Robert W. Noble

Occurrence of root effect hemoglobinsin Amazonian fishes

Abstract o 1. The Root effect was measured in hemolysates from representatives of 56 genera of Amazonianfishes. 2. Hemolysates from several species of air-breathing fishes were found to have Root effects, contraryto published hypotheses. 3. Hemolysates from Potamotrygon, a freshwater ray, exhibit a Root effect under our experimental conditions. 4. The pattern of Root effect distribution correlates positively with the distribution of choroid retiamirabile and swimbladders, but not with the distribution of swimbladder retia mirabile; it is proposed that the former is the more primitive structure which is associated with the origin of Root effect hemoglobins 5. Some of the fish hemoglobins differ spectrally from one another. The positions of the absorptionmaxima of the deoxyhemoglobins range from 553 nm (Lepidosiren paradoxa and Potamotrygon sp.) to 560 nm (Plagioscion) 6. Occurrence of Root effects is not correlated with the complexity of the hemoglobin electrophoreticpattern, although several species are found to have multiple hemoglobin systems in which the Root effect is restricted to certain components.

Functional properties of hemoglobins from deep-sea fish: correlations with depth distribution and presence of a swimbladder

The ligand binding properties of the hemoglobins of several deep-sea, bottom-living fish have been examined. These include five species of rattails (Macrouridae) and Antimora rostrata, all of which possess swimbladders, and two unrelated species without swimbladders, Bathysaurus mollis and Alepocephalus sp. All of the hemolysates of these fish exhibited the Root effect with a minimum ligand affinity at about pH 6 in the presence of organic phosphate. Under these conditions the hemolysates from fish which possess swimbladders exhibit two roughly equal populations of heme groups with markedly different ligand affinities. For the deeper-dwelling species the affinities for carbon monoxide differ by some 500-fold, the low-affinity population having a p50(CO) of 100 mmHg at 15 degrees C. This very low affinity is associated with a second-order rate constant for CO combination of the order of 10(3) M-1 X s-1. Those species without swimbladders have hemoglobins which do not have such heterogeneous binding sites, suggesting a relationship between these very-low-affinity heme groups and the pumping of oxygen into a swimbladder at high hydrostatic pressures.

Isolation and characterization of low and high affinity goat antibodies directed to single antigenic sites on human hemoglobin

Abstract A group of anti-hemoglobin antibodies from a goat immunized with human HbA1 has been fractionated according to affinity. By elution of anti-HbA1 antibodies bound to a β chain Sepharose column with a gradient of decreasing pH and increasing acetic acid concentration, the lower affinity antibodies eluted earlier from the column and were separated from the higher affinity antibodies. Fluorescence quenching titrations with HbA1 and the β chains showed the first and the last of these fractions to be directed toward single antigenic sites on the β chain. Comparisons of their interactions with several hemoglobin variants establish that they bind to different sites on the β chain. The antigenic site on the β-chain to which the high affinity fraction binds involves the β6 position since its affinity for HbS (β6 glu → val) and Hb Leiden (β6 or7 deleted) is significantly lower than its affinity for HbA1.

STRUCTURE AND OXYGEN AFFINITY OF CRYSTALLINE DES-HIS-146BETA HUMAN HEMOGLOBIN IN THE T STATE

To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146β human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146β, the COOH-terminal histidine residues of the β chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146β results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141α hemoglobin showed that removal of the COOH termini of the α chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146β in the absence and presence of inositol hexaphosphate are 2.5- and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6- and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141α than those associated with His-146β.

[9] Fish hemoglobins

Publisher Summary Fish hemoglobins exhibit the essential features of mammalian hemoglobins, cooperative ligand binding and heterotropic responses to a variety of ionic species, but they display an astounding variety of functional behaviors. These different properties are of interest as examples of evolutionary adaptation to differing physiological and environmental needs. They also offer valuable systems in which to study specific phenomena in either an exaggerated form or a simplified context. There are components of the hemoglobins of Salmonidae that exhibit neither Bohr effects nor responses to organic phosphates. Many fish hemoglobins exhibit exaggerated Bohr effects, commonly known as the “Root effect,” permitting the study of pH dependencies, which are considerably amplified relative to that observed in mammalian hemoglobins. Fish hemoglobins can exhibit widely differing ligand affinities, with the total range reported for different hemoglobins under varying conditions being greater than four orders of magnitude. Additionally, many fish hemoglobins that exhibit the Root effect lose cooperative ligand binding when they attain their minimum ligand affinity at low pH in the presence of organic phosphates. These noncooperative, low-affinity states appear to be excellent models of liganded T states and are ideal for the analysis of the origins of the differences in the ligand affinities of the two extreme quaternary states of hemoglobin—namely, the R and T states.

A possible new control mechanism suggested by resonance Raman spectra from a deep ocean fish hemoglobin

The rattail fish, Coryphaenoides armatus, lives at ocean depths of 3000 m. As an adaptation for pumping oxygen into the swim bladder against the extreme pressures at the ocean bottom, the hemoglobin from this fish at low pH exhibits an extraordinarily low affinity for ligands. In this study, continuous wave and time-resolved Raman techniques are used to probe the binding site in this hemoglobin. The findings show an association between the low-affinity material and a highly strained heme-proximal histidine linkage. The transient Raman studies reveal differences in the protein structural dynamics at pH 6 and 8. The emerging picture derived from both this and earlier studies is that in vertebrate hemoglobins the heme-proximal histidine linkage represents a key channel through which species- and solution-dependent variations in the globin are communicated both statically and dynamically to the heme to produce an extensive range of ligand binding properties. Also presented is a new model that relates both intensity and frequency of the resonance Raman band involving the iron-proximal histidine stretching mode to specific protein controlled structural degrees of freedom. There emerges from this model a mechanism whereby modifications in the proximal heme pocket can further reduce the affinity of an already highly strained T state structure of hemoglobin.

Properties of goat anti-human hemoglobin antibodies fractionated on subunit affinity columns

Abstract The antibodies to human hemoglobin A, (HbA 1 ) in the sera of two goats were fractionated on the basis of their ability or inability to bind to the isolated α and β subunits of the hemoglobin. The fractionation was carried out by a series of affinity chromatography steps and the characterization of the binding of the antibody populations thus obtained to HbA 1 , α and β revealed some predominating characteristics common to the two goat sera. In roughly proportional yields, the anti-HbA 1 in the sera were fractionated into three major catergories: antibodies which bind only to HbA 1 and β chains, antibodies which bind only to HbA 1 and α chains and antibodies which bind to HbA 1 and both α and β chains. One goat also had an antibody population that would bind HbA 1 but neither α nor β. There was, in both sera, a larger proportion of α binding control to β binding antibodies and of α−s non-cross-reacting compared to α−s cross-reacting antibodies. Upon consideration of the amino acid sequence and three-dimensional structures of both the hemoglobin antigen and the hosts own hemoglobin with respect to sequence differences and the exposure and clustering of such differences, the existence and proportions of the diffrent antibody populations can be explained.

Time-resolved resonance Raman studies of carp hemoglobin

To understand the structural basis for the cooperativity of ligand binding to hemoglobin (Hb) it is necessary to know how the quaternary structure of the protein modulates the response of the binding site to the ligand and how the ligand brings about a switch in quaternary structure. To answer these questions it is essential to determine, as a function of quaternary structure, the functionally relevant structural changes that are generated by ligand binding. Time-resolved resonance Raman spectroscopy has been used to study the ligand-induced changes in the heme pocket of R and T states of human Hbs [1-3]. These studies [1,3] have also been effective in relating reactivity to structural features of the transient species of Hb which occur immediately after photodissociation. By focusing on the frequency of the iron-proximal histidine (F8) (Fe-His) stretching mode it is seen [2,3] that ligand binding causes a change in the heme-proximal histidine linkage that results in an increased frequency for the Fe-His stretching mode in the photolyzed species (at 10 ns) relative to that of the corresponding deoxy species. For both the deoxy [4,5] and the photolyzed [2,3] species the R state Hbs have the higher frequency. Here, we show that this same type of structural response, of the binding site to the ligand as well as the sensitivity of this response to quaternary structure is also in evidence in carp hemoglobin. The liganded derivatives of this hemoglobin can be reversibly stabilized in either the R or the T configuration [6]. In addition by comparing the yields of geminate recombination [7-9] we relate this same quaternary structure-sensitive degree of freedom to the kinetic constants that contribute to cooperativity.

[48] Oxygen binding to sickle cell hemoglobin

Publisher Summary This chapter describes oxygen binding to sickle-cell hemoglobin. Sickle-cell anemia was first recognized as a distinct disease in 1910, but only during the past three decades has its molecular basis been revealed in some detail. There are many single-amino-acid modifications of human hemoglobin that alter the functional properties of the tetrameric hemoglobin molecule and thereby compromise its physiological function. The abnormal properties of hemoglobin S are more complex than this. The major abnormality in hemoglobin S is a tendency for the deoxygenated molecules to polymerize into ordered fibrous structures. The fibers can themselves align side-by-side to form crystalline arrays. Because of the large numbers of molecules incorporated into these assemblies, this polymerization reaction appears to a first approximation as a phase separation and can be viewed as a limitation in the solubility of the deoxygenated hemoglobin S molecule. However, the amount of polymerization accompanying deoxygenation is strongly dependent on the total hemoglobin concentration and, therefore, the apparent oxygen affinity is also concentration dependent. The chapter further describes the preparation of hemoglobin S and hemoglobin A for comparison studies.

Equilibrium, kinetic and structural properties of hemoglobin Cranston, an elongated β chain variant☆

Abstract Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston ‡ at 20 °C in the absence of phosphate revealed a high affinity (P50 = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Bohr effect ( −Δ log P 50 Δ pH 7–8 = 0·2 ). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity (P50 = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Bohr effect ( −Δ log P 50 Δ pH 7–8 = 0·2 ). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid (l′, l′4, of ∼ 6 × 106m−1s−1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ∼ 0·5 × 106m−1s−1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow (k, k4∼ 11 s−1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α2βAβCranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell.