Showing papers on "Myoglobin published in 1975"

Dynamics of ligand binding to myoglobin

Abstract: Myoglobin rebinding of carbon monoxide and dioxygen after photodissociation has been observed in the temperature range between 40 and 350 K. A system was constructed that records the change in optical absorption at 436 nm smoothly and without break between 2 musec and 1 ksec. Four different rebinding processes have been found. Between 40 and 160 K, a single process is observed. It is not exponential in time, but approximately given by N(t) = (1 + t/to)-n, where to and n are temperature-dependent, ligand-concentration independent, parameters. At about 170 K, a second and at 200 K, a third concentration-independent process emerge. At 210 K, a concentration-dependent process sets in. If myoglobin is embedded in a solid, only the first three can be seen, and they are all nonexponential. In a liquid glycerol-water solvent, rebinding is exponential. To interpret the data, a model is proposed in which the ligand molecule, on its way from the solvent to the binding site at the ferrous heme iron, encounters four barriers in succession. The barriers are tentatively identified with known features of myoglobin. By computer-solving the differential equation for the motion of a ligand molecule over four barriers, the rates for all important steps are obtained. The temperature dependences of the rates yield enthalpy, entropy, and free-energy changes at all barriers. The free-energy barriers at 310 K indicate how myoglobin achieves specificity and order. For carbon monoxide, the heights of these barriers increase toward the inside; carbon monoxide consequently is partially rejected at each of the four barriers. Dioxygen, in contrast, sees barriers of about equal height and moves smoothly toward the binding site. The entropy increases over the first two barriers, indicating a rupturing of bonds or displacement of residues, and then smoothly decreases, reaching a minimum at the binding site. The magnitude of the decrease over the innermost barrier implies participation of heme and/or protein. The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies. The shape of the spectrum has been determined; its existence can be explained by assuming the presence of many conformational states for myoglobin. In a liquid at temperatures above about 230 K, relaxation among conformational states occurs and rebinding becomes exponential.

Radioimmunoassay of myoglobin in human serum. Results in patients with acute myocardial infarction.

Abstract: A radioimmunoassay has been developed for the measurement of serum myoglobin in order to evaluate the time-course and frequency of myoglobinemia in patients with acute myocardial infarction. The method can detect as little as 0.5 ng of myoglobin and is not affected by hemolysis or storage of serum at -- 20 degrees C. Myoglobin was detected in all of 92 sera from normal adults and ranged between 6 and 85 ng/ml. Levels were markedly elevated in sera from 18 of 20 patients with acute myocardial infarction when samples were obtained within 12 h after hospital admission, the mean concentration being 380+/-53 ng/ml. Wehn the initial sample was drawn between 12 and 24 h after admission in another group of 20 patients with acute myocardial infarcts, the mean serum myoglobin concentration was 195+/-47 ng/ml, and 11 of these individuals had normal levels. Serial determinations performed on nine patients with acute infarction demonstrated that maximum myoglobin levels occurred within the first 8-12 h after admission and fell rapidly toward normal thereafter. The serum concentration of myoglobin in 21 additional patients admitted with chest pain but without acute myocardial infarction was 41+/-6 ng/ml. Radioimmunoassay of serum myoglobin appears to be useful and sensitive test for the early detection of myocardial infarction.

Neutron diffraction analysis of myoglobin: structure of the carbon monoxide derivative

Abstract: The locations of hydrogen and deuterium atoms and water molecules have been investigated in carbon monoxide myoglobin using neutron diffraction, and the results are compared with earlier work on metmyoglobin. Parallel real space refinements on the two molecules show relatively few changes, but do show the carbon monoxide molecule with the iron atom moving into the heme plane.

Circular dichroism studies of myoglobin and leghemoglobin.

Abstract: The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.

Kinetics of oxygen and carbon monoxide binding to synthetic analogs of the myoglobin and hemoglobin active sites

Abstract: Kinetics of reversible oxygenation and carbon monoxide complex formation of the simple heme compounds pyrroheme-N-[3-(1-imidazolyl)propyl]amide and pyrroheme-3-(3-pyridyl)propyl ester have been measured in different solvent environments. The oxygen on and off rates and equilibria of these compounds can be made to closely match those of myoglobin, of hemoglobin alpha chains, or of the various steps for hemoglobin by varying solvent environment or the basicity of the proximal base. These results suggest that the protein could alter oxygen on rates by varying the basicity of the proximal base and the off rates by changing the polarity of the distal environment.

Oxygen transport of hemoglobin in high-altitude animals (Camelidae).

Abstract: To clarify the mechanisms by which high-altitude Camelidae can adapt to hypoxia, the study of some blood characteristics were carried out in apacas and llamas. The results show that there is a peculiar dissociation curve of hemoglobin in alpacas which permits great affinity of hemoglobin for oxygen at lung level and the release of oxygen at the tissue level with a facility similar to that in man. Fetal hemoglobin was found high in adult alpacas (55 percent). Electrophoretic studies of hemoglobin showed that this pigment has two components, both of which have a very low mobility. Lactic dehydrogenase was found six times higher than in humans. RBC glucose-6-phosphate dehydrogenase was two times higher than in man living at the same altitude. Myoglobin was found to be higher than in man living at altitude. Alpacas have erythrocytes in which the amount of 2,3-DPG is approximately the same as in man. RBC are more resistent to hypotonic solutions than humans. The amount of lactic dehydrogenase, myoglobin, and glucose-6-phosphate dehydrogenase dimishes when alpacas are bought down to sea level.

Electrostatic effects in myoglobin. Application of the modified Tanford-Kirkwood theory to myoglobins from horse, California grey whale, harbor seal, and California sea lion.

Abstract: The modified Tanford-Kirkwood electrostatic theory (Shire et al., 1974a) was applied to ferrimyoglobins from the following animal species: sperm whale (Physeter catodon), horse, California grey whale (Eschrichtius gibbosus), harbor seal (Phoca vitulina), and California sea lion (Zalophus californianus). Computations were made of the overall hydrogen ion titration curves of the proteins, and of pH and ionic strength variations of ionization equilibria for individual groups in the protein, with particular reference to the hemic acid ionization of the iron bound water molecule. Coordinates and static solvent accessibility were estimated in terms of the sperm whale myoglobin structure. Where possible, theoretical results and experimental data are compared. Some comparative features of charge and ionization properties among the various myoglobins are presented.

Equilibrium and kinetics of the reaction of Aplysia myoglobin with azide.

Abstract: The present paper reports a study on the equilibria and kinetics of the acid-alkaline transition and the azide binding reaction by ferric Aplysia myoglobin. A single completely reversible spectrophotometric titration curve is found over the pH range from similar to 5 to similar to 9, with an apparent pK equals to 7.5 for the acid-alkaline transition. The kinetics of the process, followed by the temperature-jump method, gives, at pH values close to the pK of the transition, one single, well-resolved, relaxation independent of protein concentration and of type of buffer used. The pattern accords to a simple pH dependent reaction, in buffered medium, between the two forms of the protein. The results of the azide binding reaction show that the process conforms to simple equilibrium as expected for a single site protein. The measured association constant is reported as a function of pH. The kinetics of the reaction of Aplysia metMb with N3- minus shows, on the other hand, a complex behavior. The relaxation pattern is found to strongly depend on pH and ligand concentration in such a way to suggest a linkage between ligand binding and acid-alkaline transition. The system is discussed on the basis of two simplifying conditions, i.e., at low and higher pH with respect to the pK of the acid-alkaline transition. At acid pH the reaction corresponds to a single bimolecular process as expected for a simple binding reaction; at alkaline pH, the dependence of relaxation time on ligand concentration implies the existence of a rate-limiting monomolecular step. On the basis of a reaction scheme implying that binding of the ligand can only occur through the acid (aquomet) form of the protein via the displacement of the water molecule, the experimental data are quantitatively accounted for.

Comparison of the resonance Raman spectra of carbon monoxy and oxy hemoglobin and myoglobin: similarities and differences in heme electron distribution.

Abstract: With 441.6-nm excitation, which is near the Soret band, we observe that the resonance Raman spectra of hemoproteins contain not only the bands between 650 and 1700 cm-1 which arise from vibrations of the conjugated macrocycle, but also bands below 650 cm-1, some of which involve vibrations of the iron pyrrole-nitrogen bonds. The spectra of the oxygen and carbon monoxide complexes of both myoglobin and hemoglobin are sufficiently similar to those of low spin met derivatives, that the electronic distribution on the heme for both ligands can be interpreted as that of a low spin ferriheme. This agrees with an earlier interpretation, by others, of comparative optical absorption spectra and, as pointed out previously, would imply that in the complex the ligands are bound as O2- and CO-. However, band frequencies and relative intensities differ somewhat between the carbon monoxide and oxygen complexes of the same protein, which indicates differences between the details of the pi-electron distributions in the corresponding complexes.

Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

Abstract: The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.

Manganese‐substituted hemoglobin and myoglobin*

Abstract: One of the most specific means available for exploring the function of the heme iron in hemoglobin is t o examine a metal-substituted protein. Cobaltsubstituted hemoglobin (coboglobin) binds molecular oxygen and has been found to exhibit the hemoglobin allosteric linkage effects. Because the stereochemical properties of cobalt and iron differ, this retention of linkage properties has significant implications with regard t o the nature of the allosteric mechanism. Investigation of other metal-substituted hemoglobins can offer additional valuable insights into the nature of the hemoglobin allosteric mechanism and, more generally, into the overall process by which tetrameric hemoglobin (Hb) binds four ligand molecules. We have begun the investigation of manganese-substituted hemoglobin and m y ~ g l o b i n . ~ * ~ Some properties of manganese hemoglobin in both divalent (MnHb) and trivalent (MnII'Hb) states and of mixed (MnIII, Fell) hybrid hemoglobins had previously been reported.9We here report equilibrium and kinetic studies t o determine whether the conformation of MnHb is equivalent t o that of Hb, or whether the conversion of MnHb to one of its liganded or oxidized derivatives involves a T -+ R quaternary structure change and attendant allosteric linkage effects. Equilibrium redox and ligation measurements demonstrate that manganese hemoglobin possesses the heterotropic linkage properties of hemoglobin. We have employed a variety of conformational probes to verify the existence of the T -+ R conformational change that these properties imply.I3 Although MnHb does not bind oxygen, the time course of the reaction of MnHb with NO can be readily Followed and the resulting MnHb-NO is observed t o be photodissociable. Through the use of stopped flow and flash photolysis techniques, we have also demonstrated the existence of homotropic linkage in MnHb reactions. Kinetic measurements of CO binding to the hemes in mixed (Mn\", Fell) hybrid hemoglobin give evidence of chain differences in T-state hemoglobin.

5 Iron-and Copper-Containing Monooxygenases

Abstract: Publisher Summary The unique role of molecular oxygen, or dioxygen, as an electron acceptor in the respiratory chain is so predominant in cell metabolism that other functions of this molecule were not realized for a long time. Such functions are well established in the chemistry of oxygen and are referred to as oxygenation reactions. This term defines the oxidation of an organic or inorganic compound by direct introduction of one or two oxygen atoms of the dioxygen molecule. The two transition elements copper and iron are essential cofactors of oxygen utilizing enzymes, such as hemoglobin, myoglobin, hemocyanins, and erythrocruorins as biological oxygen carriers, the iron-dependent dioxygenases, cytochrome oxidase, and the peroxidases. The cofactor function of iron and copper is based on their pronounced redox properties, their ability to form various types of metal-oxygen bonds, and the wide variation of their redox properties caused by complexation with biological chelates and proteins. Conformational changes as intrinsic properties of enzyme proteins can trigger changes in the redox behavior of the metal ion or vice versa. Monooxygenases play an important role in many biosynthetic and metabolic pathways and often have regulatory properties as their molecular activities are usually low. The biological importance of monooxygenases can be summarized by the fact that no other biochemical mechanism of attack at a nonactivated carbon-hydrogen bond is known.

The complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis).

Abstract: The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.

Mean myoglobin oxygen tension during exercise at maximal oxygen uptake.

Abstract: Changes in intracellular Po2 in myoglobin containing skeletal muscle during exercise were estimated in normal nonathlete subjects from measurements of shifts of CO between blood and muscle under co...

The amino acid sequence of myoglobin from skeletal muscles of red deer (Cervus elaphus).

Abstract: Red deer myoglobin has been fragmented by restricted tryptic digestion and by treatment with cyanogen bromide. The fragments have been separated by gel permeation. The core peptide derived from cyanogen bromide cleavage have been further digested with trypsin and the resulting peptides have been separated on Dowex 1X2. All fragments have been characterized by their amino acid composition, by determination of their N-terminal sequence using automatic Edman degradation and of their C-terminal sequence following the kinetics of amino acid cleavage by carboxypeptidases A and B. The complete sequence has been found to be identical with the already known sequence of sheep myoglobin except for residue 145 which is Gln in red deer globin and Glu in sheep globin. Reinvestigation of the corresponding sequence in sheep globin has shown that residue 145 of sheep globin is also Gln.