Myoglobin Solutions Research Articles

Non-Steady-State O2 Diffusion in Metmyoglobin Solutions Studied in a Diffusion Chamber

In this study, we studied the “passive” diffusion through myoglobin solutions by determining the oxygen diffusion coefficient (DO2) and the oxygen permeability (PO2) of metmyoglobin (metMb) solutions (3–33 g · 100 mL−1) at 25°C. These oxygen diffusion parameters were determined in a diffusion chamber using a non-steady-state method and were also determined of albumin solutions (4–32 g · 100 mL−1) and distilled water for comparison. From these parameters, the oxygen solubility (αO2) could be calculated, because αO2 = PO2/DO2. Both DO2 and PO2 decreased with increasing metMb and albumin concentration. The values of DO2, PO2 and αO2 of both metMb solutions and albumin solutions were comparable with literature values of methemoglobin (metHb) and serum protein solutions. The values of the metMb solutions can be used in following studies of facilitated oxygen diffusion through myoglobin solutions.

Evidence of nonspecific surface interactions between laser-polarized xenon and myoglobin in solution.

The high sensitivity of the magnetic resonance properties of xenon to its local chemical environment and the large (129)Xe NMR signals attainable through optical pumping have motivated the use of xenon as a probe of macromolecular structure and dynamics. In the present work, we report evidence for nonspecific interactions between xenon and the exterior of myoglobin in aqueous solution, in addition to a previously reported internal binding interaction. (129)Xe chemical shift measurements in denatured myoglobin solutions and under native conditions with varying xenon concentrations confirm the presence of nonspecific interactions. Titration data are modeled quantitatively with treatment of the nonspecific interactions as weak binding sites. Using laser-polarized xenon to measure (129)Xe spin-lattice relaxation times (T(1)), we observed a shorter T(1) in the presence of 1 mM denatured apomyoglobin in 6 M deuterated urea (T(1) = 59 +/- 1 s) compared with that in 6 M deuterated urea alone (T(1) = 291 +/- 2 s), suggesting that nonspecific xenon-protein interactions can enhance (129)Xe relaxation. An even shorter T(1) was measured in 1 mM apomyoglobin in D(2)O (T(1) = 15 +/- 0.3 s), compared with that in D(2)O alone (T(1) = 506 +/- 5 s). This difference in relaxation efficiency likely results from couplings between laser-polarized xenon and protons in the binding cavity of apomyoglobin that may permit the transfer of polarization between these nuclei via the nuclear Overhauser effect.

Effects of internal filtration on the solute removal efficiency of a dialyzer.

To improve solute removal efficiency, several types of dialyzers with enhanced internal filtration were introduced for clinical application. In these dialyzers, enhanced internal filtration increased convective transport of the solute, in addition to diffusive transport. In this study, the effects of internal filtration on solute removal efficiency were examined by both analytic and experimental studies. Internal filtration is affected by blood (Q(B)) and dialysate (Q(D)) flow rates; the patient's hematocrit and plasma level of total protein; and the effective length (L(eff)), inner diameter (D), and density ratio (DR) of the hollow fibers. An analytic model was introduced for the estimation of the changes in mass and momentum along the dialyzer. It clarified the effects of these parameters on maximum internal filtration flow rate (Q(IF)) and clearance (K) of urea (60 daltons), vitamin B(12) (1,355), and myoglobin (17,000). As a result of the analytic study, Q(IF) was increased, resulting in a smaller D, a longer L(eff), and a larger DR value. Several types of dialyzers with the same cellulose triacetate membrane, produced by Toyobo Co, Ltd., Ohtsu, Japan, and Nissho Corporation, Kusatsu, Japan, were used for the experimental study. An in vitro evaluation using myoglobin solution showed the same trends as found in the analytic study. For example, a dialyzer with 150 microm of D has a 72.0 ml/min myoglobin K value, much higher than that of 53.7 ml/min for a dialyzer with 200 microm of D under constant Q(B) (300 ml/min) and DR (50%) values. Development of a dialyzer with enhanced internal filtration, however, should take the patient's safety into account, and hemolysis and endotoxin invasion from the dialysate to the patient should be avoided.

Consequences of 129Xe–1H Cross Relaxation in Aqueous Solutions

We have investigated the transfer of polarization from 129Xe to solute protons in aqueous solutions to determine the feasibility of using hyperpolarized xenon to enhance 1H sensitivity in aqueous systems at or near room temperatures. Several solutes, each of different molecular weight, were dissolved in deuterium oxide and although large xenon polarizations were created, no significant proton signal enhancement was detected in l-tyrosine, α-cyclodextrin, β-cyclodextrin, apomyoglobin, or myoglobin. Solute-induced enhancement of the 129Xe spin–lattice relaxation rate was observed and depended on the size and structure of the solute molecule. The significant increase of the apparent spin–lattice relaxation rate of the solution phase 129Xe by α-cyclodextrin and apomyoglobin indicates efficient cross relaxation. The slow relaxation of xenon in β-cyclodextrin and l-tyrosine indicates weak coupling and inefficient cross relaxation. Despite the apparent cross-relaxation effects, all attempts to detect the proton enhancement directly were unsuccessful. Spin–lattice relaxation rates were also measured for Boltzmann 129Xe in myoglobin. The cross-relaxation rates were determined from changes in 129Xe relaxation rates in the α-cyclodextrin and myoglobin solutions. These cross-relaxation rates were then used to model 1H signal gains for a range of 129Xe to 1H spin population ratios. These models suggest that in spite of very large 129Xe polarizations, the 1H gains will be less than 10% and often substantially smaller. In particular, dramatic 1H signal enhancements in lung tissue signals are unlikely.

One-step sandwich enzyme immunoassay for serum myoglobin with three monoclonal antibodies to different epitopes

Enzyme-linked sandwich immunosorbent assay for serum myoglobin with 3 monoclonal antibodies to different epitopes is described. Antibodies 13G6 and 18H2 were adsorbed on a carrier, and 14D6 antibodies were labeled with horseradish peroxidase. One-step incubation with standard myoglobin solution yielded double sandwich: adsorbed antibodies—myoglobin—labeled antibodies; the intensity of peroxidase reaction was directly proportional to myoglobin concentration. The sensitivity of the proposed method for serum myoglobin was below 5 ng/ml. The sensitivity of the method increases due to myoglobin binding with two antibodies adsorbed on the carrier. Fluctuations within and between series did not exceed 10%.

Near-Infrared Spectroscopic Measurement of Myoglobin Oxygen Saturation in the Presence of Hemoglobin Using Partial Least-Squares Analysis

Myoglobin is an important intracellular protein found in cardiac and skeletal muscle. It is involved in the intracellular transport of oxygen from the cell membrane to the mitochondria where oxidative phosphorylation takes place. The optical absorbance characteristics of myoglobin are similar to those of hemoglobin in the near-infrared spectral region. Distinguishing spectral information of myoglobin from hemoglobin should allow for determination of intracellular oxygen availability in muscle. Partial least-squares analysis is used in this report to determine the oxygen saturation of myoglobin, in the presence of hemoglobin, in vitro. Studies were performed with the use of both transmission and reflectance spectroscopic techniques. Transmission spectra of myoglobin solutions were determined with varying degrees of oxygen saturation achieved by deoxygenating the solution using E. coli. Calibration spectral data sets were developed with the use of varying concentrations of hemoglobin interference, and with varying degrees of myoglobin oxygen saturation. Reflectance spectra were obtained from myoglobin and hemoglobin solutions containing a scattering agent to mimic muscle tissue conditions. Predicted myoglobin saturation values were within 2% of the known saturation values from the use of this analysis. Partial least-squares analysis allows for accurate prediction of myoglobin oxygen saturation in the presence of hemoglobin from either transmission of reflectance near-infrared spectra.

A novel nonmetallized tip for electrospray mass spectrometry at nanoliter flow rate

We report the fabrication of a durable nonmetallized nanospray tip. This nanospray tip does not require complex preparation procedures such as chemical treatment, deposition of gold or SiOx vapor. It was fabricated by pulling a heated glass capillary of 1.1 mm internal diameter to produce a fine tip with an orifice of 10–15 μm in diameter. A 10 μm gold-plated tungsten wire was inserted through the capillary tip. This tungsten wire played a central role in the operation of this durable nanospray tip by providing electrical contact. This type of nanospray tip could withstand electrical discharges and sustained spraying of solution at nanoliter flow rate for more than 3 h. Using insulin (35 μM) and myoglobin (1 μM) solutions, useful mass spectrum could be acquired with low fmol sensitivity.

Determination of Iron−Ligand Bond Lengths in Horse Heart Met- and Deoxymyoglobin Using Multiple-Scattering XAFS Analyses

XAFS data in the range 0 ≤ k ≤ 14.5 A-1 have been obtained from frozen aqueous solutions (10 K) of horse heart myoglobin (Mb) in the Fe(III) (aqua-met) and Fe(II) (deoxy) forms. The structures of the Fe sites have been refined using both single-scattering (SS) and multiple-scattering (MS) analyses. The XAFS MS analyses yield more precise Fe−ligand bond lengths (estimated error 0.02−0.03 A) than those determined crystallographically (estimated errors ≥0.1 A). For met-Mb, the MS analysis results in an average Fe−N(pyrrole) distance of 2.05 A, an Fe−N(imidazole) distance of 2.17 A, and an Fe−O(aqua) distance of 2.08 A. For deoxy-Mb, the MS analysis results in Fe−N(pyrrole) and Fe−N(imidazole) distances of 2.06 and 2.16 A, respectively. The final XAFS R values are 18.8% and 17.8% for met- and deoxy-Mb, respectively. The robustness of the refinements was tested by varying the starting models, constraints, restraints, and k ranges.

Hindered transmembrane protein transport in hollow-fibre devices

Protein transport in the extracapillary and intracapillary spaces (ECS and ICS) as well as across the membranes of hollow-fibre devices was investigated. Regenerated-cellulose ultrafiltration membrane cartridges were loaded with solutions of myoglobin and operated in the closed-shell mode for a period of several days. The transmembrane leakage and spatial distribution of myoglobin were dependent on the protein loading, the ICS flow rate and the presence of bovine serum albumin in the ECS. A mathematical model was developed to characterise the time-dependent protein redistribution, taking into account the effects of osmotic pressure, fibre expansion under wet conditions and the fibre-free manifold regions of the ECS. According to the model, the transmembrane transport of protein can be described using one semi-empirical parameter, the membrane constant, defined as the ratio of the square of pore tortuosity to surface porosity, from which the osmotic reflection coefficient, the partition coefficient, and the diffusive and convective hindrance factors can be calculated. The experimental data were used to test the model and to find the values of the membrane constant for the system under consideration.

Spectroelectrochemical and electrochemical determination of ligand binding and electron transfer properties of myoglobin, cyanomyoglobin, and imidazolemyoglobin

Abstract Electrochemistry and spectroelectrochemistry were used to study solutions of horse myoglobin, both by itself and complexed with cyanide or imidazole, at indium-tin oxide electrodes. In the absence of ligands other than water, myoglobin exhibited slow quasi-reversible heterogeneous electron transfer kinetics, with a formal heterogeneous electron transfer rate constant ( k °′) value of 1.6(±0.4)×10 −5 cm s −1 . Differences from simple simulations in the reverse sweep (anodic) cyclic voltammograms are proposed to be due to residual amounts of dioxygen in solution and/or in the protein molecule. Those differences were not seen in the anodic waveforms of simultaneously acquired derivative cyclic voltabsorptometry (DCVA) experiments. Cyanomyoglobin exhibited faster heterogeneous electron transfer kinetics, with a k °′ value of 6.0(±1.0)×10 −4 cm s −1 . Values for the homogeneous rate constants for dissociation ( k f ) and association ( k b ) of cyanide from and to the electrochemically reduced protein were determined to be 0.08(±0.03) s −1 and 0.06(±0.03) M −1 s −1 , respectively. Imidazolemyoglobin was found to transfer electrons faster than cyanomyoglobin, with a k °′ value of 2.0(±0.5)×10 −3 cm s −1 . The values for the homogeneous rate constants k f and k b were 0.40(±0.1 5) s −1 and 1.1(±0.1) M −1 s −1 , respectively.

Dielectric Relaxation, Molecular Motion and Interprotein Interactions in Myoglobin Solution

The results of the investigation of protein molecule dynamic in solution by Time Domain Dielectric Spectroscopy are presented. The horse myoglobin solutions in wide range of concentration from 0.6% to 54% at 20°C have been investigated. The result of analysis produced in the term of dipole correlation function has shown that the obtained correlation function of macromolecule motion may be presented as sum of three components corresponding to three kinds of protein motions: anisotropic intramolecular motion, anisotropic Brownian tumbling and isotropic slow motion. We suppose that the cause of protein tumbling anisotropy and the possibility to keep slow motion is the interprotein electrostatic interactions. The characteristic time of slow motion depends on the concentration of protein and perhaps is controlled by translational diffusion. The dipole moment of myoglobin calculated by the Onsager-Oncley equation is 200D for solutions less than 10% protein concentration. It is in a good agreement with the theoretical value.

Myoglobin oxygen dissociation by multiwavelength spectroscopy.

Multiwavelength optical spectroscopy was used to determine the oxygen-binding characteristics for equine myoglobin. Oxygen-binding relationships as a function of oxygen tension were determined for temperatures of 10, 25, 35, 37, and 40 degrees C, at pH 7.0. In addition, dissociation curves were determined at 37 degrees C for pH 6.5, 7.0, and 7.5. Equilibration was achieved with a myoglobin solution, at the desired temperature and pH, and 16 oxygen-nitrogen gas mixtures of known oxygen fraction. Correction for the inevitable presence of metmyoglobin was made by using a three-component least squares analysis and by correcting the end point oxymyoglobin spectra for the presence of metmyoglobin. The PO2 at which myoglobin is half-saturated with O2 (P50) was determined to be 2.39 Torr at pH 7.0 and 37 degrees C. The myoglobin dissociation curve was well fit by the Hill equation [saturation = PO2/(PO2 + P50)].

Determination of the diffusion coefficient of myoglobin in muscle cells. Photo-oxidation and microinjection method.

Myoglobin (Mb) serves as an intracellular store for oxygen in red muscles, ensuring aerobic metabolic processes during temporary respiratory or circulatory deficits in oxygen supply. Myoglobin can also contribute to sarcoplasmic oxygen transport by loading oxygen near the capillaries, diffusing to sites where the PO2 is low and releasing the oxygen there. To what extent this myoglobin-facilitated oxygen diffusion enhances the overall oxygen transport rate primarily depends on the cellular myoglobin concentration and on the mobility of this protein within the muscle cell. Myoglobin concentrations can easily be determined but until recently no direct measurements of the diffusion coefficient (DMb) of myoglobin in intact mammalian skeletal muscle cells have been performed. In many theoretical studies dealing with facilitated oxygen diffusion, therefore, estimates of the DMbvalue representing results from self-diffusion measurements in highly concentrated myoglobin solutions have been applied instead.

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...

Diffusion coefficient of oxygen in various model layers as determined by analysis of time-dependent diffusion.

To obtain an accurate oxygen diffusion coefficient for muscle tissue, the tissue is divided into several fractions. These fractions can be represented by myoglobin solutions, protein solutions, endothelium, lipid layers, etc. For each of these fractions an oxygen diffusion coefficient can be measured in vitro in a diffusion chamber. In general, the methods for measuring oxygen diffusion across these various layers or membranes can be divided into two types; steady state and nonsteady state measurements. The steady state methods yield the oxygen permeability of a layer (p 02), which is equal to the product of the oxygen diffusion coefficient (D02) and the oxygen solubility (a02). This implies that the determination of the diffusion coefficient from steady state measurements requires accurate solubility data. These may not be available for every type of model layer. From the nonsteady state measurements, however, the diffusion coefficient can be obtained directly [1,2]. Therefore, in this study the nonsteady state method was applied to determine the D02. The model layers that were used in the experiments were plain water layers, either confined to Millipore® filters or applied on top of a porous PTFE membrane.KeywordsModel LayerSupporting LayerDiffusion ChamberSteady State MeasurementSteady State MethodThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Surface-Linked Molecular Monolayers of an Engineered Myoglobin: Structure, Stability, and Function

The maintenance of active conformation and biological function is important for the development of solid substrate immobilized biomacromolecules for material applications. A protein monolayer was obtained on SiO2 substrates by chemically linking a site-directed mutant of sperm whale myoglobin, A126C, which has a unique and reactive cysteine residue on its surface, to a thiol specific functional group on the silane-derivatized substrates. Fourier transform infrared (FTIR) spectroscopy of this protein monolayer suggests that a native-like secondary structure is retained for the immobilized myoglobin. In addition, the immobolized myoglobin retains its ability to bind carbon monoxide. Structural changes in the surface-bound protein were examined under variation of temperature, pH, urea concentration, and ethanol content by UV−vis absorption spectroscopy. The densely packed protein, roughly 60% surface coverage of the substrate, is as resilient to high temperature, low-pH environment, and high concentration of urea as myoglobin solution is at low concentration. Surprisingly, we found that after immobilization, the protein resists ethanol denaturation more efficiently than a diluted solution of 1 μM myoglobin with ethanol as the cosolvent. Intriguingly, the secondary structure of the immobilized myoglobin was preserved after 30 min incubation at 150 °C, as determined by FTIR at room temperature.

Effect of Solution Ionic Strength on Analyte Charge State Distributions in Positive and Negative Ion Electrospray Mass Spectrometry

The variability of ESMS charge state distributions was investigated for a number of analytes when the ionic strength of their solutions was altered over a wide range. Solution ionic strength was adjusted by adding varying amount of electrolytes (NH 4 OAc, CsCl or (n-C 4 H 9 ) 4 N + Cl - ) to protein solutions of myoglobin and lysozyme. Measured solution QH values were found to be nearly constant over the entire range of CsCl and (n-C 4 H 9 ) 4 N + Cl - addition, and a slight increase in QH was observed upon NH 4 OAc addition. Analyte peak intensities in positive ion ESMS reproducibly decreased as the solution ionic strength increased. However, analyte charge state distributions remained quite constant desite 4 orders of magnitude variation in electrolyte concentration

Investigation of molecular motion and interprotein interactions in solutions by TDDS: a comparison with NMR data

The results of dynamic protein behavior in solution studied by time domain dielectric spectroscopy (TDDS) are presented. The analyses for myoglobin solutions at concentrations 50, 120 and 150 mg/ml in the temperature interval from 5 to 35°C was carried out in terms of dipole correlation functions. It was found that the correlation function of the protein motion can be presented as a sum of three components corresponding to three types of protein motion: internal local motion, anisotropy rotational Brownian diffusion and translational Brownian diffusion. According to the hypothesis presented earlier, it is supposed that the reason for anisotropy of protein rotation and capability for the detection translational diffusion (slowest motion) is the mutual interprotein electrostatic steering.

Facilitated permeation of myoglobin through porous membranes: Different permeation behavior through membranes of polyoxyethylene‐ and dextran‐conjugated polyamides prepared by quasi‐living polymerization of a bicyclic lactam

AbstractIn the ultrafiltration test of a myoglobin solution through porous membranes of the ABA‐type block copolymers composed of polyamide as outer segments and polyoxyethylene (Mn = 1.9–2.0 × 104) as an inner segment, in which the values of weight fraction of the polyamide segments (W) were 0.90, 0.84, 0.82, 0.81, and 0.73, the concentration of the permeate was found to be much higher than that in feed under the pressure difference of 1–2 kg/cm2. Such singularly facilitated permeation was observed also in the test through the dense membranes of the polyamide–polyoxyethylene block copolymer with W of 0.81 and 0.73. On the other hand, neither porous nor dense membranes of the graft copolymer (W = 0.83) having a dextran stock (Mn = 1.8 × 104) and 2.6 pieces of polyamide branches showed similar facilitated permeation, although dextran was soluble in water as well as polyoxyethylene. The apparent interaction of myoglobin with dextran may be stronger than that with polyoxyethylene. © 1994 John Wiley & Sons, Inc.

A portable diffuse reflectance spectrophotometer for the rapid and automatic measurement of meat pigments

A portable reflectance spectrophotometry system, which can measure a wide range of absorbance and has an adhesive pad type fibre optic probe, was developed to improve the availability of non-invasive reflectance spectrophotometry. A Czerny-Turner type spectrometer, which can be connected to an optical fibre probe, was developed, and a linear charge coupled device (CCD) was used as its light detector to increase data acquisition speed. The scanning time of the linear CCD is automatically controlled in accordance with the reflectance of meat to increase the dynamic range of absorption. The author tried to apply this instrument to samples of meat pigments in order to demonstrate whether reflectance spectrophotometry is suitable for evaluating the freshness of meat. The author examined myoglobin solution in the form of the met-derivative in order to detect the concentration of myoglobin. The absorbance spectrum of myoglobin in the range of 0-4 mg dl-1 was successfully distinguished with this instrument.