oxidation. For all days of RD, steaks that were frozen for 0 months had higher a* values (greater redness) than steaks frozen for 6 months which typically had the lowest a* values (P < .0001). HiOxy steaks frozen for 6 months had the lowest amounts of percentage oxymyoglobin than all other frozen storage periods and myoglobin oxygenation levels on days 4-7 of RD (P < .05). The HiOxy steaks frozen for 4 and 6 months had higher percentage metmyoglobin than DeOxy and Oxy, regardless of packaging (P < .05). Delta E, discoloration, and lipid oxidation were greatest for HiOxy steaks compared to Oxy and DeOxy (P < .05). Extended storage brought about detrimental color effects for all differing levels of myoglobin oxygenation. The HiOxy steaks through the first few days of RD and frozen for under 6 months provided had bright cherry red color, similar to that of DeOxy and Oxy. However, with extended frozen storage and RD, HiOxy steaks had worse color characteristics (more discoloration) than the other myoglobin oxygenation levels., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Stability of proteins plays a significant role not only in their biological function but also in medical science and protein engineering. Since proteins are only stable in special conditions, maintaining their stability and function in biological and biotechnological applications may pose serious challenges. Osmolytes provide a general method of shielding proteins from the unfolding and aggregation caused by extreme stress on the environment. In such studies, the researchers used spectroscopic and simulation approaches to study the alterations of the myoglobin structure and stability in glycerol presence. Experimental results showed a stability improvement of the complex myoglobin-glycerol. After the addition of glycerol resulting in the initiation of hydrogen bonds and higher levels of hydrophobicity, the increase of the T m was observed. The static mode quenching observed in this study. Van der Waals forces and hydrogen bindings had a decisive and significant role concerning the stability of protein which was consistent with the modeling results. Molecular dynamics simulation showed that the glycerol presence could enhance myoglobin stability. The consistency between the theoretical studies and experimental findings demonstrates that the method proposed in this study could provide a useful method for protein-ligand complex investigations., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm 2 . ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10 -13  m 2 , providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 μL min -1 , collapsing only at flow rates >700 μL min -1 , a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hydropersulfides are reported to be good biological reductants, superior to thiols and akin to selenols. As such, they have been previously shown to reduce metalloproteins such as ferric myoglobin and ferric cytochrome c to their ferrous forms under conditions where little or no reduction from corresponding thiols is observed. Not surprisingly, the reduction of ferric myoglobin to ferrous myoglobin under aerobic conditions results in the generation of oxymyoglobin (dioxygen bound ferrous myoglobin). Previous studies have demonstrated that oxymyoglobin can also act as an oxidant with highly reducing species such as hydroxylamine and ascorbate. Considering the reducing properties of hydropersulfides, it is possible that they can also react with oxymyoglobin similarly to hydroxylamine or ascorbate. Herein, this reaction is examined and indeed hydropersulfides are found to react with oxymyoglobin similarly to other reducing species leading to a fleeting ferric myoglobin which is rapidly reduced to the ferrous form also by hydropersulfide., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Label-free proteomics was applied to understand the color and color stability development of dark cutting beef (DC). The color traits of DC (pH = 6.86) and normal beef (pH = 5.49) were determined during 7 days of display. DC had a lower redness and greater color stability, which was attributed to its higher deoxy-myoglobin content, greater oxygen consumption, as well as higher metmyoglobin reducing activity and lower lipid oxidation. A total of 28 differentially expressed mitochondrial proteins (fold change >1.8) between the two groups were identified, with 21 proteins overexpressed in DC mainly involved in oxidative phosphorylation, generation of reducing equivalent, TCA cycle and chaperones. These enhance the level of mitochondrial respiration, the stability of reducing MetMb and myoglobin and mitochondria function, leading to color characteristics of DC. Moreover, Glutaryl-CoA dehydrogenase was for the first time reported to be significantly associated with color parameters and its direct relationship with meat color is worthy investigation., Competing Interests: Declaration of Competing Interest No conflict of interest exits in the submission of this manuscript, and this manuscript has been approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described has not been published previously, and it is not under consideration for publication elsewhere., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

1 H Nuclear Magnetic Resonance (NMR) relaxometry and Dielectric Spectroscopy (DS) have been exploited to investigate the dynamics of solid proteins. The experiments have been carried out in the frequency range of about 10 kHz-40 MHz for NMR relaxometry and 10 -2 Hz-20 MHz for DS. The data sets have been analyzed in terms of theoretical models allowing for a comparison of the correlation times revealed by NMR relaxometry and DS. The 1 H spin-lattice relaxation profiles have been decomposed into relaxation contributions associated with 1 H- 1 H and 1 H- 14 N dipole - dipole interactions. The 1 H- 1 H relaxation contribution has been interpreted in terms of three dynamical processes of time scales of 10 -6 s, 10 -7 s and 10 -8 s. It has turned out that the correlation times do not differ much among proteins and they are only weakly dependent on temperature. The analysis of DS relaxation spectra has also revealed three motional processes characterized by correlation times that considerably depend on temperature in contrast to those obtained from the 1 H relaxation. This finding suggest that for solid proteins there is a contribution to the 1 H spin-lattice relaxation associated with a kind of motion that is not probed in DS as it does not lead to a reorientation of the electric dipole moment., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

This review aimed to examine the literature about blooming to determine any practical implications for meat colour and colour stability. Blooming is critical to surface colour in addition to pigment and achromatic factors and causes meat to become lighter (higher L*) and redder (higher a*) due to the formation of oxymyoglobin (OMb). Bloom depth of red meat varies in the range of about 1-12 mm due to a range of extrinsic factors notably oxygen partial pressure, temperature and time post slicing. A range of intrinsic factors also affect bloom acting via the oxygen consumption rate of mitochondria and metmyoglobin (MMb) reductase activity post mortem, such as post mortem age, muscle type, rigor temperature, ultimate pH (pHu), genotype and vitamin E status. These sources of variation have implications for measurement protocols and commercial applications for colour and colour stability. Development of a rapid method to measure bloom depth would enable assessment of bloom independently of surface colour., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019. Published by Elsevier Ltd.)

Freezing is widely used during the manufacturing process of protein-based therapeutics, but it may result in undesired loss of biological activity. Many variables come into play during freezing that could adversely affect protein stability, creating a complex landscape of interrelated effects. The current approach to the selection of freezing conditions is however nonsystematic, resulting in poor process control. Here we show how mathematical models, and a design space approach, can guide the selection of the optimal freezing protocol, focusing on protein stability. Two opposite scenarios are identified, suggesting that the ice-water interface is the dominant cause of denaturation for proteins with high bulk stability, while the duration of the freezing process itself is the key parameter to be controlled for proteins that are susceptible to cold denaturation. Experimental data for lactate dehydrogenase and myoglobin as model proteins support the model results, with a slow freezing rate being optimal for lactate dehydrogenase and the opposite being true for myoglobin. A possible application of the calculated design space to the freezing and freeze-drying of biopharmaceuticals is finally described, and some considerations on process efficiency are discussed as well., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

This review focuses on the mechanisms responsible for some the achromatic aspects of meat colour (paleness or darkness) due to light scatter from structures within the tissue. Recent investigations have highlighted the role of three key mechanisms contributing to variations in the lightness of meat: (1) Variations in the myofilament lattice spacing, and the resultant changes in myofibril diameter and muscle fibre diameter. A 20% increase in lightness (L* value) between muscles with ultimate pH of 6.1 versus 5.4 is accompanied by a 17% change in muscle fibre diameter. (2) Variations in sarcomere length, if these are associated with changes in myofilament and myofiber diameter, (3) Variations in sarcoplasmic protein distribution, including whether these are bound or precipitated onto the myofilaments, as demonstrated by an increase of 1.24 in the ratio of X-ray diffraction intensities from mass centered on the thin filaments versus thick filaments in dark (pH 6.15) versus light (pH 5.47) muscles. For clarity, the discussion of these mechanisms is principally in relation to pH and temperature at rigor (5 °C-35 °C), although the possibility of contributions from numerous other factors is acknowledged., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ultraviolet (UV) irradiation has gained interest as a decontamination method for food for several years. This study investigated how UV-C affected the microbial load of pork, inoculated with Yersinia (Y.) enterocolitica and Brochothrix (B.) thermosphacta. The initial effect as well as the effect after 1, 7 and 14 days of storage were investigated. Additionally, the meat quality parameters color, pH value, myoglobin redox form percentages and antioxidant capacity were analyzed. During storage, the bacterial load on pork was significantly reduced up to 1.2 log 10 using doses of 408 or 2040 mJ/cm 2 . In contrast to this, in vitro experiments with bacterial suspensions showed that calculated UV doses of 16.16 and 19.30 mJ/cm 2 resulted in a 3.0 log 10 reduction of Y. enterocolitica and B. thermosphacta, respectively. The analyzed meat quality parameters were not influenced by UV-C treatment. Hence, UV-C light can reduce microbial surface contamination without negatively affecting meat quality., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Meat color evolution from freshly cut to beyond shelf life, up to 40% of metmyoglobin, has been theoretically modeled using the Kubelka-Munk theory and a set of measured reference reflectance spectra of deoxymyoglobin, oxymyoglobin and metmyoglobin. Color evolution depicts characteristic color paths in CIELAB color space. During oxidation the model explains the approximately constancy of L*, b* and h ab , with variations typically hidden by sample dispersion, and the special significance of a* and C* in relation with metmyoglobin formation. CIELAB ΔE* color difference and the reflectance ratio R630/R580 are even better indicators of metmyoglobin changes during oxidation. The role of a*, C*, ΔE* and R630/R580 and their relationship during oxidation is a normal feature in the model with quantitative predictions in general agreement with literature. Results further emphasize the dangers of reporting color coordinates in different illuminants., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Isoelectric focusing (IEF) has been used for determination of meat quality with high stability analysis. However, it still suffered from time-consuming, laborious and cost-effective performances, e.g., 3 h protein extraction, more than 10 h rehydration time, 5-12 h focusing time, and imaging of protein band. To overcome these issues, a speedy extraction of colorful proteins was developed by controlling extraction and centrifugation of 0.2g sample within 10 min and 15 min respectively; a rapid analytical method was designed by using a quick array IEF with 25 min rehydration, 7 min focusing, 2 min online scanning and imaging of focused proteins. The total analytical time was well controlled within 1 h, significantly less than the traditional IEF time of 24 h. To demonstrate the proposed method, 18 chickens were classified into three groups, e.g., the normal slaughtering, death treatment underwater, and death with infection via the New castle disease (NDV) virus. The experiments demonstrated that two Mb bands with pI 6.8 and 7.4 were present in slaughtered chickens, while four other bands with pI 6.83, 6.95, 7.09, and 7.13 were observed in abnormal chicken. The additional four proteins bands were identified by western blot (WB) as hemoglobin proteins. Furthermore, array Immobilized pH Gradient (IPG) has high sensitivity (absolute LOD of Mb and Hb were 1.3 ng and 5.5 ng), fair stability (RSD values of 2.32%, 2.27%, and 1.69%) for slaughtered, drowned, NDV-infected chickens for intra-day and (2.94%, 1.66%, and 1.07%) for inter-days, and good recovery (100%, 98.25% and 99.75%). Finally, the developed method could be used for the identification of chicken meat quality with less time and small volume reagents consuming., (Copyright © 2019. Published by Elsevier B.V.)

Aims: The naturally occurring compound curcumin has been proposed for a number of pharmacological applications. In spite of the promising chemotherapeutic properties of the molecule, the use of curcumin has been largely limited by its chemical instability in water. In this work, we propose the use of water soluble proteins to overcome this issue in perspective applications to photodynamic therapy of tumors., Materials and Methods: Curcumin was bound to bovine serum albumin and its photophysical properties was studied as well as its effect on cell viability after light exposure through MTT assay and confocal imaging., Key Findings: Bovine serum albumin binds curcumin with moderate affinity and solubilizes the hydrophobic compound preserving its photophysical properties for several hours. Cell viability assays demonstrate that when bound to serum albumin, curcumin is an effective photosensitizer for HeLa cells, with better performance than curcumin alone. Confocal fluorescence imaging reveals that when curcumin is delivered alone, it preferentially associates with mitochondria, whereas curcumin bound to bovine serum albumin is found in additional locations within the cell, a fact that may be related to the higher phototoxicity observed in this case., Significance: The higher bioavailability of the photosensitizing compound curcumin when bound to serum albumin may be exploited to increase the efficiency of the drug in photodynamic therapy of tumors., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein aggregation leads to vast conformational changes and plays a key role in the pathogenesis of various neurodegenerative diseases including Alzheimer's and Parkinson's. In the current piece of work, we have explored the interaction of quinoline yellow (QY) with myoglobin (Mb) at two different pH (3.5 and 7.4). Various spectroscopic techniques such as turbidity, Rayleigh light scattering (RLS), UV-Vis absorbance, fluorescence resonance energy transfer (FRET), far UV-CD along with transmission electron microscopy (TEM) and molecular docking have been utilized to characterize dye-induced aggregation in Mb. Binding results showed that interaction between QY and myoglobin is spontaneous and static in nature with high K SV value of 2.14 × 10 4  M -1 . On the other hand, thermodynamics studies (∆H & ∆S) revealed that complex formation was driven by hydrogen and Van der Walls forces. Molecular docking analysis showed strong binding affinity (K d  = 4.95 × 10 4  M -1 ) between QY and Mb at Pro100, Ile101, Lys102, Glu105, Glu136, Arg139, Lys140, and Ala143 residues. The intrinsic fluorescence and circular dichroism studies indicated that QY induced conformational changes in Mb at pH 3.5. Turbidity and RLS studies showed aggregation of Mb in the presence of QY (0.2-5 mM). Moreover, kinetics data revealed nucleation independent aggregation of myoglobin in the presence of QY. TEM analysis further established amorphous nature of Mb aggregate induced by QY. At pH (7.4), QY was unable to induce aggregation in myoglobin; it might be due to repulsive nature of negatively charged dye and myoglobin or partially altered states of protein could be pre-requisite for binding and aggregation., (Copyright © 2019. Published by Elsevier B.V.)

Oligomerization of heme proteins is useful for construction of new materials with cooperative and systematic functions; thus, diverse methods have been applied for construction of artificial heme protein oligomers. Three-dimensional (3D) domain swapping is a protein oligomerization phenomenon that exchanges the same domain or secondary structural element between molecules. 3D domain swapping was first reported in 1994; since then many proteins have been reported to domain swap. Our research group has been showing that various heme proteins domain swap. We also found that domain swapping of heme proteins occurs at the early stage of protein folding, and utilized it to construct various heme protein assemblies, including nanorings, cages, hetero dimers with different active sites, and a ligand-binding reversible monomer-polymer system. In this review, the basics and applications of domain swapping of heme proteins are summarized., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Accumulating evidence suggests a new role for cellular heme as a signalling molecule, in which interactions with target proteins are more transient than found with traditionally-defined hemoproteins. To study this role, a precise method is needed for determining the heme-binding affinity (or dissociation constant, K d ). Estimates of K d are commonly made following a spectrophotometric titration of an apo-protein with hemin. An impediment to precise determination is, however, the challenge in discriminating between the Soret absorbance for the product (holo-protein) and that for the titrant (hemin). An altogether different approach has been used in this paper to separate contributions made by these components to absorbance values. The pure component spectra and concentration profiles are estimated by a multivariate curve-resolution (MCR) algorithm. This approach has significant advantages over existing methods. First, a more precise determination of K d can be made as concentration profiles for all three components (apo-protein/holo-protein/hemin) are determined and can be simultaneously fitted to a theoretical-binding model. Second, an absorption spectrum for the holo-protein is calculated. This is a unique advantage of MCR and attractive for investigating proteins in which the nature of heme binding has not, hitherto, been characterised because the holo-protein spectrum provides information on the interaction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A metal affinity-carboxymethyl cellulose functionalized magnetic graphene, namely MGCI-Cu composite, was prepared by successive modifications of graphene oxide nanosheets with magnetic nanoparticles, carboxymethyl cellulose (CMC), iminodiacetic acid (IDA) and then chelated with copper ions. The successful modifications of the graphene surface were demonstrated by various characterizations, and a high density of 6.17 μmol m -2 for metal affinity groups was obtained. The composite exhibited high adsorption selectivity toward histidine-rich proteins. The adsorption was governed by strong metal affinity binding force between hisitidine residues of proteins and immobilized Cu 2+ ions of MGCI-Cu composite. In particular, highly selective isolation of hemoglobin (Hb) was achieved in 0.2 mol L -1 phosphate buffer at pH 8. The adsorption capacity of Hb significantly increased to 769 mg g -1 in comparison to that of 435 mg g -1 on metal affinity modified magnetic graphene composite (MGI-Cu) without CMC modification. The adsorbed Hb molecules were recovered with a carbonate buffer (0.2 mol L -1 pH 10) containing 0.5 mol L -1 imidazole. MGCI-Cu composite displayed favorable reusability for at least four times after regeneration of the composite by edetic acid (EDTA) and Cu 2+ solution. The practical applications demonstrated that MGCI-Cu composite could highly selectively isolate Hb from human whole blood and polyhistidine-tagged recombinant protein from Escherichia coli (E. coli) lysate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Myoglobin is known to show formation of intermediate states under various environmental conditions, in spite of that, this is the first evidence of formation pre-molten globule (PMG) in myoglobin. Polyethylene glycol (PEG) of various molecular sizes shows assorted effects on different proteins. Out of too short and too long PEGs, only PEGs of optimal size interact with proteins leading to change in protein structure that form intermediate state. We are the first one to report the formation of PMG in a protein in the presence of a crowding agent. The PEG-induced intermediate state was characterized by various techniques like absorption, fluorescence, near- and far-UV circular dichroism spectroscopy, ANS binding, and dynamic light scattering measurements to be PMG. Isothermal titration calorimetry and docking studies were further carried out to delineate the mechanism of formation of PMG in myoglobin in physiological conditions. The intermediate formed due to interaction of PEG with myoglobin has physiological implications which are essential to unravel the mystery to solve the massively complicated problems involved in the proper folding of proteins in vivo. Further, outcomes from this study are expected to gain mechanistic insights on the native structure and functions of proteins under in vivo conditions., (Copyright © 2018 Elsevier B.V. All rights reserved.)