Jeffries Wyman

LINKED FUNCTIONS AND RECIPROCAL EFFECTS IN HEMOGLOBIN: A SECOND LOOK.

Publisher Summary This chapter discusses linked functions and reciprocal effects in hemoglobin. Because the components are defined thermodynamically in terms of independently variable chemical entities and, therefore, a given component may exist in any number of different forms in equilibrium with one another, these considerations show that not only do the linkage relations apply irrespective of whether the macromolecules undergo chemical change or polymerization, but also irrespective of whether the ligands themselves associate and dissociate, possibly as macromolecules. From a formal point of view the distinction between ligand and macromolecule breaks down. This principle may be useful in the application of linkage relations to antibody–antigen systems and to enzyme systems when both enzyme and substrate are proteins. Another more general, and perhaps more interesting, question is whether the dissociation process is also dependent on oxygenation, i.e., whether the protons involved are oxygen-linked. There is now strong evidence that the dissociation at acidic pH is, indeed, dependent on oxygenation.

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.

The binding potential, a neglected linkage concept

Corresponding to any one of the various thermodynamic potentials P there exists a binding potential Л which is a function of the derivatives, μ , of P with respect to the composition variables for constant values of the physical variables. This binding potential has the property that v i = ∂ JI ∂ μ i where ν 1 , is the ratio of the amount of component i to that of a reference component. By a suitable choice of P and the physical variables the μ s may be made the same as the ordinary chemical potentials. The introduction of the concept of the binding potential greatly clarifies the definition of linkage groups, linked functions, and linked sites, and suggests the construction of linkage maps. It also leads to a compact expression for the equilibrium constant for a reaction involving macromolecules which combine with several different ligands.

Regulation in macromolecules as illustrated by haemoglobin

Mηδέν ἄγαν—nothing in excess. These words of the old Greek saying might well find a place in modern Biology. The maintenance of order and balance—homeostasis, as the physiologist would have it—is an attribute of all living things. At a molecular level the study of control and regulation, involving the conjugate concepts of structure and function is a central theme of Biophysics. How is it that an enzyme, by the braking effect of its end products, is prevented from going too far, or, alternatively, by the accumulation of other metabolic substances in its environment, is brought into play? How is it that a working respiratory protein like haemoglobin, by the reciprocal action of its metabolic opposite, carbon dioxide, is aided in the taking up of oxygen in the lungs and the giving it off in the tissues? These are problems of linkage. From one point of view they may be thought of in terms of information— the communication between ligand and macromolecule and between one part of the macromolecule and another. From a different point of view they may be thought of in terms of the storage and transfer of energy, of the way in which the work of introducing ligand, the same or different, at another. The analysis may be pitched either at the phenomenological, or at the mechanistic, level; but whatever the approach it profits cosiderably from the introduction of the concept of the binding potential.

STUDIES ON FUNCTIONAL PROPERTIES OF FISH HEMOGLOBINS .2. OXYGEN EQUILIBRIUM OF 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.

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.

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.

The transition between ‘acid’ and ‘alkaline’ ferric heme proteins

We have studied the transition between the ‘acid’ and the ‘alkaline’ forms in several ferriheme proteins, which is generally attributed to the ionization of the water molecule coordinated with the heme iron in the sixth position. The different heme proteins studied (sperm whale and Aplysia myoglobins, Chironomus and human hemoglobins) have different dissociation constants, which arise mainly from heat effects. These differences are tentatively correlated to some differences in the general structure of the globin. Alkylation of sperm whale myoglobin or human hemoglobin, obtained by reaction with acetic or succinic anhydrides, leads to large changes in the pK′ of the transition. The profile of the curve relating the ΔpK′ to the percent alkylation suggests the possibility of dividing the different reactive groups of the protein into different classes on the basis of their effects on the ionization process. The substitution of hemes differing from protoheme in sperm whale myoglobin leads to significant changes in the equilibrium constant of the reaction, the pK′ ncreasing in the order proto-deutero-meso-myoglobins.