Showing papers on "Myoglobin published in 1968"

Formation of Nitric Oxide Myoglobin: Mechanisms of the Reaction with Various Reductants

Abstract: SUMMARY– The concentration, temperature and pH dependences of the formation of nitric oxide myoglobin (NOMb) from metmyoglobin nitrite (MetMb·NO2) were determined for nitrite and the reductants, ascorbic acid, cysteine, hydro-quinone, nicotinamide adenine dinucleotide (NADH) and glyceraldehyde. The reaction for all reductants except glyceraldehyde involves the production of a nitroso-reductant intermediate which breaks down to release nitric oxide. The latter forms a nitric oxide metheme complex (Fe+++) which is then reduced to the ferrous state (Fe++). With cysteine and NADH there is a second pathway which probably involves the direct reduction of MetMb NO2. Ascorbate and hydro-quinone form nitroso intermediates that are stabilized in alkali. The effects of oxygen, ethylenediaminetetraacetic acid and cytochrome c on the reaction were determined. Oxygen slows or inhibits the reaction, while the latter two have no effect on the reaction as studied.

Histochemical Demonstration of Myoglobin in Skeletal Muscle Fibres and Muscle Spindles

Abstract: MOST muscles are composed of a mixture of two types of muscle fibre which can be shown to differ in myoglobin content. Slow type 1 fibres are narrow, contain numerous mitochondria and are poor in glycogen and phosphorylase. Fast type 11 fibres are broad, contain few mitochondria and are rich in glycogen and phosphorylase1. It has been assumed that type 1 fibres are rich in myoglobin and these are termed red. Type 11 fibres are assumed to be poor in myoglobin and are termed white. Similarly, it has been assumed that the large nuclear bag fibres of muscle spindles are poor in myoglobin and that they contract rapidly. The smaller nuclear chain fibres would be expected to be rich in myoglobin and contract slowly. No comprehensive survey of the myoglobin content of muscle fibres has previously been reported. Knowledge of the precise distribution of myoglobin in extrafusal and intrafusal fibres would throw light on the function of myoglobin and muscle spindle activity.

Electron Resonance Studies of Haemoglobin Derivatives. III. Line-Width and g-Value Measurements of Acid-Met Myoglobin and of Met Myoglobin Azide Derivatives

Abstract: Details of the way in which haem plane orientations were deduced from g-value measurements on five different crystal types of acid-met myoglobin were given in parts I and II (Bennett, Gibson & Ingram 1957, and Bennett et al. 1961). This paper now summarizes more detailed measurements of line-width and g-value variations observed in both the acid-met and azide derivatives of type A myoglobin crystals. Since these crystals are those for which a detailed X-ray analysis is now available, a direct comparison of this with the electron resonance measurements can now be made. The g-values obtained for the azide derivative are first analysed, and their anisotropy and asymmetry are related to the possible orientation of the azide group itself. The line widths of the electron resonance absorptions, and their angular variations are then summarized and discussed. It is shown that their large magnitudes and rapid variation with angle can be explained in terms of a slight random misorientation of the molecular axes within the crystal, and that, due to the large g-value anisotropy in the acid-met derivative, a standard deviation of only 1.6 degrees in angular distribution is sufficient to explain the results obtained. A similar analysis is also applied to the results on the azide derivative.

The diffusion coefficient of myoglobin in muscle homogenate.

Abstract: One layer of muscle homogenate rich in myoglobin (heart muscle of the rat) and three layers of muscle homogenate poor in myoglobin (skeletal muscle of the rat) were arranged one upon the other, than reseparated after a certain time. According to the concentration change of myoglobin in the layers the diffusion coefficient of myoglobin is 1.5×10−7 cm2/sec at 20° C and 2.7×10−7 cm2/sec at 37° C. From this result it was calculated that the facilitated O2 diffusion on the basis of myoglobin diffusion amounts, at 37° C, to the free O2 diffusion at about 3 mm Hg partial pressure difference when myoglobin concentration is 2×10−7 mol (3 mg) per g wet muscle.

Lack of some muscle proteins in serum of patients with Duchenne dystrophy.

Abstract: AN'abnormal increase in the activity of several serum enzymes is one clue to the nature of the biochemical abnormality in Duchenne muscular dystrophy. The increased serum enzyme activity is thought to occur because enzyme protein molecules are released from muscle, and it has been inferred that the fundamental disorder is an abnormality of the muscle membrane which permits pathological leakage of large molecules. If this were true, it might be expected that other cytoplasmic constituents of muscle would also be found in the serum. We have been studying phosphofructokinase (PFK), phosphorylase (PPL), and myoglobin in normal human muscle diseases. Appropriate techniques were applied to the serum of patients with Duchenne dystrophy but PFK and myoglobin were not found at all and the activities of PPL were very low, as will be described in detail. Methods The patients were all ambulatory males below age 10 years, with overt manifestations of

Proton magnetic resonance studies of human cyanomethemoglobin.

Abstract: Human hemoglobin has a molecular weight of 64,500 and is composed of four subunits, two each of two types, a and F. Each subunit consists of an aor fpolypeptide chain and one protoheme IX group. In the biologically active state, each heme group contains one iron (II) ion which binds molecular oxygen in its first coordination sphere. The entire hemoglobin molecule, a232, thus contains four heme groups and binds four oxygen molecules in the fully oxygenated state. It has been fournd that the oxygen affinity is closely related to the structure of hemoglobins.' In the present paper we give a preliminary account of proton nuclear magnetic resonance (NMR) studies of human cyanomethemoglobin. From the NAMR data, electron spin densities at various positions in the heme group are derived. We indicate how this kind of N1\IR experiment can give new information about some aspects of the relations between structure and function in hemoglobin. In the NA/MR spectra of proteins, essentially all the proton resonances are observed in a narrow range which extends from the internal standard DSS (2,2dimethyl-2-silapentane-5-sulfonate) downfield to ca. -10 ppm (parts per million relative to DSS). The larger the protein molecule, the more the resoniances of the protons of the individual amino acid side chains overlap. Therefore most NMR studies are done with relatively small proteins with molecular weights less than 20,000. In paramagnetic heme proteins, local magnetic fields arising both from aromatic ring currents2' I and from the unpaired electron spins have been shown to shift certain proton resonances out of the range between DSS and -10 ppm.4-6 For these reasons the NMR spectrum of cyanomethemoglobin contains a considerable number of resolved resonances despite its high molecular weight. Experimental.-Human hemoglobin solutions were prepared from the freshly drawn blood of one of us (K. W.) by the following method. The erythrocytes were separated from the plasma by centrifugation within 15 min. after the addition of the anticoagulant (sodium oxalate), then washed four times with 1 vol of 1% NaCl. After hemolysis, the solution was subjected to 105,000 g centrifugation for 21/2 hr. The upper two thirds of the supernatant, free of any flocculus precipitate, was separated off and then further centrifuged. This procedure was repeated twice. The hemoglobin solution was dialyzed overnight at 4?C against 0.1 M phosphate buffer. Some hemoglobin solutions were also prepared by the widely used toluene method.7 The NMR spectra of cyanomethemoglobin solutions prepared by these two different methods were found not to differ noticeably. Methemoglobin was prepared by addition of a sixfold excess of K3Fe(CN)6 to the hemoglobin solution that was thenl dialyzed extensively against 0.1 M phosphate buffer. Methemoglobin was purified on the cation exchange resin Bio-Rex 70 (Bio-Rad) and concentrated by vacuum dialysis. Sodium phosphate buffer, pH 6.42, total ionic strength of 0.304, was used for the elution of methemoglobin.8 The homogeneity of isolated hemoglobin fractions was checked by starch gel electrophoresis9 with the discontinuous buffer system.10 Methemoglobin A1 was then converted to cyanomethemoglobin by the addition of a freshly prepared solution of KCN in phosphate buffer.

Immunological Studies of Duck Myoglobin

Abstract: SummaryPurified duck myoglobin was prepared by ammonium sulfate precipitation and ion-exchange chromatography. Its sedimentation constant was estimated to be approximately 1.75. A specific precipitating antiserum to duck myoglobin was prepared in rabbits. This antiserum did not form precipitates with duck plasma, hemoglobin, liver extract or with components of crude muscle extracts other than myoglobin. No immunologic differences were detectable between duck cardiac or skeletal muscle myoglobin, and none were noted between myoglobins of goose, duck, or chicken present in muscle extracts. Chicken pectoral muscle lacked myoglobin. Precipitin data suggest that three regions on the duck myoglobin molecule act as combining sites or determinants for the rabbit serum. The myoglobin content of several avian muscles was measured immunologically. Skeletal muscle of newly hatched ducklings was deficient in myoglobin content, while cardiac muscle of these immature birds contained almost one-half the adult amount 2 da...

Myoglobin and Muscular Dystrophy: Electrophoretic and Immunochemical Study

Abstract: THE muscular dystrophies are inherited diseases, presumably due to abnormality of a protein which is either completely lacking or so altered by genetic defect that it is functionally inadequate. There is no satisfactory clue to the nature of this fault, but there has been difference of opinion regarding the state of myoglobin. In 1961, Whorton and his associates 1,2 first reported that the absorption spectrum of myoglobin from dystrophic muscle is abnormal in the range of visible light, implying an abnormality of structure. Soon afterwards, Miyoshi et al 3 reported that they found no spectral abnormality in the visible range, but that there were differences in the absorption of ultraviolet light. At about the same time, Kossman et al 4 and Benoit 5,6 described rapidly migrating forms of myoglobin in starch gel electrophoresis of urine during attacks of myoglobinuria; they suggested that these were either abnormal forms of the

Electron spin resonance linewidths in met myoglobin.

Abstract: WE have published1 a detailed account of the variation of electron resonance line width in both acid met myoglobin and met myoglobin azide as a function of orientation. We explained the results in terms of a random misorientation of the molecular axes within the single crystals, and showed that, because of the large g value anisotropy in the acid met derivative, a standard deviation of only 1.6° in angular distribution is sufficient to explain the results obtained. These measurements were carried out at Q-band frequencies and the slight deviations from the expected variations were adequately explained in terms of residual broadening mechanisms such as spin–spin interaction and unresolved hyperfine structure. It can be shown1 that the misorientation produces a linewidth in an axially symmetric system given by

Conversion of δ-Amino Laevulinic Acid to Porphobilinogen by Different Tissues of the Polychaete Annelid Neoamphitrite figulus

Abstract: THE heart-body, a small spongy mass of tissue within the dorsal vessel of the terebellid polychaete Neoamphitrite figulus (Dalyell), is almost certainly concerned with synthesis of the haemoglobin of the blood. The coelomocytes also contain haemoglobin, but this is different. Kennedy and Dales1 have shown that the heart-body tissue and the coelomocytes in this species converted porphobilinogen (PBG) to uroporphyrin. Tissues were incubated with purified PBG suspended in sea water and at room temperature for varying periods and the products identified after extraction and purification by paper chromatography. Some similarly performed experiments using δ-amino laevulinic acid (ALA) did not give clear results. Subsequent work, using the method of McRae2, whereby the conversion of ALA to PBG is identified against suitable controls by the positive reaction PBG gives with Ehrlich's p-aminobenzaldehyde reagent3, has, nevertheless, given quite clear results. McRae's method was followed in detail except that to quantify the results the tissues were merely dried with acetone and weighed, for it was desired only to compare the relative ability of different tissues to convert ALA to PBG. McRae used homogenates of whole animals (Dugesia). Tissues were deep frozen and homogenized on thawing in M/15 phosphate buffer, pH 7.0, the ALA being added to provide a concentration of 400 µg/ml. of homogenate. Homogenates, suitably controlled, were incubated at 37° C for 2.5 h. The colour of the Ehrlich reaction was measured at 553 mµ after 15 min with a ‘Unicam SP 500’ spectrophotometer. While the activity of each tissue is expressed in arbitrary units, their relative activities in one typical experiment are shown in Table 1. The experiment was repeated five times with similar results. The tissues of five worms were pooled in each experiment. Table 1 shows that the heart-body is ten times more active than any other tissue determined. The body wall muscle is colourless and if it contains any myoglobin this is in a relatively low concentration and any ALAase present is insufficient to produce enough PBG to be detected by the method. On the other hand, Kennedy and Dales4 detected coproporphyrin III, which must have been formed during haem synthesis, in the body wall; whether this is formed in the skin or is deposited there, as this negative result suggests, is being investigated further. The hind stomach or gizzard has muscles of a deep red colour and some ability to convert ALA to PBG is not surprising. The ability of the coelomocytes to do so is also expected, confirming the hypothesis that they synthesize their own haemoglobin from ALA and do not receive compounds later in the biosynthetic pathway to haem. The high activity of the gut (fore stomach plus intestine) from which the contents had carefully been removed is surprising, especially because Kennedy and Dales4 found no free porphyrins in these tissues. It may be that the coelomocytes, which contain haemoglobin, arise from the coelomic epithelium of the gut wall. This is being investigated further.

Presence of Compounds containing Iron in the Digestive System

Abstract: PROBLEMS concerned with iron storage and its subsequent utilization by invertebrates have, most often, concentrated on those forms in which haemoglobin is present1–10 and in certain instances in those organisms in which haemocyanin is utilized as a respiratory pigment11. Reports have shown that when haemoglobin is utilized as a respiratory pigment it is demonstrable not only in the body fluid of the animal but also in a variety of tissues—muscle, nerve and eggs6, as well as fat cells12. In some cases, such as Daphnia magna which is actively losing haemoglobin, porphyrins are not demonstrable, but loosely bound iron is easily demonstrated histologically in the walls of the gut caeca, in the fat cells and in the maxillary glands13. In certain organisms possessing the copper blood pigment haemocyanin it has also been possible to demonstrate the presence of iron porphyrin compounds (haemochromogens) not only in the blood but also in such diverse structures as the eggs of Limulus polyphemus, the radula muscle and heart of Busycon canaliculatum (both myoglobin and cytochrome being present), cytochromes in the heart, claw and skeletal muscle of Homarus americanus, and cytochromes in many of the tisssues of Loligo peali11. Conclusions drawn from these many experiments indicate that, except for the substitution of haemocyanin for haemoglobin, oxygen utilization is essentially the same as in mammals. They also point out that these animals are utilizing iron in the synthesis of certain porphyrin compounds of the cytochrome system. It has thus been fairly well established that possession of haemoglobin as a respiratory pigment is no prerequisite for the presence of compounds containing iron in animal tissues.

The Structure of Oxygenated and Deoxygenated Myoglobin

Abstract: The proteins myoglobin and haemoglobin have as their chief physiological function the reversible combination with molecular oxygen. In this contribution we present details of X-ray crystallographic studies of the two equilibrium forms of myoglobin from the sperm whale, which are involved in this oxygenation-deoxygenation reaction.

Regional myoglobin concentration in the myocardium of the dog.

Abstract: It has been demonstrated previously that chronic hypoxia leads to an increase in myoglobin concentration. Evidence of differences in and blood flow in deep and superficial layers of cardiac muscle ...