1,721,050 research outputs found
Functional heterogeneity of the alpha and beta subunits in the association reaction between hemoglobin and carbon monoxide
No full textA technique is described for the rapid inactivation and removal of excess ferricyanide used for the non-cryogenic oxidation of the unliganded subunits of the intermediates in the association reaction between hemoglobin and carbon monoxide. Under these conditions the asymmetric oxidized intermediates, which dissociate into non-identical dimers, disproportionate into their parent tetramers and four species, Hb+, HbCO, alpha 2+ beta 2CO, alpha 2CO beta 2+, are isolated by non-cryogenic isoelectric focusing. The relative concentrations of species alpha 2CO beta 2+ and alpha 2+ beta 2CO measure the overall distribution of the ligand between the alpha and beta subunits in the association reaction. At 20 degrees C in 0.1 M KCl, pH 7, preferential CO binding to the beta subunits was observed, in agreement with observations made by the cryogenic technique for the isolation of the intermediates [M. Perrella, N. Davids and L. Rossi-Bernardi, J. Biol. Chem. 267 (1992) 8744]
Gaetano M. Perrella, C. M., I luoghi santi, Collection Monografie del Collegio Alberoni, n° 15, 1936
No full textAmann E. Gaetano M. Perrella, C. M., I luoghi santi, Collection Monografie del Collegio Alberoni, n° 15, 1936. In: Revue des Sciences Religieuses, tome 17, fascicule 4, 1937. pp. 502-504
Understanding mechanisms in a cooperative protein : the CO ligation intermediates of hemoglobin
No full textHemoglobin is a regulatory component of the oxygen transport to the tissues, and for decades has been a prototype to develop new strategies for the study of the structure/function relationships in proteins. One of the most difficult, and so far, unattained objectives of hemoglobin research has been the study of the hemoglobin molecules in a state of partial ligation with oxygen, or intermediates, as a means of testing theories of cooperativity. A cryogenic technique has been developed for the isolation, identification and quantification of the reaction intermediates of hemoglobin and CO, which in many aspects is a close approximation to the physiological ligand. The technical features that are crucial for the evaluation of the significance of the experimental data obtained using this technique and various approaches to the analysis of the data are reported. The discussion points out the importance of accessing direct information on the nature and concentrations of the intermediates in solution to clarify mechanisms of cooperativity as opposed to the less informative studies of the bulk properties of the solution
Crucial role of ligation analogues in the understanding of hemoglobin cooperativity
No full textThe functional/structural analysis of the ligation intermediates is the most powerful approach to the study of hemoglobin cooperativity. Carbon monoxide is as cooperative as O2 in the interaction with hemoglobin and the distributions of its ligation intermediates under equilibrium and dynamic conditions have been experimentally determined using cryogenic techniques. The analyses of such distributions have contributed to test theoretical models of cooperativity. However, conclusive evidence on the mechanisms of cooperativity requires the direct study of the functional properties of specific intermediates, which is not possible using O2 or CO. Ligation analogues, such as the complex of cyanide with the heme in the ferric state and the metal substituted hemoglobins, are indispensable tools for cooperativity studies provided that O2, CO and other ligation analogues share the same basic mechanisms. We have shown that this is true for CO and the cyanomet ligation analogue. This finding adds significance to our studies of the Bohr effects of the cyanomet ligation intermediates. In this paper we review our work on the CO and cyanomet intermediates. The conclusion drawn from such studies is that the basic mechanism of hemoglobin cooperativity contains elements of both the concerted and sequential models. The interaction of one ligand molecule with deoxy hemoglobin does not promote an equilibrium between T and R quaternary structures. A T→R switch seems to occur upon binding the second ligand molecule. However, a definite statement in this regard cannot be made until the problem of the thermodynamic equivalence of the diliganded intermediates is definitely clarified. Lastly, the tertiary structures of the unliganded subunits in the R and T structures are functionally different
The hemoglobin cyanomet ligation analogue and carbon monoxide induce similar allosteric mechanisms
No full textCurrent thermodynamic models of protein cooperativity predicting sigmoidal ligand equilibrium curves differ in the assumptions regarding the structural/functional properties of the intermediate ligation states. Quantitative information on the intermediates cannot be extracted from the equilibrium curves, but must be obtained from direct studies of the intermediates. Since the intermediates are intrinsically unstable species, ligation analogues with reduced mobility are indispensable tools for cooperativity studies provided that the tertiary/quaternary changes triggered by the ligation analogue are similar to those observed using the physiological ligands. We demonstrate that the valency exchange reactions occurring in mixtures of deoxy and cyanomethemoglobin yield non-random distributions of deoxy/cyanomet intermediates that resemble those observed in the equilibrium with carbon monoxide. Previous and new data using the analogue, in agreement with the studies of the CO intermediates, indicate that the mechanism of hemoglobin cooperativity is neither purely concerted nor sequential nor combinatorial, but contains some elements of each of these models
Modulation of the association reaction between hemoglobin and carbon monoxide by proton and chloride
No full textA cryogenic technique for the isolation of the ligation intermediates in the association reaction between hemoglobin and carbon monoxide at 20 degrees C [Perrella, M., Davids, N., and Rossi-Bernardi, L. (1992) J. Biol. Chem. 267, 8744-8751] was used to study the effects of proton and chloride concentrations on the rates of the stepwise reactions. The reaction rate was observed to increase continuously in the course of the ligation process, yet the acceleration of the reaction after the binding of two ligand molecules, observed previously in 100 mM KCl, pH 7, was not observed at other pH values. At pH 6.3, such an acceleration occurred after the binding of three ligands, and at pH 8.5, a large acceleration was observed after the binding of the first ligand molecule. Greater CO binding to the beta chains was observed under all conditions, as in the previous study. The functional heterogeneity of the chains in the first ligation step increased with pH. The chloride concentration did not influence the distribution of the ligand between the alpha and beta chains at pH 6.3 and 8.5. At pH 7, less binding to the alpha chains was observed at 7 mM chloride with respect to 100 mM. The nature of the biliganded component isolated at pH 7 in 100 mM KCl and unresolved by the cryogenic technique was studied using a combination of cryogenic and noncryogenic isoelectric focusing. This component was a mixture of intermediates (alpha beta) (alpha CO beta CO), about 65%, and (alpha beta CO) (alpha CO beta), about 35%. The experimental data were compared with the distributions of intermediates calculated according to the Monod kinetic model assuming rapid and concerted transitions between two quaternary structures at each ligation step. The model provided a qualitative fit of the observed distributions of intermediates at acidic and neutral pH. A large discrepancy between the experimental observations and the predictions of the model was found at alkaline pH. The mechanism of the association reaction is discussed in the light of the available information on the tertiary/quaternary structures of the intermediates, as obtained from the studies of the deoxy/cyanomet model of ligation
The association reaction between hemoglobin and carbon monoxide as studied by the isolation of the intermediates : implications on the mechanism of cooperativity
No full textThe concentrations of the intermediates in the association reaction between human hemoglobin and CO at 20 degrees C, pH 7, under conditions of negligible dissociation of the ligand, were measured by cryogenic techniques. The monoligated species were predominant at all values of overall ligand bound studied. The analysis of the experimental data assuming a scheme of four consecutive reactions indicated that the binding rates increased in a continuous fashion. A significant acceleration after the binding of the second molecule of ligand occurred in the presence of 0.1 M KCl, but not with the addition of an excess of inositol hexaphosphate, indicating that major functional, and possibly structural, transitions occur at the diligated state. Differences in the concentrations of the intermediates in the same state of ligation were observed under all conditions. The analyses of the data on the basis of schemes of multiple pathways of reaction indicated that the beta subunits reacted about 1.5 times faster than the alpha subunits in the first ligation reaction. After the addition of inositol hexaphosphate, the alpha subunits reacted about 1.5 times faster than the beta subunits in the first ligation step, but the overall rate of the first CO binding step was unchanged
Hybrid formation for liganded hemoglobins A and C at subzero temperatures
Full text linkThe kinetics of formation of the asymmetric carbon-monoxyhemoglobin hybrid (αβ)(A)(αβ)(C) from the parent molecules α2β2(A) and α2β2(C) have been studied by electrophoresis at subzero temperatures (down to -40°C) using as supporting media gels of acrylamide/methylacrylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid formation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH>10, t=-40°C, the hybrid between α2β2(A) and α2β2(C) is stable for several hours. A rapid thermal quenching of a mixture of α2β2(A) and α2β2(C) prevented hybrid formation during the time required to separate the 2 molecules
The kinetics of the reaction between NO and O2 as studied by a novel approach
No full textThe kinetics of the reaction between NO and O2 was determined by measuring the time course of the decrease in the concentration of NO with a quench-flow technique. NO and O2 were mixed rapidly and reacted for periods of time varying from 10 to 50 s. A second rapid mixing with a solution containing an excess of deoxyhemoglobin and sodium hydrosulfite trapped free NO as nitrosylhemoglobin and reduced O2. The spectrum of the mixture of deoxy- and nitrosylhemoglobin was recorded within 30 s from the second mixing, before any appreciable dissociation of NO from the protein, by means of a flow-cell mounted on-line with the quench-flow apparatus. The amount of NO not consumed in the auto-oxidation reaction was calculated from the proportion of nitrosylhemoglobin in the mixture. As NO and O2 bind deoxyhemoglobin at comparable rates and NO is oxidized to nitrate by oxyhemoglobin, the ratio of hemoglobin/(NO + O2) had to be optimized to avoid the interference of this oxidation reaction. The kinetics was first and second order with respect to O2 and NO, respectively and third order overall with a rate constant k = 4 x kaq = 4 x 2.23 (+/- 0.26) x 10(6) M-2 s-1 at 20 degrees C, invariant in the pH range 7-9, in agreement with published values obtained by different methodologies
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