Myoglobin Solutions Research Articles

Dissociation of heme from gaseous myoglobin ions studied by infrared multiphoton dissociation spectroscopy and Fourier-transform ion cyclotron resonance mass spectrometry

Detachment of heme prosthetic groups from gaseous myoglobin ions has been studied by collision-induced dissociation and infrared multiphoton dissociation in combination with Fourier-transform ion cyclotron resonance mass spectrometry. Multiply charged holomyoglobin ions (hMbn+) were generated by electrospray ionization and transferred to an ion cyclotron resonance cell, where the ions of interest were isolated and fragmented by either collision with Ar atoms or irradiation with 3 mum photons, producing apomyoglobin ions (aMbn+). Both charged heme loss (with [Fe(III)-heme]+ and aMb(n-1)+ as the products) and neutral heme loss (with [Fe(II)-heme] and aMbn+ as the products) were detected concurrently for hMbn+ produced from a myoglobin solution pretreated with reducing reagents. By reference to Ea = 0.9 eV determined by blackbody infrared radiative dissociation for charged heme loss of ferric hMbn+, an activation energy of 1.1 eV was deduced for neutral heme loss of ferrous hMbn+ with n = 9 and 10.

UV‐Visible and Electrochemical Monitoring of Carbon Monoxide Release by Donor Complexes to Myoglobin Solutions and to Electrodes Modified with Films Containing Hemin

AbstractThis study reports on the evaluation of the CO donating behavior of tricarbonyl dichloro ruthenium(II) dimer ([Ru(CO)3Cl2]2) and 1,3‐dimethoxyphenyl tricarbonyl chromium (C6H3(MeO)2Cr(CO)3) complex by UV‐visible technique and electrochemical technique. The CO release was monitored by following the modifications of the UV‐visible features of MbFe(II) in phosphate buffer solution and the redox features of reduced Hemin, HmFe(II), confined at the surface of a vitreous carbon electrode. In the latter case, the interaction between the hemin‐modified electrode and the released CO was seen through the observation of an increase of the reduction current related to the FeIII/FeII redox process of the immobilized porphyrin. While the ruthenium‐based complex, ([Ru(CO)3Cl2]2), depended on the presence of Fe(II) species to release CO, it was found that the chromium‐based complex released spontaneously CO. This was facilitated by illuminating and/or simple stirring of the solution containing the complex.

Thermodynamic study of metabolic reactions in an aqueous protein solution

The time dependences of the thermal power of aqueous myoglobin solutions were measured by microcalorimeter at 298.15 K. Exothermic reactions occurred in aqueous myoglobin solutions due to the metabolism of aerobic microbes, and these roughly consisted of four phases. The generation times obtained were about (55±5) min for the logarithmic exothermal reaction phase. The total energies were considerably dependent on the amount of oxygen present, suggesting strongly that the exothermic reaction was caused by aerobic microbes. The apparent thermal metabolic rates were positively dependent on the concentration of myoglobin, probably because of the effects of myoglobin as a food source and/or as a donor of oxygen.

Denaturation of Lysozyme and Myoglobin in Laser Spray

In laser spray, the tip of an electrospray capillary is irradiated with a continuous CO(2) laser beam. Here, we report results from a modified laser spray method that employs a relatively low laser irradiance level. With a laser power of approximately 2 W and a focal spot size ( approximately 0.3 mm), which covered the entire front surface of the electrospray capillary, the irradiance was approximately 3 x 10(3) W/cm(2). This resulted in a quiescent and smooth vaporization of aqueous solutions. This "evaporation-mode" laser spray method yielded the best results so far obtained in our laboratory with laser-irradiated electrospray, producing higher and more stable signals. The method was applied to the analysis of aqueous solutions of lysozyme and myoglobin. Mass spectra were obtained as a function of laser power from 0 W (electrospray) to approximately 2 W. The spray generated at the tip of the stainless steel capillary was observed with a CCD camera. With increase of laser power, the droplets in the spray became finer and the Taylor cone became progressively smaller. The strongest ion signals were recorded when the sample solution protruded only slightly from the tip of the capillary. A broadening of the lysozyme charge-state distribution, attributable to protein unfolding, was observed with a laser power of 2 W. No denaturation of myoglobin took place up to a laser power of 1.6 W. However, a sudden onset of denaturation was observed at 1.8 W as a broadening of the myoglobin charge distribution and the appearance of apo-myoglobin peaks. These findings demonstrate that laser spray is capable of dissociating the noncovalent complexes selectively without breaking covalent bonds.

Mechanistic Roles of Ser-114, Tyr-155, and Lys-159 in 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni, a short chain dehydrogenase/reductase, catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. A catalytic triad of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR has been proposed based on structural analysis and sequence alignment of the short chain dehydrogenase/reductase family. The 3alpha-HSD/CR-catalyzed reaction has not been kinetically analyzed in detail, however. In this study, we combined steady-state kinetics, site-directed mutagenesis, and pH profile to explore the function of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR-catalyzed reaction. The catalytic efficiency of wild-type and mutants S114A, Y155F, K159A, and Y155F/K159A is 4.3 x 10(7), 7.3 x 10(4), 1.7 x 10(4), 2.4 x 10(5), and 71 m(-1)s(-1), respectively. The values of pKa on kcat/Km for the wild-type, S114A, Y155F, K159A, and Y155F/K159A are 7.2, 7.4, 8.4, 9.1, and 10.2, respectively. Mutant S114A/Y155F exhibits a pH-independent profile with 10(-5) times of wild-type activity at pH 10.5. The activity decreases as the pH lowers, which indicates that a functional group with an apparent pKa of 7.2 is involved in the general base catalysis for wild-type 3alpha-HSD/CR. The pKa shift to 9.1 for mutant K159A suggests the role of Lys-159 is to lower the pKa of the residues involved in the general base catalysis. Because pH dependence is observed for both S114A and Y155F mutants and pH independence is observed in S114A/Y155F, Tyr-155 may be important as a general base catalysis in the wild-type, whereas Ser-114 may act as a general base on mutant Y155F to catalyze the reaction.

EFFECT OF HEATING ON RESIDUAL CARBON MONOXIDE CONTENT IN CO-TREATED TUNA AND MYOGLOBIN

Carbon monoxide (CO) can be used by food manufacturers to retain the red color of meat or dark fish prior to vacuum packaging or in the modified atmosphere packaging. Yellowfin tuna steak (c. 1.5 cm) was treated with CO gas, then packaged in a vacuum bag and heated at a constant temperature ranging from 50 to 100C for 30 min. A myoglobin (Mb) solution was prepared from tuna dark muscle and purged with CO gas for comparison. The released CO concentration and the residual CO in the tuna flesh or Mb solution were measured by gas chromatography. The higher level of CO concentration in the vacuum bag from CO-treated tuna was found for conditions with temperatures above 80C. The content of residual CO in the CO-treated tuna steaks after heating was much higher than that in untreated flesh. Regardless of the temperature of heating, a ratio of 58-82% CO was released from Mb solution after heating. This suggested that MbCO was not completely dissociated by heating at temperatures ranging from 50 to 100C.

Over 1000-fold protein preconcentration for microliter-volume samples at a pH junction using capillary electrophoresis

An effective protein preconcentration technique specifically designed for microliter-volume samples is presented. The preconcentration is based on the capturing of protein in its isoelectric point (p I) within an applied electric field, using a pH junction created by a discontinuous buffer system. The buffers were chosen to selectively preconcentrate proteins of neutral p I, myoglobin in this case, while removing other proteins with acidic or basic p Is. For the suppression of electro-osmotic flow (EOF) and protein adsorption, the capillary inner wall was modified with a zwitterionic phospholipid bilayer coating. A preconcentration factor of up to 1700 was obtained for a 1 μg/mL solution of myoglobin. The preconcentration was completed in approximately 20 min. Several sample introduction conditions were presented to accommodate sample volume from one to a few hundreds of microliters. The final volume of the preconcentrated sample band was estimated to be approximately 5 nL.

Non-thermal effects of electromagnetic fields at mobile phone frequency on the refolding of an intracellular protein: myoglobin.

Non-thermal effects induced by exposure to microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, have been observed on the refolding kinetics of the heme binding site in an intracellular protein: tuna myoglobin, starting from acidic conditions. We have selected myoglobin because it can be considered a good model to study protein interactions with MW-EMF for its well-known high-resolution crystallographic structure. Myoglobin solutions at pH 3.0 were subjected to 3 h exposure to microwave field (with a specific absorption rate of 51 +/- 1 mW/g); the heme site refolding has been followed by measuring the molecular absorption in the Soret spectral region and the data were fitted to a bi-exponential model. The kinetics of exposed samples appear to be slowered by MW-EMF action. Moreover, the tryptophanyl lifetime distribution of the exposed protein, as deduced by the analysis of the fluorescence emission decay from its single tryptophan, appears sharper if compared to non-exposed protein samples. This observation suggests that the presence of MW-EMF could affect the propensity of protein molecules to populate specific conformational substates among which myoglobin molecules fluctuate at acidic pH. Changes in the structural fluctuation caused by MW perturbation can affect differently the aggregation process that occurs competitively during the protein folding, so representing a potential risk for protein "misfolding." These data suggest that MW-EMF could have also biochemical and, consequently, biological effects on eukaryotic cells that are still under investigation.

Poly(dimethylsiloxane) microchip: microchannel with integrated open electrospray tip.

A polymer microchip with an open tip for electrospray mass spectrometry is presented. The tip consists of a groove with parallel walls where a droplet can form at the end surface. A lid covers the whole chip except at the microchannel tip, which is left open. Poly(dimethylsiloxane) (PDMS) microchips were cast using a nickel mould which in turn was replicated from a dry etched silicon wafer. Tips with microchannel widths of around 50 microm could easily be replicated. Since the tip had no cover, the assembly of microchip and cover was simplified. A total ion current variation of 5% during 300 s was achieved for a 1 microM myoglobin solution. The non-complex design of the cover makes it suitable for versatile tests of chip prototypes. The nickel mould was found to be useful for PDMS microstructure fabrication. Also, such a robust mould allows casting electrospray tips in more rigid thermoset materials.

Study of ultrafiltration of a single and binary protein solution in a thin spiral channel module

The aim of this paper is to demonstrate the factors influencing a protein filtration process. Therefore single and mixed solutions of lysozyme and myoglobin were ultrafiltered through a hydrophilic membrane (cellulose acetate type) with a molecular weight cutoff (MWCO) of 30 kDa. The proteins are of similar molar mass and size. Filtration was done using a thin spiral channel module (TCF10). The change in transmission and flux was evaluated and the data of the single protein solutions compared with the binary protein mixture. Further, the effects of Dean vortex formation, was evaluated. From the collected data the effects of the parameters dominating the filtration process were assessed. By comparing the experimentally obtained mass transfer coefficients with literature data, it can be seen that the curved channel led to an increased mass transfer as compared with classical modules. For both proteins, a Sherwood correlation was derived and based on these data a separation process was modeled. Results obtained from separation experiments correlated well with the developed model. It could be shown that the separation of proteins by ultrafiltration is possible, but for proteins bearing similar molar masses it is a difficult task. Separation factors up to 2.5 were achieved. As expected the selectivity increases with the crossflow velocity and decreasing transmembrane pressure (TMP). The separation process requires careful choice of the pH value and the ionic strength. Further, attention has to be paid to the membrane stability and protein solubility while varying the pH value.

Dual-function microanalytical device by in situ photolithographic grafting of porous polymer monolith: integrating solid-phase extraction and enzymatic digestion for peptide mass mapping.

Microfluidic devices with a dual function containing both a solid-phase extractor and an enzymatic microreactor have been prepared, and their operation has been demonstrated. The devices were fabricated from a 25-mm-long porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith prepared within a 50-microm-i.d. capillary. This capillary with a pulled 9-12-microm needle tip was used as a nanoelectrospray emitter coupling the device to a mass spectrometer. Photografting with irradiation through a mask was then used to selectively functionalize a 20-mm-long portion of the monolith, introducing reactive poly(2-vinyl-4,4-dimethylazlactone) chains to enable the subsequent attachment of trypsin, thereby creating an enzymatic microreactor with high proteolytic activity. The other 5 mm of unmodified hydrophobic monolith served as micro solid-phase extractor (microSPE). The dual-function devices were used in two different flow directions; concentration of myoglobin that was absorbed from its dilute solution, followed by elution and digestion or digestion, followed by concentration. Operations in both directions afforded equal sequence coverage. Different volumes of myoglobin solution ranging from 2 to 20 microL were loaded on the device. Very high sequence coverages of almost 80% were achieved for the highest loading. Despite the very short length of the extractor unit, the device operated in the digest-solid-phase extraction direction also enabled the separation of peaks that mostly contained undigested protein and peptides.

Electrochemical and electrocatalytic properties of myoglobin and hemoglobin incorporated in carboxymethyl cellulose films

Protein-CMC films were made by casting a solution of myoglobin (Mb) or hemoglobin (Hb) and carboxymethyl cellulose (CMC) on pyrolytic graphite electrodes. In pH 7.0 buffers, Mb and Hb incorporated in CMC films gave a pair of well-defined and quasi-reversible cyclic voltammetric peaks at about −0.34 V vs. SCE, respectively, characteristic of heme Fe III/Fe II redox couples of the proteins. The electrochemical parameters such as apparent standard heterogeneous electron transfer rate constants ( k s) and formal potentials (E°′) were estimated by square wave voltammetry with nonlinear regression analysis. In aqueous solution, stable CMC films absorbed large amounts of water and formed hydrogel. Scanning electron microscopy of the films showed that interaction between Mb or Hb and CMC would make the morphology of dry protein-CMC films different from the CMC films alone. Positions of Soret absorbance band suggest that Mb and Hb in CMC films retain their secondary structure similar to the native states in the medium pH range. Trichloroacetic acid, nitrite, oxygen, and hydrogen peroxide were catalytically reduced at protein-CMC film electrodes.

Myoglobin in crowded solutions: structure and diffusion

Abstract We present a neutron scattering study of the structure and diffusion of myoglobin solutions at high concentration. The protein–protein structure factor was determined by small angle neutron scattering (SANS) measurements using mean spherical analysis (MSA), and the intermediate scattering function was measured by neutron spin-scho spectroscopy. We observe the cross-over between self-diffusion at high Q and collective diffusion below the structure factor maximum. The self-diffusion coefficient decreases exponentially with concentration. The collective diffusion coefficient is shown to increase at low Q due to direct interactions.

Determination of carbon isotope ratios in amino acids, proteins, and oligosaccharides by inductively coupled plasma-mass spectrometry

Carbon isotope ratios ( 12C/ 13C) are measured for aqueous solutions of tryptophan, myoglobin, and β-cyclodextrin using C + ions from an inductively coupled plasma (ICP) and a prototype twin quadrupole mass spectrometer (MS). 13C/ 12C ratios can be determined with a relative standard deviation (RSD) of ∼1%. This precision is close to the limiting value predicted by counting statistics (1.16%). Spectral interference on 13C +, presumably from 12C 1H +, comes from the incomplete dissociation of myoglobin and/or β-cyclodextrin, but not tryptophan. Decreasing the aerosol gas flow rate slightly from that which yields maximum signal eliminates this 12C 1H + interference. The count rate of the minor isotope ( 13C +) can be artificially enhanced by increasing the voltage of the 13C + detector, and/or by shifting the ion beam splitter offset from the central axis. Instrumental modifications to the MS that improve the sensitivity are also described.

Performance Evaluation of a Dialyzer Equipped with a Hydrophilic Fiber Spacer to Enhance Internal Filtration

Dialyzers equipped with a hydrophilic fiber spacer have been newly introduced in order to enhance internal filtration. The spacer is composed of a dialysis membrane bundle with several turns of the fibers wounding around it. Swelling of the spacer by absorption of water reduces the cross-sectional area of the dialysate stream and increases the pressure drop (ΔPD) on the dialysate side of the dialyzer. In addition to diffusive transport, the internal filtration induced by increasing ΔPD enhanced convective transport of the solute through the dialysis membrane, especially of relatively large molecular weight substances. We examined the performance of the newly introduced dialyzer in an experimental study using an aqueous solution of myoglobin and assessed the solute removal characteristics of the dialyzer with an analytical model. Three dialyzers with the spacer at different positions were used. The 40 mm wide spacer was inserted at the inlet (40 mm inlet), the center (40 mm central), and the outlet (40 mm outlet) of the dialyzer. The results of the experimental study showed that all dialyzers containing the spacer had a higher ΔPD than the control dialyzer (FB-150UH, Nipro Corp.). On the other hand, the 40 mm central dialyzer yielded higher myoglobin clearance than the 40-mm-inlet and the 40-mm-outlet dialyzers. The 40 mm central dialyzer had a higher average transmembrane pressure (TMP), because it had enough length before and after the spacer.

高吸水性ゲル粒子を利用した２成分系タンパク質溶液の分画と濃縮の同時操作

A new technique has been developed for separating target protein selectively from protein mixtures using superabsorbent crosslinked hydrogels which have a drastic change in volume by absorbing water. In the process, the gel particles swell by absorbing water in an aqueous solution of complex solutions of BSA and myoglobin, and these proteins are simultaneously fractionated by the principle of polyelectrolyte precipitation resulting from electrostatic interactions between the oppositely charged protein and the gel. The experimental data clearly demonstrated that the concentration ratio of BSA markedly increases with the dosage of the gel, while that of myoglobin decreases, resulting in a high degree of fractionation of BSA and myoglobin with the hydrogels. In addition, the variation over time of the concentration ratio of BSA was successfully evaluated by using the equation of motion of the gel network on the basis of the kinetics of swelling of the spherical gel.

Effect of gamma-irradiation on the molecular properties of myoglobin.

To elucidate the effect of gamma-irradiation on the molecular properties of myoglobin, the secondary and tertiary structures, as well as the molecular weight size of the protein, were examined after irradiation at various irradiation doses. Gamma-irradiation of myoglobin solutions caused the disruption of the ordered structure of the protein molecules, as well as degradation, crosslinking, and aggregation of the polypeptide chains. A SDSPAGE study indicated that irradiation caused initial fragmentation of the proteins and subsequent aggregation, due to cross-linking of the protein molecules. The effect of irradiation on the protein was more significant at lower protein concentrations. Ascorbic acid protected against the degradation and aggregation of proteins by scavenging oxygen radicals that are produced by irradiation. A circular dichroism study showed that an increase of the irradiation decreased the alpha-helical content of myoglobin with a concurrent increase of the aperiodic structure content. Fluorescence spectroscopy indicated that irradiation increased the emission intensity that was excited at 280 nm.

Mechanisms of Pink Color Formation in Irradiated Precooked Turkey Breast Meat

ABSTRACT: Precooked turkey breast meat was aerobically packaged or vacuum‐packaged and irradiated at 0, 2.5, or 5.0 kGy. CIE color, reflectance, oxidation‐reduction potential (ORP), gas production, and lipid oxidation were determined at 0, 7, and 14 d. Irradiation increased redness of vacuum‐packaged meat, and the redness was distinct and stable under vacuum. Irradiation decreased ORP and produced carbon monoxide (CO). This indicated that the pink color was caused by the heme pigment‐CO complex formation. The reflectance of meat and the absorption spectra of myoglobin solution supported the assumption that denatured CO‐myoglobin is the pigment in irradiated precooked turkey breast.

Contribution of solvent water to the solution X-ray scattering profile of proteins

A theoretical framework is presented to analyze how solvent water contributes to the X-ray scattering profile of protein solution. Molecular dynamics simulations were carried out on pure water and an aqueous solution of myoglobin to determine the spatial distribution of water molecules in each of them. Their solution X-ray scattering (SXS) profiles were numerically evaluated with obtained atomic-coordinate data. It is shown that two kinds of contributions from solvent water must be considered to predict the SXS profile of a solution accurately. One is the excluded solvent scattering originating in exclusion of water molecules from the space occupied by solutes. The other is the hydration effect resulting from formation of a specific distribution of water around solutes. Explicit consideration of only two molecular layers of water is practically enough to incorporate the hydration effect. Care should be given to using an approximation in which an averaged electron density distribution is assumed for the structure factor because it may predict profiles considerably deviating from the correct profile at large K.

Critical flux in ultrafiltration of myoglobin and baker’s yeast

Critical fluxes were determined by constant flux ultrafiltration (UF) experiments under laminar flow conditions. The experiments were performed by using hydrophilic C30G and hydrophobic GR51 ultrafiltration membranes and dilute myoglobin solutions and baker’s yeast suspensions as model colloids. Solution concentration, pH and cross-flow were investigated. The critical flux increased with increasing flow velocity and decreasing solute concentration. The regenerated cellulose C30G membrane exhibited higher critical fluxes than the polysulphone GR51 membrane. The highest critical flux was obtained at pH 8 in the presence of repulsive electrostatic forces between the molecules and the surface of the membrane and the lowest at the isoelectric points of the colloids. In the case of baker’s yeast below the critical flux, the flux was about the same as the pure buffer solution flux showing a strong form of the critical flux. This also occurred with the C30G membrane at low concentrations of myoglobin except at pH 6 when a weak form of the critical flux was measured. With the GR51 membrane, the permeate flux deviated from the pure buffer solution flux even at the lowest fluxes.