Bovine Myoglobin Research Articles

Rapid and Efficient Proteolysis for Protein Analysis by an Aptamer-Based Immobilized Chymotrypsin Microreactor

Efficient protein digestion is a key step for successful mass spectrometry identification. However, traditional in-solution digestion suffers some drawbacks, such as autolysis of protease, long analysis times and lack of control. Recently, specific single-stranded nucleic acids, aptamers, screened from random sequence pools, have been performed high affinity for targets. In this paper, we have developed a novel enzyme reactor, which immobilized chymotrypsin based on aptamer-grafted silica beads. Mixed proteins, which consist of bovine serum albumin, myoglobin, and cytochrome c, were used as samples, to evaluate the digestion performance of the enzymatic reactor. With the use of this novel tool, proteins were digested in 40 min to an extent similar to that achieved with soluble enzyme at 37°C after 16 h. Moreover, enzymatic reactor regeneration was carried out through chymotrypsin elution and re-immobilization. The advanced characteristics of the aptamer-based chymotrypsin reactor demonstrated that aptamers could serve as novel materials for rapid and efficient enzyme immobilization and application in protein studies.

Hierarchical LiMn2O4 microspheres for high rate lithium ion batteries, and direct electrochemistry and electrocatalysis

Hierarchical LiMn2O4 microspheres were prepared using urchin-like α-MnO2 as a self-sacrificial template. As the cathode material for lithium ion batteries, the electrochemical properties were characterized by cyclic voltammetry and charge/discharge experiments. The results indicated that the LiMn2O4 microspheres had high discharge capacity and excellent cycle stability. Furthermore, a new electrochemical biosensor was constructed by immobilization of bovine myoglobin (Mb) and LiMn2O4 in a thin film of chitosan (CTS) on the surface of carbon ionic liquid electrode (CILE). Electrochemical behaviors of Mb entrapped in the film were investigated by cyclic voltammetry. A pair of well-defined redox peaks appears, which indicates a fast electron transfer rate of Mb with the underlying CILE. The electron transfer coefficient and electron transfer rate constant of Mb were calculated to be 0.47 and 1.04s−1, respectively. The Mb modified electrode showed good electrocatalytic ability toward the reduction of trichloroacetic acid (TCA) with a detection limit of 0.16mmolL−1 (3σ).

Immobilization of Antibodies on Magnetic Carbonaceous Microspheres for Selective Enrichment of Lysine‐acetylated Proteins and Peptides

AbstractLysine acetylation is a dynamic and reversible modification, which has been proved to be a key posttranslational modification in cellular regulation. However, the low amounts of the acetylated proteins could hardly be detected before enrichment. In this study, for the first time, antibody‐immobilized magnetic carbonaceous microspheres were developed for selective enrichment of acetylated proteins and peptides. At first, standard proteins composed of acetylated bovine serum albumin, myoglobin, α‐casein and ovalbumin were used as model proteins to verify the enrichment efficiency. Then, the synthesized peptide was employed to confirm the selectivity of the method. Besides, the antibody‐immobilized magnetic particles were successfully applied to analyze mouse mitochondrial proteins. After database search, 29 acetylated sites in 26 proteins were identi?ed.

Site E14 in Hemoglobins and Myoglobins: A Key Residue That Affects Hemin Loss and Lipid Oxidation Capacity

Fish hemoglobins (Hbs) frequently contain glycine at site E14 while mammalian Hbs contain larger residues (e.g., alanine and serine). These differences were examined by creating structural variants at E14 using recombinant bovine myoglobin (Mb) as a model heme protein that contains alanine at E14. The Ala(E14)Gly mutation increased k(ox) and hemin loss 3-fold and 45-fold, respectively. Glycine at E14 creates a channel for solvent to enter the heme crevice, which enhances autoxidation and hemin loss rates. Hydration of the proximal heme pocket facilitates hemin loss because protonation of the proximal histidine weakens the linkage of the imidazole group to the iron atom of the hemin moiety. Ala(E14)Gly promoted lipid oxidation in washed fish muscle more rapidly during iced storage compared to wild type Mb at pH 5.7. This suggested that the rapid hemin loss from Ala(E14)Gly accelerated lipid oxidation. Ala(E14)Ser and Ala(E14)Val had little effect on k(ox) but somewhat accelerated net hemin loss. These studies suggest that enhanced access of solvent to the heme crevice of many fish Hbs at site E14 facilitates rapid hemin loss and moderately accelerates autoxidation. This likely is part of the reason fish Hbs promote lipid oxidation much more effectively compared to mammalian Hbs.

Immobilization of Chymotrypsin on Silica Beads Based on High Affinity and Specificity Aptamer and Its Applications

Aptamers with high affinity and specificity to targets, bring new approaches to immobilizing proteins or enzymes. In this work, a group of single-stranded DNA aptamers specific for chymotrypsin were obtained by SELEX method in vitro. After investigation and characterization of all aptamers, AptC.1 (abbreviation for the aptamer with the highest affinity for chymotrypsin) was selected and grafted onto silica matrix with the help of glutaraldehyde as linker, and used subsequently to immobilize chymotrypsin. Specifically, it is shown in experiment that, 12.65 µg of chymotrypsin could be immobilized on 10 mg of AptC.1-Silica in 10 mM pH 8.0 borate solutions, and the activity of immobilized enzyme was not inhibited. Bovine serum albumin, myoglobin and cytochrome c were introduced to investigate the enzymatic performance of prepared immobilized chymotrypsin reactor. All these results demonstrated that aptamer could serve as a potential medium for the immobilization of proteins or enzymes.

Preparation and evaluation of an enzymatic microreactor based on HILIC matrix for digestion and identification of proteins

In the present study, a new enzymatic microreactor is developed by immobilizing trypsin on the matrix-HILIC (hydrophilic interaction chromatography). The influences of different types of buffer solutions (borate, Tris·HCl, and phosphate), their concentrations and pH values on the immobilizing rate and the activity of immobilized trypsin were investigated. The optimal condition for off-line immobilization of trypsin on matix-HILIC was Tris·HCl buffer (20 mmol/L, pH 8.0), and then, under the optimal condition, an enzymatic microreactor was prepared by trypsin dynamically immobilized on HILIC matrix which was packed in a Peeksil column. It can be found that the microreactor possessed high hydrolytic activity according to the hydrolysis results of N-α-benzoyl-DL-arginine P-nitroanilide (BAPNA), a substrate of trypsin. Moreover, bovine serum albumin (BSA), cytochrome C and myoglobin as standard proteins were chosen to characterize the enzymolysis of the microreactor demonstrating the high enzymolysis efficiency by this enzymatic microreactor. Besides, the deactivated trypsin can be easily eluted from the matrix-HILIC to re-immobilize trysin, which is highly cost-effective in the experiment. All the results show that the new enzymatic microreactor associated with HPLC-MS techniques can realize online enzymolysis of proteins, and the time to analyze and identify the proteins is greatly shortened.

Improved Current-Monitoring Method for Low Electroosmotic Flow Measurement in Modified Microchip

Current-monitoring method is a widely used approach to measure electroosmotic flow (EOF) in microchip, but low and zero EOF is difficult to be measured. In this report, the mechanism of current-monitoring method was explained with Kohlraush regulation function principle, and an improved current-monitoring method was developed for low EOF measurement with tilting microchip. Fluid flow in the channel was accelerated with the help of hydrostatic pressure generated by tilting microchip, the time of dilute solution displacing the concentrated one in channel was shortened. EOF could be calculated according to the time difference between twice experiments under two different applied voltages by tilting microchip. Low even zero EOF could be measured by this improved current-monitoring method. Three modified microchips were characterized to verify the method. EOF in microchannels modified with poly(vinyl alcohol), bovine serum albumin and myoglobin were 0.27 ± 0.05, 0.16 ± 0.05 and −0.45 ± 0.04 × 10−4 cm2 V−1 s−1, respectively.

Characterization and kinetics studies of water buffalo (Bubalus bubalis) myoglobin

It is generally accepted that dry-aged buffalo (B. bubalis) meat becomes dark faster than bovine (B. taurus) meat, discouraging consumer purchase. We have investigated whether this faster darkening process might depend on structural and/or kinetic differences between buffalo and bovine myoglobins (Mbs). To this end, we have purified to homogeneity buffalo Mb from Longissimus dorsi muscle and obtained both its Mr (17,034.5) and the complete amino acid sequence, which, compared with the bovine one, showed three amino acid substitutions: Dbo141Ebu, Abo19Tbu and Abo117Dbu. As revealed by the 3D structure, they were located on the surface of the protein, far from the heme binding pocket, and did not cause appreciable structural changes. Autoxidation rates of purified buffalo and bovine myoglobins at 37 °C, pH 7.2, were almost identical (0.052±0.001 h−1 and 0.054±0.002 h−1, respectively), as were their oxygen-binding Kd values (3.7±0.1 μM and 3.5± 0.1 μM, respectively). These data indicate that the structure of the heme pockets in the two proteins is similar, with similar functional properties. Moreover, the percent of MetMb values in the purified buffalo and bovine samples, after the same time from slaughtering, were almost identical (57% and 47%, respectively). The results presented here suggest that the faster darkening of buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393±0.005 g/100 g) and consequently its MetMb content, which is almost twice as high as bovine meat (0.209±0.003 g/100 g), and likely other factors.

Characterization and kinetics studies of water buffalo ( Bubalus bubalis) myoglobin

The colour of buffalo ( Bubalus bubalis L.) meat is darker than bovine meat. Since meat colour depends on the concentration of myoglobin (Mb) and its oxidation state, we have determined the main structural and functional properties of buffalo Mb. Buffalo Mb was purified from longissimus dorsi muscles and its molecular mass determined by ESI Q-TOF mass spectrometry. The molecular mass 17,034.50 was 86.20 Da higher than the bovine Mb. This was confirmed by analysing its primary structure, using a combined approach based on Edman degradation and MALDI-TOF mass spectrometry. Comparing the amino acid sequences of both Mbs, we found three amino acid differences out of 153 amino acid residues. One is a conservative substitution (D bov141E buf) , and the other two (A bov19T buf and A bov117D buf) are nonconservative. These amino acid substitutions are unlikely to cause structural changes because they are located far from the heme binding pocket, as revealed by the 3D structure of buffalo Mb elaborated by homology modelling. Stability analyses show no difference with the bovine Mb for helix E and only minor differences in the stability values for helices A and G. Moreover, autoxidation rates of purified buffalo and bovine myoglobins at 37 °C, pH 7.2, were almost identical, 0.052 ± 0.001 h − 1 and 0.054 ± 0.002 h − 1 , respectively, as were their oxygen-binding Kd values, 3.7 ± 0.1 μM and 3.5 ± 0.1 μM, respectively. The percent of MetMb values were almost identical. The results presented here suggest that the darker buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393 ± 0.005 g/100 g of tissue) and consequently MetMb, which are almost twice as high as bovine meat (Mb: 0.209 ± 0.003 g/100 g of tissue).

Redox Instability Induced by 4-Hydroxy-2-nonenal in Porcine and Bovine Myoglobins at pH 5.6 and 4 °C

Myoglobin (Mb) redox stability affects meat color and is compromised by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Pork lipids are generally more unsaturated and would be expected to oxidize readily and produce more oxidation products than beef. Supranutritional supplementation of vitamin E improves Mb redox stability of beef but not pork. The present study investigated HNE-induced redox instability in porcine and bovine myoglobins at 4 degrees C and pH 5.6. Oxymyoglobin (OxyMb) was incubated with HNE (0.075 mM porcine OxyMb + 0.5 mM HNE; 0.15 mM bovine OxyMb + 1.0 mM HNE). In porcine Mb, only monoadducts formed via Michael addition were detected after 72 h, whereas in bovine Mb both mono- and diadducts were identified. LC-MS-MS identified four histidine residues (His 36, 81, 88, and 152) of bovine Mb that were readily adducted by HNE, whereas in porcine Mb only two histidine residues (His 24 and 36) were adducted. These results suggested that the primary structure of bovine Mb predisposes it to greater nucleophilic attack by HNE and subsequent adduction than is suffered by porcine Mb.

Occurrence and nature of equine and bovine myoglobin dimers.

In commercial samples of equine myoglobin and samples of equine and bovine myoglobin prepared in the laboratory, a small amount of the protein was present as an aggregate. The presence of the myoglobin aggregate could be demonstrated by gel filtration on Sephadex G-100 Superfine, which also provided a means of isolating it. Gel filtration on Sephadex G-100 showed the molecular weights of the equine and bovine moyglobin aggregates to be about 35000 and 34000 respectively, thus supporting the hypothesis that they are dimers. This was confirmed for the equine myoglobin by ultracentrifugation measurements. The exact nature of the dimer cannot be specified. The conditions required for its formation in vitro and the fact that no traces of dimer could be detected in fresh muscle preclude the possibility of its natural occurrence. The spectral properties of the equine myoglobin dimer and its sedimentation and diffusion behaviour in the ultracentrifuge indicate, however, that the configuration of native monomeric myoglobin is essentially preserved in the dimer. Like their respective monomers, the dimers of equine and bovine myoglobin were shown by isoelectric fractionation to be microheterogeneous.

A new approach for sequencing human IRS1 phosphotyrosine-containing peptides using CapLC-Q-TOFmicro

Reversible phosphorylation of proteins functions as a biological switching network for activation and inhibition of downstream biological processes. Since phosphorylations of these sites are often transient processes, and hence sub-stoichiometric, systematic characterization of phosphorylation sites is a formidable challenge. In this work, a new approach was developed to pinpoint phosphotyrosine sites on tyrosine-containing peptides. This required (1) the development of a new and highly sensitive nano-electrospray assembly and (2) validation of the concept that the specificity and detection limit for trace levels of phosphotyrosine immonium ion in peptide mixtures from protein digests can be increased by varying the collision energy. With our method, an automatic tandem mass spectrometric analysis of peptides eluted from a C(18) capillary liquid chromatographic column is triggered by a positive confirmation of phosphotyrosine immonium ion in a time-of-flight mass spectrometric survey. The approach was tested by analyzing the phosphorylation of human IRS-1 peptides that interact with the Src-homology 2 domain and mixtures of these peptides with tryptic digests of bovine serum albumin and horse heart myoglobin.

Effects of reducing agents on premature browning in ground beef

This study was performed to investigate the effects of food-grade reducing agents on counteracting premature browning (PMB) and to determine the relative heat stability of different redox states of bovine myoglobin (Mb). Sodium erythorbate (SE), erythorbic acid (EA), sodium ascorbate (SA), ascorbic acid (AA) and ascorbyl palmitate (AP) were added to ground beef (15% fat) at a concentration of 2.3 mM; patties were prepared and stored at 4 °C and at −18 °C. Surface redness ( a* values), lipid oxidation and total reducing activity (TRA) were measured on raw beef, and a* values recorded from cooked internal surfaces. Bovine Mb was purified from biceps femoris muscle and heat capacity was measured using differential scanning calorimetry. All reducing agents decreased lipid oxidation and increased TRA relative to controls ( p < 0.05). In general, SE and SA were more effective at maintaining red color in cooked ground beef patties than other reducing agents ( p < 0.05). The temperature at peak heat capacity, T m, of metmyoglobin appeared lower than that of oxymyoglobin and deoxymyoglobin. Reducing agents can maintain Mb in the reduced state, and their addition to ground beef may be one method of preventing PMB.

Induction of redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal.

The redox stability of myoglobin (Mb) is compromised by many factors, including lipid oxidation and its products. 4-Hydroxy-2-nonenal (HNE) is an alpha,beta-unsaturated aldehyde derived from the oxidation of omega-6 polyunsaturated fatty acids and is highly reactive and cytotoxic. Our objective was to study potential binding of HNE to Mb and determine how it affects redox stability. OxyMb (0.15 mM) was incubated with HNE (1 mM) at 4, 25, and 37 degrees C at pH 7.4 or 5.6. Samples were analyzed for MetMb formation and by Western blot analyses, LC-MS, LC-MS-MS, circular dichroism (CD), and differential scanning calorimetry (DSC). MetMb formation increased with increasing temperature and was greater at pH 5.6 than at pH 7.4 (P < 0.05). At 37 degrees C, HNE accelerated oxidation at pH 7.4 but not at pH 5.6 (P < 0.05). At both 25 and 4 degrees C, HNE accelerated oxidation at pH 7.4 and 5.6 (P < 0.05). LC-MS revealed the covalent binding of HNE to Mb at both pH values via Michael addition, while Western blot analysis indicated that HNE was bound to histidine (HIS) residues. LC-MS-MS identified six histidine residues of Mb that were readily adducted by HNE, including the proximal (HIS 93) and distal (HIS 64) histidine associated with the heme group. Secondary structure differences between control Mb and Mb incubated with HNE were not detected by CD. However, DSC revealed a decreased T(m) for Mb reacted with HNE at pH 7.4, indicating Mb tertiary structure was altered in a manner consistent with destabilization. These results suggest that HNE accelerates bovine skeletal muscle OxyMb oxidation in vitro by covalent modification at histidine residues.

Myoglobin and cytochrome b5: a nuclear magnetic resonance study of a highly dynamic protein complex.

The transient complex of bovine myoglobin and cytochrome b(5) has been investigated using a combination of NMR chemical shift mapping, (15)N relaxation data, and protein docking simulations. Chemical shift perturbations observed for cytochrome b(5) amide resonances upon complex formation with either metmyoglobin (Fe(III)) or carbon monoxide-bound myoglobin (Fe(II)) are more than 10-fold smaller than in other transient redox protein complexes. From (15)N relaxation experiments, an increase in the overall correlation time of cytochrome b(5) in the presence of myoglobin is observed, confirming that complex formation is occurring. The chemical shift perturbations of proton and nitrogen amide nuclei as well as heme protons of cytochrome b(5) titrate with increasing myoglobin concentrations, also demonstrating the formation of a weak complex with a K(a) in the inverse millimolar range. The perturbed residues map over a wide surface area of cytochrome b(5), with patches of residues located around the exposed heme 6-propionate as well as at the back of the protein. The nature of the affected residues is mostly negatively charged contrary to perturbed residues in other transient complexes, which are mainly hydrophobic or polar. Protein docking simulations using the NMR data as constraints show several docking geometries both close to and far away from the exposed heme propionates of myoglobin. Overall, the data support the emerging view that this complex consists of a dynamic ensemble of orientations in which each protein constantly diffuses over the surface of the other. The characteristic NMR features may serve as a structural tool for the identification of such dynamic complexes.

Endocytosis of ferritin and hemoglobin by the trabecular endocardium in swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L. (Poecilidae: Teleostei)

The cardiac atrium and ventricle of swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L., are spongious, consisting of muscle trabeculae covered by endocardial cells. The cardiac trabecular endocardium is able to take up and store large amounts of horse-spleen ferritin and bovine hemoglobin from the blood stream. No such uptake was registered in endocardial cells lining the cardiac valves, atrio-ventricular junction and ventriculo-bulbar junction. The trabecular endocardium in these species seems to be unable to accumulate latex beads or bovine myoglobin, cytochrome C and holotransferrin from the blood stream. It is proposed that the trabecular endocardium in these species is able to clear the blood stream of some types of waste macromolecules; i. e. this tissue may have a scavenger function. The present results indicate that the uptake of foreign ferritin in bony fish endocardium can be clearly demonstrated at the light microscopic level in deparaffined sections by means of acid ferrocyanide or Mallory solutions. A similar uptake of hemoglobin is demonstrated by means of Mallory stain.

Anesthetic-like Interactions of Nitric Oxide with Albumin and Hemeproteins: A MECHANISM FOR CONTROL OF PROTEIN FUNCTION

Noncovalent bonding interactions of nitric oxide (NO) with human serum albumin (HSA), human hemoglobin A, bovine myoglobin, and bovine cytochrome c oxidase (CcO) have been explored. The anesthetic nitrous oxide (NNO) occupies multiple sites within each protein, but does not bind to heme iron. Infrared (IR) spectra of NNO molecules sequestered within albumin, with NO present, support the binding of NO and NNO to the same sites with comparable affinities. Perturbations of IR spectra of the Cys(34) thiol of HSA indicate NO, NNO, halothane, and chloroform can induce similar changes in protein structure. Experiments evaluating the relative affinities of binding of NO and carbon monoxide (CO) to iron(II) sites of the hemeproteins led to evidence of NO binding to noniron, nonsulfur sites as well. With HbA, IR spectra of cysteine thiols and/or the iron(II) N-O stretching region denote changes in protein structure due to NO, NNO, or CO occupying noniron sites with an order of decreasing affinities of NO > NNO > CO. Loss of NO from some, not all, noniron sites in hemeproteins is very slow (t(1/2) approximately hours). These findings provide examples in which NO and anesthetics alter the structure and properties of protein similarly, and support the hypothesis that some physiological effects of NO (and possibly CO) result from anesthetic-like noncovalent bonding to sites within protein or other tissue components. Such bonding may be involved in mechanisms for control of oxygen transport, mitochondrial respiration, and activation of soluble guanylate cyclase by NO.

Hydrogen bonding interaction of the amide group of Asn and Gln at distal E7 of bovine myoglobin with bound-ligand and its functional consequences

Asn and Gln with an amide group at γ- and δ-positions, respectively, were substituted for distal His-E7 of bovine myoglobin to establish a system where hydrogen bonding interaction between the distal residue and bound-ligand can be altered by changing donor–acceptor distance. Two mutant myoglobins showed nearly identical 1H-NMR spectral pattern for resolved heme peripheral side-chain and amino acid proton signals and similar two-dimensional NMR connectivities irrespective of cyanide-bound and -unbound states, indicating that the heme electronic structure and the molecular structure of the active site are not affected by a difference in one methylene group at the E7 position. Chemical exchange rate of Asn-E7 N δH proton in met-cyano myoglobin is larger than that of Gln-E7 N ϵH proton by at least two orders of magnitude, suggesting a considerable difference in the strength of hydrogen bond between the E7 side-chain and bound-ligand, due to the differential donor–acceptor distance between the two mutants. Thus a comparative study between the two proteins provides an ideal system to delineate a relationship between the stabilization of bound-ligand by the hydrogen bond and myoglobin’s ligand affinity. The Asn-mutant showed a faster dissociation of cyano ion from met-myoglobin than the Gln-mutant by over 30-fold. Similarly, oxygen dissociation is faster in the Asn-mutant than in the Gln-mutant by ∼100-fold. Association of cyanide anion to the mutant met-myoglobin was accelerated by changing Gln to Asn by a 4-fold. Likewise, oxygen binding was accelerated by ∼2-fold by the above substitution. The present findings confirm that hydrogen bonding with the distal residue is a dominant factor for determining the ligand dissociation rate, whereas steric hindrance exerted by the distal residue is a primary determinant for the ligand association.

Immunochemical quantification of myoglobin heat denaturation: Comparative studies with monoclonal and polyclonal antibodies

Solutions of bovine myoglobin in 0.1 M‐phosphate buffer, pH 6.0, were heated over the temperature range 55–85°C for 60 min. Their immunological reactivity was tested after 0.2 μm filtration using polyclonal antibodies in the single radial immunodiffusion assay (SRID) and six monoclonal antibodies (MAbs) in a competitive and a sandwich ELISA. Reproducible results were obtained in the SRID assay (coefficient of variation (CV) = 4.0%), with a mid‐point denaturation of 79.0 ± 0.4°C. Similar results were obtained with two MAbs (mid‐point denaturation at 79.4 ± 0.5°C and 78.3 ± 0.8°C for competitive and sandwich ELISAs respectively), but the CV% values were higher (8 and 13% respectively). With the remaining four MAbs, quantification was unreliable as immunoreactivity increased during heating with large inter‐assay variations. A careful control of MAb immunoreactivity in situations covering all possible denaturation states of the test protein is an absolute prerequisite to their use in ELISAs for the immunoquan...

Epitopic analysis and quantification of bovine myoglobin with monoclonal antibodies.

Seven rat monoclonal antibodies (MAb) for bovine myoglobin were produced. Five antibodies reacted with surface-absorbed myoglobin whereas the two remainders reacted only in a sandwich type ELISA. The ability of different antibodies to bind simultaneously to myoglobin was examined by competition and additivity experiments and three noncompeting epitope regions were found. A two-site enzyme immunoassay was developed and allowed quantification of 30 ng/ml bovine myoglobin. These antibodies should be valuable tools in comparative studies for immunological reactivity of mammalian myoglobins and for myoglobin measurement in serum and urine of myopathic animals.