Rate Of CO Binding Research Articles

Nature of carbon monoxide binding of iron(II)-bleomycin

The CO vibrations of the Fe(II)-CO adduct complexes of bleomycin (BLM) and its synthetic analog(PYML) have been determined at 1973 and 1980 cm −1, respectively. The model PYML-Fe(II) complex ( p 1 2 CO = 3.8 × 10 −2 torr) binds CO with much greater affinity than the native BLM-Fe(II) complex ( P 1 2 CO = 2.2 × 10 −1 torr). Of special interest is the fact that the rate of CO rebinding to the ferrous BLM complex ( k = 7.7 × 10 2 M −1↓ s −1) is clearly slower than that to the corresponding PYML complex ( k = 7.4 × 10 3 M −1 · s −1). The steric effect of the sugar portion of BLM molecule appears to have an important influence on the present low affinity of CO binding and slow rate of CO rebinding. Iron-bleomycin CO affinity Photolysis Steric effect

Flash photolytic studies of carbon monoxide binding to the ferrous chains of [Mn(II),Fe(II)] hybrid hemoglobins: kinetic mechanism for the early stages of hemoglobin ligation.

Flash photolysis is employed to investigate the kinetics of CO recombination to the ferrous chains of [Mn(II),Fe(II)] hemoglobin (Hb) hybrids. At low pH (6.6), Hb remains predominantly in the T quaternary state for the first two CO ligation steps, when binding to either the alpha chains or beta chains. At elevated pH, CO binding to the alpha chains produces a larger degree of T to R conversion than binding to the beta chains, in support of earlier equilibrium measurements. This study provides the full pH dependence of the CO binding rate constants for both alpha- and beta-Fe chains within the T state and at elevated values of pH gives the R-state rate constants for the monoliganded analogues. The data can be analyzed within the context of a two-state model for Hb cooperativity, but they give clear evidence for slow quaternary structure interconversion at the monoliganded level.

Laser photolysis study of conformational change rates for hemoglobin in viscous solutions

Rates for the R leads to T conformational change of deoxyhemoglobin formed by laser photolysis of carboxyhemoglobin were determined from CO rebinding observed in three solution systems with viscosities between 1 and 6 cP. Experiments were carried out at 20 degrees C and pH 8.3 in solutions consisting of borate buffer containing various amounts of sucrose, glycerol, or ethylene glycol. As in the case of earlier experiments in borate buffer (Sawicki and Gibson, 1976, J. Biol. Chem., 251:1533-1542), a simple two-state allosteric model which takes into account tetramer-dimer dissociation was found to give a good description of all experimental results. Using measured values for the R- and T-state CO-binding rate constants and the tetramer-dimer dissociation constant, values for the conformational change rate were determined by fitting this model to the experimental data. These rates were compared with Gavish's transient strain model (Gavish, 1978, Biophys. Struct. Mech., 4:37-52), which predicts an inverse dependence of conformational change rate on viscosity. Although fair agreement is found for hemoglobin in sucrose/borate solutions, in glycerol/borate and ethylene glycol/borate solutions, conformational change rate falls off much more rapidly with increasing viscosity than predicted by the model.

Reactivity of ferrous heme proteins at low pH.

The pH rate profile of sperm whale myoglobin (Mb) reacting with CO has been confirmed to follow the behavior previously reported (Giacometti, G.M., Traylor, T.G., Ascenzi, P., Brunori, M., and Antonini, E. (1977) J. Biol. Chem. 252, 7447-7448), and appears to be different from that obtained by others. The pH investigation has been extended to the CO-binding rates of Chironomus thummi thummi erythrocruorin and Aplysia limacina Mb, whose pH rate profile is different from that of sperm whale Mb. Besides the "base dissociation" mechanism previously invoked, the iron atom-heme plane distance is discussed as a possible determinant of the CO reactivity in these monomeric heme proteins.

An investigation of the allosteric functioning of the haemoglobin of the cane toad, Bufo marinus

1. 1. Cane toad haemoglobin shows co-operative equilibrium CO binding at pH 7.0 with n = 2.6. 2. 2. Flash photolysis studies show that at high pH the R form of the protein is stabilised whilst low pH and the presence of IHP favours the T form. 3. 3. The combination of stopped-flow and flash photolysis measurements allows estimation of the association and dissociation rates of CO binding for the two forms of the protein. 4. 4. The protein functioning can be described by a modified two state model and mathematical modelling allows the estimation of the allosteric equilibrium constant for this haemoglobin.

An investigation of the co-operative binding of carbon monoxide to the haemoglobin of the carpet shark Cephaloscyllium

1. 1. Carpet Shark haemoglobin shows co-operative equilibrium CO binding at pH 7.0 ( n = 1.8). 2. 2. Flash photolysis shows that the T form of the protein is stabilised at low pH and in the presence of Inositol Hexaphosphate (I.H.P.). 3. 3. At high pH the R form of the protein is stabilised and exhibits kinetic heterogeneity, assigned to different chain reactivities. 4. 4. Stopped flow and flash photolysis under various conditions allows estimates to be made of the association and discociation rates of CO binding for the two forms of the protein. 5. 5. The functionality of the protein, with regard to CO binding, can be described by a modified two state model.

Effects of solvent composition and viscosity on the rates of CO binding to heme proteins.

The rate of CO binding to myoglobin increases 4-fold, from 5 X 10(5) M-1 s-1 to 2 X 10(6) M-1 s-1, in going from 0 to 80% glycerol in phosphate buffer at pH 7, 20 degrees C. Under the same conditions, the rate of CO binding to protoheme decreases monotonically from about 1 X 10(8) M-1 s-1 to 2 X 10(7) M-1 s-1. The kinetic behavior of protoheme at neutral pH is that expected for a diffusion-controlled reaction. Increasing solvent viscosity causes a decrease in the observed second order rate constant. In contrast, the behavior of the myoglobin indicates quite clearly that internal, nondiffusive processes are limiting the speed of the reaction. The rate enhancement is due to an increase in the standard chemical potential of the ligand molecule as the polyalcohol concentration is increased. Both types of behavior are observed for ligand binding to protoheme in 0.1 N NaOH; first an increase and then a decrease in rate is observed as the concentration of glycerol is increased. At low glycerol concentrations, the reaction rate is limited by a first order process. At high concentrations, the rate becomes diffusion-controlled and exhibits a dependence on the reciprocal of the solvent viscosity. The data for all these conditions have been analyzed empirically in terms of a single free energy barrier and more specifically in terms of a consecutive reaction scheme.

The reaction of hemoglobin Zürich with oxygen and carbon monoxide.

The present paper reports a spectroscopic and kinetic study of the reaction with oxygen and carbon monoxide of (a) the abnormal hemoglobin Zürich (beta 63, E7 His leads to Arg), (b) its isolated abnormal chains (beta ZH), and (c) a reconstituted hybrid containing cobalt instead of iron on the normal alpha chains (Co alpha)2-(Fe beta ZH)2. The abnormal beta ZH chains, isolated from hemoglobin Zürich (HbZH) tetramer, display very peculiar spectral properties in the Soret region which were determined, for the oxy and deoxy derivatives, by kinetic difference spectra. The spectral properties of abnormal chains are maintained in the native and reconstituted hybrid tetramer. The rate constants for CO and O2 binding to isolated beta ZH chains were compared with those of the normal alpha and beta chains, It is confirmed that the CO combination rate constant to beta ZH is much higher than that of normal chains, whereas that for O2 is similar for the normal and abnormal chains. The CO binding rate constant for Fe beta ZH chains in the hybrid tetramer is the same as in the native HbZH molecule, i.e. much higher than that of normal chain. The new data are fully consistent with the sequential mechanism for CO binding previously proposed by us; on the other hand, on the basis of the spectral and kinetic results, and contrary to what has been suggested by other authors, a sequential binding of the ligand is excluded in the case of O2.

Binding of carbon monoxide to isolated hemoglobin chains.

Binding of carbon monoxide to the separated alpha and beta chains of hemoglobin, with and without bound p-mercuribenzoate, has been measured at temperatures from 5 to 340 K for times 2 mus to 1 ks using flash photolysis. All four proteins exhibit three different rebinding processes. The data are interpreted by a model in which the carbon monoxide, moving from the solvent to the binding site at the ferrous heme iron, encounters three barriers. The temperature dependences of the three processes yield activation enthalpies and entropies for the three barriers for all four proteins. Binding at temperatures below about 200 K is nonexponential, implying that the innermost barrier has a distribution of activation enthalpies. The distributions for the four proteins have been determined. At temperatures below 30 K, the CO binding rates approach finite low-temperature limits; binding thus proceeds by quantum-mechanical tunneling. Invoking a simple model, the widths of the innermost barriers are extracted from the measured tunneling rates. The experimental parameters are correlated with structural features of the hemoglobin chains and compared with previously published data on myoglobin and protoheme. A correlation is established between the height of the innermost barrier and the equilibrium CO pressure.

The kinetics of carbon monoxide binding to partially reduced methemoglobin

The pulse radiolysis technique has been used to study the kinetics of the CO binding to partially reduced methemoglobin. Experiments with horse heart metmyoglobin show that this technique gives results which are in good agreement with those obtained by other methods. The kinetics of the CO binding to partially reduced methemoglobin show two phases, whose amplitudes appear to depend on the degree of reduction in such a way that they can be attributed to methemoglobin molecules with one or two reduced heme groups. In the presence of inositol hexaphosphate the rate of CO binding to partially reduced methemoglobin decreases strongly. With inositol hexaphosphate a slight biphasic behavior is observed independent of the degree of reduction.

Kinetic studies on the five principal components of normal adult human hemoglobin

The five principal components of human hemoglobin (Ala, Alb, Alc, Ao, and A2) have been isolated by column chromatography and by preparative isoelectric focusing in gels. The isoelectric points and a number of kinetic parameters have been determined for each hemoglobin. The greatest kinetic differences are found in the binding of CO to the deoxy conformation. At pH 7, A0 and A2 are nearly identical in their overall reaction with CO, whereas the initial lag phase characteristic of crude hemolysate and A0 is greatly reduced in Ala and Alc and is essentially absent in Alb. The general effect of p-mercuribenzoate bindind on CO association is to magnify kinetic differences among the hemoglobins, diminish the initial lag phase, and increase the overall rate of CO binding. Hemoglobin Ala is anomalous in that the overall CO binding rate actually decreases after reaction with the mercurial. In terms of an Adair model with four association constants the rate constant for the binding of the first molecule of CO (1l') showed the greatest variation among the five hemoglobins, with A0 having the smallest constant, and Alb the largest. For the native hemoglobins, 1l' for Alb was more than twice that for A0; for the mercurated hemoglobins, the difference was greater than threefold. Raising the pH form 7 to 8 increases 1l' for all hemoglobins, but Ala is anomalous in having a slower overall rate for CO binding at the higher pH. At pH 9, the time course of CO binding is biphasic for all hemoglobins, with A0, the fastest, and Ala, the slowest, differing by nearly threefold in rate. The equilibrium constant for the tetramer-dimer equilibrium was determined by flash photolysis. The largest dissociation constant occurs for Ala and is 4.4 times that for A0, and 5.6 times that for Alc, the least dissociated of the hemoglobins. The overall oxygen dissociation reaction is biphasic for Ala and Alb, with the two phases differing by a factor of 5; the dissociation reactions for the other three hemoglobins appear essentially monophasic. The kinetics of dissociation of the first oxygen molecule from oxyhemoglobin are very similar for all five hemoglobins, as are the association kinetics for CN-minus and N3-minus binding to the five methemoglobins.

Horse hemoglobins containing deutero- and mesoheme; Functional and structural studies

Summary Preliminary equilibrium, kinetic, and crystallographic investigations are reported for horse hemoglobin reconstituted with deutero- and mesoheme. Both modified hemoglobins exhibit lower n values, higher oxygen affinities, and more rapid rates of CO binding than native horse hemoglobin. Initial crystallographic results indicate that these derivatives are isomorphous with native horse metHb, thus allowing difference Fourier methods to be applied to structure determination.

Nonequivalence of the α and β Chains in the Reaction of Hybrid-Heme Hemoglobin with Carbon Monoxide

Abstract The rates of binding and dissociation of CO and hybrid-heme hemoglobins, in which one chain contains protoheme and the other chain meso- or deuteroheme, were measured at each heme site independently of changes at the partner chain site. The rates were also determined on isolated chains. The binding reaction of CO to hybrid-heme hemoglobins follows an autocatalytic process characteristic of a heme-heme interaction, and the initial and final values of the apparent second order rate constants of this reaction were obtained. The rate constant of the dissociation of the CO molecule from saturated hemoglobins was also estimated by the NO replacement method. All of these rate constants were compared with those determined for isolated chains containing the corresponding heme. For isolated chains, the rate of CO binding to β chains is usually larger than that to α chains containing the same heme, and the rate of CO dissociation from β chains is slower than that from the corresponding α chains. In hybrid-heme hemoglobins, when the rates were compared with those of corresponding isolated chains, CO binding to the β chains was slowed down more than that to the α chains. Moreover, the rate of CO dissociation from β chains was increased, while that from α chains was decreased. It was suggested that a conformational change occurs upon formation of a tetramer molecule (composed of α and β chains) which changes β chains from a more accessible state to the CO molecule than α chains to a less accessible state with respect to both the binding and dissociation processes.

Conditions Restricting Allosteric Transitions in Carp Hemoglobin

Abstract Below pH 5.6, in the presence of endogenous polyphosphates, carp hemoglobin has a very low ligand affinity, and ligand binding is noncooperative, the values of n in the Hill equation being 0.75 and 1.0 for oxygen and carbon monoxide, respectively (Tan, A. L., De Young, A., and Noble, R. W. (1972) J. Biol. Chem. 247, 2493–2498). It has been postulated that under these conditions the molecule remains in the low affinity deoxy conformation even when liganded. Raising the pH above 5.6 or removing the organic phosphates converts carp hemoglobin to a molecule that exhibits the usual cooperative ligand binding. Ligand affinity and the degree of cooperativity are phosphate- and pH-dependent. Above pH 8.2 in the presence of organic phosphates and above pH 7.5 in their absence, the ligand-binding properties approach those characteristic of a molecule that again remains in a single conformation, only this time with a high ligand affinity. Whenever ligand binding was cooperative, the rate of CO recombination with partially liganded hemoglobin produced by partial flash photolysis was faster than that with fully unliganded hemoglobin produced by full flash photolysis. Under conditions when the molecule was thought to remain in one structure, the rates of CO recombination upon full and partial flash photolysis were equal. The time course of the conformation change in carp hemoglobin relative to ligand binding has been examined by measuring the rate of release of a fluorescent polyphosphate analogue during CO binding. At low pH there was a marked lag between dye release and CO binding. This lag decreased and was finally reversed with increased pH in agreement with our other results. Kinetic studies of CO combination at wave lengths near the Soret isosbestic point revealed a difference in the rate of CO binding to α and β chains of carp hemoglobin. Although our results for the most part can be explained on the basis of a simple two-state model of hemoglobin, computer fits of the time course of the CO combination reaction could not be achieved with such a simplified scheme, but rather required intra-dimer interactions.

Studies on Ligand Binding to Hemoglobins from Teleosts and Elasmobranchs

Abstract Hemoglobins from three sharks, the porbeagle (Lamna nasus), the dusky (Carcharhinus obscurus), and the mako (Isurus oxyrinchus), and four bony fishes, the big-eye tuna (Thunnus obesus), the swordfish (Xiphias gladius), the carp (Cyprius carpio), and the smallmouth bass (Micropetrus dolomieu), have been examined by a variety of techniques to determine both their molecular structure and their kinetic and equilibrium ligand-binding properties. For all species multiple hemoglobins were found in the hemolysates. These hemoglobins are tetrameric and exhibit cooperative ligand binding. The main components of the hemolysates from the oceanic species consist of either three or four electrophoretically separable globin chains. However, the hemolysates behave as if they contain only two distinct functional components which appear to be present within the same tetrameric molecule. These two types of ligand-binding sites exhibit different kinetic and spectral properties for the carbon monoxide-binding reaction and for the displacement of oxygen by carbon monoxide. At 20° the differences between the rates of oxygen dissociation from these two components are largest for the hemoglobins of the mako shark (22.7 s-1 and 1.86 s-1) and those of the swordfish (28.2 s-1 and 4.8 s-1). Similar albeit smaller differences were observed for porbeagle, dusky, big-eye tuna, and carp hemolysates. The carbon monoxide-binding reactions of these hemolysates were also separable into a fast and slow component with all species showing a rapid reaction on the short wave length side of the 425 nm isosbestic point and a slower reaction on the long wave length side. With the possible exception of big-eye tuna hemoglobin, all of the fish and shark hemoglobins appear to interact with organic phosphates with a stoichiometry of 1 mole bound per mole of hemoglobin tetramer. At neutral pH inositol hexaphosphate lowers the rate of CO binding to swordfish and bass hemoglobins by a factor of 10 to 20 but appears to affect only half of the heme sites. Although less dramatically, this organic phosphate also lowers the rate of CO binding to the shark hemoglobins. The oxygen equilibrium curves of porbeagle and big-eye tuna hemoglobins change shape with temperature, but the concentration of oxygen at 50% saturation for the former is independent of temperature and that of the latter varies only slightly in going from 35 to 5°. The basis of this unusually small temperature dependence lies in a differential temperature response of the two types of functional components which appear to be present within the same tetramer. For big-eye tuna hemoglobin, the rate of oxygen dissociation for one of the components is the same at 3° as it is at 20°, approximately 50 s-1. For porbeagle hemoglobin, the rate of ligand association to one of the components present in the deoxygenated hemolysate is 36 times smaller at 3° than at 20°. Both of these responses produce unusually low affinity species at 3°.

The reaction of inositol hexaphosphate with hemoglobin

The rate of oxygen dissociation from the fully liganded hemoglobin tetramer (Hb 4(O 2) 4) is increased by the presence of inositol hexaphosphate from approximately 50/sec to 180/sec at 22°. It follows from this observation that inositol hexaphosphate is bound by oxygen-saturated human hemoglobin. This result is in marked contrast to absence of an effect of 2,3-diphosphoglycerate or pyridoxal phosphate on the rate of oxygen dissociation from Hb 4(O 2) 4. Inositol hexaphosphate does not, however, modify the rate of CO dissociation from Hb 4(CO) 4 but it does decrease the rate of CO binding to Hb 4(CO) 3 by about seven-fold compared to the phosphate-free system.