Eraldo Antonini

HEMOGLOBIN AND MYOGLOBIN.

Publisher Summary This chapter describes hemoglobin and myoglobin. Hemoglobin and myoglobin are, among all proteins, ones that have been, and are, most actively studied; an enormous number of papers have been published over the past hundred years on all aspects of their properties and behavior. The study of these proteins has gone beyond the interest in their physiological role as oxygen carriers because they represent ideal models for investigating the properties of proteins in general, especially of enzymes. Correspondingly, current knowledge of the structure and function of hemoglobin and myoglobin is far greater than that available for any other protein. In spite of this, however, many questions still remain unsolved regarding the exact molecular mechanisms involved in the function of these proteins. Before discussing the properties of respiratory heme proteins, it is necessary to briefly describe some properties and reactions of their prosthetic group, mainly because the characteristic physiological functions of these proteins arise from the intrinsic reactivity of the heme.

Studies on the functional properties of fish hemoglobins: II. The oxygen equilibrium of the isolated hemoglobin components from trout blood

Homogeneous components of trout hemoglobin (Salmo irideus) have been isolated by column chromatography. The oxygen equilibrium of the two main components has been investigated. The oxygen affinity and the shape of the ligand equilibrium curve is independent of pH for component I. On the other hand, component IV is characterized by a very large Bohr effect, to which a considerable change in the shape of the oxygen equilibrium curve with pH is associated. The different oxygen-binding behavior of the isolated components can explain data obtained with the whole blood and in particular the contribution of the various proteins to the Root effect. The dependence on pH of the apparent ΔH for oxygenation has been measured for both components. Component I is characterized by a fairly low (ΔH ~ −3 kcal/mole) and pH independent value of the enthalpy change, while for component IV the apparent ΔH decreases from ~ −14 kcal/mole at pH near 9 to ~ −7 kcal/mole at pH 7.

[5] Preparation and properties of apohemoglobin and reconstituted hemoglobins

Publisher Summary This chapter describes the procedures for the isolation of globin in the native state and for its reconstitution with metalloporphyrins. Globins are isolated from the corresponding hemoproteins by the removal of the heme moiety at acid pH, under controlled experimental conditions. The quality of the globin samples obtained can be checked by titration experiments with hemin (ferric protoheme) and by studying the properties of the reconstituted hemoproteins. There are two known methods for the preparation of globin from the holoprotein; both are based on the decreased affinity of the heme for globin at acid pH values. The first method employs acid–acetone at low temperature to split the heme group from the globin, which precipitates and is subsequently redissolved in water. A second method is based on heme extraction in methyl ethyl ketone at acid pH; in this case, the apoprotein remains dissolved in the aqueous layer. Further purification of the globin is accomplished similarly in both methods. The chapter presents the physicochemical properties of globin.

Enzyme catalysed reactions in water - Organic solvent two-phase systems

Abstract Enzyme-catalysed transformations carried out in two-phase systems consisting of water and a water-immiscible organic solvent are discussed. The systems appear advantageous when substrates poorly soluble in water, such as steroids, are used. The methodology also makes possible the use of hydrolytic enzymes for the synthesis of ester bonds. Several applications of such two-phase systems are illustrated. The criteria that must be taken into consideration for selecting the most suitable organic solvents and the operational conditions are discussed.

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: Structural and functional properties

The bivalve mollusc Scapharca inaequivalvis contains in the coelomic fluid erythrocytes with a dimeric (HbI) and a tetrameric (HbII) hemoglobin like the other members of the arcid family. The tetrameric protein is made up from two types of polypeptide chain, while the dimeric protein is made from a single type of chain which differs from the other two in terms of molecular weight and isoelectric point. The optical and circular dichroism spectra show that the heme environment in HbI and HbII resembles that of vertebrate hemoglobins, although distinctive features are present in the deoxygenated derivative. p]The dimeric HbI in the pH range 6 to 9 does not change its association state upon deoxygenation, while the tetrameric HbII polymerizes as indicated by the appearance of a fast peak in the sedimentation velocity patterns. The dependence of the areas and sedimentation coefficients of the fast and slow peaks on protein concentration is characteristic of a rapidly established association-dissociation equilibrium between tetramers and polymers higher than octamers. The pH, ionic strength and temperature dependence of polymer formation indicate that both hydrophobic and ionic interactions stabilize the polymers. The functional properties of HbI and HbII differ. HbI shows co-operative oxygen binding (h = 1·5) and a constant oxygen affinity (p12 = 7.8 mm Hg) over the pH range 5.5 to 9.5. HbII likewise shows co-operativity in oxygen binding (h = 2·0). Its oxygen affinity at neutral and alkaline pH values is slightly lower (p12 = 9.1 mm Hg) than that of the dimeric protein, but becomes higher at pH values below 6.5 due to the presence of an acid Bohr effect. At high protein concentrations, under conditions of extensive polymerization of the deoxygenated derivative, the oxygen affinity is lowered and co-operativity slightly increased. Both phenomena require that the oxygen affinity of the polymer be lower than that of the tetramer, consistent with the predictions of linkage theory.

Nuclear magnetic resonance quadrupole relaxation studies of chloride binding to human oxy- and deoxyhaemoglobin.

Abstract Chloride binding to oxy-, carbon monoxy- and deoxyhaemoglobin and to myoglobin has been measured directly in quadrupole relaxation experiments on the excess line-width of the nuclear magnetic resonance signals of 35 Cl − associated with its binding to the protein. The measurements, which have been extended over a wide range of conditions, suggest that in haemoglobin there are at least two classes of chloride binding sites. The high affinity sites are oxygen linked and over the range 0.1 to 2.5 m -NaCl deoxyhaemoglobin binds more chloride ions than liganded haemoglobin. In contrast no such difference exists between the corresponding myoglobin derivatives. The difference in chloride binding between oxy- and deoxyhaemoglobin may be correlated with the conformational transitions associated with ligand binding in haemoglobin and is reflected in the effect of chloride on the oxygen equilibrium. Competition experiments with ATP indicate that the high affinity chloride binding sites correspond to those for the organic phosphates.

THE PROPERTIES AND INTERACTIONS OF THE ISOLATED ALPHA AND BETA CHAINS OF HUMAN HAEMOGLOBIN. 3. OBSERVATIONS ON THE EQUILIBRIA AND KINETICS OF THE REACTIONS WITH GASES.

The oxygen equilibrium of the isolated α and β ohains of human haemoglobin and of their mixtures has been studied; also the kinetics of their reactions with carbon monoxide. The experiments were carried out both on the chains as they were directly obtained by the preparative procedure, with bound parachloro-mercuribenzoate (i.e. as PMB† compounds) and after removal of the mercurial (i.e. as SH compounds). The oxygen equilibrium of the isolated α or β chains with or without PMB, shows absence of haem-haem interactions, absence of Bohr effect, and high oxygen affinity; the β SH system behaves essentially like haemoglobin H. In contrast, in the oxygen equilibrium of mixtures of the α and β chains, with or without PMB, haem-haem interactions and a Bohr effect are present; the ( α SH + β SH ) system behaves essentially like normal human haemoglobin. The velocity constant for combination with carbon monoxide of the isolated chains with or without PMB is twentyfold, or more, greater than that of normal human haemoglobin; when α and β chains, with or without PMB, are mixed together, the velocity of combination with carbon monoxide falls to a value similar to that of normal haemoglobin. The absorption spectrum in the Soret region of the deoxy derivative of the isolated chains differs characteristically from that of their mixture, which is the same as that of normal haemoglobin. The results indicate that the functional interactions in haemoglobin require the simultaneous presence of the two kinds of polypeptide chain, α and β .

Studies on the structure of hemoglobin: III. Physiochemical properties of reconstituted hemoglobins

Abstract Artificial hemoglobins have been reconstituted from native human globin and the following hemes: mesoheme, deuterheme, chloroheme (or Spriographis heme) and hematoheme. All these differ from protoheme in the side chains of the porphyrin ring at position 2 and 4. Some physical and physiochemical properties of thse hemoglobins have been determined. The spectral properties were similar to those of protohemoglobin. The sedimentation coefficients of all these hemoglobins had values near to 4.0 S. The stability to heat and alkali denaturation decreased in the order: proto-, meso-, deutero-, and hematohemoglobin. The O 2 equilibrium of these unnatural hemoglobins also differed from that of protohemoglobin, being characterized by a decrease of the heme-heme interactions. The results have been correlated with the decrease in affinity of the various modified hemes for globin and suggest that the vinyl groups of the heme play an important role in determining the conformation of the hemoglobin molecule.

Studies on erythrocruorin. I. Physicochemical properties of earthworm erythrocruorin.

Abstract Erythrocruorin has been purified by preparative ultracentrifugation from the blood of Lumbricus. The protein is homogeneous in the ultracentrifuge and in gel electrophoresis. The sedimentation coefficient is 59.6, and the molecular weight by light scattering is 3.45 × 106 daltons. The protein has 2.67% heme iron corresponding to a minimum molecular weight of ~23 × 103 daltons. The full protein molecule thus contains 144 hemes arranged in a subunit structure similar to that of other erythrocruorins and of Spirographis chlorocrun.

The properties and interactions of the isolated α and β chains of human haemoglobin: I. Sedimentation and electrophoretic behaviour

The α and β chains of human haemoglobin A 1 may be isolated by starch block electrophoresis or by chromatography after treatment of the protein with p -mercuribenzoate. The chains so isolated do not show any free SH groups; however, after treatment with cysteine or thioglycolate, between 1 and 2 SH groups per β chain and 1 per α chain become titratable. The chains before and after such treatment are designated α PMB† or β PMB and α SH or β SH respectively. Ultracentrifugal analysis shows that the isolated α SH and α PMB chains are both monomeric; in contrast the β PMB chains tend to polymerize. The β SH chains are heterogeneous but the major component appears to be a tetramer and may be identified with haemoglobin H. A mixture of α PMB and β PMB chains in equal amounts is homogeneous and has a sedimentation coefficient in the neighbourhood of 3. A similar mixture of α SH and β SH chains behaves like haemoglobin A both in the ultracentrifuge and in starch gel electrophoresis. Certain systems which are homogeneous in the centrifuge and on Sephadex show multiple bands in electrophoresis, which implies differences beyond those due to molecular weight. These results are discussed in terms of the interactions between the chains under various conditions.