LXV.—The reduction of triphenylhalogenomethanes

112

The use of subunit dissociation as a means of probing intersubunit contact energy changes which accompany cooperative ligand binding has been studied for the case of human hemoglobin. An analysis is presented delineating the information that can be obtained from the linkage relationships between ligand binding and subunit dissociation of hemoglobin tetramers into dimers. The analysis defines (a) the variation of the saturation function, Y., with total protein concentration, (b) the variation of the subunit dissociation constant zK2 with ligand concentration (X) and (c) the correlations between changes in dimer-dimer contact energy and the sequential ligand binding steps. Sensitivity of the linkage functions has been explored by numerical simulation. It is shown that subunit dissociation may appreciably affect oxygenation curves under usual conditions of measurement and that relying solely on either 2K2 or Y may lead to incorrect pictures of the energetics, whereas the combination defines the system much more exactly.The correlation of energy changes and quaternary structure changes with the sequence of oxygen binding steps is a central problem in hemoglobin research. The major transformations in quaternary structure upon oxygenation are known to occur via changes in the intersubunit contact region separating (a1131) and (a2132) dimer pairs (1, 2). It is here that stabilizing interactions, present in the "constrained" deoxy quaternary structure, are abolished upon oxygenation. A determination of the amount of energy expended within this contact region at each stage of oxygenation is of crucial importance to understanding the mechanism of cooperativity. There is now considerable evidence that the dissociation of tetramer at neutral pH occurs via cleavage along the same contact region to form (a#) dimer pairs (3)(4)(5)(6)(7)(8)

mentioning

confidence: 99%

The Linkage Between Oxygenation and Subunit Dissociation in Human Hemoglobin

Ackers¹,

Halvorson²

1974

112

“…Based on this observation, the effectiveness and the rapidity of stopping the kinetic process were tested by examining the extent of incorporation of *C into C2R2 within 5, 10, 15, and 20 sec after the addition of concentrated buffer to give a final concentration of 50 mM Tris-HCl containing 2 mM 2-mercaptoethanol at pH 7.5. Regardless of the extent of the disproportionation process, i.e., different periods of time at the low ionic strength, more than 80% of the radioactivity was always found as *C. Moreover, no decrease in radioactivity as *C was observed in the interval between 5 and 20 sec. Analysis of the gels for the total amount of C2R2, C2R3, and C along with those from a control experiment without *C showed that the distribution of products was not affected by the addition of *C after the ionic strength was increased.…”

mentioning

confidence: 93%

“…Of these, hemoglobin has been studied most extensively. The dissociation constants for the tetramer-dimer equilibria have been evaluated for hemoglobin in both the oxy and deoxy states (4)(5)(6)(7)(8)(9), and the changes in the intersubunit contact energies have been related to the functional properties of the protein (10,11). In this paper we present an approach aimed at providing comparable information for the regulatory enzyme, aspartate transcarbamoylase (ATCase; carbamoyl- (12).…”

mentioning

confidence: 99%

Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamoylase

Subramani

Bothwell

Gibbons

et al. 1977

The cooperativity and feedback inhibition exhibited by the It is generally recognized that the homotropic and heterotropic effects exhibited by allosteric enzymes are attributable to ligand-promoted conformational changes whereby the proteins are converted from a constrained or low-affinity state to a relaxed form having a higher affinity for substrates (1, 2). Since these conformational changes are mediated by alterations in the subunit interactions, it is important to determine the strengths of the intersubunit bonding domains and their perturbation by ligands (3). Despite the evident need for such data, quantitative estimates of ligand effects are available for only a few proteins. Of these, hemoglobin has been studied most extensively. The dissociation constants for the tetramer-dimer equilibria have been evaluated for hemoglobin in both the oxy and deoxy states (4-9), and the changes in the intersubunit contact energies have been related to the functional properties of the protein (10, 11). In this paper we present an approach aimed at providing comparable information for the regulatory enzyme, aspartate transcarbamoylase (ATCase; carbamoyl- (20).Since the dissociation of ATCase into free subunits could not be measured directly, we initiated studies of subunit exchange as an alternative, sensitive approach for estimating the strengths of the intersubunit bonding domains. Very little exchange of either C or R subunits occurred even in the presence of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which promotes the conformational transition of ATCase to the relaxed state (20,21). Studies were conducted, therefore, on the effect of this active-site ligand on the bonding domains in ATCase-like molecules lacking one R subunit (22)(23)(24)(25). This R-deficient species, designated C2R2, which exhibits both cooperativity and inhibition by CTP (22,23), is an intermediate in both the in vitro assembly of ATCase from subunits and the dissociation of the enzyme (24,26

“…[Dissociation of deoxy-Hb can be neglected (34).] Protein concentrations were low, typically [h] = 11 ,uM.…”

Section: Methodsmentioning

confidence: 99%

Redox equilibria of liganded forms of methemoglobin.

Bull¹,

Hoffman²

1975

We have examined the redox equilibria of azidomethemoglobin (low-spin) and fluoromethemoglobin (high-spin). We have derived a modified Hill equation which includes the tetramer-dimer equilibrium of the oxidized form, and also generalized the two-state model to incorporate ligand binding to the ferriheme. The pH dependence of the redox Hill's constant for fluoromethemoglobin is the same as that for methemoglobin, demonstrating that this dependence and the marked cooperativity achieved (n = 2.2)are not coupled to changes of the ferriheme spin state. The redox Hill's constant for azidomethemoglobin, however, is as large as the oxygenation Hill's constant (n -2.7) and is also roughly pH independent.A chief source of the ongoing interest in ligand binding to hemoglobin (Hb) rests in the allosteric properties which result from its tetrameric nature (1, 2). In constructing a detailed scheme for the ligation process of Hb, the question can be formulated as to the existence and nature of an "allosteric trigger" (3-7), the mechanism by which the ligation (or oxidation) of five-coordinate, ferroheme induces conformational changes within the protein.The Hb tetramer can be regarded as having essentially two quaternary structures, T (unliganded) and R (liganded) (8-11). The most widely held mechanism for controlling the conformational equilibrium between R and T has been proposed by Perutz (3-7). Following suggestions of Williams (12) and Hoard (13,14), it includes a proposed coupling between protein conformation and the spin-state of iron: the five-coordinate high-spin (hs) Fe(II) of an unliganded heme appears to lie about 0.7 A out of the mean porphyrin plane, and is considered to be held in a stressed state by tension exerted through the protein. Upon oxygenation, the Fe atom becomes low-spin (ls) and appears to move into the porphyrin plane. The resultant movement then couples to the protein through motion of the proximal histidine.The cooperativity of the redox reaction varies markedly with pH and with the presence of organic phosphates (15)(16)(17), in contrast to the virtually constant cooperativity of ligation (1, 2). Methemoglobin (met-Hb), however, also exhibits a tertiary structure variability which is spin-state related (3-7): the high-spin Fe(III) in aquomet-Hb is slightly outof-plane, and thus in an intermediate structure, while the Fe(III) in hydroxymet-Hb is substantially low-spin and should more nearly lie in the porphyrin plane. It has been suggested the pH dependence of the redox cooperativity has two causes, the pH dependence of the met-Hb spin state and of salt bridges between the subunits (8-7, 17).Although a spin-state coupling contribution is quite appealing in its directness and simplicity, our results for oxygenation of coboglobin (cobalt-substituted Hb) and the redox equilibrium of manganese-substituted hemoglobin nevertheless suggested that Hb cooperativity is thot coupled to the spin-state of either unliganded (18-22) or oxidized (23, 24) forms. Evidence against stress in CoHb has also be...