Heme Protein Myoglobin Research Articles

Myoglobin Coadsorbed on Electrodes from Microemulsions Provides Reversible Electrochemistry and Tunable Electrochemical Catalysis

The iron heme protein myoglobin (Mb) coadsorbs with surfactant onto glassy carbon, pyrolytic graphite, and platinum electrodes from microemulsions of sodium dodecyl sulfate or cetyltrimethylammoniu...

EXHAUSTIVE INCREMENTAL RUNNING AND CYCLING INDUCE OXIDATIVE MODIFICATIONS TO HEMOGLOBIN

Various markers are available to establish the occurrence of oxidative stress in exercise. A relatively under-investigated source of free radicals and oxidative stress involves the heme proteins hemoglobin (Hb) and myoglobin (Mb). Autoxidation of Hb and Mb is a constant source of superoxide in the body, and further reactions may produce hydrogen peroxide, protein bound radicals, and ferryl iron, which are strong oxidants. Oxidatively modified heme (OxH) is one of the products of these reactions, and might be useful as a specific marker of heme-induced oxidative stress in exercise. PURPOSE To determine the effects of two modes of exhaustive incremental exercise on blood levels of OxH. METHODS Ten untrained subjects (5 males, 5 females; 20±1 yrs, 75.2±20.0kg, 175±13cm) performed continuous incremental running test to exhaustion; eight of the subjects performed an additional cycling test one week later. Power output for the cycling test was calculated from individual running VO2-data to produce similar test durations in both tests. Venous blood samples were taken before and directly after exercise, and analyzed for OxH (as a percentage of total heme). Differences between pre- and post-exercise, values, and between cycling and running data were analyzed using paired-sample t-tests. RESULTS Average VO2peak was significantly higher for the running max-test (43.0±7.2 ml·kg −1·min −1) compared to the cycling max-test (41.4±7.5 ml·kg −1·min −1; P < 0.05). Small but significant increases in OxH levels were observed in both the running test (0.0400±0.0058% vs. 0.0431±0.0072%, P < 0.05), and the cycling test (0.0427±0.0062% vs. 0.0450±0.0058%, P < 0.05). No differences were observed between the change in OxH levels in running vs. cycling. CONCLUSION The results of the present study indicate that OxH can be used to demonstrate exercise-induced oxidative stress, and suggest a possible mechanistic role of heme proteins in oxidative stress.

Interaction of porphyrins with heme proteins--a brief review.

Two important porphyrins, protoporphyrin IX and hematoporphyrin IX, derivatives of which form the basis of photosensitization in the photodynamic therapy of cancer treatment, interact with two physiologically important heme proteins hemoglobin and myoglobin. The extent and modality of these interactions vary with the state of aggregation of the two porphyrins. Upon binding with these proteins, both the drugs change the protein conformations and release the heme-bound oxygen from the oxyproteins. At the same time, the peroxidase activities of these proteins are potentiated due to the protein-porphyrin complexation, as is found in case of horseradish peroxidase also. The effect of porphyrins on heme proteins should be given due consideration in elucidating the details of the mechanism of porphyrin actions in therapy.

Investigations of amplitude and phase excitation profiles in femtosecond coherence spectroscopy

We present an effective linear response approach to pump–probe femtosecond coherence spectroscopy in the well-separated pulse limit. The treatment presented here is based on a displaced and squeezed state representation for the nonstationary states induced by an ultrashort pump laser pulse or a chemical reaction. The subsequent response of the system to a delayed probe pulse is modeled using closed form nonstationary linear response functions, valid for a multimode vibronically coupled system at arbitrary temperature. When pump–probe signals are simulated using the linear response functions, with the mean nuclear positions and momenta obtained from a rigorous moment analysis of the pump induced (doorway) state, the signals are found to be in excellent agreement with the conventional third-order response approach. The key advantages offered by the moment analysis-based linear response approach include a clear physical interpretation of the amplitude and phase of oscillatory pump–probe signals, a dramatic improvement in computation times, a direct connection between pump–probe signals and equilibrium absorption and dispersion lineshapes, and the ability to incorporate coherence associated with rapid nonradiative surface crossing. We demonstrate these aspects using numerical simulations, and also apply the present approach to the interpretation of experimental amplitude and phase measurements on reactive and nonreactive samples of the heme protein myoglobin. The role played by inhomogeneous broadening in the observed amplitude and phase profiles is discussed in detail. We also investigate overtone signals in the context of reaction driven coherent motion.

Investigations of Coherent Vibrational Oscillations in Myoglobin

The technique of femtosecond coherence spectroscopy (FCS) is applied to the heme protein myoglobin. Photostable samples of deoxy myoglobin (Mb) and photochemically active samples of the nitric oxide adduct (MbNO) are investigated. The pump-induced change in the probe transmittance for both samples displays coherent oscillations that, when transformed into the frequency domain, are in agreement with the resonance Raman spectrum of deoxy Mb. This indicates that the coherences associated with the photoreactive sample (MbNO) arise from the rapidly changing forces appearing in the crossing region(s) between the reactant and product state potential energy surfaces. The relative phase and amplitude of the Fe−His vibration, associated with the sole covalent linkage between the heme and the protein, are analyzed as a function of sample state and pump/probe carrier frequency. The dependence of the phase on carrier frequency is found to be significantly different for the “field driven” coherence in Mb and the “reaction driven” coherence in MbNO. In MbNO we observe a dip in amplitude and a phase flip near 439 nm for the Fe−His mode, whereas in deoxy Mb we observe a nearly constant phase and amplitude for this mode across the Soret absorption band. These observations are shown to be in good agreement with a simple theoretical model of the pump−probe experiment. Finally, we present recent observations of strong low-frequency oscillations, occurring near 40 cm-1 in both species and near 80 cm-1 for MbNO.

Application of femtosecond coherence spectroscopy to the observation of nuclear motions in heme proteins and transparent solutions

The technique of femtosecond coherence spectroscopy (FCS) was applied to simple transparent organic fluids (chloroform and fenchone) and light-absorbing heme proteins (myoglobin derivatives). This study demonstrates that time-domain coherence measurements are in good agreement with standard frequency-domain Raman and resonance Raman scattering experiments. The unique capability of FCS to detect the low-wavenumber nuclear motions driven by the pumping fields and by the electronic state changes associated with photoinduced chemical reactions is also demonstrated. The latter response cannot be probed using traditional Raman spectroscopy. In all studies of myoglobin derivatives a low-wavenumber mode was observed near 40 cm−1. A sequence of low-wavenumber modes was also observed near 80, 120 and 160 cm−1 that could be an indication of an overtone sequence. Copyright © 2000 John Wiley & Sons, Ltd.

A Thermodynamic Study of Azide Binding to Myoglobin

The visible absorption spectrum associated with the heme protein myoglobin changes as a result of azide binding to the iron center. Therefore, a titration with azide can be monitored and concentrations of the myoglobin-azide adduct quantified. From these ligand binding data, the dissociation constant and number of binding sites can be determined using the Scatchard method for analysis. This laboratory project can be completed in two 3-hour lab periods. Evaluation of other thermodynamic properties is possible by exploring the temperature dependence of these binding data. A third lab period is required for students to probe the temperature dependence.

Ultrafast Spectroscopy of Laser-driven Shock Waves in Molecular Materials

The “nanoshock technique” for generating shock waves in molecular materials at high repetition rates is described. Ultrafast spectroscopy is used to study shock waves in crystalline anthracene, a high explosive NTO, and a molecular nanomachine, the heme protein myoglobin.

Salt and pH effects on electrochemistry of myoglobin in thick films of a bilayer-forming surfactant

Salt concentration and pH of external solutions were shown to control the electrochemistry of the heme protein myoglobin (MbFe(III)H 2O) in stable, ordered films of didodecyldimethylammonium bromide (DDAB). Protonation of aquometmyoglobin (MbFe(III)H 2O) in these films precedes electron transfer from electrodes, causing formal potentials to shift negative as pH increases from 5 to 8. At pH > 8, MbFe(III)H 2O dissociates to MbFe(III)OH, which is reduced directly at the electrode at higher rates than MbFe(III)H 2O. Corelations of voltammetric data with FT-IR spectra suggested that at pH < 4.6, an unfolded form of Mb resides in the films and is reduced directly. The concentration of salt in solution influences electrochemical properties of Mb-DDAB films by its influence on Mb conformation and by effects on interfacial Donnan potentials. NMR indicated strong binding of anions to Mb within DDAB films. Bound anions may neutralize positive charge on Mb's surface so that it can reside in a partly hydrophobic environment, as postulated on the basis of previous ESR and linear dichroism studies.

Proton-Coupled Electron Transfer from Electrodes to Myoglobin in Ordered Biomembrane-like Films

Voltammetry and visible and infrared spectroscopy were used to explore protonation equilibria coupled to electron transfer between electrodes and the heme protein myoglobin (Mb) in thin liquid crystal films of didodecyldimethylammonium bromide (DDAB) and phosphatidylcholines (PC). Mb conformation and heme iron ligation in the films were controlled by the pH of the external solution. Acid−base equilibrium models successfully explained pH dependencies of Soret band absorbances, formal potentials, electron transfer rate constants, and electroactive surface concentrations of Mb in the films. A pKa1 of 4.6 in the Mb−lipid films is associated with protonation of histidine residues in hydrophobic regions of the Mb structure, possibly involving the proximal histidine bound to iron and/or the distal histidine in the heme pocket. At pH < 4.6, a partly unfolded molten globule form of Mb predominates in the films and is reduced directly. Native metmyoglobin [MbFe(III)−H2O] appears to be the major species in films between pH 5.5 and 8. In this pH range, protonation of MbFe(III)−H2O occurs prior to electron transfer, and a protonated form which may be a kinetic conformer accepts the electron. MbFe(III)−OH is formed in the films at pH > 9, and its one-electron reduction is also coupled to protonation.

A method for probing the topography and interactions of proteins: footprinting of myoglobin.

We describe a procedure for mapping residues on the surface of a protein molecule to its sequence, using a scheme that is analogous to nucleic acid footprinting. The protein is end labeled radioactively and subjected to limited proteolysis, and the products are analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. The method is tested with the heme protein myoglobin and applied to mapping the (unknown) surface of the molecule lacking the heme group: apomyoglobin. Sites of protein-protein interaction can be identified, as illustrated by footprinting the association between myoglobin and an anti-myoglobin monoclonal antibody.

Enhanced electron transfer for myoglobin in surfactant films on electrodes

The Fe(III)/Fe(II) redox couple of the heme protein myoglobin (Mb) gave standard electron-transfer rate constants about 1000-fold larger in liquid crystal films of didodecyldimethyl ammonium bromide (DDAB) on pyrolytic graphite (PG) electrodes than in aqueous solutions. Electron-transfer rates of Mb were also enhanced in films of soluble cationic and anionic surfactants adsorbed on PG. Results suggest a role for strongly adsorbed surfactant at electrode-film interfaces, which may prevent adsorption of macromolecular impurities which can block electron transfer. Mb-DDAB films were prepared by spontaneous insertion of Mb from solution into water-insoluble cast films of DDAB

1H-NMR study of reduced heme proteins myoglobin and cytochrome P450.

The 1H-NMR spectra of deoxymyoglobin and reduced cytochrome P450 are analyzed by NOE spectroscopy. Progress has been made in the assignment of the hyperfine-shifted signals of deoxymyoglobin. The nuclear longitudinal-relaxation-time values indicate short electron-relaxation times whereas Curie relaxation contributes significantly to the signals linewidths. For reduced cytochrome P450 the linewidths are larger due to the Curie-relaxation contribution in a large protein. Therefore, the spectral information is poor. The electron-relaxation rates are discussed in terms of possible electronic structure.

Tetrapyrroles as inhibitors of normal cartilage metabolism: relative potency of different compounds.

The present study was designed to explore the role of different tetrapyrroles as inhibitors of cartilage metabolism. We studied the effects of tetrapyrroles on the incorporation of [35S]sulfate into proteoglycans, [14C]-leucine into protein, and [3H]uridine into the RNA of normal cartilage from two different vertebrate classes using the embryonic chicken pelvic rudiment bioassay and the hypophysectomized rat costal cartilage bioassay, both very sensitive to cartilage growth factors and growth inhibitors. We compared the relative potencies of the following compounds: both metalloporphyrins (heme and chlorophyllin), linear tetrapyrroles (bilirubin), and heme proteins (hemoglobin, myoglobin, and cytochrome c). Hemoglobin and heme were the most potent inhibitors of rat cartilage metabolism, and bilirubin was a far more potent inhibitor of embryonic chick cartilage metabolism. Chlorophyllin had moderate inhibitory activity, especially on chick cartilage, whereas cytochrome c was inactive in these bioassays. Surprisingly, myoglobin was relatively ineffective despite its close similarity to heme and hemoglobin. The bilirubin-induced inhibition of sulfate incorporation into chick cartilage was partially prevented when glutathione was included in the incubation medium, suggesting that a free-radical mechanism may be involved. There were significant differences in the sensitivity of the two cartilages studied, indicating there may be species-dependent sensitivity to different tetrapyrroles.

Investigation of protein induced changes of the electronic structure of iron in heme proteins by 1H NMR spectroscopy

The chemical shifts of the proton magnetic resonances from the heme methyl groups provide information on the electronic structure of the heme iron and on protein-heme interactions. The 1H NMR data of heme model compounds and of heme proteins (myoglobin, hemoglobin, cytochrome c and cytochrome c peroxidase) have been analyzed.

Picosecond spectroscopy of some metalloporphyrins

The energy relaxation process of metal-free protoporphyrin IX dimethyl ester and its compounds with Cu(II), Ag(II), Ni(II), Pd(II), Pt(II), Zn(II), Fe(III) and the heme proteins myoglobin, hemoglobin and cytochrome c were studied by dual-beam picosecond spectroscopy. Excitation at 530 nm, which is in the β visible absorption band for these compounds, revealed that both the metal-free porphyrin and the Zn(II) porphyrin have lifetimes for S 1 which are longer than 1 ns, in keeping with their known fluorescence properties. From the time-resolved spectra after excitation, absorption due to S 1, T 1 and S 0 could be identified. In addition, a low-lying singlet d-d level was seen for the Ni(II) porphyrin. While the Pt(II) and Pd(II) compounds have intersystem crossing times of less than 8 ps and 20 ps respectively, the Ni(II) compound relaxes from S 1 to S d in about 10 ps and from S d to S 0 in 260 ps. The paramagnetic Cu(II) and Ag(II) species have intersystem crossing from 2S 1 to 2T 1 in less than 8 ps followed by an equilibrium of the 2T 1 and 4T 1 states, requiring about 450 ps for the Cu(II) compound and about 12 ps for the Ag(II) compound. An anomalous lengthening of the decay time with increasing excitation intensity was observed for the copper but not for the silver porphyrin. The Fe(III) porphyrins relax with rates faster than 6 ps, which was the resolving time of this experiment. No differences between heme proteins and Fe(III) protoporphyrins in solution were observable within this time resolution.

Photodissociation of ligands from heme and heme proteins. Effect of temperature and organic phosphate.

The effect of temperature on ligand photodissociation from protoheme and the heme proteins hemoglobin (Hb) and myoglobin (Mb) has been examined. The quantum yield of photodissociation (phi) is greater at 40 degrees than at 0 degrees; in general, larger increases are seen in the less photosensitive complexes, while phi does not change in the most photosensitive complexes. The ratio of phi at 40 degrees to phi at 0 degrees is 1.8 for HbCO, 2.3 for n-butyl isocyanide Hb, 2.7 for HbO2, and 1.3 for HbNO, with initial phi values of 0.38, 0.26, 0.028, and 0.003, respectively. This pattern of quantum yield increases is seen in protoheme as well as Hb and Mb ligand photolysis. The allosteric effector inositol hexaphosphate increases the quantum yield of lignad photolysis from hemoglobin. As with temperature, inositol hexaphosphate addition has a larger effect on complexes of low quantum yield; phi increases 1.2-fold for HbCO and 2.2-fold for HbO2 at 0 degrees. The results are discussed in terms of a model containing a photoexcited intermediate (Phillipson, P.E., Ackerson, B.J., and Wyman, J. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1550-1553).

Effect of pH on the Phosphorescence of Tryptophan, Tyrosine, and Proteins

Whereas the fluorescence of proteins has been extensively investigated, in particular by Weber and co-workers, the phosphorescence has been little investigated. This paper reports studies of the phosphorescence of a representative group of proteins and aromatic amino acids. The previously discovered quenching of the fluorescence of tyrosine and tryptophan in acid and alkaline pH is accompanied by an enhancement of the phosphorescence. The quenching of tyrosine fluorescence in proteins at neutral pH and by formate ion at neutral pH is accompanied by a quenching of the phosphorescence. Interpretation of these phenomena depends on a series of assumptions concerning the excited states of tyrosine and tyrosinate ion, which are shown in Figs. 6 and 7. Basically, tyrosine and tyrosinate ion are assumed to have π, π* states at lower energies than n, π* states. In tyrosinate ion the 3n, π* state is assumed to lie below the first excited singlet π, π* state. This facilitates singlet to triplet conversion (intersystem crossing) and accounts for the quenching of the fluorescence and the enhancement of the phosphorescence of tyrosinate ion as compared to tyrosine. An even more speculative assumption concerning the 3n, π* state is made to explain the fact that in tyrosine hydrogen-bonded to bases both the fluorescence and phosphorescence are quenched. In the heme proteins myoglobin and hemoglobin, neither fluorescence nor phosphorescence is seen at neutral or alkaline pH. When the heme is separated, the residual protein exhibits normal emission. In neutral solution the emission is quenched by energy transfer from the excited singlet states of tyrosine and tryptophan to the excited states of the heme. In alkaline solution, on the other hand, reasons are given for believing that the transfer is either from the excited triplet or from singlet states of the acids to the excited singlets of the heme.