Clara Fronticelli

Modulation of oxygen affinity in hemoglobin by solvent components: Interaction of bovine hemoglobin with 2,3-diphosphoglycerate and monatomic anions☆

In the absence of Cl- in Hepes buffer at pH 7.4, the oxygen affinity of bovine and human hemoglobin is equally sensitive to 2,3-diphosphoglyceric acid. The low oxygen affinity measured for bovine hemoglobin at physiological salt concentration can be explained by the high affinity of Cl- anions for oxygen-linked sites that are absent in human hemoglobin. Bovine hemoglobin can discriminate between the different halogens in the sense that different halide concentrations are necessary to produce the same P50. Competition experiments indicate that the halogens interact with the same oxygen-linked sites. In agreement with the different affinities for halides, the Bohr effect of bovine hemoglobin is larger in the presence of Cl- than in that of Br- and there is good agreement between the number of protons and anions exchanged with the solvent upon oxygenation of bovine hemoglobin.

A possible new mechanism of oxygen affinity modulation in mammalian hemoglobins

Bovine red cells do not contain appreciable amounts of 2,3-diphosphoglycerate (2,3-DPG). Bovine hemoglobin, however, has a particular sensitivity to chloride ions and as a result it can attain oxygen affinity values lower than those measured for human hemoglobin in the presence of 2,3-DPG. The interaction of bovine hemoglobin with anions is modulated by the hydrophobic characteristics of the protein. Comparison of the hydropathy plots of primate and ruminant hemoglobins indicates constant regions of opposite hydrophobicity, which have fixed amino acid differences. A model is proposed for explaining the regulation of oxygen affinity by chlorides, as an alternative to the classic modulation by 2,3-DPG.

Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

A plasmid analogous to the one described by Nagai and Thogersen (Nature,309, 810–812, 1984) has been constructed for the expression of globins inE. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX-β-globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when thepH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic β-globin which can be folded in the presence of cyanohemin and native α-chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.

Molecular engineering of a polymer of tetrameric hemoglobins

We have engineered a recombinant mutant human hemoglobin, Hb Prisca β(S9C+C93A+C112G), which assembles in a polymeric form. The polymerization is obtained through the formation of intermolecular SS bonds between cysteine residues introduced at position β9, on the model of Hb Porto Alegre (β9Ser → Cys) (Bonaventura and Riggs, Science 1967;155:800–802 ). Cβ93 and Cβ112 were replaced in order to prevent formation of spurious SS bonds during the expression, assembly, and polymerization events. Dynamic light scattering measurements indicate that the final polymerization product is mainly formed by 6 to 8 tetrameric hemoglobin molecules. The sample polydispersity Q = 0.07 ± 0.02, is similar to that of purified human hemoglobin (Q = 0.02 ± 0.02), consistent with a good degree of homogeneity. In the presence of strong reducing agents, the polymer reverts to its tetrameric form. During the depolymerization process, a direct correlation is observed between the hydrodynamic radius and the light scattering of the system, which, in turn, is proportional to the mass of the protein. We interpret this to indicate that the hemoglobin molecules are tightly packed in the polymer with no empty spaces. The tight packing of the hemoglobin molecules suggests that the polymer has a globular shape and, thus, allows estimation of its radius. An illustration of an arrangement of a finite number of tetrameric hemoglobin molecules is presented. The conformational and functional characteristics of this polymer, such as heme pocket conformation, stability to denaturation, autoxidation rate, oxygen affinity, and cooperativity, remain similar to those of tetrameric human hemoglobin. Proteins 2001;44:212–222.

Bovine hemoglobin as a potential source of hemoglobin-based oxygen carriers: crosslinking with bis(2,3-dibromosalycyl)fumarate

Reaction in anaerobic conditions of bovine hemoglobin with bis(2,3-dibromosalycyl)fumarate resulted in new derivatives with P50 in excess of 40 mmHg, as determined at 37 degrees C in 0.15 M Cl- at pH 7.4. Although the chromatographic preparations indicated some heterogeneity of the reacted material, the proteins obtained were homogeneous with regard to sedimentation velocity, which showed the presence of only nondissociable tetrameric species. SDS gel electrophoresis showed the presence of a new band with a mobility corresponding approximately to a molecular mass of 32 kDa, indicating the presence of covalent intramolecular crosslinks between subunit pairs. Chromatographic analyses indicated that both alpha and beta chains were chemically modified. The retention times in rats of the crosslinked hemoglobin was 10-times longer than that of untreated hemoglobin.

Time-resolved emission spectra of hemoglobin on the picosecond time scale

We used front-face illumination to examine the steady-state and time-resolved emission from the intrinsic tryptophan emission of human hemoglobin (Hb). Experimental conditions were identified which eliminated all contributions of scattered light. The sensitivity obtained using front-face optics was adequate to allow measurement of the wavelength-dependent frequency response of the emission to 2 GHz. The intensity decays displayed pico- and nanosecond components in the emission at all wavelengths from 315 to 380 nm. The contribution of the picosecond component decreased from 72 to 37% over this range of wavelengths. Frequency-domain measurements were used to calculate the time-resolved emission spectra and decay-associated emission spectra. These spectra indicate that the picosecond components of the emission display maxima near 320 nm, whereas the nanosecond components are centered at longer wavelengths near 335 nm. The nanosecond components appear to be due to residual impurities which remain even in highly purified samples of Hb. However, we cannot eliminate the possibility that some of these components are due to Hb itself.

Cysteines β93 and β112 as Probes of Conformational and Functional Events at the Human Hemoglobin Subunit Interfaces

Abstract Three variants of tetrameric human hemoglobin, with changes at the α 1 β 2 / α 2 β 1 -interface, at the α 1 β 1 / α 2 β 2 -interface, and at both interfaces, have been constructed. At α 1 β 2 / α 2 β 1 -interface the β 93 cysteine was replaced by alanine ( β C93A), and at the α 1 β 1 / α 2 β 2 -interface the β 112 cysteine was replaced by glycine ( β C112G). The α 1 β 2 interface variant, β C93A, and the α 1 β 1 / α 1 β 2 double mutant, β (C93A+C112G), were crystallized in the T-state, and the structures determined at 2.0 and 1.8A resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of detectable changes in the tertiary folding of the protein or in the T-state quaternary assembly. At the β 112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional characteristics of β C112G are essentially those of human hemoglobin A. At the β 93 site, water molecules do not replace the cysteine side chain, and the alanine substitution increases the conformational freedom of β 146His, weakening the important interaction of this residue with β 94Asp. As a result, when Cl − is present in the solution, at a concentration 100mM, the Bohr effect of the two mutants carrying the β 93Cys→Ala substitution, β C93A and β (C93A+C112G), is significantly modified being practically absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between β 94Asp and Cl − in the salt link with β 146His in T-state hemoglobin. These results point to an interplay between the β His146- β Asp94 salt bridge and the Cl − in solution regulated by the Cys present at position β 93, indicating yet another role of β 93 Cys in the regulation of hemoglobin function.

Human carboxyhemoglobin at 2.2 A resolution: structure and solvent comparisons of R-state, R2-state and T-state hemoglobins.

The three-dimensional structure and associated solvent of human carboxyhemoglobin at 2.2 A resolution are compared with other R-state and T-state human hemoglobin structures. The crystal form is isomorphous with that of the 2.7 A structure of carboxyhemoglobin reported earlier [Baldwin (1980). J. Mol. Biol. 136, 103-128], whose coordinates were used as a starting model, and with the 2.2 A structure described in an earlier report [Derewenda et al. (1990). J. Mol. Biol. 211, 515-519]. During the course of the refinement, a natural mutation of the alpha-subunit, A53S, was discovered that forms a new crystal contact through a bridging water molecule. The protein structure shows a significant difference between the alpha and beta heme geometries, with Fe-C-O angles of 125 and 162 degrees, respectively. The carboxyhemoglobin is compared with other fully ligated R-state human hemoglobins [Baldwin (1980). J. Mol. Biol. 136, 103-128; Shaanan (1983). J. Mol. Biol. 195, 419-422] with the R2-state hemoglobin [Silva et al. (1992). J. Biol. Chem. 267, 17248-17256] and with T-state deoxyhemoglobin [Fronticelli et al. (1994). J. Biol. Chem. 269, 23965-23969]. The structure is similar to the earlier reported R-state structures, but there are differences in many side-chain conformations, the associated water structure and the presence and the position of a phosphate ion. The quaternary changes between the R-state carboxyhemoglobin and the R2-state and T-state structures are in general consistent with those reported in the earlier structures. The location of 238 water molecules and a phosphate ion in the carboxyhemoglobin structure allows the first comparison of the solvent structures of the R-state and T-state structures. Distinctive hydration patterns for each of the quaternary structures are observed, but a number of conserved water molecule binding sites are found that are independent of the conformational state of the protein.

Design of Recombinant Hemoglobins for Use in Transfusion Fluids

Molecular biology has been applied to the development of hemoglobin-based oxygen carrier (HBOC) proteins that can be expressed in bacteria or yeast. The transformation of the hemoglobin molecule into an HBOC requires a variety of modifications for rendering the acellular molecule of hemoglobin physiologically acceptable when transfused in circulation. Hemoglobins with different oxygen affinities can be obtained by introducing mutations at the heme pocket, the site of oxygen binding, or by introducing surface mutations that stabilize the hemoglobin molecule in the low-oxygen-affinity state. Modification of the size of the heme pocket is also used to hinder nitric oxide depletion and associated vasoconstriction. Introduction of cysteine residues on the hemoglobin surface allows formation of intermolecular bonds and formation of polymeric HBOCs. These polymers of recombinant hemoglobin have the characteristics of molecular size, molecular stability, and oxygen delivery to hypoxic tissue suitable for an HBOC.

The dimer-tetramer equilibrium of recombinant hemoglobins. Stabilization of the α1β2 interface by the mutation β(Cys112 → Gly) at the α1β1 interface

Abstract The dimer-tetramer association constants of several recombinant human hemoglobins (in the CO form) have been measured by differential gel filtration. Recombinant human hemoglobin prepared from recombinant β-chains, and mutant hemoglobins where the substitution was on the surface, β(Thr4 → Asp), in the heme pocket, β(Val67 → Thr), at the 2,3-DPG binding site, β(Val1 → Met + His2del), had a twofold smaller association with respect to natural hemoglobin. In a mutant at the α 1 β 2 1 interface, β(Cys93 → Ala), the association constant was decreased three-fold. Conversely, in a mutant at the α 1 β 1 interface, β(Cys112 → Gly), the association constant was two- and four-fold increased with respect to natural and recombinant human hemoglobin. These differences are energetically very small, consistent with the correct folding of the recombinant hemoglobins. The stabilization of the tetrameric structure by a mutation at the α 1 β 1 interface indicates that structural changes at this interface can be propagated through the protein to the α 1 β 2 interface and, thereby, exert an effect on the allosteric equilibrium.