Thermodynamic aspects of the linkage between binding of chloride and oxygen to human hemoglobin

Haire, Robert N.; Hedlund, Bo E.

doi:10.1073/pnas.74.10.4135

Cited by 27 publications

(15 citation statements)

References 20 publications

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“…This shifts the R/T equilibrium in favor of the T-state and consequently lowers the overall affinity to oxygen (8,13). The well known Bohr effect and results reported for Cl Ϫ , also influencing the oxygen affinity of the T-state (14,15), show that, in a broader sense, H ϩ and Cl Ϫ can also be considered as being members of the family of allosteric effectors.…”

mentioning

confidence: 92%

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Schay

Smeller

Tsuneshige

et al. 2006

Journal of Biological Chemistry

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The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R-and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl ؊ , 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (␣FeO 2 ) 2 -(␤Zn) 2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70 -200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl ؊ . We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.Hemoglobin (1) is a tetrameric protein, which plays a vital role in the transport of oxygen. It consists of two dimers of ␣ and ␤ subunits that reversibly bind and release oxygen (1). The description of this cooperative phenomenon has been most frequently derived from the Monod-Wyman-Changeux (MWC) 2 two-state allosteric model (2) that attributes cooperativity to a rapid equilibrium between two conformations of distinct oxygen affinity of the whole tetramer. These distinct states are the fully unliganded T-state and the fully ligated R-state. Szabo and Karplus (3) modified the two-state model incorporating the stereochemical mechanism suggested by Perutz (4) for the T to R switch, and introduced ligation-induced tertiary changes within the T-state. In this extended model (MWC-SK), it was proposed that cooperativity still works through a ligation-induced shift in the equilibrium of states T and R, but the model attributed importance in the conformational switch to certain changes at the inter-and intrasubunit interfaces.

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mentioning

confidence: 92%

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Schay

Smeller

Tsuneshige

et al. 2006

Journal of Biological Chemistry

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“…The concentration of chloride ion was adjusted with NaCl to give a final concentration of 0.1 M. While there were differences in the amount of sodium in solution the amount of chloride, the stronger effector, remained constant under all conditions (26,27). Hb was deoxygenated and dithionite was added anaerobically to a final concentration of 2 mM to ensure complete anaerobicity.…”

Section: Methodsmentioning

confidence: 99%

“…These modifications to the N terminus should have minimal effect on a completely recombinant Hb since the allosteric role of these residues is primarily associated with the amino group rather than the side chain (25,26). We have purified recombinant human Hb coexpressed in E. coli to homogeneity and examined the physical properties, using optical spectroscopy and EPR, and compared them to native Hb.…”

Section: ␤1mentioning

confidence: 99%

Tetrameric Hemoglobin Expressed in Escherichia coli

Hernan

Sligar

1995

Journal of Biological Chemistry

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Recombinant ␣ 2 ␤ 2 tetrameric Hb expressed and assembled in Escherichia coli has been characterized extensively. Electrospray mass spectrometry and optical and electron paramagnetic resonance spectroscopy suggest that the overexpressed protein is identical to native human Hb. Although the functional properties of this recombinant Hb are nearly identical to native Hb, crucial differences exist between the two molecules. The recombinant Hb expressed in E. coli has a lower Hill coefficient even though oxygen equilibrium binding studies indicate cooperative binding. The most significant difference observed between the recombinant and native Hb is the loss of oxygen affinity regulation by 2,3-diphosphoglyerate and protons. CO binding to the deoxy tetramer was found to be biphasic with both phases sensitive to the presence of allosteric effectors. The recombinant chains were isolated, and the ligand binding properties demonstrated that the recombinant chains behave in a similar fashion to native ␣ ؊sh and ␤ ؊sh . To investigate whether the chains were capable of forming a well behaved tetramer, the isolated chains were reassembled into a tetramer and purified to homogeneity. Oxygen binding properties of the reassembled recombinant Hb now show an increased Hill coefficient of 2.5, close to, but still slightly lower than, that observed for native Hb. Additionally, reassembly of recombinant Hb produces a protein that is subject to regulation by allosteric effectors. Furthermore, CO binding to the reassembled recombinant deoxy tetramer was found to be monophasic under all conditions. Hb has long been studied as a model compound for many biochemical phenomena and continues to be the object of intense work to elucidate the molecular details of protein-protein recognition, allosteric regulation, ligand binding and dynamics, spectroscopy, energetics of cooperativity, and structure-function relationships (1-3). X-ray structures of liganded (R), unliganded (T) states, and with several intermediate species have been solved to high resolution and have provided much information on the detailed characterization of the mechanism of action by Hb (4 -7). In addition to mechanistic and structurefunction studies, Hb has grown in popularity for use in clinical applications as a potential blood substitute (8). These efforts, along with the possibility of engineering specific and novel properties into the molecule, have prompted the development of many recombinant Hb expression systems. We have investigated a purified recombinant human Hb using a coexpression system in Escherichia coli (9) in order to determine whether a completely assembled recombinant tetrameric Hb constitutively expressed in E. coli is fully functional.The first expression of individual human Hb chains in E. coli was reported by Nagai and Thøgerson (10). Their expression system involved producing ␤-globin as an insoluble fusion protein, which was solubilized, purified, and cleaved with factor X a to produce the correct N terminus. The ␤-globin was then reconstituted an...

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“…From in vitro studies of the relation between 02 binding and Cl-activity, it has been thought that some 1.6 chlorides bind to stabilize the deoxy or T form (1)(2)(3)(4), although this stoichiometry is puzzling when one attempts to locate it on the x-ray structure (4,5). We recently reported (6) that the oxygen affinity of hemoglobin correlates with water activity in the presence of several neutral solutes.…”

mentioning

confidence: 99%

Reevaluation of chloride's regulation of hemoglobin oxygen uptake: the neglected contribution of protein hydration in allosterism.

Colombo

Rau²,

Parsegian³

1994

Proc. Natl. Acad. Sci. U.S.A.

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We have measured hemoglobin oxygen uptake vs. the partial pressure of oxygen, with independently controlled activities of chloride and water. This control is effected by combining different concentrations of NaCl and sucrose in the bathing solution to achieve: (i) water activities were varied and Cl-activity was flxed, (ii) both water and Clactivities were varied with a traditional NaCl titration, or (us) Cl-activities were varied and water activity was fixed by adding compensating sucrose. Within this analysis, the C1-regulated loading of four oxygens can be described by the reaction HbCl-+ 4 2 + 65 H20 Hb40265H20 + Cl-.The dissociation of a neatly integral chloride, rather than the nonintegral 1.6 chlorides inferred earlier from simple salt titration, demonstrates the need to recognize the potentially large contribution from chanes in water activity when titrating weakly binding solutes. The single-chloride result might simplify structural considerations of the action of Cl-in hemoglobin regulation.Among the "allosteric effectors" regulating hemoglobin oxygen affinity, C1-has long been known to have a significant physiological role. From in vitro studies of the relation between 02 binding and Cl-activity, it has been thought that some 1.6 chlorides bind to stabilize the deoxy or T form (1-4), although this stoichiometry is puzzling when one attempts to locate it on the x-ray structure (4, 5). We recently reported (6) that the oxygen affinity of hemoglobin correlates with water activity in the presence of several neutral solutes. We inferred a change of some 60-65 waters in protein hydration when hemoglobin goes from the deoxy T state to the fully oxygenated R state. The water activity in physiological salt solutions is comparable to the water activities used with the neutral solutes that showed the contribution of protein hydration. Therefore, we now have reexamined the regulatory action of chloride, explicitly including changes in protein hydration and the dependence ofwater activity on added salt. We now find that an alternate description of the data has only a single allosteric Cl-(Ancl-= -1.02 ± 0.02) directly linked with oxygenation during the deoxy-to-oxy transition, while some 65.2 ± 2.4 additional water molecules bind to the protein.Most, perhaps all, weakly binding effectors such as chloride necessarily work at high enough concentrations that their presence also significantly changes the activity of water. Ligand regulation ofhemoglobin and other allosteric proteins should be reevaluated with regard for the activity of water and molecular hydration, as first anticipated by Tanford (7). MATERIALS AND METHODSFreshly drawn human blood was washed three times in isotonic salt solutions and the erythrocytes were then lysed with distilled water. Debris was removed by centrifugation (Sorvall RC-5B, 27,000 X g for 1 h at 4QC). The resulting hemoglobin solution supernatants were then eluted over a PD 10 Sephadex G-25M (Pharmacia) column equilibrated with 0.1 M NaCl/0.05 M Tris-HCl, pH 7.5. The protein s...

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Thermodynamic aspects of the linkage between binding of chloride and oxygen to human hemoglobin

Cited by 27 publications

References 20 publications

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Tetrameric Hemoglobin Expressed in Escherichia coli

Reevaluation of chloride's regulation of hemoglobin oxygen uptake: the neglected contribution of protein hydration in allosterism.

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