Subunit hybridization studies of partially ligated cyanomethemoglobins using a cryogenic method

Perrella, Michele; Benazzi, Louise; Shea, Madeline A.; Ackers, Gary K.

doi:10.1016/0301-4622(90)80064-e

Cited by 58 publications

(57 citation statements)

References 12 publications

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“…3 indicate that a high proportion of hybrid was attained after 2-3 h, although not yet at equilibrium. In contrast, the rate of hybridization between HbS and CNHbA 0 , yielding intermediate 21 is slower (17), and valency exchange controls under these conditions indicated a high proportion of exchange, in agreement with the measurements of Shibayama et al (7). Therefore we could not confirm the data previously published on the BE of species 21 (5).…”

Section: Methodscontrasting

confidence: 62%

The Bohr Effect of Hemoglobin Intermediates and the Role of Salt Bridges in the Tertiary/Quaternary Transitions

Russo

Benazzi

Perrella

2001

Journal of Biological Chemistry

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These observations suggest that ligation of a subunit brings about a tertiary structure change of hemoglobin in the T quaternary structure, which breaks some salt bridges, releases hydrogen ions, and is signaled across the dimer interface in such a way that ligation of a second subunit in the adjacent dimer promotes the switch from the T to the R quaternary structure. The rupture of the salt bridges per se does not drive the transition.A vast amount of data on the structure/function of human hemoglobin in solution apparently supports the mechanism of a concerted transition between two quaternary structural states in the course of ligand binding, in agreement with the Monod-Wyman-Changeaux model (1). Due to cooperativity, the end states largely prevail on species in a partial state of ligation under equilibrium conditions, masking the functional properties of the intermediate species. This is demonstrated by the close agreement between the isotherms of CO binding calculated from the experimental distributions of the CO ligation intermediates according to the Monod-Wyman-Changeaux model and the alternative Koshland-Nemethy-Filmer model, which assumes transitions through intermediate structural/ functional states (2), as shown by Perrella and Di Cera (3). The functional/structural studies of the intermediates still provide the most critical test for any model of cooperativity. Such studies are difficult because of the high rates of the dissociation and association reactions of the physiological ligand, the complexity of the intermediate ligation states Fig. 1, and the instability of tetrameric hemoglobin. The partially liganded hemoglobin tetramers reversibly dissociate into dimers faster than the rate of resolution of the separation techniques (4), and dimer rearrangement reactions occur under nonequilibrium conditions, as depicted in Fig. 2. In a previous study of the Bohr effect of the intermediate ligation states (5), the problem of the ligand mobility was circumvented by using cyanide bound to the ferric subunits to mimic ligation and a cryogenic technique to determine the proportion of any asymmetrical hybrid species in equilibrium with the respective symmetrical parental species (6). This information was needed to calculate the contribution of each species from the total Bohr effect of a mixture of hybrid and parental species. The study of the pH dependence of the Bohr effects of the mono-and diliganded intermediates revealed the absence of additivity of the effects, an important clue to the mechanism of tertiary/quaternary transitions in ligand binding to hemoglobin. However, the discovery by Shibayama et al. (7) that the cyanomet intermediates undergo valency exchange has made such studies questionable. We have now repeated the measurement of the Bohr effect of the mono-and some diliganded species under conditions of slight or negligible valency exchange, confirming the results of the previous study.Using the same technical approach we have measured the decrease in Bohr hydrogen ions in hemoglobin derivati...

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Section: Methodscontrasting

confidence: 62%

The Bohr Effect of Hemoglobin Intermediates and the Role of Salt Bridges in the Tertiary/Quaternary Transitions

Russo

Benazzi

Perrella

2001

Journal of Biological Chemistry

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“…Analysis of this distribution showed that the hemoglobin tetramer occupies a third allosteric state in addition to those of the unlgated (1) ates plus the 2 end-state species) are shown in Fig. 1 for conditions of pH 7.4 and 21.50C (13,15). The initial determination ofthis distribution (13) revealed several striking features.…”

Section: Introductionmentioning

confidence: 99%

“…[8][9][10][11][12], further progress has awaited experimental resolution of the actual energetics of cooperativity for the 10 microstates of ligation. The recently resolved distributions (13)(14)(15) and related findings (16,17) provide a necessary information base for deducing the rules that connect tertiary and quaternary switching with hemoglobin cooperativity. A remaining crucial step is to determine the molecular nature of the intermediate allosteric species revealed by the microstate distributions.…”

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Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

Daugherty

Shea

Johnson

et al. 1991

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

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The 10 ligation species of human cyanomethemoglobin were previously found to distribute into three discrete cooperative free energy levels according to a combinatorial code (i.e., dependent on both the number and configuration of ligated subunits). Analysis of this distribution showed that the hemoglobin tetramer occupies a third allosteric state in addition to those of the unlgated (1) ates plus the 2 end-state species) are shown in Fig. 1 for conditions of pH 7.4 and 21.50C (13,15). The initial determination ofthis distribution (13) revealed several striking features.(i) The 10 species, each with a structurally unique combination of ligated and unligated subunits, distribute into three discrete cooperative free energy levels (i.e., the free energy relative to species 01 after subtraction of intrinsic binding free energies). Procedurally, the cooperative free energies are calculated from experimentally determined free energies of dimer-tetramer assembly, as illustrated by Fig. 2. (ii) The doubly ligated tetramers distribute into two separate levels, which rules out the 2-state Monod-WymanChangeux mechanism (see ref.16 for a rigorous analysis).This feature precludes all purely concerted n-state mechanisms since they require that cooperative free energies depend solely on the number of ligands bound and not on the specific configuration of ligated subunits. By contrast, the actual distribution (Fig. 1) indicates a dramatic combinatorial aspect to the apparent switching code-i.e., dependence on both the number and configuration of ligated sites (see ref. 17 for detailed discussion).tTo whom reprint requests should be addressed. 1110The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…The solution oxygen binding curves contain relatively little information, and numerous models have been proposed over the years that are consistent with such data (Antonini & Bmnori, 1971;Imai, 1982;Wyman & Gill, 1990). The investigation of tetramerdimer dissociation equilibria has focused on artificial intermedi-ates, particularly those containing the cyanide complex of oxidized hemes (Smith & Ackers, 1985;Perrella et al, 1990a;. This work has led to the conclusion that the twostate MWC model is incompatible with the experimental results (Ackers & Smith, 1987;Ackers, 1990;Ackers et al, 1992).…”

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confidence: 99%

“…Several such experiments have been conducted over the past several years, including determination of the equilibrium ligation state distribution by low-temperature electrophoresis (Perrella et al, 1990a;Perrella & Denisov, 1995), oxygen binding curves of hemoglobin encapsulated in silica gels (Shibayama & Saigo, 1995), and nanosecond-millisecond ligand binding and conformational kinetics . Perhaps the most incisive "new" experiment is the measurement of oxygen binding to the T quaternary structure in single crystals of hemoglobin (Mozzarelli et al, 1991;Rivetti et al, 1993aRivetti et al, , 1993bKavanaugh et al, 1995;Bettati et al, 1996).…”

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Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals

et al. 1997

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Abstract:In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a. Biochemistry 32:2888-2906 suggested that there are high-and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (

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Subunit hybridization studies of partially ligated cyanomethemoglobins using a cryogenic method

Cited by 58 publications

References 12 publications

The Bohr Effect of Hemoglobin Intermediates and the Role of Salt Bridges in the Tertiary/Quaternary Transitions

The Bohr Effect of Hemoglobin Intermediates and the Role of Salt Bridges in the Tertiary/Quaternary Transitions

Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals

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