Multiplicity of interactions between dromedary hemoglobin and solvent components. A structural and functional study

The interaction of dromedary hemoglobin with various solvent components [2-(p-chlorophenoxy)-2-methylpropionic acid (CFA), 2,3-bisphospho-D-glycerate (glycerate-2,3-P2) and chloride] has been studied. 1. CFA greatly lowers the oxygen affinity of dromedary hemoglobin. 2. The oxygen-linked CFA binding sites are probably located in the deoxy derivative at the alpha cleft, while in the oxy form and in the presence of two other effectors (glycerate-2,3-P2 and chloride) additional, structurally and possibly functionally relevant binding site(s) should be considered. 3. Both CFA and glycerate-2,3-P2 stabilize the deoxy-like tertiary structure in the oxy derivative. 4. Chloride appears to be fundamental to obtain quaternary structural changes. 5. Interaction energy, retained in the protein when the three ligands (CFA, glycerate-2,3-P2 and chloride) are bound to the oxy form, favours intermediates not stable if only one or two allosteric effector(s) is (are) present on the protein. 6. The oxygen affinity appears to be related to both tertiary and quaternary structural changes, while cooperatively is largely invariant with solvent conditions. In conclusion, the functional properties of dromedary hemoglobin do not depend in any simple way on the variety of stabilized conformations.     

Allosteric interactions in sipunculid and brachiopod hemerythrins

Chemical and spectroscopic consequences of allosteric interactions for ligand binding to sipunculid (Phascolopsis gouldii) and brachiopod (Lingula reevii) hemerythrins (Hrs) have been investigated. Possible allosteric effectors for homotropic effects in sipunculid Hrs have been examined, but only reduction in ligand affinity is observed without cooperativity. In contrast to sipunculid Hr, L. reevii Hr binds O2 cooperatively in the pH range 7-8 and exhibits a Bohr effect. Spectroscopic comparisons of the sipunculid and brachiopod Hrs show no significant differences in the active site structures; therefore, modulation of oxygen affinity is attributable to effects linking the site to quaternary structural changes in the octamer. Oxygen equilibria can be fit with a conformational model incorporating a minimum of three states, tensed (T), relaxed (R), and an R-T hybrid. Resonance Raman spectra of L. reevii oxyHr show a shift in the peroxo stretching frequency when the pH is lowered from pH 7.7 (predominantly R oxyHr) to pH 6.3 (a mixture of R, T, and R-T hybrid), but P. gouldii Hr does not have a frequency shift under the same conditions. In contrast to hemoglobins, ligand binding to the deoxy and met forms is noncooperative for brachiopod (and sipunculid) Hrs. It is thus suggested that conformational changes in the protein are linked to the oxidation state change that accompanies oxygenation of the coupled binuclear iron site (deoxy [FeIIFeII]----oxy [FeIIIFeIII]). The total allosteric energy expended in oxygenation is about 1.4 kcal/mol, and such a shift is possible in the relaxed-tense conversion with relatively limited constraints of the iron coordination environment via the protein quaternary structure. The mechanism of cooperativity in the binuclear copper oxygen carrier hemocyanin is discussed in light of these results.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

A stopped-flow study of the cupric ion oxidation of adult-human haemoglobin.

The addition of excess Cu2+ to adult human haemoglobin leads to the production of alpha 2(2+) beta 2(3+), in both the oxy and deoxy forms of the protein. Stopped-flow studies of the oxidation process yields apparent second-order rate constants of 196M-1 X S-1 and 41M-1 X S-1 for the deoxy and oxy forms respectively. The rate of the deoxy-form oxidation is linearly dependent on [Cu2+], whereas that of the oxy form is rate-limited above 2 mM to 0.11 S-1. Arrhenius activation energies of the two processes are almost identical at 91 kJ X mol-1, as are the activation enthalpies of 89 kJ X mol-1. The activation entropies show small differences, being 31 entropy units and 48 entropy units for the oxy and deoxy forms respectively. ATP and glycerate 2,3-bisphosphate at saturating concentrations do not affect the rate of oxidation of the oxy form, but halve the rate found for the deoxy form. These data are discussed in terms of the previously proposed mechanism of oxidation in which slow Cu2+ binding is followed by rapid electron transfer.     

Circular dichroism and spin-label studies of carp hemoglobin

Circular dichroism (c.d.) spectra were obtained for deoxy, oxy, carboxy, nitrosyl, aquomet and azidomet derivatives of carp hemoglobin. The spectra of the hemolysate and its two major components are virtually identical. Binding of diatomic ligands induces large changes in the 287 nm ellipticity. In the case of oxygen binding this change appears to be proportional to the free energy of co-operation. The changes of L-band ellipticity and Soret rotational strength with ligation reflect tertiary structural alterations and bear no relationship to quaternary transitions. The c.d. results indicate that carp deoxyhemoglobin has very similar tertiary and quaternary structures between pH 6·4 and 8·0, whereas the oxyhemoglobin undergoes continuous conformational adjustment in response to pH changes. The effect of inositol hexaphosphate on c.d. spectra is much smaller than it is on the functional properties. Electron paramagnetic resonance spectra of iodoacetamide nitroxide label are sensitive to ligation, the label is probably attached to Cys142β.     

A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.     

A plausible sequence of the conformational changes of hemoglobin induced by oxygenation.

Recent experimental data of oxygen equilibrium constants of human adult hemoglobin, which are measured over a wide range of oxygen pressures, are analyzed successfully from the viewpoint that the change in the molecular structure of hemoglobin induced by oxygenation is considered individually at each stage of oxygenation. Then, a simple phenomenological rule, which explains quantitatively the values of the four Adair constants with only three parameters, is found for hemoglobin under normal physiological conditions. The temperature dependence of these parameters suggests a sequence of the conformational changes such that until the third stage of oxygenation the conformational changes occur within the deoxy quaternary structure and at the fourth stage of oxygenation the deoxy quaternary structure is altered to the oxy one. The effects of pH and phosphate compounds on the Adair constants are discussed, and a possible modification and extension of the rule is suggested. The connection between the rule and the molecular structures of deoxy- and oxyhemoglobin is also discussed.     

Comparison of the binding of Na+ and Ca2+ to bovine alpha-lactalbumin

alpha-Lactalbumin is a metal-binding protein which binds Ca2+- and Na+-ions competitively to one specific site, giving rise to a large conformational change of the protein. For this reason, the enthalpy change of binding Ca2+ to apo-alpha-lactalbumin (delta Ho) is strongly dependent on the concentration of Na+ ions in the medium. From that relationship a molar enthalpy of -145 +/- 3 kJ X mol-1 is calculated for the Ca2+-binding at pH 7.4 and 25 degrees C, while a delta Ho of -5 +/- 3 kJ X mol-1 is found to substitute a complexed Na+ by a Ca2+-ion. These measurements also allowed us to calculate a binding constant for Na+ of 195 +/- 18 M-1. The molar enthalpy of Na+-loading was found to be -142 +/- 3 kJ X mol-1, a value very close to delta Ho of the binding of Ca2+ to alpha-lactalbumin. Both enthalpy changes in binding Ca2+ and Na+ are independent of the protein concentration. These exothermic values are in agreement with the hypothesis that both Na+- and Ca2+-ions are able to induce the same conformational change in alpha-lactalbumin upon which hydrophobic regions are removed from the solvent, yielding a less hydrophobic protein. The latter is confirmed by means of affinity measurements of the hydrophobic fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulphonate](bis-ANS) to alpha-lactalbumin. The association constant (Ka) decreased from (6.6 +/- 0.5) X 10(4) M-1 in the absence of NaCl to (2.7 +/- 0.2) X 10(4) M-1 in 75 mM NaCl, while the maximum intensity (Imax) of the binary bis-ANS-alpha-lactalbumin complex remained constant at 0.44 +/- 0.02 (arbitrary units). The Ka value of bis-ANS for Ca2+-alpha-lactalbumin was determined at (1.7 +/- 0.2) X 10(4) M-1 and Imax was 0.43 +/- 0.02 (arbitrary units). The difference in hydrophobicity between the two conformational states of the protein was further demonstrated by adsorption experiments of both conformers to phenyl-Sepharose. Apo-alpha-lactalbumin, hydrophobically bound to phenyl-Sepharose, can be eluted by adding Ca2- or Na+-solutions.     

Recombinant hemoglobins with low oxygen affinity and high cooperativity

By introducing an additional H-bond in the alpha(1)beta(2) subunit interface or altering the charge properties of the amino acid residues in the alpha(1)beta(1) subunit interface of the hemoglobin molecule, we have designed and expressed recombinant hemoglobins (rHbs) with low oxygen affinity and high cooperativity. Oxygen-binding measurements of these rHbs under various experimental conditions show interesting properties in response to pH (Bohr effect) and allosteric effectors. Proton nuclear magnetic resonance studies show that these rHbs can switch from the oxy (or CO) quaternary structure (R) to the deoxy quaternary structure (T) without changing their ligation states upon addition of an allosteric effector, inositol hexaphosphate, and/or reduction of the ambient temperature. These results indicate that if we can provide extra stability to the T state of the hemoglobin molecule without perturbing its R state, we can produce hemoglobins with low oxygen affinity and high cooperativity. Some of these rHbs are also quite stable against autoxidation compared to many of the known abnormal hemoglobins with altered oxygen affinity and cooperativity. These results have provided new insights into the structure-function relationship in hemoglobin.     

Comparison of cyclic nucleotide binding to adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate-dependent protein kinases.

Binding sites for [3H]cAMP on purified regulatory dimers of type II A-kinase (II-R2) are independent as assessed by equilibrium binding (KD = 6 +/- 1.3 nM at pH 7.2, 25 degrees; nH = 1.0) and by the lack of effect of unlabeled cAMP on dissociation rate (kd = 1.0 X 10(-3) sec -1 at pH 7.2, 25 degrees). In contrast, binding sites for [3H]cGMP on purified G-kinase displayed positively cooperative interactions in both equilibrium and dissociation assays with convex upward Scatchard plots, an nH of 1.6 and a dissociation rate (kd = 6.2 X 10(-3) sec-1 at pH 6.8, 0 degree) which was slowed by excess unlabeled cGMP (kd = 1.13 X 10(-3) sec-1 at pH 6.8, degree). Calculated transition state free energies of dissociation revealed that dissociation of nucleotide from G-kinase in the presence of cGMP was restrained by an energy barrier (20.8 kcal.mol-1) similar to that of II-R2 (20.9 kcal.mol-1), whereas dissociation from G-kinase without excess nucleotide occurred more easily (18.9 kcal.mol-1).     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

The conformational and dynamic basis for ligand binding reactivity in hemoglobin Ypsilanti (beta 99 asp-->Tyr): origin of the quaternary enhancement effect

Hemoglobin Ypsilanti (HbY) is a stable tetrameric hemoglobin that binds oxygen with little or no cooperativity and with high affinity [Doyle, M. L., et al. (1992) Proteins: Struct., Funct., Genet. 14, 351-362]. It displays an especially large quaternary enhancement effect. An X-ray crystallographic study [Smith, F. R., et al. (1991) Proteins: Struct., Funct., Genet. 10, 81-91] of the carboxy derivative of this hemoglobin (COHbY) revealed a new quaternary structure that partially resembles the recently described R2 structure [Silva, M. M., et al. (1992) J. Biol. Chem. 267, 17248-17256]. Very little is known about either the solution phase conformations of the liganded and deoxy forms of HbY or the molecular basis for the large quaternary enhancement effect (Doyle et al., 1992). In this study, near-IR absorption, Soret-enhanced Raman, and UV (229 nm) resonance Raman spectroscopies are used to probe the liganded and deoxy derivatives of HbY in solution. Nanosecond time-resolved near-IR absorption measurements are used to expose the relaxation properties of the photoproduct of COHbY. Time-resolved (Soret band) absorption is used to generate the geminate and solvent phase ligand rebinding curves for photodissociated COHbY. The spectroscopic results indicate that COHbY has an R-like conformation with respect to both the proximal heme pocket and the hinge region of the alpha 1 beta 2 interface. The deoxy derivative of HbY has spectroscopic features that are very similar to those observed for species assigned to the deoxy R or half-liganded R conformations of human adult hemoglobin (HbA). The 10 ns to 100 micros relaxation properties of the photoproduct of COHbY are distinctly different from those of HbA in that for HbY, little if any tertiary or quaternary relaxation is observed. The near-absence of relaxation in the HbY photoproduct explains the differences in the geminate and solvent phase CO recombination between HbA and HbY. The impact of the conformational and relaxation properties of HbY on the geminate rebinding process forms the basis of a model that accounts for the large quaternary enhancement effect reported for HbY (Doyle et al., 1992). In addition, the spectroscopic data and the X-ray crystallographic results explain the slow relaxation for HbY and the near-absence of cooperative ligand binding for this protein based on the behavior of the penultimate tyrosines.     

Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins

A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, base-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol(-1), n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer-dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (deltaGc)--the energy barrier between the allosteric transition from deoxy to oxy hemoglobin.     

Thermodynamic analysis of heparin binding to human antithrombin.

The binding of heparin to human antithrombin III (ATIII) was investigated by titration calorimetry (TC) and differential scanning calorimetry (DSC). TC measurements of homogeneous high-affinity pentasaccharide and octasaccharide fragments of heparin in 0.02 M phosphate buffer and 0.15 M sodium chloride (pH 7.3) yielded binding constants of (7.1 +/- 1.3) x 10(5) M-1 and (6.7 +/- 1.2) x 10(6) M-1, respectively, and corresponding binding enthalpies of -48.3 +/- 0.7 and -54.4 +/- 5.4 kJ mol-1. The binding enthalpy of heparin in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.3) was estimated from TC measurements to be -55 +/- 10 kJ mol-1, while the enthalpy in Tris buffer (0.02 M, 0.15 M NaCl, pH 7.3) was -18 +/- 2 kJ mol-1. The heparin-binding affinity was shown by fluorescence measurements not to change under these conditions. The 3-fold lower binding enthalpy in Tris can be attributed to the transfer of a proton from the buffer to the heparin-ATIII complex. DSC measurements of the ATIII unfolding transition exhibited a sharp denaturation peak at 329 +/- 1 K with a van 't Hoff enthalpy of 951 +/- 89 kJ mol-1, based on a two-state transition model and a much broader transition from 333 to 366 K. The transition peak at 329 K accounted for 9-18% of the total ATIII. At sub-saturate heparin concentrations, the lower temperature peak became bimodal with the appearance of a second transition peak at 336 K. At saturate heparin concentration only the 336 K peak was observed. This supports a two domain model of ATIII folding in which the lower stability domain (329 K) binds and is stabilized by heparin.     

Binding characteristics of a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine, to bovine serum albumin as measured by fluorescence

The interaction between a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine and bovine serum albumin was investigated by fluorescence measurements. The apparent binding constants were obtained at various pHs assuming the equivalence and independence of the interaction sites on the protein from the fluorescence titration curves. The maximum binding was attained at pH 8.0, and the apparent binding constant was (5.28 +/- 0.13).10(5) M-1 with one binding site per albumin molecule. Thermodynamic parameters were also determined from the van't Hoff plot of the apparent binding constants at pH 7.5. The free energy change, enthalpy change and entropy change were -7.70 +/- 0.09 kcal.mol-1, -4.59 kcal.mol-1 and 10.2 e.u., respectively.     

Transient kinetics of oxygen dissociation from ferrous subunits of iron-cobalt hybrid hemoglobins. The principal reaction controlling the co-operativity

The oxygen dissociation constants from Fe subunits in the half-ligated intermediate states of Fe-Co hybrid hemoglobins, alpha(Fe-O2)2 beta(Co)2 and alpha(Co)2 beta(Fe-O2)2, have been determined as functions of pH, temperature and inositol hexaphosphate. The oxygen dissociation rates from alpha(Fe-O2)2 beta(Co)2 are estimated to be more than 1300 s-1 for the deoxy quaternary state (T-state) and less than 3 s-1 for the oxy quaternary state (R-state) at 15 degrees C in 50 mM-Tris or Bis-Tris buffer containing 0.1 M-Cl-, while those of alpha(Co)2 beta(Fe-O2)2 are more than 180 s-1 and less than 5 s-1 for the T and R-states, respectively. The pH dependence of the oxygen dissociation rate from Fe subunits is large enough to be accounted for by the R-T transition, and implies that those half-ligated intermediate hybrids mainly exist in the R-state at pH 8.8, and in the T-state at pH 6.6, while other studies indicated that the half-ligated hybrids are essentially in the R-state at pH 7. Large activation energies of the oxygen dissociation process of 19 to 31 kcal/mol determined from the temperature dependence suggest that the process is entropy-driven.     

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional and spectroscopic characterization of half-liganded iron-zinc hybrid hemoglobin: evidence for conformational plasticity within the T state

Functionally distinct conformations of HbA (human adult hemoglobin) were probed using deoxy and diliganded derivatives of symmetric Fe-Zn hybrids of HbA. To expand the range of accessible structures, different environments were utilized including solution, sol-gel encapsulation, and crystals. Further structural and functional modulation was achieved by the addition of allosteric effectors. Functional characterization included oxygen affinity measurements, CO combination rates, and geminate and bimolecular CO recombination, after photodissociation. The conformational properties were studied using visible resonance Raman spectroscopy as a probe of local tertiary structure at the iron-containing hemes and UV resonance Raman spectroscopy as a probe of elements of the globin known to be sensitive to quaternary structure. The combined results show a pattern in which there is a progression of conformational and functional properties that are consistent with a picture in which the T quaternary structure can accommodate a range of tertiary conformations (plasticity). At one end of the distribution is the equilibrium deoxy T state conformation that has the lowest ligand reactivity. At the other end of the distribution are T state conformations with higher ligand reactivity that exhibit "loosened" T state constraints within the globin including the alpha(1)beta(2) interface and reduced proximal strain at the heme.     

Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

Pressure denaturation of beta-lactoglobulin. Different stabilities of isoforms A and B, and an investigation of the Tanford transition.

Beta-lactoglobulin, the main whey protein in bovine milk, exists in several isoforms of which the most abundant are isoforms A and B. We have previously reported the denaturation of beta-lactoglobulin A by hydrostatic pressure [Valente-Mesquita, V.L., Botelho, M.M. & Ferreira, S.T. (1998) Biophys. J. 75, 471-476]. Here, we compare the pressure stabilities of isoforms A and B. These isoforms differ by two amino-acid substitutions: Asp64 and Val118 in isoform A are replaced by glycine and alanine, respectively, in isoform B. Replacement of the buried Val118 residue by the smaller alanine side-chain is not accompanied by significant structural rearrangements of the neighbouring polypeptide chain and creates a cavity in the core of beta-lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (DeltaVunf) of - 49 +/- 8 mL.mol-1 and -75 +/- 3 mL.mol-1, and unfolding free energy changes (DeltaGunf) of 8.5 +/- 1.3 kJ.mol-1 and 11.3 +/- 0.4 kJ.mol-1 were obtained for isoforms A and B, respectively. The volume occupied by the two methyl groups of Val118 removed in the V118A substitution is approximately 40 A3 per monomer of beta-lactoglobulin, in excellent agreement with the experimentally measured difference in DeltaVunf for the two isoforms (DeltaDeltaVunf = 26 mL.mol-1, corresponding to approximately 43 A3 per monomer). Thus, the existence of a core cavity in beta-lactoglobulin B may explain its enhanced pressure sensitivity relative to beta-lactoglobulin A. beta-Lactoglobulin undergoes a reversible pH-induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of beta-lactoglobulin A at pH 7 and 8. Unfolding free energy changes of 8.5 +/- 1.3 and 8.3 +/- 0.3 kJ.mol-1 were obtained at pH 7 and 8, respectively, showing that the thermodynamic stability of beta-lactoglobulin is identical at these pH values. Interestingly, DeltaVunf was dependent on pH, and varied from -49 +/- 8 mL.mol-1 to -68 +/- 2 mL.mol-1 at pH 7 and 8, respectively. The large increase in DeltaVunf at pH 8 relative to pH 7 appears to be associated with an overall expansion of the protein structure and could explain the increased pressure sensitivity of beta-lactoglobulin at alkaline pH.