Bovine Myoglobin Research Articles

The effect of pH and aging on mitochondrial reduction of bovine myoglobin's affinity for oxygen

In skeletal muscles, mitochondria have been shown to decrease the oxygen affinity of myoglobin. In this study, we investigated whether the mitochondrial function of decreasing myoglobin affinity for oxygen persists and operates at the final pH of postmortem bovine skeletal muscle. The oxygen affinity and myoglobin consumption in the presence of mitochondria obtained from fresh and wet-aged beef were evaluated and compared at pH 5.1, 5.6, and 5.7. The results showed that mitochondria obtained from fresh beef preserved myoglobin oxygen consumption and affinity interference, whereas those obtained from wet-aged beef did not. Oxygen consumption and affinity interference were mostly absent at pH 5.1 and were higher at pH 5.7 than those at pH 5.6. Our findings suggest that mitochondria contribute both to an increase in the oxygen affinity of myoglobin in aged meat and a decrease in the oxygen affinity of myoglobin in high-pH meat, such as dark-cutting beef.

Moo-re Gene-edited Animals Get Regulatory Clearance: U.S. FDA Approves Genetically Engineered Cattle for Human Consumption

Moo-re Gene-edited Animals Get Regulatory Clearance: U.S. FDA Approves Genetically Engineered Cattle for Human Consumption

Loading Effect of Chitosan Derivative Nanoparticles on Different Antigens and Their Immunomodulatory Activity on Dendritic Cells.

Drug carrier nanoparticles (NPs) were prepared by the polyelectrolyte method, with chitosan sulfate, with different substituents and quaternary ammonium chitosan, including C236-HACC NPs, C36-HACC NPs, and C6-HACC NPs. To evaluate whether the NPs are suitable for loading different antigens, we chose bovine serum albumin (BSA), ovalbumin (OVA), and myoglobin (Mb) as model antigens to investigate the encapsulation effect of the NPs. The characteristics (size, potential, and encapsulation efficiency) of the NPs were measured. Moreover, the NPs with higher encapsulation efficiency were selected for the immunological activity research. The results showed that chitosan derivative NPs with different substitution sites had different loading effects on the three antigens, and the encapsulation rate of BSA and OVA was significantly better than that of Mb. Moreover, the NPs encapsulated with different antigens have different immune stimulating abilities to DCS cells, the immune effect of OVA-coated NPs was significantly better than that of BSA-coated NPs and blank NPs, especially C236-HACC-OVA NPs. Furthermore, we found that C236-HACC-OVA NPs could increase the phosphorylation level of intracellular proteins to activate cell pathways. Therefore, C236-HACC NPs are more suitable for the loading of antigens similar to the OVA structure.

Interplays of electron and nuclear motions along CO dissociation trajectory in myoglobin revealed by ultrafast X-rays and quantum dynamics calculations

Ultrafast structural dynamics with different spatial and temporal scales were investigated during photodissociation of carbon monoxide (CO) from iron(II)-heme in bovine myoglobin during the first 3 ps following laser excitation. We used simultaneous X-ray transient absorption (XTA) spectroscopy and X-ray transient solution scattering (XSS) at an X-ray free electron laser source with a time resolution of 80 fs. Kinetic traces at different characteristic X-ray energies were collected to give a global picture of the multistep pathway in the photodissociation of CO from heme. In order to extract the reaction coordinates along different directions of the CO departure, XTA data were collected with parallel and perpendicular relative polarizations of the laser pump and X-ray probe pulse to isolate the contributions of electronic spin state transition, bond breaking, and heme macrocycle nuclear relaxation. The time evolution of the iron K-edge X-ray absorption near edge structure (XANES) features along the two major photochemical reaction coordinates, i.e., the iron(II)-CO bond elongation and the heme macrocycle doming relaxation were modeled by time-dependent density functional theory calculations. Combined results from the experiments and computations reveal insight into interplays between the nuclear and electronic structural dynamics along the CO photodissociation trajectory. Time-resolved small-angle X-ray scattering data during the same process are also simultaneously collected, which show that the local CO dissociation causes a protein quake propagating on different spatial and temporal scales. These studies are important for understanding gas transport and protein deligation processes and shed light on the interplay of active site conformational changes and large-scale protein reorganization.

The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues

Non-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

The Molecular Magic of “Meatless” Meats: Structural and Sequence Similarities between Soy Leghemoglobin and Bovine Globins

Plant‐based proteins are becoming more prevalent in the modern day due to humanity’s fight against climate change. How do scientists produce plant‐protein that tastes similar to animal‐protein? When comparing bovine hemoglobin (2qss), soy leghemoglobin (2GDM) and bovine myoglobin (2qss), the proteins differ by location, number of heme proteins, oxygen affinity and backbone alignment. Hemoglobin is found in red blood cells of animals. More specifically, myoglobin is found in the red blood cells of muscle tissue in animals. Leghemoglobin, however, is the byproduct of the symbiotic relationship between root nodules on legumes and nitrogen‐fixing bacteria that lives on them. However, all of these proteins are linked by the common molecule, heme. Plant‐based “meat” manufacturers suggest that this heme group, that is embedded in each of these globins, is responsible for this “meaty” taste. Additionally, the presence of umami‐inducing amino acids was investigated and glutamic acid surface prevalence related to flavor. Using the suite of structural analysis applications in the Protein Data Bank, such as FATCAT and fast2BLAST, the structural similarities between bovine hemoglobin, especially the heme itself, bovine myglobin and soy leghemoglobin can be illustrated, both quantitatively and qualitatively (e.g. 3D‐printed models).

A novel diazoresin/polystyrene-poly(ethylene oxide) covalent capillary coating for the analysis of proteins by capillary electrophoresis

A novel method for the preparation of covalently linked capillary coatings of polystyrene-poly (ethylene oxide) (PS-PEO) was demonstrated by using photosensitive diazoresin (DR) as coupling agents. Layer by layer (LBL) self-assembly membrane of DR and PS-PEO based on hydrogen bonding firstly was fabricated on the inner wall of capillary, then with the treatment of UV light through the unique photochemistry reaction of DR, the hydrogen bonding was converted into covalent bonding. The covalently bonded coatings have the ability of inhibiting protein adsorption on the inner surface of capillary, and thus a baseline separation of Lysozyme (Lys), cytochrome c (Cyt-c), bovine serum albumin (BSA), ribonuclease A (RNase A) and myoglobin (Mb) was achieved by using capillary electrophoresis (CE). Compared with bare capillary or non-covalently bonded DR/PS-PEO coatings, the covalently linked DR/PS-PEO capillary coatings improved the CE separation performance for proteins; what’s more, it exhibited good stability and repeatability. This method can provide a green and easy way to make the covalently coated capillaries for CE, replacing of the old way which using highly toxic and moisture sensitive silane coupling agent by DR in the covalent coating preparation.

81 Species-specificity in metmyoglobin reduction

Abstract Myoglobin is the primary sarcoplasmic protein responsible for meat color. Previous research has reported that myoglobin oxidation is species-specific. Metmyoglobin reducing activity is an inherent property to limit myoglobin oxidation. However, limited research has determined species specificity in metmyoglobin reducing properties. The objective of current study was to compare metmyoglobin reducing properties of eight different species such as beef, porcine, bison, deer, emu, equine, goat, and sheep in vitro. Myoglobin was isolated from eight different species via ammonium sulfate precipitation. The pH of the myoglobin was adjusted by passing through a column pre-calibrated with 50 mM phosphate buffer at pH 5.6. All species myoglobin were converted to metmyoglobin, and the metmyoglobin reduction was determined by two different approaches, non-enzymatic metmyoglobin reducing activity (NMRA) and oxidation-reduction potential (ORP). In the first method, NADH (electron donor), EDTA, and methylene blue (electron carrier), were added in a cuvette and increase in absorbance at 582 nm was monitored using a UV-Vis spectrophotometer. In the second method, the ability of the heme to get reduced was determined using an RedoxSys analyzer, in which electron was directly transferred to heme. The NMRA and ORP experiments were replicated five times. The data were analyzed using the Mixed Procedure of SAS, with species as the fixed effect. There were species-specific differences (P < 0.05) in NMRA and ORP. Bovine myoglobin had the greatest (P < 0.05) NMRA compared with sheep, equine, goat, deer, bison, pork, and emu. There were no differences (P > 0.05) in NMRA among equine, goat, deer, bison, pork, and emu. ORP studies indicated that beef and porcine myoglobins had the greatest ability to get reduced (P < 0.05) compared with other species. Hence, use of different techniques and approaches will help to elucidate the mechanistic basis of metmyoglobin reduction.

46 Species-specificity in metmyoglobin reduction

Abstract Myoglobin is the primary sarcoplasmic protein responsible for meat color. Previous research has reported that myoglobin oxidation is species-specific. Metmyoglobin reducing activity is an inherent property to limit myoglobin oxidation. However, limited research has determined species specificity in metmyoglobin reducing properties. The objective of current study was to compare metmyoglobin reducing properties of eight different species such as beef, porcine, bison, deer, emu, equine, goat, and sheep in vitro. Myoglobin was isolated from eight different species via ammonium sulfate precipitation. The pH of the myoglobin was adjusted by passing through a column pre-calibrated with 50 mM phosphate buffer at pH 5.6. All species myoglobin were converted to metmyoglobin, and the metmyoglobin reduction was determined by two different approaches, non-enzymatic metmyoglobin reducing activity (NMRA) and oxidation-reduction potential (ORP). In the first method, NADH (electron donor), EDTA, and methylene blue (electron carrier), were added in a cuvette and increase in absorbance at 582 nm was monitored using a UV-Vis spectrophotometer. In the second method, the ability of the heme to get reduced was determined using an RedoxSys analyzer, in which electron was directly transferred to heme. The NMRA and ORP experiments were replicated five times. The data were analyzed using the Mixed Procedure of SAS, with species as the fixed effect. There were species-specific differences (P < 0.05) in NMRA and ORP. Bovine myoglobin had the greatest (P < 0.05) NMRA compared with sheep, equine, goat, deer, bison, pork, and emu. There were no differences (P > 0.05) in NMRA among equine, goat, deer, bison, pork, and emu. ORP studies indicated that beef and porcine myoglobins had the greatest ability to get reduced (P < 0.05) compared with other species. Hence, use of different techniques and approaches will help to elucidate the mechanistic basis of metmyoglobin reduction.

Methyl and methylene vibrations response in amino acids of typical proteins in water solution under high-frequency electromagnetic field

ABSTRACTThe aim of this paper was to study the response of methyl (CH3) and methylene (CH2) vibration bands in amino acids of some typical proteins diluted in bidistilled water solution after exposure to a high-frequency electromagnetic fields using Fourier Transform Infrared (FTIR) Spectroscopy. Hemoglobin in H2O solution and bovine serum albumin and myoglobin diluted in different D2O solutions were exposed for 4 h to a power density of 0.95 W/m2 at the frequency of 1750 MHz, emitted by operational mobile phones Nokia model 105 and Samsung model GT-E1270, in order to study the response of stretching vibrations of CH3 and CH2 that are in amino acids of those proteins. The main result was that CH3 stretching bands increased significantly in myoglobin in D2O solution because this protein is represented by a single protein chain so that the torque induced by the applied field is larger than that induced on the other two proteins. Otherwise, CH2 stretching vibrations decreased in intensity significantly for all exposed proteins. This result can be explained as well, assuming that, given a fixed volume, a decrease of population of CH2 occurred after exposure because of the alignment of proteins α-helices along the direction of the applied electromagnetic field.

Quantification of species-specific meat proteins in cooked and smoked sausages using infusion mass spectrometry

Label-free quantification combined with high-resolution infusion-based mass spectrometry (MS) was evaluated to authenticate ‘horse sausages’ made from horse meat and pork. Four types of industrially processed sausages, including cooked horse meat, pork and beef, and their mixtures were analysed. Quantitation and evaluation of the species composition were based on a set of 11 species-specific meat proteins and 14 unique heat-stable peptide markers. Using infusion MS, the highest distinguishing value was found in four proteins, namely, horse myosin-7 (MYH7_HORSE) and horse myoglobin (MYG_HORSE), porcine myosin-4 (MYH4_PIG) and bovine myoglobin (MYG_BOVIN). The limit of detection was 5% (w/w) for pork and beef in the three-component matrix and 1% (w/w) for horse meat. The proteins’ abundance was computed using a peak intensity measurement technique for precursor ions, based on the extracted ion currents/intensities of precursor ions. The procedure enabled discrimination between horse meat, pork and beef proteins, as well as estimation of the relative changes in protein abundance in all the examined samples. Substantial differences in the abundance of specific proteins were obtained from the pure meat samples, three-component mixtures and commercial sausages. The method may be useful in the preliminary screening of protein-rich food samples, aimed at fraud detection and estimation of the overall level of adulteration.

Resonant interaction between electromagnetic fields and proteins: A possible starting point for the treatment of cancer

ABSTRACTSamples of human hemoglobin, bovine serum albumin, lysozyme and myoglobin were used as prototype of proteins to investigate their response to exposure to high frequency electromagnetic fields (HF-EMFs), in order to study possible application to the treatment of cancer.To this aim, Fourier-transform infrared spectroscopy was used in the infrared region. The most evident result which appeared after 3 h exposure to HF-EMFs was a significant increase in intensity of the Amide I band and of CH2 bending vibrations, showing that the proteins aligned toward the direction of the field. In addition, proteins’ unfolding and aggregation occurred after exposure to HF-EMFs. These findings can be explained assuming a resonance interaction between the natural frequencies of proteins and HF-EMFs, which can induce iperpolarization of cells. Given that cancerous tissues were found to have natural frequencies different from natural frequencies of normal tissues, we can hypothesize to irradiate cancerous tissues using EMFs at natural frequencies of cancer cells, causing resonant interaction with cellular membrane channels, inducing increasing of ions’ flux across cellular channels and damaging the cellular functions of cancer cells.

A novel diazoresin/rapamycin covalent capillary coating for the separation of biomacromolecules by capillary electrophoresis

ABSTRACTCovalent capillary coatings of diazoresin (DR) and rapamycin (RAPA) were prepared by a layer-by-layer self-assembly. DR worked as the coupling agent. Under ultraviolet light, the corsslinking was carried out by covalent bonds between DR and RAPA. The covalent capillary coatings could suppress protein adsorption. The baseline separation of lysozyme (Lys), cytochrome c (Cyt-c), bovine serum albumin (BSA) and myoglobin (Mb) could be achieved within 17 minutes by using capillary electrophoresis (CE). The covalent capillary coatings exhibited good chemical stability and repeatability. In the synthesis, highly toxic moisture-sensitive silane has been replaced by DR. The method provides an environmentally friendly way to prepare the covalent capillary coating.

Attributes of lipid oxidation due to bovine myoglobin, hemoglobin and hemolysate

Bovine hemolysate was purified by size exclusion chromatography, and one high molecular weight protein was detected relative to the hemoglobin (Hb) fraction. Purified Hb promoted lipid oxidation in washed muscle slightly but significantly better than hemolysate, which may have been due to the absence of catalase and peroxiredoxin in the purified Hb. Purified Hb auto-oxidized to metHb more rapidly than Hb in the hemolysate (P<0.05). OxyHb promoted lipid oxidation in washed muscle more effectively compared to oxyMb (P<0.05). This was ascribed to hemin, released from metHb, promoting lipid oxidation more readily than oxidative forms of Mb that retain their protoporphyrin moiety. A 3:1 ratio of Mb:Hb promoted lipid oxidation similarly compared to adding a 1:1 ratio of Mb:Hb to washed muscle. Lipid oxidation products due to Hb-mediated lipid oxidation were elevated 60-fold at pH 6.3 compared to pH 6.7.

Species-Specificity in Myoglobin Oxygenation and Reduction Potential Properties

The objective was to compare oxygenation and reduction potential properties of bovine and porcine myoglobins in-vitro. Cyclic voltammetry and homology-based myoglobin modeling were used to determine the species-specific effects on myoglobin reduction potential and oxygenation properties at pH 5.6, 6.4, and 7.4. At all pHs, porcine myoglobin had greater (P = 0.04) oxygen affinity than bovine myoglobin. For both species, oxygen affinity was higher at pH 6.4 &gt; pH 7.4 &gt; 5.6 (P = 0.0002). Myoglobin reduction potential for both species was affected by pH (P &lt; 0.0001). The redox potentials became more negative as pH increased, indicating a proton-coupled electron transfer. There were no differences (P = 0.51) between species in reduction potential properties of heme. Homology-based myoglobin modeling indicated that the porcine myoglobin has a shorter distance between the distal histidine and heme than does bovine myoglobin. The variation in amino acid composition between bovine and porcine myoglobin could be partially responsible for differences in oxygen affinity.

Protein Adsorption Behavior of Hydroxyapatite during Hydrothermal Synthesis

The surface structure of Hydroxyapatite (HAp) particles during hydrothermal synthesis and their protein adsorption behavior was investigated. The HAp particles were prepared by mixing calcium acetate solution and diammonium hydrogen phosphate solution by hydrothermal synthesis. When the temperature of mixture were heated up to 120°C, 150°C and 180°C, the HAp particles were collected during hydrothermal synthesis. The adsorption properties of proteins onto HAp were studied using three types of proteins: bovine serum albumin (BSA), myoglobin (MGB) and lysozyme (LSZ). Surface analysis by BET revealed that their pore volumes were decreased by increasing synthesis temperature. The adsorbed amount of BSA and LSZ per unit milligrams showed no obvious difference in all of the HAp particles prepared with synthesis temperature. In contrast, the amount of MGB adsorbed onto HAp particles synthesized at 120°C, 150°, and 180°C shows the decreasing with an increasing synthesis temperature. This result suggests that the adsorbed amount of MGB was decreased by decreasing pore volume of HAp particles.

Detection of ruminant meat and bone meal in feeds by sandwich ELISA with monoclonal antibodies.

A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies directed against a synthetic peptide with an amino-acid sequence related to the C-terminus of bovine myoglobin and the whole molecule of sodium dodecyl sulphate (SDS)-denatured bovine myoglobin was adapted for detecting bovine myoglobin in contaminated feeds. The ELISA employed bovine meat extract of a known myoglobin concentration as a calibration standard and had an limit of detection (LOD) of 3.54 ng/ml and an limit of quantification (LOQ) of 11.0 ng/ml corresponding to 0.022% and 0.067% (wt/wt) bovine meat-and-bone-meal (MBM) mixed in 20-fold-diluted feed extracts, respectively. A cut-off threshold of 20.6 ng/ml bovine myoglobin was set to simplify ELISA and facilitate quick assessment of test results without a tedious calibration process. The ELISA was able to detect bovine MBM in artificially prepared model feeds, mixed botanical feeds, mixed botanical feeds with skimmed milk, fish meal, pork meal and pork/chicken meal at 0.1% (wt/wt). It was also able to detect sheep MBM in test feeds, but showed no reactivity to swine MBM, chicken MBM, skimmed milk or gelatine of bovine origin. The advantages of this method are the quick and easy extraction protocol of proteins from test feeds, using 100 mM sodium sulphide and 0.6% sodium dodecyl sulphate in the extraction solution and the effective detection of bovine and sheep MBM at 0.1% (wt/wt).

Access of Hydrogen-Radicals to the Peptide-Backbone as a Measure for Estimating the Flexibility of Proteins Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

A factor for estimating the flexibility of proteins is described that uses a cleavage method of “in-source decay (ISD)” coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The MALDI-ISD spectra of bovine serum albumin (BSA), myoglobin and thioredoxin show discontinuous intense ion peaks originating from one-side preferential cleavage at the N-Cα bond of Xxx-Asp, Xxx-Asn, Xxx-Cys and Gly-Xxx residues. Consistent with these observations, Asp, Asn and Gly residues are also identified by other flexibility measures such as B-factor, turn preference, protection and fluorescence decay factors, while Asp, Asn, Cys and Gly residues are identified by turn preference factor based on X-ray crystallography. The results suggest that protein molecules embedded in/on MALDI matrix crystals partly maintain α-helix and that the reason some of the residues are more susceptible to ISD (Asp, Asn, Cys and Gly) and others less so (Ile and Val) is because of accessibility of the peptide backbone to hydrogen-radicals from matrix molecules. The hydrogen-radical accessibility in MALDI-ISD could therefore be adopted as a factor for measuring protein flexibility.

An enzyme reactor based on aptamer modified microfluidic chip for protein analysis

As a kind of recognition molecule, aptamer has been studied and applied widely in numerous science fields in recent years. Immobilized enzymatic reactor has drawn much attention because of its striking advantages, such as high digestion efficiency and ease in coupling with the separation and detection systems. In this study, a novel microfluidic enzymatic chip, which immobilized trypsin based on aptamer, was prepared and proposed. An online analysis platform, which consisted of an aptamer-based chip and high performance liquid chromatography tandem mass spectrometry, was established by using a 6-port valve and applied to protein analysis. The enzymatic capacity and stability performance of chip reactor were characterized by using mixed protein sample, which consisted of bovine serum albumin (BSA), myoglobin (Mb) and cytochrome c (Cyt. c). The sample digestion time of the chip reactor was about 5.76 s while 1 microL/min of flow rate was adopted; and moreover, 5 ng of Mb was identified successfully with the sequence coverage of 37%. Furthermore, the sequence coverages and the relative standard deviations were 44.3% and 6.5% for BSA, 65.0% and 2.7% for Mb, 62.0% and 5.6% for Cyt. c respectively when 500 ng digest of mixed proteins were analyzed in three runs. According to experimental results, the online analysis platform possesses the ability of high sensitivity and good stability, which can provide a promising tool for rapid and high-throughput proteomics study in the near future.

Determination of the Binding Parameters between Proteins and Luminol by Chemiluminescence Using Flow Injection Technique

The interaction behavior of bovine serum albumin (BSA), lysozyme (LYS), myoglobin (MB), and catalase (CAT) with luminol, respectively, was first studied by chemiluminescence (CL) using flow injection (FI) technique based on the fact that the studied proteins can enhance the CL intensity of luminol. A FI-CL model of protein-luminol interaction, lg[(I0−I)/I]=1/nlg[P]+1/nlgKa+2lgn, was constructed, and the interaction parameters of BSA, LYS, MB, and CAT with luminol were determined accordingly. The binding constants Ka are in the descending order of CAT &gt; MB &gt; LYS &gt; BSA at the level of 105 to 107 L mol−1, and the number of binding sites n of luminol to BSA or LYS is around 2 and to MB or CAT is around 1. The results of thermodynamic parameters (ΔH, ΔS, and ΔG) showed that the binding processes of luminol to the four proteins are spontaneous mainly through the hydrophobic force.