The covalent structure of dog myoglobin.

The primary structure of the myoglobin of the domestic dog (German shepherd) was studied. Tryptic and thermolytic peptides were compared with the sequence of other known myoglobins; the stepwise automatic Edman's degradation of the whole globin and also the chymotryptic digestion of the median fragment obtained by CNBr cleavage completed this sequence. Comparison of the established dog myoglobin structure with those from other carnivora shows 16 differences versus badger, 20 versus harbour seal and 15 versus California sea lion.     

1H NMR study of labile proton exchange in the heme cavity as a probe for the potential ligand entry channel in myoglobin

The exchange rates of heme cavity histidine nitrogen-bound protons in horse and dog metcyanomyoglobins have been determined at 40 degrees C as a function of pH by 1H NMR spectroscopy. They were compared to the results reported for the sperm whale homologue [Cutnell, J. D., La Mar, G. N., & Kong, S. B. (1981) J. Am. Chem. Soc. 103, 3567-3572]. The rate profiles suggest that the exchange follows EX2-type kinetics, and the relative rate values favor a penetration model over a local unfolding model. It was found that the behavior of protons located on the proximal side of the heme is similar in the three proteins. The distal histidyl imidazole NH, however, shows a highly accelerated hydroxyl ion catalyzed rate in horse and dog myoglobins relative to that in sperm whale myoglobin. NMR spectral and relaxational characteristics of the assigned heme cavity protons indicate that the global geometry of the heme pocket is highly conserved in the ground-state structure of the three proteins. We propose a model that attributes the different distal histidine exchange behavior to the relative dynamic stability of the distal heme pocket in dog or horse myoglobin vs. sperm whale myoglobin. This model involves a dynamic equilibrium between a closed heme pocket as found in metaquomyoglobin [Takano, T. (1977) J. Mol. Biol. 110, 537-568] and an open pocket as found in phenylmetmyoglobin [Ringe, D., Petsko, G. A., Kerr, D. E., & Ortiz de Montellano, P. R. (1984) Biochemistry 23, 2-4].(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 145–151 (antigenic site 5) of myoglobin

Monoclonal antibodies of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any protein carrier) synthetic peptide representing region 145–151 of sperm whale myoglobin (SpMb) and their cross-reactions with eight Mb variants were determined. Five Mbs—bottle-nose dolphin myoglobin (BdMb), pacific common dolphin myoglobin (PdMb), horse myoglobin (HsMb), dog myoglobin (DgMb), and badger myoglobin (BgMb)—have an identical sequence in that region. Nevertheless, these Mbs exhibited very different cross-reactivities. BdMb and PdMb exhibited cross-activities which were comparable to that of the reference antigen, SpMb; while the reactivity of HsMb was remarkedly decreased, DgMb and BgMb showed almost no cross-reactions with these mAbs. Since the region 145–151 has an identical sequence in all the five Mbs, it is concluded that the differences in their antigenic reactivities with anti-region 145–151 mAbs are due to the effects of amino acid substitutions outside the region 145–151. Another pair of myoglobins, echidna myoglobin (EdMb) and chicken myoglobin (ChMb), have the same sequence in that region, but reacted very differently with anti-region 145–151 mAbs. The reactivity and affinity of EdMb were substantially decreased while those of ChMb were almost completely absent, relative to SpMb. It is concluded, contrary to popular assumptions, that when an amino acid substitution influences the binding of a protein variant to a mAb, it is not necessary for that substitution to be an actual contact residue (i.e., a residue within the antigenic site where the mAb binds). Such effects, which are often very drastic, could be due to indirect influences of the substitution on the chemical and binding properties of the site residues. Furthermore, residues which had been postulated, on the basis of these assumptions, to constitute discontinuous antigenic sites in SpMb, were found [from the present studies and those recently reported with mAbs against the other four antigenic site of Mb (regions 15–22, 56–62, 94–100, and 113–120 of SpMb)] to merely be exerting indirect effects on the known five antigenic sites of Mb. The effects of substitutions, which can happen even in the absence of conformational changes, are determined by many factors, such as the chemical nature of the substitution, its environment, its distance from the site, and the nature of the site residue(s) being affected.     

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species.

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.     

1H NMR resonance assignments in a paramagnetic heme protein by two-dimensional spectroscopy: heme resonances in equine met-azido myoglobin

Specific heme protons for the majority of resonances in the downfield resolved region of equine met-azido myoglobin have been assigned using solely the two-dimensional 1H NMR experiments NOESY and COSY. Metazido myoglobin provides a useful test case for the applicability of these techniques to paramagnetic proteins for the following reasons. First met-azido myoglobin is a mixed spin-state protein, with significantly shorter relaxation times and broadened lines relative to pure low-spin systems (eg., met-cyano myoglobin). Second, met-azido hemoglobin and met-azido myoglobin are important as models for the physiological forms of hemoglobin. Third, a few sperm whale met-azido myoglobin resonances have been previously assigned, which permits a comparison of assignments for these similar proteins, and a check of the method presented here.     

Adsorption of the protein antigen myoglobin affects the binding of conformation-specific monoclonal antibodies.

Five monoclonal antibodies against sperm whale myoglobin have been used to investigate the physical state of the antigen adsorbed onto a polydimethylsiloxane surface. The binding of each antibody is sensitive to the antigen's conformation in solution while the locations of the antigenic sites on the myoglobin molecule for three of the antibodies have been determined (Berzofsky, J.A., G.K. Buckenmeyer, G. Hicks, F.R.N. Gurd, R.J. Feldmann, and J. Minna. 1982. J. Biol. Chem. 257:3189-3198). The binding of the fluorescein isothiocyanate-labeled IgG and Fab antibodies to previously adsorbed myoglobin has been observed using total internal reflection fluorescence. Three of the antibodies bind specifically to surface-adsorbed myoglobin with affinities at least 50% relative to myoglobin in solution whereas two of the antibodies show affinities for the surface-adsorbed myoglobin diminished by at least two orders of magnitude relative to myoglobin in solution. The specific loss of certain antigenic determinants on the adsorbed myoglobin, coupled with the retention of others, indicates a nonrandom adsorption of the myoglobin molecules.     

Electrochemical immunoanalysis of cardiac myoglobin

Methods of myoglobin determination based on electrochemical analysis by means of analysis of electrochemical parameters of modified electrodes have been proposed. The method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of this hemoprotein. The electrode surface was modified by a membrane-like synthetic didodecyldimethylammonium bromide (DDAB), gold nanoparticles and antibodies to human cardiac myoglobin the electrochemical reduction of myoglobin heme was registered provided that the antigen (myoglobin) was present in the samples. The reaction of myoglobin binding to antibodies immobilized on the electrode surface was also registered using electrochemical impedance spectroscopy. The study of electro analytical characteristics revealed high specificity and sensitivity of the developed method. The biosensor was characterized by low detection limit and a high working range of the detected concentrations from 17.8 to 1780 ng/ml (from 1 to 100 nM). The method of myoglobin determination based on a signal of gold nanoparticles has also been proposed. The signal was detected with stripping voltammetry. There was a change in the cathodic peak area and the peak height of gold oxide reduction for the electrodes with antibodies and the electrodes with the antibody-myoglobin complex.     

Structural and functional aspects of the heart ventricle myoglobin of bluefin tuna

The heart ventricle myoglobin of bluefin tuna has been purified to an apparent homogeneity. The amino acid analysis has revealed only a limited number of substitutions between the myoglobins of yellowfin and bluefin tuna. The alpha-helix content of tuna myoglobin has been found considerably lower than that of mammalian myoglobin. No correlation has been discovered between the conformational stability and alpha-helix content. Denaturation experiments have shown that the whole structure of tuna myoglobin results from the interaction of two structural units which represent the product of independent folding processes. The structure of tuna myoglobin has been found more open and disorganized than that of sperm whale. This result has been related to the low content of electrostatic interactions and explained in terms of evolutive adaptations.     

Structural characterization of non-native states of sperm whale myoglobin in aqueous ethanol or 2,2,2-trifluoroethanol media

The effects of aqueous ethanol or 2,2,2-trifluoroethanol media on the structure of sperm whale myoglobin have been investigated by absorption, CD, and NMR spectra. The structural properties of myoglobin such as heme environments, helix contents, protein folding, and interactions between heme and the protein moiety have been sharply manifested in these spectra. The characterization demonstrated that alcohol-induced conformational change of myoglobin depends on the nature of alcohol and its concentration. It was shown for the first time that, upon the alcohol-induced denaturation of myoglobin, heme is released from partially denatured protein of which helix contents is altered by only about 20% relative to that of native state. Myoglobin has shown to unfold and refold reversibly by controlling the alcohol concentration. Novel methods for the preparation of apomyoglobin and in situ reconstitution of apomyoglobin with heme, based on the alcohol-induced denaturation of the protein, were presented.     

Crystal structure of myoglobin form a synthetic gene

Crystal have been grown of myoglobin produced in Escherichia coli from a synthetic gene, and the structure has been solved to 1.9 Å resolution. The space group of the crystals is P6, which is different from previously solved myoglobin crystal forms. The synthetic myoglobin is essentially identical to myoglobin isolated from sperm whale tissue, except for the retention of the initiator methionine at the N-terminus and the substitution of asparagine for aspartic acid at position 122. Superposition of the coordinates of native and synthetic sperm whale myoglobins reveals only minor changes in the positions of main chain atoms and roeientation of some surface side chains. Crystals of variant of the “synthetic” myoglobin have also been grown for structural analysis of the role of key amino acid residues in ligand and specificity.     

X-ray crystal structure of a recombinant human myoglobin mutant at 2.8 A resolution

We have grown crystals in trigonal space group P3(2)21 of a mutant human myoglobin, aquomet form, in which lysine at position 45 has been replaced by arginine and cysteine at position 110 has been replaced by alanine. Suitable crystals of native recombinant human myoglobin have not been obtained. We have used the molecular replacement method to determine the X-ray crystal structure of the mutant at 2.8 A resolution. At the present stage of refinement, the crystallographic R-value for the model, with tightly restrained stereochemistry, is 0.158 for 5.0 to 2.8 A data. As expected, the overall structure is quite similar to the sperm whale myoglobin structure. Arginine 45 adopts a well-ordered conformation similar to that found in aquomet sperm whale myoglobin.     

Assessment of the functional diversity of human myoglobin

Myoglobin is presumably the most studied protein in biology. Its functional properties as a dioxygen storage and facilitator of dioxygen transport are widely acknowledged. Experimental evidence also implicates an essential role for myoglobin in the heart in regulating nitric oxide homeostasis. Under normoxia, oxygenated myoglobin can scavenge excessive nitric oxide, while under hypoxia, deoxygenated myoglobin can reduce nitrite, an oxidative product of nitric oxide, to bioactive nitric oxide. Myoglobin-driven nitrite reduction can protect the heart from ischemia and reperfusion injury. While horse and mouse myoglobin have been previously described to reduce nitrite under these conditions, a comparable activity has not been detected in human myoglobin. We here show that human myoglobin is a fully functional nitrite reductase. To study the role of human myoglobin for nitric oxide homeostasis we used repeated photometric wavelength scans and chemiluminescence based experiments. The results revealed that oxygenated human myoglobin reacts with nitrite-derived nitric oxide to form ferric myoglobin and that deoxygenated human myoglobin acts as a nitrite reductase in vitro and in situ. Rates of both nitric oxide scavenging and nitrite reduction were significantly higher in human compared to horse myoglobin. These data extend the existing knowledge about the functional properties of human myoglobin and are the basis for further translational studies towards the importance of myoglobin for nitric oxide metabolism in humans.     

The myoglobin of primates. I. Hylobates agilis (gibbon)

The myoglobin of a gibbon has been compared with that of man. There was only one difference, residue 23 (B4) which is Gly in man was found to be Ser in the gibbon. Two corrections have also been made to the previously published sequence of human myoglobin (A.E. Romero Herrera and H. Lehmann, Nature, New Biol., 232 (1971) 149).     

The myoglobin of rodents:Lagostomus maximus (viscacha) andSpalax ehrenbergi (mole rat)

The primary sequences of the myoglobins of two rodents (the South American viscacha and the Mediterranean mole rat) have been determined. Both myoglobins exhibit one polymorphism. The two rodent sequences have been compared with each other and with other known myoglobins. The myoglobin of the viscacha is similar to those of the diving mammals and penguin in having a high arginine content. Among mammalian sequences, the arginines at positions 77 (in one of the viscacha myoglobins) and 79 have been found only in the myoglobin from viscacha. Mole rat myoglobin has a lysine at position 31, where arginine or serine is found in all other known vertebrate myoglobins.     

The myoglobin of rodents:Lagostomus maximus (viscacha) andSpalax ehrenbergi (mole rat)

The primary sequences of the myoglobins of two rodents (the South American viscacha and the Mediterranean mole rat) have been determined. Both myoglobins exhibit one polymorphism. The two rodent sequences have been compared with each other and with other known myoglobins. The myoglobin of the viscacha is similar to those of the diving mammals and penguin in having a high arginine content. Among mammalian sequences, the arginines at positions 77 (in one of the viscacha myoglobins) and 79 have been found only in the myoglobin from viscacha. Mole rat myoglobin has a lysine at position 31, where arginine or serine is found in all other known vertebrate myoglobins.     

Myoglobin structure and regulation of solvent accessibility of heme pocket

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.     

Molecularly imprinted polymers (MIP) in electroanalysis of proteins

The review summarizes current knowledge on the main approaches used for creation of high affinity polymer analogs of antibodies (known as molecularly imprinted polymers, MIP) applicable for electroanalysis of functionally important proteins such as myoglobin, troponin T, albumin, ferritin, lysozyme, calmodulin. The main types of monomers for MIP preparation as well as methods convenient for analysis of MIP/protein interactions, such as surface plasmon resonance (SPR), nanogravimetry with the use of a quartz crystal resonator (QCM), spectral and electrochemical methods have been considered. Special attention is paid to experimental data on electrochemical registration of myoglobin by means of o-phenylenediaminebased MIP electrodes. It was shown that the imprinting factor calculated as a ratio of the myoglobin signal obtained after myoglobin insertion in MIP to the myoglobin signal obtained after myoglobin insertion in the polymer lacking the molecular template (NIP) is 2–4.     

High electric field dielectric studies of aqueous myoglobin solutions.

High field dielectric measurements of the Piekara factor deltaepsilon/E2 have been carried out for a range of concentrations of horse heart myoglobin in water at 293K. Using the literature value for the dipole moment of myoglobin and the established theory for the classical orientational dipolar non-linear effect predicts a value of deltaepsilon/E2 one order of magnitude greater than that for water. The measured effect, however, was found to be one order of magnitude less than for water. This difference is explained as being most probably due to the existence of antiparallel molecular dipole pairs in the myoglobin solution. The possibility of a positive deltaepsilon due to a field induced conformational change of the myoglobin cannot, however, be ruled out.     

Predictability of weak binding from X-ray crystallography: inhaled anesthetics and myoglobin

Xenon and dichloromethane are inhalational anesthetic agents whose binding to myoglobin has been demonstrated by X-ray crystallography. We explore the thermodynamic significance of such binding using differential scanning calorimetry, circular dichroism spectroscopy, and hydrogen-tritium exchange measurements to study the effect of these agents on myoglobin folding stability. Though specific binding of these anesthetics might be expected to stabilize myoglobin against unfolding, dichloromethane actually destabilized myoglobin at all examined concentrations of this anesthetic (15, 40, and 200 mM). On the other hand, xenon (1 atm) stabilized myoglobin. Thus, dichloromethane and xenon have opposite effects on myoglobin stability despite localization in comparably folded X-ray crystallographic structures. These results suggest a need for solution measurements to complement crystallography if the consequences of weak binding to proteins are to be appreciated.