Interaction Of Myoglobin Research Articles

Physicochemical and conformational studies on interaction of myoglobin with an amino-acid based anionic surfactant, sodium N-dodecanoyl sarcosinate (SDDS)

Physicochemical and conformational studies on the interaction between horse myoglobin, a monomeric water soluble heme protein and sodium N-dodecanoyl sarcosinate (SDDS), an amino-acid based anionic surfactant have been performed at different pHs using surface tension, viscosity, UV–vis and fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry and differential scanning calorimetry. Myoglobin shows surface activity, according to the surface tension measurement. Protein forms aggregates with surfactant molecules and becomes unfolded with increasing SDDS concentration. Again, when concentration of myoglobin increases, the binding ratio decreases; whereas heme changes its spin and coordination number with increasing surfactant concentration. α-Helicity of myoglobin decreases with increase in concentration of surfactant, while β-sheet and random coil appear. The temperature of transition of myoglobin and the enthalpy of thermal denaturation also change at higher concentration of SDDS. The myoglobin-SDDS interaction at different pH, ranging from 5 to 8 has been studied and it shows that pH has very little effect on the conformational change of the myoglobin-SDDS system.

Synergistic Effect of Distal Polar Interactions in Myoglobin and Their Structural Consequences.

In the L29F variant of myoglobin (Mb), the coordination of oxygen (O2) to the heme Fe atom is stabilized by favorable electrostatic interactions between the polar Fe-O2 moiety and the multipole of the phenyl ring of the Phe29 side chain (Phe29 interaction), in addition to the well-known hydrogen bond (H-bond) between the Fe-bound O2 and the 64th residue (distal H-bond; Carver, T. E.; Brantley, R. E., Jr.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem. 1992, 267, 14443-14450). The O2 and carbon monoxide (CO) binding properties and autoxidation of the L29F/H64L and L29F/H64Q variants reconstituted with a series of chemically modified heme cofactors were analyzed and then compared with those of native Mb, and the L29F, H64Q, and H64L variants similarly reconstituted with the chemically modified heme cofactors in order to elucidate the relationship between the Phe29 interaction and the distal H-bond that critically contributes to stabilization of Fe-bound O2. We found that the Phe29 interaction and distal H-bond act cooperatively to stabilize the Fe-bound O2 in such a manner that the Phe29 interaction strengthens with increasing strength of the distal H-bond. Comparison of the functional properties between the L29F and H64L variants indicated that the synergistic effect of the two interactions decreases the O2 dissociation and autoxidation rate constants of the protein by factors of ∼1/2000 and ∼1/400, respectively. Although the CO binding properties of the proteins were not greatly affected by the distal polar interactions, their synergistic effects were clearly and sharply manifested in the vibrational frequencies of the Fe-bound C-O stretching of the proteins.

Colloids versus solution state adsorption of proteins: Interaction of Myoglobin with supported lipid bilayers

This study examines adsorption of Myoglobin (Mb) in solution and as colloid on supported lipid bilayers of neutral phospholipids and a mixture of neutral+cationic lipids formed on gold coated quartz in a Quartz crystal microbalance (QCM). Results indicate that thin adsorbed films of Mb in solution and as colloids, show atleast 3 steps in the interaction with the bilayers: i) An initial strain of a viscoelastic film ii) Entrained water that moves in and out of the adsorbed film and iii) The coupled load from the bulk liquid which increases the strain of the film. These three components constitute an effective viscoelastic film which is rigidly coupled to the QCM. Grazing incidence XRD (GIXD) shows that the bilayer head group remains nearly undisturbed for Mb solution with pure (neutral) and (neutral+cationic) mixtures, whereas for the colloids there is an increase in head group thickness with neutral and decrease in the case of mixture. Unsaturation in the alkyl tails in the neutral lipid resulting in flexible disordered bilayers and more entrained water in the cationic system results in these changes. The sensitivity of QCM-D, makes it useful to study real-time monitoring of bilayer structural robustness cytotoxicity, drug delivery and lipid self-assembly.

The Influence of Redox‐Active Transition Metal Containing Micro‐ and Nanoparticles on the Properties of Representative Bioinorganic Reaction Systems

The use of nanomaterials in biological applications requires a better understanding of the effect of nanoparticles on the activity of proteins and related molecules. We have examined the influence of environmental particulate matter (PM) on the interaction of myoglobin and microperoxidase‐11 as model systems with the small molecules O2 and NO, respectively. The influence of PM on the reaction of dioxygen with deoxymyoglobin to form oxymyoglobin, and subsequently metmyoglobin, was studied for its essential role in living organisms. Samples of urban PM (SRM 1648a) were used as standard reference material. Also the influence of Fe2O3 nanoparticles on the stability of the investigated protein was studied. The obtained results show that binding and release of dioxygen by deoxymyoglobin may be affected by the presence of PM especially over longer periods of time. Similar studies were performed on the nitrosylation of microperoxidase‐11 as a model for the catalytic center of enzymes, for which the influence of the standard reference material, its water‐soluble extract, as well as metal oxide nanoparticles (Fe2O3 and ZnO), were studied. The reported results show that the presence of PM had no influence on the kinetics of the nitrosylation of the model microperoxidase under the selected experimental conditions.

Interaction of Myoglobin with Cationic and Nonionic Surfactant in Phosphate Buffer Media

Interaction of myoglobin with cationic surfactants hexadecyltrimethylammonium bromide (HTAB), gemini surfactant 16-2-16 (dimethylene-1,2-bis(hexadecyldimethylammonium bromide)) and nonionic surfactant Mega 10 (N-decanoyl-N-methylglucamine) have been studied in phosphate buffer at pH 7.4 using surface tension, UV–visible, fluorescence, and circular dichroism spectroscopies and differential scanning calorimetry. With increasing concentration of HTAB, metal ion of the heme group changes its spin states; but in case of 16-2-16 and Mega 10, spin change does not occur. Fluorescence spectra clearly denote the unfolding process in HTAB media. With increasing HTAB concentration, α-helicity of myoglobin decreases with the appearance of β-sheet and random coil more rapidly than other two surfactants. Melting temperature of myoglobin is reduced drastically upon interaction with HTAB than their corresponding gemini and nonionic surfactants.

Utility of heme analogues to intentionally modify heme–globin interactions in myoglobin

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme–globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized.Proton NMR spectroscopy revealed that loose heme–globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron–histidine bond. The full rotational rate was estimated to be about 1400s−1 at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme–globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005mmHg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin.Heme–globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics – the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Interaction of myoglobin with oleic acid

Previous studies have shown that palmitate (PA) can interact with myoglobin (Mb). The present study has investigated the interaction of the more soluble unsaturated fatty acid, oleic acid (OA). Indeed, 1H NMR measurements of the Mb signal during OA titration also show signal changes consistent with specific and non-specific binding. At OA:Mb ratio<4:1, OA perturbs selectively the 8-heme methyl signal, consistent with a local and specific fatty acid-protein interaction. As OA:Mb ratio increases from 4:1 to 40:1, all hyperfine shifted MbCN signals decrease, consistent with a non-selective, global perturbation of the protein. The pH titration analysis indicates that the observed OA methylene signal in the presence of Mb reflects a non-specific interaction and does not originate from a shift in the lamella–micelle equilibrium. Given the OA interaction with Mb, a fatty acid flux model suggests that Mb can play a fatty acid transport role under certain physiological conditions.

Evaluation of medicine effects on the interaction of myoglobin and its aptamer or antibody using atomic force microscopy.

The effects of medicine on the biomolecular interaction have been given increasing attention in biochemistry and affinity-based analytics since the environment in vivo is complex especially for the patients. Herein, myoglobin, a biomarker of acute myocardial infarction, was used as a model, and the medicine effects on the interactions of myoglobin/aptamer and myoglobin/antibody were systematically investigated using atomic force microscopy (AFM) for the first time. The results showed that the average binding force and the binding probability of myoglobin/aptamer almost remained unchanged after myoglobin-modified gold substrate was incubated with promazine, amoxicillin, aspirin, and sodium penicillin, respectively. These parameters were changed for myoglobin/antibody after the myoglobin-modified gold substrate was treated with these medicines. For promazine and amoxicillin, they resulted in the change of binding force distribution of myoglobin/antibody (i.e., from unimodal distribution to bimodal distribution) and the increase of binding probability; for aspirin, it only resulted in the change of the binding force distribution, and for sodium penicillin, it resulted in the increase of the average binding force and the binding probability. These results may be attributed to the different interaction modes and binding sites between myoglobin/aptamer and myoglobin/antibody, the different structures between aptamer and antibody, and the effects of medicines on the conformations of myoglobin. These findings could enrich our understanding of medicine effects on the interactions of aptamer and antibody to their target proteins. Moreover, this work will lay a good foundation for better research and extensive applications of biomolecular interaction, especially in the design of biosensors in complex systems.

Effect of artificial and natural phospholipid membranes on rate of sperm whale oxymyoglobin autooxidation

We were the first to show that MbO2 deoxygenation in the cell occurs only upon interaction of myoglobin with mitochondrial membrane, which must be accompanied by changes in the heme cavity conformation of the protein and its affinity for the ligand. Under aerobic conditions, some changes in the equilibrium O2 dissociation constant (Kdis) can be detected by changes of the rate of MbO2 autooxidation, i.e. spontaneous turning it into metMb (kox), as far as a direct correlation between Kdis and kox is experimentally shown. In this work, we studied the effect on MbO2 autooxidation rate of phospholipid liposomes from neutral soybean phosphatidylcholine (lecithin) and from negatively charged 1-palmitoyl-2-oleylphosphatidylglycerol (POPG) at various phospholipid/MbO2 ratios from 25 : 1 to 100 : 1, and also the effect of rat liver mitochondria at concentration of 1 and 2mg/ml mitochondrial protein (at 22 and 37°C). In all cases, kox was found to increase due to interaction of the protein with phospholipid membranes. The effect of negatively charged liposomes from POPG on kox is significantly greater than that of neutral lecithin liposomes. At the POPG/MbO2 molar ratio of 25 : 1, MbO2 autooxidation rate is almost 25-fold increased compared to the control, whereas in the presence of 50-fold molar excess of lecithin, kox is only ~10 times higher (10mM buffer, pH7.2, 22°C). With the same phospholipid/MbO2 ratio of 100 : 1, kox is 7 times higher for the POPG than for lecithin liposomes. In the presence of mitochondria inhibited by antimycin A, kox grows proportionally to their concentration (about 10-fold per 1mg/ml of mitochondrial protein), and practically does not change after adding superoxide dismutase in the reaction mixture. The kox value decreases markedly at high ionic strength, thus suggesting an important role of coulombic electrostatics in the myoglobin-mitochondrial interaction. The increase in the autooxidation rate of MbO2 (and hence its Kdis) due to the interaction with phospholipid membranes points to decreasing affinity of myoglobin for oxygen, which facilitates O2 detachment from MbO2 at physiological p02 values.

Fluorescence studies on the interaction of myoglobin with mitochondria

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO(2)) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6-8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO(2) and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO(2) and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6-8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K(m)) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K(m) on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K(m) values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.

Two-dimensional IR spectroscopy of protein dynamics using two vibrational labels: a site-specific genetically encoded unnatural amino acid and an active site ligand.

Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform infrared (FT-IR) and 2D IR spectroscopic experiments on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ∼1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chemical nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The experiments demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein.

Electrochemical immunoanalysis of cardiac myoglobin

Method targeting the direct monitoring of myoglobin based on analysis of electrochemical parameters of modified electrodes were proposed. Method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of hemeprotein. Myocardial infarction biomarker myoglobin was quantified at biological level using screen printed electrodes modified with gold nanoparticles stabilized with didodecyldimethylammonium bromide (DDAB) and antibodies. Proposed method did not require signal enhancement and amplification and also labeled secondary antibodies. Electro analysis has high specificity and sensitivity. Myoglobin -antibodies interaction was studied also with electrochemical impedance spectroscopy. Sensor has low detection limit and broad diapason of working concentrations (17.8 ng/ml-1780 ng/ml; 1 nM-10 nM). Method based on gold nanoparticles detection on the surface of electrodes was treated for myoglobin identification. AuNP worked as an electrochemical sensing platform: the oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. The difference of cathodic peak area and peak high of gold oxide reduction in the case of electrodes with antibodies and electrodes with antibodies-myoglobin complex, was registered.

Spectroscopy and Stopped-Flow/Fluorescence Studies on the Interactions ofMyoglobin and Its Mutant (D60K) with Surfactants

Spectroscopy and Stopped-Flow/Fluorescence Studies on the Interactions ofMyoglobin and Its Mutant (D60K) with Surfactants

Interaction of myoglobin with poly(methacrylic acid) at different pH in their layer-by-layer assembly films: An electrochemical study

Myoglobin (Mb), with net positive surface charges at pH 5.0, was successfully assembled into layer-by-layer films on various solid surfaces with poly(methacrylic acid) (PMAA) at different pH, designated as {PMAA(pH 5.0)/Mb} n, {PMAA(pH 6.5)/Mb} n, and {PMAA(pH 8.0)/Mb} n, respectively. As a weak polycarboxylic acid with pK a = 6 - 7, PMAA carried different negative charges at different pH due to different ionization degree of its carboxylic acid groups. Quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV) were used to monitor and confirm the assembly of {PMAA/Mb} n films. All the results showed that the adsorption amount of Mb in each bilayer had an “unexpected” sequence of {PMAA(pH 5.0)/Mb} n > {PMAA(pH 6.5)/Mb} n > {PMAA(pH 8.0)/Mb} n, which could be explained by the formation of soluble complex of PMAA-Mb at pH 8.0 and the cooperative effect of hydrogen bonding and induced electrostatic interaction between Mb and PMAA at pH 5.0. The influence of ionic strength in exposure solution and in Mb adsorbate solution was investigated, and the results supported the above explanations. The {PMAA/Mb} n films provided a suitable microenvironment for Mb to retain its near-native structure and transfer electron with underlying electrodes. The reversible CV peak pair for Mb Fe(III)/Fe(II) redox couple could be used to catalyze reduction of hydrogen peroxide electrochemically, showing the potential applicability of the films as the new type of biosensors or bioreactors based on the direct electrochemistry of Mb. The electrochemical and electrocatalytic behaviors of protein layer-by-layer films with weak polyelectrolytes could thus be controlled by adjusting the solution pH of weak polyelectrolytes.

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the E m,7 of dimethylester heme-substituted Mb (DME-Mb) ( E m,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased ∼40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and Δ S° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA) 2− is increased > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10 3 M −1 s −1 with Δ S ‡ = −13(1) cal/mol/deg and Δ H ‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA) 2− demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the 1H NMR spectra of native and DME-metMb that reveal shifts in p K a values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues.

Adsorption of Myoglobin onto Porous Zirconium Phosphate and Zirconium Benzenephosphonate Obtained with Template Synthesis

Porous zirconium phosphate (P-ZrP) and zirconium benzenephosphonate (P-ZrBP) were prepared in the presence of an anionic surfactant acting as a template. Poorly crystalline materials with a P/Zr molar ratio equal to 2 and having a relatively high surface area and micro/mesoporosity have been obtained. The interaction of myoglobin with the two types of surfaces, the hydrophobic P-ZrBP and the hydrophilic P-ZrP, was investigated, and the adsorption isotherms were determined at different pH and temperature values. A model was proposed for the mechanism of the interaction of the protein with the surface based on the shape of the adsorption isotherm and the physical-chemical properties of myoglobin. The pH has been found to be an important parameter for determining the maximum adsorption capacity of P-ZrBP and P-ZrP for myoglobin molecules because of the changes that occur in the type and net charge of the protein surface as the pH of the medium changes. Protein binding affinity and capacity increase when the temperature is increased. This phenomenon occurs because myoglobin varies its conformation at high temperature with an increase in the exposed hydrophobic region. This process causes a stronger hydrophobic interaction between the protein and the adsorbent and reduces the repulsion between the adsorbed molecules. Studies on the activities of the obtained biocomposites are in progress.

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002, 16, 1454–1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, “harsh” conditions at the ion source are required to disrupt any potential gas- phase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme–protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation.

Structural Origins of pH and Ionic Strength Effects on Protein Stability: Acid Denaturation of Sperm Whale Apomyoglobin

A recently developed approach to calculate the pH dependence of protein stability from three-dimensional structure information is applied to the analysis of acid denaturation of sperm whale apomyoglobin. The finite difference Poisson-Boltzmann method is used to calculate p Ka values and these are used to obtain titration curves for the folded protein as well as for compact intermediates. The total electrostatic free energy change involved in apomyoglobin unfolding is then evaluated. Calculations are carried out of the unfolding free energy of the native (N) and the compact intermediate (I) of apomyoglobin relative to the unfolded state (U) over a range of pH at various ionic strengths. The contributions from key ionizable groups to the unfolding process are discussed. For the acid-induced partial unfolding of apomyoglobin near pH 5, the transition from N to I is found to be driven by three histidines that are exposed when the B, C, D and E helices unfold. Similarly, the unfolding of the compact intermediate I consisting of the A, G and H helices is driven primarily by a few carboxylic acids with low pKa values in the compact state. This picture is in contrast to the view which attributes acid denaturation to electrostatic repulsion resulting from the build up of positive charge. In fact, charge-charge interactions in myoglobin are found to be attractive at all pH values where the protein unfolds. pH-dependent changes in these interactions contribute to acid denaturation but other electrostatic effects, such as hydrogen bonding and solvation, are important as well. The effect of increasing ionic strength on unfolding is attributed to the decrease of attractive charge-charge interactions which destabilize the N state relative to I, but stabilize the I state relative to U by reducing the p Ka shifts of a few critical carboxylic acids. The I state is found to be more stable than U at neutral pH thus accounting for its presence as an intermediate on the protein folding pathway. Our results have implications for the origins of compact intermediates or "molten globule" states.

The formation of free radicals by cardiac myocytes under oxidative stress and the effects of electron-donating drugs.

The interaction of myoglobin with H2O2 leads via a two-electron oxidation process to the formation of ferryl myoglobin. Metmyoglobin is more readily activated than oxymyoglobin to the ferryl states, which are capable of inducing peroxidative damage to membranes. E.p.r. and optical spectroscopic studies show that the thiol-containing compounds N-(2-mercaptopropionyl) glycine and N-acetylcysteine and the trihydroxamate desferrioxamine attenuate these processes by reducing the ferryl myoglobin species to metmyoglobin, with the formation of thiyl radicals and the desferrioxamine nitroxide radical respectively. Biochemical investigations of the potential for myoglobin in ruptured myocytes to be involved in radical generation, when under oxidative stress, and of the nature of the resulting species, were also undertaken. E.p.r. spectroscopic studies revealed the formation of a radical species which is capable of inducing membrane lipid peroxidation. The interaction of the thiol compounds and desferrioxamine with components of myocardial tissue under these conditions results in the generation of thiol-derived radical species and the desferrioxamine nitroxide radical respectively. These data, along with those obtained using optical spectrocopy, support the assignment of the identity of the radical species generated from the myocytes as the ferryl myoglobin radical.

Variations of electrostatic interactions in myoglobin probed by UVRR spectroscopy. Effect of iron ligand and pH on tryptophan and tyrosine vibrations

The present work investigates the variations of electrostatic interactions within the myoglobin molecule associated with azide heme binding and pH variations. Far ultraviolet (223 nm) resonance Raman spectroscopy of the tryptophan and tyrosine residues, along with acid-base titration measurements, have been used to monitor variations in the protein matrix. With previously determined mode assignments, it is shown that the Trp and Tyr residues of the globin moiety are influenced by the charge spatial distribution. Upon ligand binding or under various pH conditions, the polar interactions inside the protein appear to be modulated by the electric field generated by the charge array. It is concluded that the binding site properties of myoglobin can be modulated by the charge spatial distribution within the protein, even in the absence of measurable conformational changes of the bulk.