Low-affinity Ligands Research Articles

Functional consequences of ligand-linked dissociation in hemoglobin from the sea cucumber molpadia arenicola

It has been established that Molpadia hemoglobin tends to dissociate into subunits as oxygen is bound. The kinetics and equilibria of carbon monoxide and ehtylisocyanide binding reported here show a dependence on protein concentration that supports the conclusions that the aggregated hemoglobin has a lower ligand affinity than the dissociated subunits. This is true for the isolated D-chain as well as for the unfractionated hemolysate that contains four distinct polypeptide chains (A-D). This indicates that even homopolymers of Molpadia hemoglobin have lower ligand affinity than the dissociated subunits. At high protein concentration hemolysates of Molpadia hemoglobin show slight cooperativity. The time course of ligand binding to the deoxy D-chain also suggests cooperative interactions, The low affinity of the aggregated state may have a different molecular explanation than in human hemoglobin were tetramers of identical subunits (as in Hb H) show high oxygen affinity. The absence of tyrosine and histidine at the C-tremini of the Molpadia D-chains also suggests a different stabilization of the low affinity deoxy state. An additional functional difference between Molpadia hemoglobin and human hemoglobin is that organic phosphate do not alter the ligand affinity of the sea cucumber hemoglobin.

Structural states and transitions of carp hemoglobin.

The wide ligand affinity range previously observed for carp hemoglobin is bounded at both extremes by regions of constant affinity. Within these regions, pH, organic phosphates, and the extent of ligand binding have no effect on the measured affinity and the cooperativity of ligand binding is greatly reduced or absent. The rates of CO recombination to fully and partially unliganded carp hemoglobin, under various organic phosphate and pH conditions, are shown to reflect this behavior. Constant kinetic rates are seen to directly correspond to the regions of constant affinity. Therefore, these are taken to be single protein conformations, one of high and one of low ligand affinity. In the simplest view, these conformations represent the R and T states of a two-state model, and most of the properties of carp hemoglobin are explained quite well within this framework. Increases in either hydrogen or phosphate ion concentrations favor the stabilization of the low affinity structure of even fully liganded carp hemoglobin. We have studied the structural transition from high to low affinity by monitoring the absorption spectra of carp hemoglobins at constant pH as a function of organic phosphate concentration. We find that different spectra are induced in both carp methemoglobin and cyanomethemoglobin by inositol hexaphosphate addition. Furthermore, the dependence of the magnitude of the spectral changes on pH and organic phosphate concentration is the close agreement with that predicted from studies of the ligand binding properties of the molecule.

Gelation of sickle hemoglobin: III. Nitrosyl hemoglobin

Sickle cell nitrosyl hemoglobin was examined for gelation by an ultracentrifugal method previously described (Briehl & Ewert, 1973) and by birefringence. In the presence of inositol hexaphosphate gelation which exhibited the endothermic temperature dependence seen in gels of deoxyhemoglobin S was observed by both techniques. In the absence of inositol hexaphosphate no gelation was observed, nor did nitrosyl hemoglobin A exhibit gelation. On the assumption that gelation is dependent on the deoxy or T (low ligand affinity) as opposed to the oxy or R (high ligand affinity) quaternary structure this supports the conclusion that nitrosyl hemoglobin S in inositol hexaphosphate assumes the T structure, in contrast to the other liganded ferrohemoglobin derivatives oxy and carbon monoxide hemoglobin. Assuming further that the quaternary structures and isomerizations are the same in hemoglobins A and S it can also be concluded that nitrosyl hemoglobin A in inositol hexaphosphate assumes the T state. Since no gelation was seen in stripped nitrosyl hemoglobin S, inositol hexaphosphate serves to effect an R to T switch in this derivative. Thus R-T isomerization in nitrosyl hemoglobin occurs without change in ligand binding at the sixth position of the heme group confirming the conclusion of Salhany (1974) and Salhany et al. (1974). Lowering of the pH toward 6 favors gelation of NO hemoglobin S as it does of deoxy and aquomethemoglobin S (Briehl & Ewert, 1973,1974), consistent with a favoring of the T structure due to strengthening of the interchain salt bridges and the binding of inositol hexaphosphate and/or changes in site-to-site interactions on which gelation depends.

The effects of inositol hexaphosphate on the allosteric properties of two beta-99-substituted abnormal hemoglobins, hemoglobin Yakima and hemoglobin Kempsey.

Hemoglobins (Hb) Yakima and Kempsey were purified from patients' blood with diethylaminoethyl cellulose column chromatography. The oxygen equilibrium curves of the two hemoglobins and the effects of organic phosphates on the function were investigated. In 0.1 M phosphate buffer, Hill's constants n for Hb Yakima and Hb Kempsey were 1.0 to 1.1 at the pH range for 6.5 to 8.0 and the oxygen affinities of both the mutant hemoglobins were about 15 to 20 times that of Hb A at pH 7.0. The Bohr effect was normal in Hb Yakima and one-fourth normal in Hb Kempsey. In the presence of inositol hexaphosphate, the oxygen affinities to Hb Yakima and Hb Kempsey were greatly decreased, and an interesting result revealed that these hemoglobins showed clear cooperativity in oxygen binding. Hill's constant n in the presence of inositol hexaphosphate was 1.9 for Hb Kempsey and 2.3 for Hb Yakima at pH 7.0. The cooperativities of these mutant hemoglobins were pH-dependent, and Hb Kempsey showed high cooperativity at low pH (n equal 2.1 at pH 6.6) and low cooperativity at high pH (n equal 1.0 at pH 8.0). Hb Yakima showed similar pH dependence in cooperativity. In the presence of inositol hexaphosphate, Hb A showed a pH-dependent cooperativity different from those of Hb Yakima and Hb Kempsey, namely, Hill's n was the highest in alkaline pH (n equal 3.0 at pH 8.0) and decreased at lower pH (n equal 1.5 at pH 6.5). 2,3Diphosphoglycerate bound with the deoxygenated Hb Yakima and Hb Kempsey, however, had no effect on the oxygen binding of these abnormal hemoglobin. The pH-dependent cooperativity of alpha1beta2 contact anomalous hemoglobin and normal hemoglobin was explained by the shifts in the equilibrium between the high and low ligand affinity forms.

Spectral-kinetic heterogeneity in reactions of nitrosyl hemoglobin.

When NO replaces CO in hemoglobin A in the presence of inositol hexaphosphate, the time course is heterogeneous in contrast to stripped hemoglobin A, where it is homogeneous. If nitrosyl hemoglobin is mixed with inositol hexaphosphate in the stopped-flow apparatus, an extra spectral change is observed which is the cause of the spectral-kinetic heterogeneity in the CO replacement reaction. At wavelengths isosbestic for this extra spectral change, the time courses show an accelerating rate of CO dissociation. On the other hand, the same reaction for NES-des-Arg hemoglobin (hemoglobin reacted with N-ethylmaleimide and carboxypeptidase B) in the presence of inositol hexaphosphate is homogeneous and slow, and shows isosbesty. High-resolution nuclear magnetic resonance spectra indicate that adult nitrosyl hemoglobin in the presence of inositol hexaphosphate is in the low ligand affinity state, thus offering a structural basis for the acceleration observed in the rate of CO dissociation. Hemoglobin Kansas, in the presence of inositol hexaphosphate, which starts and finishes the reaction in the low affinity state, shows a rate of CO dissociation about nine times faster than stripped hemoglobin A. We conclude from these results that (i) the CO to NO replacement reaction can include a functionally important change in the overall conformation of fully liganded hemoglobin, depending on solution conditions and protein type; (ii) the extra spectral change observed for nitrosyl hemoglobin is not the functionally dominating conformational change, but is a secondary effect within the low ligand affinity protein structure; and (iii) the functional properties of heme ligands are largely controlled by two conformational states of the protein, as seen by nuclear magnetic resonance spectroscopy.

Morphologic changes associated with combined BCNU and radiation therapy in glioblastoma multiforme

The guinea pig tracheal contractile serotonergic receptor shows pharmacological similarity to both 5-hydroxytryptamine2 (5-HT2) and 5-hydroxytryptamine1C (5-HT1C) receptors. The present in vitro study utilizes 5-methyltryptamine (5-MT), a high-affinity 5-HT1C receptor ligand, and dipropyl-5-carboxamidotryptamine (DP-5-CT), a low-affinity 5-HT1C receptor ligand, as tools to probe the role of 5-HT1C vs. 5-HT2 receptors in tracheal contractility. Both tryptamines contracted trachea with lower potency (-log EC50 < 5) than 5-HT (-log EC50 = 6.98). Maximum contraction to both 5-MT and DP-5-CT was only 50 to 80% of the maximum response to 5-HT, suggesting that these compounds were partial agonists relative to 5-HT. Guinea pig tracheal contraction to 5-HT, 5-MT and DP-5-CT was inhibited by 1-(1-naphthyl)piperazine (1-NP, 30 nM). The apparent antagonist dissociation constants (KB) for 1-NP were similar when 5-HT (-log KB, K = 8.70 +/- 0.17) or 5-MT (- log KB = 8.40 +/- 0.12) were used as agonists. Although 1-NP also blocked tracheal contraction to DP-5-CT, solubility limitations with DP-5-CT did not permit calculation of a KB value for 1-NP. Nevertheless, these data indicate that 5-MT and DP-5-CT interact with the same receptor as 5-HT. As antagonists, both 5-MT and DP-5-CT inhibited tracheal contraction to 5-HT, indicating interaction with the same receptor as 5-HT and confirming partial serotonergic agonist activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic and Equilibrium Properties of Hemoglobin Kansas

Abstract The reactions of hemoglobin Kansas (102 β Asn → Thr) have been examined by kinetic and equilibrium methods. The behavior of the T state toward oxygen, derived from oxygen binding and oxygen pulse experiments, appears similar to that of hemoglobin A. The behavior of the R tetramer is difficult to separate from that of the dimer (the dissociation constant, K4,2, for oxyhemoglobin Kansas is about 70 µm as against 1.5 µm for hemoglobin A), but correlation of kinetic and equilibrium data suggests that the R tetramer of hemoglobin Kansas has a lower affinity for oxygen than that of hemoglobin A. Carbon monoxide binding is slower than for hemoglobin A, chiefly because of a lower rate of binding by the β chains. Comparisons between carbon monoxide binding and release of 8-hydroxypyrene-1,3,6-trisulfonic acid, and reaction with p-hydroxymercuribenzoate suggest that a conformation change (T → R transition) occurs on ligand binding as with hemoglobin A. Detailed examination of the dissociation reactions from the liganded dimers of oxygen and of carbon monoxide shows abnormally rapid dissociation from the β chain. The hemoglobin-haptoglobin reaction shows that deoxyhemoglobin Kansas is too slightly dissociated to give a measurable reaction. This result is consistent with an abnormally low ligand affinity of the R form. It is suggested that the differences between hemoglobin Kansas and hemoglobin A are due, at least in part, to differences between the R forms, and that an R → T transition occurs normally in hemoglobin Kansas. The allosteric effector 2,3-diphosphoglycerate lowers the affinity of hemoglobin Kansas for oxygen, but the effect is much smaller than for hemoglobin A when measured at high concentrations of hemoglobin. The data are most consistent with the assumption that the binding constant for 2,3-diphosphoglycerate at the primary site on tetrameric, liganded hemoglobin Kansas is only slightly lower than that for the unliganded form.

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.

Relation between allosteric effects and changes in the energy of bonding between molecular subunits

Allosteric effects (dependencies of the ligand affinity of certain specific ligand binding sites on the state of other ligand binding sites) which occur in a protein with multiple subunits but not in the isolated subunits can all be expressed in terms of changes in the inter-subunit bonding energy that must occur during ligand attachment. Co-operative allosteric interactions such as the heme-heme interaction in hemoglobin require that the change in inter-subunit bonding energy accompanying the attachment of a ligand be not uniform but depend rather on the extent to which the protein is already liganded. It is suggested that changes in the energy of subunit bonding may be the primary co-operative parameter. With these considerations in mind, the equilibrium and kinetic constants for the reactions of hemoglobin and of the isolated chains of hemoglobin with oxygen and carbon monoxide are compared and some conclusions are drawn about the structural basis of the heme—heme interaction. At pH 7 and 20 °C the interaction energy between the chains in hemoglobin decreases by 8 kcal./mole hemoglobin when the molecule is saturated with ligand. Since the ligand reactivity of fully liganded hemoglobin is very similar to that of the isolated chains, it is postulated that in deoxyhemoglobin the chains exist in a strained conformation which has a low ligand affinity but permits a higher inter-chain interaction energy. The presence of a ligand raises the free energy of this conformation and therefore reduces the interchain bonding energy.